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Notes: Background.  Detection of Helicobacter pylori infection in atrophic body gastritis (ABG) is difficult, as during progression of body atrophy, H. pylori disappears.Aim.  To increase the diagnostic yield of detection of active H. pylori infection in atrophic body gastritis patients by using noninvasive tests such as 13C-Urea Breath Test (13C-UBT) and H. pylori stool antigen test (HpSA) would be useful.Patients.  27 consecutive patients with newly-diagnosed atrophic body gastritis (19F/7M, age 27–73 years).Methods.  Gastroscopy with biopsies (antrum n = 3, body n = 3) and histology according to updated Sydney system, H. pylori IgG serology, 13C-UBT, and HpSA.Results.  All tests used in the diagnosis of H. pylori infection were in agreement in 9/27 atrophic body gastritis patients (33.3%), being all positive in four (14.8%) and all negative in five patients (18.5%). Ten of the 27 (37%) patients were Giemsa stain-positive and serology-positive (group I). Seventeen of the 27 (63%) patients were Giemsa stain-negative: 5/17 with positive serology (group II) and 12/17 with negative serology (group III). In group I, 5/10 (50%) were 13C-UBT positive and 4/10 (40%) HpSA positive. In group II, two patients were 13C-UBT positive, but all were HpSA negative. Also in group III, all patients were HpSA negative, but one had a positive 13C-UBT.Conclusions.  In atrophic body gastritis patients, neither 13C-UBT nor HpSA per se add useful information regarding active H. pylori infection, but these noninvasive tests may be important in combination with histology and serology to define the H. pylori status in some atrophic body gastritis patients.

Notes: Background.  Because of the increasing resistance to clarithromycin and metronidazole, two of the antibiotics used for the eradication of Helicobacter pylori, new therapeutic alternatives are needed. The aim of this study was to determine the efficacy of a randomized, comparative trial of 7 vs. 14-day triple treatment with rabeprazole, ofloxacin and amoxicillin for H. pylori eradication.Material and Methods.  The present authors studied 76 dyspeptic patients infected with H. pylori diagnosed by both histology and a rapid urease test. Patients were randomized to receive rabeprazole (20 mg b.i.d.), plus ofloxacin (400 mg b.i.d.) and amoxicillin (1000 mg b.i.d.) for 7 days (group 1) vs. 14 days (group 2) and were followed by 6 weeks. Eradication was assessed 4 weeks after completing the course of study treatment by the 14C-urea breath test. Per protocol and intention-to-treat eradication rates were determined.Results.  For the intention to treat analysis, the eradication rate was 62.2% for group 1 and 92.3% for group 2 (p = .004). For the per protocol analysis, eradication rate for group 1 was 63.9% and for group 2 was 97.3% (p = .001).Conclusions.  Triple therapy with rabeprazole, amoxicillin and ofloxacin by 14 days was efficient for H. pylori eradication and therefore deserves further study. The same regimen prescribed for 7 days had a significantly lower and unacceptable cure rate and should not be used.

Notes: Background.  Duodenal ulcer in adults chronically infected with Helicobacter pylori is associated with a polarized T-helper cell type 1 (Th1) mucosal immune response, with a predominantly immunoglobulin G2 (IgG2) systemic specific response. It has been suggested that children colonized by H. pylori also produce a mucosal Th1 response, but there are few studies that have measured IgG subclass responses in children with duodenal ulcer.Materials and methods.  Seven children with endoscopically proven duodenal ulcer and H. pylori infection and 18 children with biopsy proven H. pylori infection but no duodenal ulcer had relative concentrations of IgG subclass responses (IgGsc) against H. pylori antigens measured by ELISA. Eighteen IgG seropositive adults acted as controls. The range of antigens recognised by IgG1 and IgG2 subclass responses were investigated by Western blots.Results.  There were no differences in mean IgGsc responses between children with or without duodenal ulcer. Adults produced an IgG2 predominant response. Western blots showed no qualitative differences in antigens recognised by IgG1 or IgG2.Conclusion.  Children with duodenal ulcer, in contrast to adults, produce an IgGsc response consistent with a mucosal Th2 response to H. pylori regardless of the presence of duodenal ulceration. This suggests that disease causation amongst children with H. pylori associated duodenal ulceration may not be dependant upon a mucosal Th1 biased response.

Notes: Data are accumulating on the association between Helicobacter pylori infection and idiopathic thrombocytopenic purpura (ITP) and the significant increase in platelet count after bacterial eradication. The aim of this review was to consider the studies so far published on H. pylori infection and ITP in order to evaluate a possible correlation between these two conditions. A review of the literature showed that 278 out of the 482 ITP patients investigated (58%) were positive for H. pylori infection and that the bacterium was eradicated in 88% of cases. Eradication therapy was accompanied by a complete or partial platelet response in approximately half the cases. Overall, these data show that H. pylori eradication in patients with ITP is effective in increasing platelet count. However, because the studies so far published are few, are sometimes controversial and involve small series of patients, further studies on larger numbers of patients with longer follow-up are needed to confirm these preliminary findings.

Notes: Background.  Data on the efficacy of eradication treatment for Helicobacter pylori gastritis in children are scarce.Aim.  To evaluate the efficacy of triple therapy with lansoprazole plus amoxicillin and tinidazole vs. dual therapy with amoxicillin and tinidazole in a double-blind randomized multicentre trial, and the usefulness of eradication in terms of long-term symptom resolution.Subjects.  We enrolled 43 consecutive children undergoing endoscopy for upper gastrointestinal dyspepsia with H. pylori gastritis. They underwent a 13C-urea breath test, completed a 2-week symptom diary card, and were randomized. Treatment was given in a Redidose box (Redidose Company Ltd., Brighton, UK) containing either lansoprazole-amoxicillin-tinidazole (triple therapy) or placebo plus amoxicillin-tinidazole (dual therapy) for 1 week. The completion of a 2-week symptom diary card and the performance of a breath test were repeated 6 weeks and 6 months after the end of therapy. One to two years later, a structured telephone interview was conducted with 36 of the children.Results.  According to the breath test, 6 weeks after the end of therapy H. pylori was eradicated in 15 of 22 children on triple therapy [68.2%; 95% confidence interval (CI) = 45–88] and in 15 of 21 children on dual therapy (71%; 95% CI = 48–89; not significant), and 6 months after the end of therapy it was eradicated in 16 of 22 children on triple therapy (72.7%) and in 15 of 21 children on dual therapy. Six months after therapy, symptoms were analysed in 11 H. pylori-positive and 31 H. pylori-negative children, and it was found that dyspeptic symptoms had disappeared or improved in both groups, with no difference between them. One to two years later, 36 children were interviewed. Epigastric pain had recurred in three of 26 H. pylori-negative and in seven of 10 H. pylori-positive children (p = .001); in three of the latter, pain was severe and required additional treatment.Conclusion.  One-week triple or dual therapy with two antibiotics achieved similar eradication rates. Soon after treatment, symptoms disappeared or improved in most children irrespective of eradication, but epigastric pain recurred in the majority of the still-infected children within 2 years.

Notes: Background.  Infection with Helicobacter pylori is associated with an increased risk of gastric cancer. Several studies have indicated that the association differs with strain type. We aimed to find out if infection with strains lacking the virulence factor CagA is linked to gastric cancer risk.Materials and methods.  In a hospital-based case–control study, we collected sera from 100 case patients with a newly diagnosed gastric adenocarcinoma and 96 control patients with diseases unrelated to H. pylori status. Antibodies to H. pylori were analyzed by enzyme-linked immunosorbent assay (ELISA), and antibodies to CagA were detected by immunoblot. Logistic regression was used to obtain odds ratios (ORs) as estimates of relative risk, adjusted for potential confounding.Results.  Among the case patients, 81% were ELISA positive and 86% had antibodies to CagA. The corresponding numbers among the controls were 58% and 55%, respectively. ELISA positivity was associated with an increased risk of gastric adenocarcinoma compared to ELISA negativity (OR for gastric cancer regardless of site 3.9, 95% CI 1.9–8.2). The OR was 7.4 (95% CI 3.3–16.6) for CagA-positive relative to CagA-negative subjects. Among ELISA-positive subjects the presence of CagA antibodies increased the risk 3.6 times (95% CI 1.2–11.1). ELISA-positive CagA-negative infections were associated with a fourfold increased risk (OR = 4.2, 95% CI 1.0–17.0) compared to no infection (ELISA-negative and CagA-negative).Conclusions.  Although patients with antibodies to CagA have the greatest risk of developing gastric cancer, those with CagA-negative infections run a significantly greater risk than uninfected persons.

Notes: Abstract:  Mammary fistulas occurring in males are extremely rare, the first being described in 1974 by Tedeschi et al. (1). Our detailed review of the world literature has revealed a total of just 20 cases of mammary fistulas in males; a total which includes three cases of mammary fistulas in infants and three cases associated with human immunodeficiency virus (HIV) infection. Mammary fistulas develop between the lactiferous ducts and areolar skin. Altered duct anatomy and microarchitecture may predispose a patient to mammary fistulas. This discussion focuses on mammary fistulas in males where smoking is a likely etiologic factor, and therefore examines a clinical rarity with just 14 reported cases in the literature. The treatment of mammary fistulas in males is the same as for females once the diagnosis has been made.

Notes: Abstract:   The goal of this study was to evaluate the periareolar injection of technetium 99m sulfur colloid to identify axillary sentinel nodes and compare the number of sentinel lymph nodes identified with preoperative lymphoscintigraphy to intraoperative biopsy using a handheld gamma probe. A total of 104 consecutive patients diagnosed with invasive breast cancer participated in this prospective study, with 81 patients receiving an intradermal periareolar injection and 23 patients receiving an intradermal peritumoral injection of filtered technetium 99m sulfur colloid. Preoperative lymphoscintigraphy was performed for sentinel node mapping and localization. In addition to selective sentinel node biopsy, axillary dissection was performed on all patients to determine false-negative rates. Routine histologic staining was performed on all identified nodes, along with immunohistochemical staining of sentinel nodes negative on initial routine staining. With an intradermal periareolar injection, the sentinel node identification rate was 91.4% (74/81), axillary metastatic rate 35.1% (26/74), sentinel node positive only 61.5% (16/26), and false negative 3.8% (1/26). With an intradermal peritumoral injection, the sentinel node identification rate was 91.3% (21/23), axillary metastatic rate 42.9% (9/21), sentinel node positive only 88.9% (8/9), and false negative 0% (0/9). A total of 241 sentinel nodes were identified with biplanar lymphoscintigraphy and 173 sentinel nodes were harvested during surgery, yielding a 28.2% increase in sentinel nodes identified with lymphoscintigraphy. This study demonstrates that intradermal periareolar injection of filtered technetium 99m sulfur colloid is successful in identifying axillary sentinel nodes with a low false-negative rate. Preoperative lymphoscintigraphy aids in the identification and surgical planning of sentinel node biopsy and provides an objective measure of surgical performance. 

Notes: Abstract:   Determinants of prognosis were studied in patients with breast cancer with histologically proven tumor extension to the skin without clinical evidence of distant metastases (i.e., pT4b N0–3 M0). Data were collected retrospectively on 77 consecutive patients diagnosed in one community teaching hospital over the period from 1980 to 1995. The prognostic factor of tumor size showed a 5-year survival rate for patients with a tumor ≤3 cm of 81% compared to 45% for patients with tumors larger than 3 cm (p = 0.002). Achievement of complete remission resulted in a 5-year survival rate of 66%, compared to 27% when complete remission was not achieved (p = 0.005). Another important prognostic factor was the development of local-regional recurrence: the 5-year survival rates for patients with and without local-regional recurrence were 39% and 87%, respectively (p 〈 0.001). Development of local-regional recurrence was also significantly related to tumor size (p = 0.02). Pathologic tumor size and the achievement of complete remission and local-regional control appear to be the most important prognostic factors for survival in patients with pT4b breast cancer without distant metastases. We conclude that the finding of a pT4b breast cancer does not always imply a dismal prognosis, especially for those patients with a tumor ≤3 cm. A favorable prognosis can be expected when treatment is effective in achieving complete remission and in preventing the development of local-regional recurrence. 

Notes: Abstract:   The sentinel lymph node biopsy (SLNB) procedure is an alternative method for assessing the axillary lymph node (ALN) status in patients with breast cancer. The SLNB carries the risk of a false-negative result, with patients harboring positive ALNs in the face of a negative SLNB examination. In addition, the significance of a SLNB with cells identified only with keratin or with deposits less than 0.2 mm remains unresolved. We analyzed our SLNB data over the past 5 years in order to determine the relationship between SLN tumor burden and ALN tumor burden. Pathology files for the past 5 years at Magee-Womens Hospital were searched for all SLNB cases that had an axillary lymph node dissection (ALND). Each SLNB case was reviewed and tabulated for breast tumor size, SLN tumor size, and largest tumor size in the ALND. Correlation and frequency distribution were performed for the status of all SLNs and ALNDs. Patterns of lymph node metastasis were recorded and the sizes of the SLN metastases were reported according to the recent Philadelphia Consensus Conference on Sentinel Lymph Nodes and the revised American Joint Committee on Cancer (AJCC) staging. SLN metastases were classified as immunohistochemistry (IHC) positive if only single keratin-positive cells or clusters were present and were not observed with standard tissue stains, as submicrometastatic (SMM) if tumors were less than 0.2 mm (excluding IHC positive), as micrometastatic if tumors were larger than 0.2 mm but ≤2 mm, or as macrometastatic if tumors were larger than 2 mm. A total of 445 patients had both SLNB and ALND. Fifty percent (224/445) of cases were SLN positive, including 58 SLN positive/ALN positive cases and 166 SLN positive/ALN negative cases. Of the 221 patients in the SLN-negative group, 4 were ALN positive (false-negative SLN). The incidence of SLN metastases increased with tumor stage, with the percentage of SLN positives as follows: T1a, 2.1%; T1b, 10.9%; T1c, 51.7%; and T2, 35.3%. There were 4 of 41 patients (10%) with SLNs that were IHC positive that had macrometastases in a solitary ALN. Three of 22 patients (13.6%) that were SMM positive had ALN macrometastasis in a solitary ALN. Four of 49 patients (8.1%) with micrometastatic SLNs had a solitary positive ALN, 3 of which were macrometastases (6.1%). Overall a total of 10 of 112 patients (9.0%) with traditionally defined SLN micrometastases of 2.0 mm or less had a solitary ALN macrometastasis. The vast majority (90%) of these macrometastases were found with T1c and T2 breast tumors. There was a significant difference in the means of SLN tumor sizes for the SLN-positive/ALND-negative (4.5 mm) versus SLN-positive/ALND-positive (10.1 mm) patients, although the range of SLN tumor sizes within each group were similar. There is an increasing incidence of SLN-positive and ALN-positive cases with increasing T stage. Overall in this series, 9% of patients with SLN metastases ≤2 mm had a solitary axillary macrometastasis. Ninety percent of these metastases occurred with T1c/T2 breast tumors, indicating the important codependence of T stage. Overall there is a subset of patients who are IHC positive, SMM positive, or micrometastatic positive with ALNs that are macrometastatic who are at risk of harboring axillary macrometastases. Keratin IHC of breast SLNs is useful for defining these subsets. 

Notes: Abstract:   The increased interest in the use of neoadjuvant chemotherapy for patients with locally advanced or large tumors at initial presentation necessitates the recognition of sequelae of this therapy. This article describes the interval appearance of malignant, linear-branching, microcalcifications during neoadjuvant chemotherapy for locally advanced breast cancer. Mammographically the malignant microcalcifications appeared only in the region of the breast where the primary tumor was found. Pathologically only a subpopulation of malignant cells responded to the chemotherapy, demonstrating viability of the majority of the tumor, with cell death only in the subpopulation. With the increased use of neoadjuvant chemotherapy, it is important to recognize previously undescribed mammographic findings secondary to this therapeutic approach. 

Notes: Abstract:   By 2010, the majority of approximately 1.5 million annual new cases of breast cancer will be diagnosed in women in countries with limited resources. Public health approaches to medical problems emphasize the importance of practical, limited toxicity and very inexpensive interventions. While clinical trials in Western countries are testing the concept of breast cancer prevention, they are not defining useful public health approaches. Early detection of breast cancer using mammography, while effective, is a high-technology, expensive approach. Adjuvant systemic and radiation therapies are increasingly expensive; careful consideration of efficacy and cost-efficacy data appear warranted. Public health perspectives thus suggest that many current “standard” approaches to breast cancer in Western countries cannot help the majority of women in the world. 

Notes: Abstract:  Delay in diagnosis of breast cancer is the leading cause for malpractice lawsuits against physicians. Most cases are tried under civil law and, more specifically, the tort of negligence which defines departures from conduct of a reasonable and prudent physician under similar circumstances. The role of both the clinician and imager, separately and in concert, needs to be understood with respect to accomplishing early diagnosis and avoiding potential legal exposure. An understanding of basic legal concepts as they apply to medical practice should provide health care providers a perspective from which to apply their skills and avoid unnecessary legal exposure. 

Notes: Abstract:  Axillary sentinel lymph node biopsy (SLNB) has been adopted as a suitable alternative to traditional level I and II axillary dissection in the management of clinically node-negative (N0) breast cancers. There are two current techniques used to identify the sentinel node(s): radiopharmaceutical, technetium sulfur colloid, and isosulfan blue dye (used in the United States) and technetium-labeled albumin and patent blue dye (used in Europe). (The labeled albumin is not U.S. Food and Drug Administration [FDA] approved in the United States.) SLNB to replace axillary dissection should only be performed by surgeons and patient management teams with appropriate training and experience. Although both radiocolloid and blue dye are used together by most surgeons, and training should be in both techniques, some experienced surgeons use one or the other almost exclusively. In addition, surgical pathologists must recognize the need to examine these small specimens with great care, using a generally adopted protocol. Imprint cytology or frozen sections may be used, followed by additional sections for light microscopy. Immunochemical staining with cytokeratin or other techniques to identify “submicroscopic” metastasis is often used, but the results should not be used to influence clinical decisions with respect to adjuvant therapy. “Failed” SLNB implies the surgeon's failure to identify the sentinel nodes, in which case a complete dissection is performed. A “false-negative” SLNB implies the finding of metastasis in the excised sentinel nodes by light microscopy after a negative frozen section examination. Whether a false-negative SLNB mandates completion axillary dissection is controversial, with clinical trials currently under way to answer this question. Although SLNB was initiated to accompany breast-conserving treatment, it is equally useful in patients undergoing mastectomy. It is more difficult to perform with mastectomy. When using blue dye only, SLNB may require a separate incision because of time constraints between injection and identification of the blue-stained nodes; radiocolloid usually does not. Completion axillary dissection after false-negative SLNB is more difficult after mastectomy. SLNB is a useful procedure that may save 70% of women with clinically negative (N0) axillae and all of those with pathologically negative axillae from the morbidity of complete axillary dissection. Ideally the sentinel nodes should be able to identified in more than 95% of patients, with a false-negative rate of less than 5%. Until these rates can be achieved consistently, however, surgeons should not abandon traditional axillary dissection. 

Notes: Abstract:  Women at risk of being undertreated for breast cancer include women who are older, from minority groups, from lower socioeconomic backgrounds, and those without health insurance or insured by Medicaid. Recent reviews of the cancer care experience of medically underserved populations indicate that breast cancer care may be even less optimal for these populations than the majority of women. These are the same women who may experience difficulty obtaining access to medical care once they are diagnosed with breast cancer. Indirect proof of problems with access is manifested as higher recurrence rates of breast cancer and differences in breast cancer-specific survival among medically underserved women. Multiple factors have been shown to affect access to medical care, and therefore quality of care, including patient-level factors, provider-level factors, and health system factors. This article reviews the current state of these factors in explaining breast cancer care in medically underserved women.

Notes: Abstract:  It has been established that hormone replacement therapy (HRT) increases breast tissue density on mammography in up to 30% of women receiving treatment. The effects of selective estrogen receptor modulators (SERMs) on breast tissue have received limited attention, although there have been several reports of tamoxifen decreasing mammographic tissue density in some women undergoing adjuvant or prophylactic breast cancer treatment. We report a case of a premenopausal woman treated with tamoxifen for 5 years whose mammographic density decreased while on tamoxifen and returned to her baseline density following termination of the drug. A regression of breast tissue may be reflective of sensitivity to tamoxifen and possibly, indicative of therapeutic benefit associated with treatment. Furthermore, induction of a more radiolucent pattern by tamoxifen may independently benefit women by enhancing mammographic detection. The clinical significance of resumption of a dense breast pattern following discontinuation of tamoxifen remains to be determined. 

Notes: Abstract:  The mismatch repair (MMR) gene plays a key role in the correction of DNA damage, and the loss of MMR has been implicated in resistance to a variety of chemotherapeutic drugs. The purpose of this study was to assess whether the reduced expression of hMLH1 and/or hMSH2 affects the chemotherapeutic responsiveness of sporadic invasive ductal carcinoma of the breast. Immunohistochemical studies were performed on 71 histologic specimens of breast cancer taken from the patients treated with surgery and subsequent cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) or cyclophosphamide, Adriamycin, and 5-fluorouracil (CAF) chemotherapy for stage II or III primary breast cancer. Single-strand conformational polymorphism polymerase chain reaction (PCR-SSCP) and sequencing were carried out in 16 patients. A combined immunoreactivity score (hMLH1-IS and hMSH2-IS) was calculated by multiplying the staining grade by the intensity score. Positive expression (〉4) of hMLH1-IS and hMSH2-IS were 57.7% and 60.6%, respectively, and complete losses of hMLH1 and hMSH2 were observed in 4.2% of patients. Of the patients with advanced cancer with lymph node metastasis, those having a low hMLH1-IS had a significantly higher failure rate with the CMF regimen than those having a high hMLH1-IS (p = 0.03). No significant difference was noted in chemotherapeutic response according to hMLH1 and hMSH2 expression in the CAF group. Both hMLH1-IS (p = 0.03) and progesterone receptor (PR) status (p = 0.03) were well correlated with CMF chemotherapy response in breast cancers with lymph node metastasis. Our study shows that a lack of hMLH1 expression may play a role in drug resistance, especially in the CMF group, and immunohistochemical assay for MMR protein can be used as a convenient tool for evaluating chemotherapeutic response in patients with breast cancer. 

Notes: Abstract:  Timely reporting of mammogram results helps to reduce anxiety for women with negative results and speeds up diagnosis or treatment in the case of abnormal results. The goal of this project was to improve the rate at which Virginia mammography centers provide a written report to women in lay terms within 30 days of a mammogram. The project included six intervention and five control mammography centers. The baseline period was prior to when new regulations in the Mammography Quality Standards Act (MQSA) took effect in April 1999. The remeasurement period was after April 1999. Data were obtained from abstraction of mammography reports and patient notification letters from a sample of patients with negative and abnormal mammography results at each mammography center. Each intervention mammography center received a notebook that included numerous tools on systems for patient notification and tracking, baseline notification rates and other abstracted information, biopsy recommendations, sample results letters, and a copy of the MQSA. For negative mammograms, the intervention group in aggregate increased from 24% at baseline to 79% at remeasurement in their rate of notification within 30 days. The control group increased from 25% to 46%. For abnormal mammograms, increases were from 35% to 85% and from 25% to 58%, respectively. The intervention group's increases were considerably higher, suggesting an effect due to the interventions that involved technical assistance, education, and system change. All increases were statistically significant (p 〈 0.01). At baseline, three intervention centers and two controls had policies for written notification. All 11 had policies at remeasurement. However, only two of the five control centers could provide clear supporting documentation for dates of notification at remeasurement. Mammography centers can benefit from guidance in the form of intervention materials specifically designed to address the MQSA sections that apply to patient notification, tracking, and positive predictive value of biopsy recommendations.

Notes: Abstract:  Primary granulocytic sarcoma (GS) is a rare entity, and even more unusual is the presence of primary GS of the breast. We describe such a case and report on the 19 cases of primary breast GS in the literature. Primary GS presents most commonly in skin and lymph nodes, therefore when it presents in the breast, misdiagnosis is a common problem. Primary breast GS is misdiagnosed most frequently as lymphoma or sarcoma. Histologic testing and immunostains are essential to provide the proper diagnosis. It appears that early initiation of systemic acute myelogenous leukemia (AML)-type chemotherapy is beneficial and may delay or avert the development of AML in bone marrow and blood. 

Notes: Abstract: Patients with neurofibromatosis type I or von Recklinghausen disease develop neurofibromas in the subcutaneous tissues, including the breast. There are two German reports of massive neurofibromatosis of the breast, but few reports of massive neurofibromatosis of the breast in the English literature. We present an unusual case of neurofibromatosis of the breast with large neurofibromas predominantly noted protruding from both nipple-areolar complexes.

Notes: Abstract:  Although the relationship of various breast lesions and cancer risk is complex, and is compounded by the uniqueness of each patient, numerous clinical investigations have validated the implications of specific lesions and future risk. While variations in lesion cytology, histology, and molecular biology present a challenge to the clinician in determining disease risk and formulating a management strategy for individual patients, research has yielded several guidelines. These include patient variables, such as age and menopausal status, as well as information concerning the lesion itself. Ductal carcinoma in situ (DCIS) has emerged as the central lesion against which others must be compared. Accurate diagnosis of DCIS is integral in determining the risk of local or regional future malignancy.

Notes: Abstract:  An intriguing, yet highly logical area of research for breast cancer therapy is the exploration of growth factor pathways. Areas of investigation include epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) targeting. Although results of these studies to date have been less than promising, they have yielded important insights into critical factors that must be taken into consideration when devising growth factor-targeted therapies. Even though the results of EGFR and VEGF studies have not met with expectations, several preclinical models assessing new combinations of biologic agents with either hormonal therapy or chemotherapy are beginning to show promise.

Notes: Abstract:  Three areas of development in the surgical management of breast cancer received significant attention in 2003—breast-conserving surgery, sentinel lymph node (SLN) biopsy, and ductal lavage. Provocative investigations focusing on these controversial aspects of surgical care were presented at major national oncology meetings throughout the year. The recently published 20-year updates by the National Surgical Adjuvant Breast and Bowel Project (NSABP) and the Italian National Cancer Institute confirm the survival equivalence of breast-conserving surgery and mastectomy in early stage disease. Data reveal, however, that this strategy is underutilized in the United States when compared with other countries. A meta-analysis of close to 70 published trials on the use of SLN biopsy has revealed an overall SLN identification rate of greater than 90%, with a false-negative rate of 8.4%. Two major controversies remain to be resolved: Is there a subset of sentinel node-positive patients who may safely avoid complete axillary lymph node dissection? What is the best way integrate lymphatic mapping into neoadjuvant chemotherapy protocols? The strength of ductal lavage as a risk assessment adjunct is related to the ability to detect cellular atypia, a feature associated with a three- to fivefold increased risk for breast cancer. This technique continues to be rigorously evaluated in a number of ongoing studies.

Notes: Abstract:  Among the endocrine factors associated with breast cancer, estrogens are considered to play a central role in human breast carcinogenesis. Breast cancer risks are increased by long-term exposure to estrogens. Zeranol (Ralgro) is a nonsteroidal agent with estrogenic activity that is used as a growth promoter in the U.S. beef and veal industry. To determine whether zeranol and estradiol-17β play a role in the neoplastic transformation of human breast and to compare the estrogenic potency of zeranol to that of estradiol-17β in human breast, we treated human breast epithelial cell MCF-10A with different doses of zeranol or estradiol-17β for 10 repeated treatment cycles. By utilizing the doubling time assay, soft agar assay, and reverse transcriptase polymerase chain reaction (RT-PCR) assay, we showed that 10 repeated estradiol-17β or zeranol treatment cycles to MCF-10A cells decrease the doubling time of the cells by 30 to 40% and stimulate colony formation in soft agar and induce estrogen receptor β (ER-β) mRNA expression, all of which are not dose related in our tested dose range. Furthermore, we show that zeranol and estradiol-17β have a similar potency in the stimulation and inhibition of gene expressions in human breast cancer cell line MCF-7 by RT-PCR. These results indicate that both zeranol and estradiol-17β can induce human breast epithelial cell neoplastic transformation with similar potency in the long-term exposure through the oxidation-reduction (redox) pathway and/or ER-β-mediated pathway. 

Notes: Abstract:  Clinical and mammographic features of membranous fat necrosis (MFN) may simulate breast malignancy and tissue sampling is essential for accurate diagnosis. The aim of our study was to evaluate the clinical and imaging findings in these patients. Retrospective review of the records of breast biopsies (n = 1200) during the 5-year period 1998 to 2002 revealed eight (0.67%) cases of histologically proven MFN. Seven of the eight patients had a history of breast trauma or surgery. Seven patients underwent mammography: normal in two, a mass with curvilinear calcifications in one, and heterogeneous calcifications in four. Four patients underwent surgical excision of a palpable mass, one patient had complete excision of calcifications with large core biopsy technique, and three patients had stereotactic vacuum-assisted mammotome biopsy (VAMB). MFN should be included in the differential diagnosis of lesions in a breast with previous trauma or surgery. A minimally invasive diagnostic procedure should be considered in order to avoid excessive excisional surgery. 

Notes: Abstract:  The goal of this study was to determine the frequency of alternative therapy use in postmenopausal women with early stage breast cancer who were enrolled in a randomized clinical trial designed to determine the value of breast irradiation after treatment with breast-conserving surgery and tamoxifen. A questionnaire was given to 300 patients, ages 52 to 90 years, after completion of radiation therapy (if any). Of the 290 respondents, 78 (27%) had used some form of alternative therapy. Of these, 60.3% started after the diagnosis of breast cancer. Users of alternative therapies were significantly younger than nonusers (67.0 ± 8.4 years versus 70.0 ± 8.7 years, p = 0.009) and they used a median of one type of therapy per person (range 1–13). Users of alternative therapies were more likely to have experienced symptoms (stiffness, pain, numbness, or swelling) in the ipsilateral shoulder or arm after treatment of their breast cancers compared to nonusers (odds ratio [OR] = 2.0, p = 0.02). This relationship between alternative therapy use and symptoms was strongest in the group who started alternative therapies after breast cancer diagnosis (OR = 2.1, p = 0.05). On multivariate analysis, younger age and radiotherapy treatment were related to alternative therapy use. In conclusion, 27% of patients with early stage breast cancer used alternative therapy. Users were more likely to be younger and to experience shoulder or arm symptoms after breast-conserving surgery with radiation. 

Notes: Abstract:  This investigation was undertaken to assess the risk to the embryo/fetus associated with sentinel lymph node biopsy and lymphoscintigraphy of the breast performed in pregnant patients. Approximately 92.5 MBq (2.5 mCi) of filtered Tc-99m sulfur colloid was injected peritumorally the day before surgery in two nonpregnant women with breast cancer. The whole-body distribution of the radiopharmaceutical was evaluated using a gamma camera 1 hour after injection. We then calculated the absorbed dose to the embryo/fetus for three theoretical extreme scenarios of biodistribution and pharmacokinetics: 1) all of the injected radiopharmaceutical remains in the breast and is eliminated only by physical decay; 2) all of the injected radiopharmaceutical is instantaneously transported to the urinary bladder, where it remains and is eliminated only by physical decay; and 3) the injected radiopharmaceutical behaves as though it were administered intravenously, that is, it has the biodistribution and pharmacokinetics of Tc-99m sulfur colloid injected for a liver/spleen or bone marrow scan. The fetal radiation absorbed dose was then estimated for two Tc-99m dosages: 18.5 MBq (0.5 mCi) and 92.5 MBq (2.5 mCi). The Medical Internal Radiation Dosimetry (MIRD) program was used to estimate the absorbed doses to the embryo/fetus for the first two scenarios. Published data were used to calculate the doses for the third scenario. A single breast is not among the source organs in the MIRD program, so the heart was used as a surrogate in the first scenario. In the two breast cancer patients, whole-body gamma-camera images obtained 1 hour after radiopharmaceutical injection revealed no radioactivity except in the vicinity of the injection site. In the theoretical scenarios, with 92.5 MBq, the highest absorbed doses to the embryo/fetus were as follows: scenario 1, 7.74 × 10−2 mGy at 9 months of pregnancy; scenario 2, 4.26 mGy during early pregnancy; and scenario 3, 0.342 mGy at 9 months of pregnancy. The maximum absorbed dose to the fetus of 4.3 mGy calculated for the worst-case scenario is well below the 50 mGy that is believed to be the threshold absorbed dose for adverse effects. Thus breast lymphoscintigraphy during pregnancy appears to present a very low risk to the embryo/fetus. 

Notes: Abstract:  Intramedullary spinal cord metastases (ISCMs) are very rare, but can cause devastating complications from underlying breast cancer. We report the case of a woman with known metastatic breast cancer and progressive neurologic deterioration caused by an ISCM. The epidemiology, pathogenesis, clinical presentation, diagnostic considerations, and therapeutic options are discussed. 

Notes: Abstract:  Idiopathic granulomatous mastitis is a rare disease of the breast. Clinically and radiologically it may mimic breast carcinoma. Awareness of surgeons, pathologists, and radiologists is essential to avoid unnecessary mastectomies. Data regarding 24 patients with histologically confirmed idiopathic granulomatous mastitis treated at our center over 8 years were analyzed. The mean age of patients was 34.3 years. Breast lump was the most common presentation. The right breast was affected in 16 cases. Four patients were pregnant at the time of presentation. Lactation within 6 months of presentation was documented in four patients. Two patients used contraceptives pills. A clinical suspicion of malignancy was present in 17 cases. Mammography was performed in 16 patients and showed focal asymmetrical dense lesions in 9, well-circumscribed opacity in 4, spiculated lesion in 1, and was normal in 2. Fine-needle aspiration was performed in 17 patients, of which 2 were reported as malignant. Wide local excision was the mainstay of treatment. One patient underwent mastectomy upon clinical, mammographic, and repeated cytologic findings consistent with malignancy, and the final histology confirmed idiopathic granulomatous mastitis with no evidence of malignancy. Four patients developed recurrence after a mean follow-up of 31.2 months. A greater awareness of the rare entity of idiopathic granulomatous mastitis is mandatory to avoid unnecessary mastectomies. Clinical, radiologic, and even cytologic findings are sometimes confused with malignancy. To the best of our knowledge, our patient who developed the disease at the age of 11 years is the youngest reported case. 

Notes: Abstract:  A randomized, double-blind, placebo-controlled, multicenter clinical trial was conducted with 111 otherwise healthy euthyroid women with a history of breast pain. Patients had to document moderate or severe breast pain by recording a score ≥5 on a visual analog scale (VAS) of pain for ≥6 days per cycle and had to present with fibrosis involving at least 25% of both breast surfaces. Subjects could not be effectively treated with more conservative measures such as local heat or nonprescription analgesics. There was not a statistically significant difference in the dropout rate for patients on placebo (11.8%), 1.5 mg/day (31.3%), 3.0 mg/day (18.4%), or 6.0 mg/day (25%) of molecular iodine for 6 months. Physicians assessed breast pain, tenderness, and nodularity each cycle; patients assessed breast pain and tenderness with the Lewin breast pain scale at 3-month intervals and with a VAS at each cycle. A statistically significant improvement (p 〈 0.01) associated with dose was observed in the Lewin overall pain scale for all treated groups compared to placebo. Reductions in all three physician assessments were observed in patients after 5 months of therapy in the 3.0 mg/day (7/28; 25%) and 6.0 mg/day (15/27; 18.5%) treatment groups, but not the 1.5 mg/day or placebo group. Patients recorded statistically significant decreases in pain by month 3 in the 3.0 and 6.0 mg/day treatment groups, but not the 1.5 mg/day or placebo group; more than 50% of the 6.0 mg/day treatment group recorded a clinically significant reduction in overall pain. All doses were associated with an acceptable safety profile. No dose-related increase in any adverse event was observed. 

Notes: Abstract:  The proto-oncogene c-kit encodes a transmembrane tyrosine kinase growth factor receptor. Stem cell factor, the receptor ligand, plays an important role in the development of certain neoplasms. c-kit is selectively and competitively bound by STI-571, a newly developed tyrosine kinase inhibitor. Several investigators report conflicting results concerning its expression, especially in malignant breast lesions. The objective of this study was to better characterize the expression of c-kit within the spectrum of breast epithelium (normal breast epithelium, nonneoplastic lesions, and breast carcinoma). Seventy-seven randomly selected breast tissue samples, each containing normal breast epithelium (21), invasive breast carcinoma (41), in situ breast carcinoma (29), papilloma (8), fibroadenoma (5), fibrocystic change (11), and/or metastatic breast carcinoma (4), were immunostained with polyclonal rabbit antihuman c-kit (Dako, Carpenteria, CA) at a dilution of 1:200. The staining was interpreted as negative if no cells were immunoreactive, weak positive if 5% of the cells were immunoreactive, and positive if more than 5% of the cells were immunoreactive. Appropriate positive and negative controls were used. The observed staining was cytoplasmic, with highlighting of the nuclear membrane. Normal breast epithelium was positive in all cases. More than half of the cases of hyperplastic changes and benign neoplasms (fibroadenoma and papilloma) were positive. Only 10% of invasive and in situ carcinomas showed positivity for c-kit. c-kit is consistently expressed in normal breast epithelium, variably expressed in benign breast lesions, and poorly expressed in breast carcinoma. These data suggest that c-kit may play a role in breast tumor progression and may therefore have diagnostic, prognostic, and therapeutic implications. 

Notes: Abstract:  We reviewed a rare breast infection occurring 4 months after nipple piercing. Clinical examination suggested carcinoma. Mycobacterium fortuitum was eventually isolated after surgical biopsy and debridement. Antibiotic therapy was initiated intravenously using two drugs and oral therapy was continued for 6 months. A contralateral mycobacterial lesion emerged and was excised along with a residual fibrotic nodule at the original biopsy site. When adequate sampling of a complex and suspicious breast mass is benign and initial bacterial cultures are sterile, mycobacterial infection should be considered, particularly when there is a history of previous nipple piercing procedures. 

Notes: Abstract:  This retrospective study assesses the presentation of breast cancer patients who died of disease. The goal was to estimate the proportion of patients whose cancer was detected by a screening mammogram, yet still proved fatal, and to characterize contemporary fatal breast cancers. Patients who died of breast cancer between 1995 and 2001 and were treated at three hospitals with complete, accessible information were identified using the Rhode Island Department of Health database and State Cancer Registry. Patients were classified as routinely screened, true interval, off-program, or unscreened. The median maximum diameter of fatal cancers was 2.5 cm. Sixty-nine percent of patients had lymph node metastases and 19% had stage IV disease. Fifty-seven percent were unscreened and 12% were routinely screened, with 2.5 cm and 1.5 cm median tumor diameters, respectively. Fifty-two percent of unscreened patients and no routinely screened patients had stage III or IV disease. Fifteen percent of fatal cancers were interval cancers. Only 27% of patients who died of breast cancer were participants in routine screening programs in Rhode Island, where 84% of women receive regular mammograms. This study complements randomized, population-based trials demonstrating a significant mortality reduction with an invitation to participate in screening. 

Notes: Abstract:  A 56-year-old woman who came in for screening mammography was found to have extensive unilateral calcification of her left breast which had developed since her previous screening mammogram. The calcification had a ductal and lobular appearance. Possible known etiologies are discussed, but these do not explain the appearance in this case, implying that the cause is idiopathic. 

Notes: A problem with tissue-engineered skin is clinical failure due to delays in vascularization. The aim of this study was to explore a number of simple strategies to improve angiogenesis/vascularization using a tissue-engineered model of skin to which small vessel human dermal microvascular endothelial cells were added. For the majority of these studies, a modified Guirguis chamber was used, which allowed the investigation of several variables within the same experiment using the same human dermis; cell type, angiogenic growth factors, the influence of keratinocytes and fibroblasts, mechanical penetration of the human dermis, the site of endothelial cell addition, and the influence of hypoxia were all examined. A qualitative scoring system was used to assess the impact of these factors on the penetration of endothelial cells throughout the dermis. Similar results were achieved using freshly isolated small vessel human dermal microvascular endothelial cells or an endothelial cell line and a minimum cell seeding density was identified. Cell penetration was not influenced by the addition of angiogenic growth factors (vascular endothelial growth factor and basic fibroblast growth factor); similarly, including epidermal keratinocytes or dermal fibroblasts did not encourage endothelial cell entry, and neither did mechanical introduction of holes throughout the dermis. Two factors were identified that significantly enhanced endothelial cell penetration into the dermis: hypoxia and the site of endothelial cell addition. Endothelial cells added from the papillary surface entered into the dermis much more effectively than when cells were added to the reticular surface of the dermis. We conclude that this model is valuable in improving our understanding of how to enhance vascularization of tissue-engineered grafts.

Notes: Restitution is the process by which superficial interruptions in the gastrointestinal mucosa are repaired by the flattening and spreading of epithelial cells surrounding the damage. During this process, mucosal epithelial cells undergo extensive reshaping and cytoskeletal remodeling. K+ channels, located primarily on the basolateral surface of gut epithelial cells, are central to both actin polymerization, via their control of membrane potential, and cell volume regulation. We questioned whether K+ channels are involved in restitution using an in vitro model of intestinal epithelium, monolayers of the human colon carcinoma cell line T84. We report that pharmacologic K+ channel inhibition accelerates wound healing in T84 cell monolayers. Both Ca++-dependent and constitutively active channels are involved, as indicated by the sensitivity to clotrimazole, charybdotoxin, and barium. The ability of clotrimazole to accelerate wound resealing was also observed in Caco-2 cell sheets. Pharmacologic stimulation of K+ channel activity had no effect on the repair rate. Analysis of the resealing process by time lapse and confocal microscopy revealed that K+ channel inhibitors abolished the initial wound retraction, briefly accelerated the repair rate, and altered the shape of the cell sheet abutting the injury during the early phase of resealing. We hypothesize that K+ channel inactivation interrupts the coregulation of f-actin polymerization and volume control that is initiated by the healing process.

Notes: Increasing evidence implicates excessive reactive oxygen species (ROS) generation and ROS-derived degradation products in the pathogenesis of many skin diseases. While numerous attempts have been made to identify prognostic biomarkers of wound healing in skin, these have met with limited success. This study examined the profiles of various oxidative stress biomarkers, namely total protein carbonyl content (from protein oxidation), malondialdehyde content (from lipid peroxidation), and the total antioxidant capacities, in acute wound fluid (n= 10) and chronic wound fluid (n= 12), using a rapid, noninvasive collection technique. Protein carbonyl content was quantified spectrophotometrically and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting, following 2,4-dinitrophenylhydrazine derivitization. Malondialdehyde levels were similarly quantified, following N-methyl-2-phenylindole derivitization. Total antioxidant capacity was determined via wound fluid inhibition of cytochrome C reduction by a superoxide radical flux. Acute wound fluid contained higher protein carbonyl content than chronic wound fluid, particularly evident following sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blot analysis under nonreducing and reducing conditions (p 〈 0.001 and p 〈 0.02, respectively), related to significantly higher protein levels (p = 0.0005) in acute wound fluid. Human serum albumin (∼66 kDa) was identified as the most prominent protein oxidized in both acute and chronic wound fluid, which may contribute to the reduced albumin and total protein levels in chronic wound fluid. No significant difference (p 〉 0.1) in malondialdehyde levels or total antioxidant capacities were determined between acute and chronic wound fluids, although chronic wound fluid exhibited significantly higher total antioxidant capacities (p 〈 0.005), accounting for variations in wound fluid protein content. These findings suggest an adaptation in the antioxidant profiles of chronic wound fluid to counteract the loss of consumed antioxidants in the chronic wound environment. This study highlights the roles of ROS/antioxidants in skin wound healing, their possible involvement in chronic wounds and the potential value of ROS-induced biomarkers in wound healing prognosis.

Notes: A previous study showed that topical exposure to bioelastic-thromboxane synthetase inhibitor-matrix resulted in local tissue concentrations of thromboxane synthetase inhibitor sufficient for thromboxane synthetase inhibition. The objective of this research was to use an animal model to determine if a dressing having controlled release of thromboxane synthetase inhibitor (dazmegrel) could be used to prevent tissue breakdown over pressure points, i.e., lesion at the assistive device–skin interface. The animal model studies utilized the greyhound, a dog that has thin skin, angular conformation, limited body fat and is predisposed to pressure ulcers similar to those occurring in humans. The model uses a short-limb walking cast on one pelvic limb with the severity of the dermal pressure lesions induced over the medial malleolus controlled by the amount of padding in the cast and length of time the cast is in place. The bioelastic matrix loaded with dazmegrel provided protection from shearing and pressure skin injury over the medial malleolus, as evidenced by a decrease in epidermal abrasion/ulceration as measured with planimetry. Histopathologic evaluation of the skin over the medial malleolus indicated a protective function of the bioelastic matrix as measured as lower numbers of neutrophils, lymphocytes, and decreased collagen density compared to such numbers when no bioelastic matrix was present. These studies provided evidence that bioelastic-thromboxane sythetase inhibitor- matrix helps in preventing or reducing the severity of pressure lesions, e.g., assistive device–skin interface wounds.

Notes: Clinicians now recognize that both aerobic and anaerobic microorganisms have the ability to degrade or damage host tissue at a wound site through the production of a variety of enzymes and toxins. Silver-containing dressings offer one method for controlling this polymicrobial wound bioburden, and research efforts are currently ongoing to determine their efficacy against aerobic, anaerobic, and antibiotic-resistant microorganisms. The current study aimed to determine the antimicrobial activity of a new silver-containing Hydrofiber® dressing (AQUACEL® Ag) on both aerobic and anaerobic microorganisms, using the zone-of-inhibition method. This method provides a measure of the ability of the dressing to make available a sufficient concentration of silver to have an antimicrobial effect. To some extent this test mimics the clinical use of the dressing and predicts its microbicidal activity at the wound–dressing interface. The results show that the silver-containing dressing makes silver available at a dressing–agar interface at a concentration that is effective against a broad range of aerobic, anaerobic, and antibiotic-resistant microorganisms. In the context of wound healing, the results showing antimicrobial activity against antibiotic-resistant microorganisms are particularly important, as the control and eradication of these organisms is a major concern within the health care profession.

Notes: The MRL mouse has been shown to display an epimorphic regenerative response after ear hole punching leading to complete closure within 30 days and cartilage regrowth. The regenerative capacity of the MRL has also been seen after a severe cryoinjury to the heart leads to complete healing without scarring and functional myocardium. The wound healing ear hole closure response that occurs in MRL mice has been shown to be genetically controlled. We have previously identified 11 quantitative trait loci (QTL) that govern healing in an intercross of (MRL × C57BL/6 J) mice. However, it is desirable to use another poorly healing mouse strain to elucidate the full range of genetic factors that affect this important process. In the current study, we have used an inbred subspecies of the mouse, M. castaneus, and have confirmed a number of loci identified previously. In addition, we report three new healing QTL. Furthermore, in this strain combination, we note a strong sexual dimorphism also observed in the MRL × C57BL/6 cross, both in the healing trait and in the QTL that control it.

Notes: We previously reported methods for sterilizng human skin for clinical use. In a comparison of γ-irradiation, glycerol, and ethylene oxide, sterilization with ethylene oxide after treatment with glycerol provided the most satisfactory dermis in terms of structure and its ability to produce reconstructed skin with many of the characteristics of normal skin. However, the use of ethylene oxide is becoming less common in the United Kingdom due to concerns about its possible genotoxicity. The aim of this study was to evaluate peracetic acid as an alternative sterilizing agent. Skin sterilized with peracetic acid was compared with skin sterilized using glycerol alone or glycerol with ethylene oxide. The effect of subsequently storing peracetic acid sterilized skin in glycerol or propylene glycol was also examined. Acellular dermal matrices were produced after removal of the epidermis and cells in the dermis, processed for histological and ultrastructural analysis, and the biological function was evaluated by reconstitution with keratinocytes and fibroblasts. Results showed that sterilized acellular matrices retained the integrity of dermal structure and major components of the basement membrane. There were no overall significant differences in the ability of these matrices to form reconstructed skin, but peracetic acid alone gave a lower histologic score than when combined with glycerol or propylene glycol. We conclude that peracetic acid sterilization followed by preservation in glycerol or propylene glycol offers a convenient alternative protocol for processing of human skin. It is suggested that this sterile acellular dermis may be suitable for clinical use.

Notes: Leptin is a pleiotropic cytokine constitutively expressed in adipose tissue and upregulated by hypoxia in sites of tissue injury and in the placenta. Leptin has been demonstrated to play a role in normal and pathological wound healing and it is gene is acutely expressed in experimental wounds. Angiogenesis is among the most salient and well-documented biological actions of leptin. Therefore it was hypothesized that leptin may play in the modulation of wound neovascularization. Using a murine model of experimental wounds the modulation of wound angiogenesis was investigated by treatment of the wounds to either recombinant leptin or a neutralizing anti-leptin antibody. The parameters measured were: a)woundblood flow using laser Doppler imaging; b)vesseldensity by counting the number of CD-31 positive vessels in wound sections; c)angiogenic transcriptionalprofiling using quantitative real-time RT-PCR; and d)validation of transcriptionalprofiling by immunohistochemistry and immunoblotting. The treatment of wounds with leptin had a marked effect in wound blood flow that was most significant at 24hours post-wounding with average increase of 55% with respect to the controls. The effect diminished significantly by 72 hours (24%) and became negligible thereafter. Leptin also accelerate wound neovascularization by 24 hours where the number of small caliber vessels averaged 30/mm2 in leptin treated vessels and 13/mm2 in the controls (n = 10; P 〈 0.005). However, the number of vessels measured by 72 hours not significantly different between controls and leptin-treated wounds. In addition transcriptional profiling reveled changes in gene expression of molecules related to angiogenesis such as VEGF, PlGF, Tie-1, Tie-2, Flt-1, Flk-1, FGF-2 and endothelial markers like CD-31, endoglin and eNOS. Overall, time course analysis throughout the healing process showed that leptin treatment induced an early shift in the expression of many of the analyzed transcripts. The most salient changes could be observed as early as 6 hours post-wounding and included: TSP-1 (+8.7); FLK-1(+9); Ang-2 (+18); CD31(+5.3); endoglin (+7.6); eNOS (+4.2); FGF-2 (+4.3); etc. Leptin mRNA was also significantly increased (+10) as well as the long-form of its receptor ObR-b (+9). Conversely, when wounds were exposed to anti-leptin antibodies, healing progression was impaired and changes in the transcriptional profile showed diminished expression of several genes related to wound neovascularization. Some examples by 24 hours: Endoglin(−2.5); Flk-1(−2.8); eNOS (−5.1); Ang-2 (−5.1); CD31(−3); VEGF-A (−3.6). In conclusion, leptin had and early and significant effect in modulating wound neovascularization. The effect of treatment of wounds with exogenous leptin did not elicit hypervascularization of the wound and appeared to only accelerate what appeared to be a self-limited process of wound angiogenesis.Funded by a Cedars-Sinai grant from the Skirball-Kenis Center for Plastic and Reconstructive Surgery.

Notes: Introduction: Burn depth, based on the hemodynamic alterations that occur following a thermal insult, can be assessed in a rapid, non-invasive, and nondestructive fashion using near infrared (NIR) spectroscopy. NIR has the capability to determine the difference between superficial and full thickness burn injuries. Methods: Sixteen burn patients admitted to an adult regional burn center were studied and evaluated with the NIR point and imaging devices. Non-burned skin adjacent to the burn site was used as the control. NIR measurements were compared between superficial (8 wounds), full thickness (8 wounds) burn wounds and control sites. Results: NIR was able to easily detect an increase in oxyhemoglobin (68.3%, p 〈 0.05), oxygen saturation (4.8%, p 〈 0.05%) and total hemoglobin (91.3%, p 〈 0.05) which typically occurs with superficial burn injuries. Full thickness injuries experienced a substantial drop in oxyhemoglobin (88.8%, p 〈 0.05), oxygen saturation (79.1%, p 〈 0.05) and total hemoglobin (77.5%, p 〈 0.05) in comparison to control sites. Conclusions: These results confirm that NIR spectroscopy can successfully distinguish between superficial and full thickness burn injuries. The second phase of this study will involve determining the depth of indeterminant burn wounds and this preliminary data will also be presented. Acknowledgement: National Research Council of Canada

Notes: Introduction: One of the earliest events in wound healing and scar formation is the inflammatory phase. Deficient or excessive inflammatory responses result in aberrant healing. NFK-b is a transcription factor that regulates key genes responsible for multiple intra-nuclear inflammatory cytokines. When bound to IK-B, an inhibitory protein, this factor is in an inactive state. We studied the wound healing effects of irreversibly inhibiting the activation of NFK-b by continuously infusing adenoviral activated IK-B. Methods: Seventy-five male Sprague-Dawley rats underwent subcutaneous implantation of polyvinyl alcohol sponge-osmotic pump constructs. The pumps were filled with a solution containing a genetically altered adenovirus containing the DNA for continuously activated IK-B protein. 50 μl of viral transport media containing 5 × 109 PFU of virus were delivered to each individual sponges daily. Controls included pumps filled with normal saline in one group or filled with sham virus in another group. Sponges were harvested on days 1,3,5, and 7 post-implantation. The sponges were analyzed for TNF-α and nitric oxide (NO) levels, as index of inflammation, as well as for hydroxyproline (OHP) content at seven days, an index of collagen deposition. Results: Treatment with the IK-B virus resulted in significantly higher levels of OHP after seven days when compared to normal saline and sham virus treated sponges (654 ± 81 vs 546 ± 109 vs 498 ± 123 μg OHP/100 mg sponge, P 〈 0.05 by ANOVA). Wound fluid NO concentration was significantly lower in the IkB group after 5 days (38 ± 15 vs 55 ± 12 vs 71 ± 27 μM, P 〈 0.05 by chi-square). There were no differences in sponge TNF-α concentrations on days 1,3, and 5. Conclusions: Treatment of wound sponge granulomas with IK-B increases the amount of collagen deposition after seven days. Reduction of inflammation by inhibiting NF-KB may be a possible mechanism of action, as reflected in decreased NO wound content.

Notes: Effective blockade of the pluripotent cytokine TGF-beta as a means of cutaneous scar reduction is a strategy with great potential. This desired effect may be achieved through the overexpression of mutant TGF beta receptors within the wound milieu. Our goal was to examine the effects of dominant negative mutant TGF-beta receptor II (dnTGFRII) protein expression in a well-established rabbit ear model of hypertrophic scarring. Serial injections of a retroviral construct encoding a truncated TGFβRII and the marker green fusion protein (pMSCV-rIIdn-GFP) were performed in 7mm punch wounds at day 10 and day 14 (two-day injection group) or day 8, 10, 12 (three-day injection group) post wounding. Delivery of a null vector (pMSCV-GFP) at the same time points served as a negative control. Histomorphometric analysis of wounds harvested at day 28 revealed a statistically significant reduction (33%) in the scar elevation index in 2-day treated and a more modest reduction in SEI (17.5%) in the 3-day treated arm compared to null-treated controls. Confocal microscopy confirmed stable transfection of the construct in both peri-wound tissue as well as rabbit dermal fibroblasts transfected in vitro. Optimization of this novel application in retroviral gene therapy could lead to effective anti-scarring strategies.

Notes: This study was to investigate the effect of cyclic mechanical stretching on superoxide and nitric oxide production in human patellar tendon fibroblasts (HPTFs) and to establish the role of superoxide and p38 MAPK in stretching-induced α-smooth muscle actin (α-SMA) expression. When HPTFs were grown in deformable silicone dishes and subjected to 8% cyclic, uniaxial stretching, it was found that HPTFs markedly increased the production of superoxide and nitrite but not nitric oxide. And, cyclic stretching of HPTFs increased phosphorylation of p38 MAPK with increasing stretching magnitude. In addition, stretching duration also influenced the expression of phosphorylated p38 MAPK, where the dependence followed a pattern of cellular response in p38 MAPK activation consistent with previous studies using human mesangial cells and rat 3Y1 fibroblasts. For short stretching durations (∼15 min), the expression of phosphorylated p38 MAPK increased rapidly but decreased for longer stretching durations. Furthermore, in HPTFs treated with superoxide dismutase (SOD), which converts superoxide anions to hydrogen peroxide and an oxygen molecule, levels of stretching-induced p38 MAPK phosphorylation and α-SMA expression decreased compared to stretched fibroblasts not treated with SOD. Similarly, cells treated with SB202190, which specifically inhibits p38 MAPK activation, also decreased α-SMA expression levels. Therefore, these results suggest that superoxide regulates stretching-induced α-SMA expression via p38 MAPK activation in HPTFs subjected to cyclic stretching conditions.

Notes: Hyperbaric oxygen (HBO) is used adjunctively to treat chronic, non-healing wounds. Increased nitric oxide (NO) production during HBO is suggested as an important mechanism that promotes enhanced wound healing. The purpose of this study is the documentation of NO production during and following HBO treatments (txs). Diabetic and non-diabetic patients receiving HBO therapy (20 txs; 2.0ATA × 90 min) provided wound fluid and fasting plasma and urine specimens for nitrate (NOx) determinations. Nitrate measurements were obtained prior to HBO (baseline), after 10 and 20 txs, and at one and four weeks following HBO completion. In patients healing after HBO baseline plasma NOx ranged between 17.7 and 33.20 μM. At 10 txs plasma NOx increased 〉50% to between 35.8–43.5 μM. At 20 txs plasma NOx decreased to between 22.3–35.5 μM. Healing diabetic patient plasma NOx increased after HBO reaching 170% of baseline at one month (57.8 ± 0.15 μM). Plasma NOx of healing non-diabetic patients decreased to baseline levels at one month after HBO. Urine NOx values mirrored those of plasma NOx; wound fluid NOx elevations were delayed compared to plasma and urine. Conversely, non-healing HBO patients did not demonstrate the early, significant elevations of plasma NOx after 10 txs. This study documents, for the first time, an early (10 txs), significant elevation of plasma and urine NOx after HBO that is associated with successful healing in diabetic and non-diabetic patients. A similar early increase in plasma and urine NOx was not observed in patients not responding to HBO therapy. These findings suggest that HBO-mediated increased NO production plays a critical role in the correction of impaired wound healing in selected patients. Furthermore, early plasma and urine NOx determinations after HBO therapy may prove valuable in the prediction of the clinical outcome of treatment.

Notes: Biofilms are a combination of microorganisms and extrapolysaccharide matrices. Our laboratory has previously demonstrated that biofilms are present in both acute and chronic wounds. Once biofilms are established, phagocytosis and diffusion of antibiotics are impaired thus contributing to antimicrobial resistance due to increased bacterial virulence. The purpose of this study was to determine the efficacy of two topical antimicrobial agents on partial thickness wounds containing Staphylococcus aureus biofilms. All wounds were inoculated with 106 CFU/ml and covered for 48 hrs under a polyurthene film to promote biofilm formation. Wounds were divided into three treatment groups; triple antibiotic ointment (Polymyxin B sulfate, bacitracin zinc, neomycin), mupirocin cream and untreated control. Wounds were treated twice daily. Wounds were cultured for bacterial quantitation at 24, 48, 72, 96, and 120 hrs. Significant reduction in CFU/ml was observed only after several days of treatments. This finding supports the antimicrobial resistance that occurs when bacteria live within biofilms. Our previous studies demonstrated that both of these agents were able to completely eliminate planktonic S. aureus(106) at the early time points. This study demonstrates that when bacterial biofilms are established in wounds there is a longer response time for topical antimicrobial activity suggesting bacterial resistance.

Notes: Introduction: Pseudomonas aeruginosa, remains a serious cause of nosocomial infection and septic mortality in burn patients particularly when nosocomially acquired. Our purpose is to investigate the morbidity and mortality associated with nosocomial infection with an aminoglycoside resistant Pseudomonas and associated costs compared to a group of patients with similar severity of burn injury that did not acquire resistant Pseudomonas during hospitalization. Methods: Using a TRACS burn database, patients treated at our institution with Pseudomonas resistant to gentamicin were identified and case-matched to controls for age (±5 years), TBSA(±5%), admission year (±5 years) and presence of inhalation injury. Patients who died 〈48 hours after injury were excluded. Data examined included demographics, number of days to onset of positive Pseudomonas culture and gentamicin resistance, as well as antibiotic use and cost. Results: 42 patients admitted to our unit between 1980 and 2001 were identified with gentamicin resistant Pseudomonas. Patients in the resistant Pseudomonas(Ps) group were similar in age (39.2 ± 3.2 vs 40.4 ± 3.3 years), TBSA (47.5 ± 3.7 vs 48.9 ± 3.7%), extent of full thickness injury (37.0 ± 3.9 vs 30.3 ± 3.2%) and presence of inhalation injury (62.8% vs 55.0%) compared to controls. There was a significant increase in the mortality rate in the Ps group (39.5 vs 5.0%, p 〈 0.001)(paired t test) compared to controls and the morbidity in terms of length of stay, increased in the Ps group (73.1 ± 13.2 vs 55.8 ± 8.1 days). Ventilatory days (22.6 ± 5.1 vs 8.2 ± 2.4, p 〈 0.05), number of surgical procedures (4.5 ± 0.6 vs 2.9 ± 2.5, p 〈 0.05), and amount of blood products used (packed cells 47.9 ± 7.8 vs 18.6 ± 3.3, p 〈 0.01)(platelets 10.5 ± 2.9 vs 0.5 ± 0.3, p 〈 0.01) were all significantly higher in the Ps group compared to control. Costs associated with antibiotic requirements were also significantly higher in the Ps group ($3,191.90 ± 848.00 vs $613.60 ± 145.50, p 〈 0.01). Conclusions: Our data demonstrate that nosocomial infection in burn patients with aminoglycoside-resistant Pseudomonas is associated with significantly higher morbidity and mortality and cost of care. Increased resource consumption in terms of length of stay, number of surgical procedures, amount of blood products, and antibiotic costs did not prevent significantly higher mortality rates when compared to control patients who avoided infection with the resistant organism. Thus, prevention, identification and eradication of nosocomial Pseudomonas infection are critical for cost-effective, successful burn care.

Notes: Introduction: Integra™ has gained acceptance as a dermal replacement for large burns with limited donor sites. Integra™ vascularization takes between 14 and 21 days. Accelerating the vascularization of Integra™ could improve “take” rates and potentially decrease the obligatory waiting period between application and autografting. Hypothesis: Application of negatively charged methacrylic acid (MAA) beads will enhance vascularization of Integra™ Methods Male Wistar rats (n = 11) had two 2 × 3 cm contra lateral dorsal full thickness wounds excised and grafted using Integra™. The MAA beads were applied under the Integra™ topically on the wound bed. Three experimental treatments were compared: Group 1- high-dose MAA beads, Group 2- low-dose MAA beads, Group 3- Integra only. Laser Doppler Imaging (LDI) to assess perfusion, as well as digital photography was done on days 7,10 and 14. Tissue samples for H + E and Factor VIII histological staining were collected on day 14. Results: The average “take” was 99%(p 〈 0.05) for high-dose MAA beads, 98%(p 〈 0.05) for low-dose MAA beads and 82% in the Integra™ only group at day 7. At day 7 (p 〈 0.01) and day 10 (p 〈 0.05), the low-dose MAA group had significantly greater perfusion than the Integra™ only group. There were no statistically significant differences at day 14. Microvessel density (MVD) counts revealed a 〉40% increase in the number of vessels in both the low-dose MAA (p 〈 0.05) and high-dose MAA (p 〈 0.05) groups when compared to the Integra™ only group. There was no difference in LDI perfusion or MVD counts between low and high-dose MAA groups. Conclusion: Factor VIII staining revealed enhanced angiogenesis in Integra™ treated with low and high-dose negatively charged MAA beads. Low-dose negatively charged MAA beads improved and accelerated the vascularization of Integra™ in this rodent model.

Notes: The importance of stromal-epithelial interactions in wound healing is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the TGF-ß family. TGF-ß1, ß2 and ß3 isoforms signal by sequentially binding to the TGF-ß type II and type I receptors, respectively. We address the role of TGF-ß signaling in dermal fibroblasts using a conditional fibroblastic TGF-ß type II receptor knockout mouse model (termed FßKO). We found that the loss of TGF-ß signaling in the dermal fibroblasts results in accelerated excision-wound closure compared with similar wounds in wild type mice. The mechanism of the altered rate of re-epitheliaization in the FßKO mice was examined with regard to keratiocyte motility and proliferation. The migration of keratinocytes through collagen I coated 8 μm pore filters in the presence or absence of fibroblast-conditioned media was tested. These experiments showed increased keratinocyte migration when incubated with FßKO dermal fibroblast conditioned media compared to media conditioned in wild type fibroblasts. Immuno-histochemical staining of paraffin embedded intact skin indicated both wild type and FßKO mice had similar low levels of keratinocyte proliferation, based on Ki67 staining. In healing wounds, only the distal wound edges of wild type mice were proliferative. In contrast, the FßKO mice exhibited elevated proliferation across the length of the wound, including the leading edge of epithelial closure. Together our results suggest TGF-ß signaling by the dermal fibroblasts suppresses re-epithelialization of excision wounds by regulating keratinocyte motility and proliferation through paracrine mechanisms. 
 Funding: DOD BC99184 and NIH CA85492.

Notes: Experimentally induced wounds in animal models are useful in gaining a better understanding of the cellular and molecular processes of wound healing and in the initial evaluation of the safety and effectiveness of potential therapeutic agents. However, studying delayed healing has proven difficult in animals, whose wounds heal within a few days. In this report, we describe a novel method for establishing mouse wounds that require up to more than three weeks for complete closure, and we show the validity of this model in Smad3 null mice, which are known to display accelerated healing. Full-thickness wounds, measuring 0.3 by 1.0 cm, were made down to fascia on the dorsal aspect of the mouse tail in Smad3 KO mice and control littermates, approximately 1 cm distal to the body of the animal. The wounds were left to heal by secondary intention and were assessed histologically by computerized planimetry for wound closure at various times after wounding. These wounds in wild-type mice displayed delayed healing, with full closure occurring between 14 and 25 days after wounding. Complete closure of similar wounds in Smad3 null mice healed 30% faster (p 〈 0.01). By immunostaining with ki67, a marker for proliferation, Smad3 null animals also showed increased proliferation of dermal wound cells. Cultured dermal fibroblasts from Smad3 null mice showed increased baseline DNA synthesis and, interestingly, enhanced response to TGF-β1. By Western blot analysis, Smad3 null mice fibroblasts showed a compensatory increase in MAPK phosphorylation in response to TGF-β1, suggesting that MAPK overcompensation together with loss of Smad3 may be involved in the modulation of faster healing. We conclude that this novel tail wounding model can be useful for studying delayed or prolonged wound closure.Experimentally induced wounds in animal models can be useful in gaining a better understanding of the cellular and molecular processes of wound healing. Such models have also proven themselves valuable in the initial evaluation of the safety and effectiveness of potential therapeutic agents targeted for chronic non-healing wounds. (Gottrup, Agren et al. 2000). However, no ideal animal model exists which reliably reproduces delayed healing. In the mouse, a mammal whose genome has been completely cloned and which is easily manipulated genetically, wounds normally heal within a few days, and with a great deal of contraction. (Morris, Wu et al. 1997; Gottrup, Agren et al. 2000) There are models utilizing either genetically altered and inbred mice with certain characteristics that cause delayed healing. (Carmeliet 1995) However, it would be useful to have wound healing models that are applicable to most wild type mice used as controls and which would have a large enough window of observation before healing occurs. In this report, we describe a novel method for studying delayed healing in mice. This method utilizes full-thickness wounds made down to fascia on the dorsal and mostly hairless aspect of the mouse tail. The wounds are left to heal by secondary intention and assessed histologically by computerized planimetry for wound closure at various times after wounding. In this first report, the validity of the model was determined by studying control littermates and Smad3 null mice, which have been shown to display accelerated healing. The results shown here suggest that this is a useful model for studying delayed healing in mice.

Notes: Introduction: Adenoviral gene transfer of Angiopoietin-1 (AdAng1) recruits endothelial progenitor cells (EPCs) and improves diabetic wound healing. A suggested mechanism for EPC mobilization from the bone marrow (BM) is mediated through MMP-9 and stem cell factor (SCF). We hypothesize that Ang-1 recruits EPCs to diabetic wounds via an MMP-9 dependent mechanism. 
Methods: Lethally irradiated mice were reconstituted with BM from transgenic TIE-2/LacZ mice. After engraftment, diabetes was induced with steptozotocin. 8 mm wounds were created in BM transplanted (BMT)(n = 12) or MMP-9 knockout (KO)(n = 12) mice and treated with 1 × 108 PFU of AdAng1, AdGFP or PBS. At 7 days wounds were analyzed for epithelial gap, vessel density, and EPCs. Serum levels of VEGF, proMMP9 and SCF were assessed. Data are expressed as mean ± SEM 
Results: In diabetic BMT wounds, AdAng1 results in improved reepithelialization (Ang1 2.3 ± .2 mm; GFP 3.9 ± .2; PBS 4.0 ± .1 p 〈 .0001) neovascularization (Ang1 6.8 ± .3Caps/Hpf; GFP 3.0 ± .4; PBS 2.9 ± .3 p 〈 .0001) and EPC recruitment (Ang1 5.3 ± .4 EPCs/Hpf; GFP 2.1 ± .3; PBS 2.2 ± .3 p 〈 .0001). AdAng1 treatment results in increased levels of proMMP-9 (Ang1 9.7 ± .8 ng/ml; GFP 6.3 ± .9; PBS 6.4 ± .4 p 〈 .01) and SCF (Ang1 265 ± 28 pg/ml; GFP 119 ± 16; PBS159 ± 12 p 〈 .001). In MMP9 KO mice, AdAng1 accelerates reepithelialization (Ang1 3.2 ± .1 mm; GFP 4.1 ± .2; PBS 3.8 ± .2; p〈.) but has no significant effect on neovascularization (Ang1 4.1 ± .5 Caps/HPF; GFP 3.9 ± .4; PBS 3.9 ± .6), EPC recruitment (Ang1 2 ± .3 EPC/Hpf; GFP 1.5 ± .3; PBS 1.6 ± .2) or SCF levels (Ang1 83 ± 5 pg/ml; GFP 83 ± 2; PBS 88 ± 6). 
Conclusions: The effects of Ang-1 on EPC recruitment and neovascularization are dependent on MMP-9. Our data support the hypothesis that MMP-9 enables SCF to permit mobilization of EPCs.

Notes: Cytomodulin (CM-1) is a synthetic heptapeptide. Human dermal fibroblasts cultured in the presence of CM-1 showed increased expression of transforming growth factor–ß(TGF-ß) and collagen I and decreased expression of MMP-1. TGF-β is involved in numerous vital processes in wound healing, and the rate of wound repair is influenced by its overexpression at the site of injury. To establish the effect of CM-1 in wound healing, we administered several concentrations of CM-1 to incisional wounds and to unwounded skin of collagen-luciferase transgenic mice. Luciferase bioluminescence served as a quantitative reporter of CO1A2 transcription. Imaging was performed to measure the luciferase activity on these mice at different time points after administration of 1, 10 and 100 ug CM-1. The highest amount (100 μg) showed the highest level of collagen expression both in unwounded and wounded skin. In another experiment, we used a scrambled peptide as a control and administered the peptides both topically and by injection. CM-1 was effective in stimulating luciferase expression both by topical and intradermal administration. To determine the effect of CM-1 on wound strength, we measured the tensile strength in incisional wounds of wild type C57B6 mice at 10 d. Histological analysis was also performed to assess the collagen content and organization of the tissue. The data showed a statistically significant increase in tensile strength and collagen content at 10 and 100 μg doses. These results illustrate the utility of CM-1 as a novel, low-molecular weight vulnerary agent.

Notes: It has been evidenced that the fat may have a potential to secret some growth factors or be a source of stem cells. So, we explore the effects of fat on healing of porcine skin wounds so as to provide a new method for clinical skin wound repair after injury.Forty-eight full-thickness skin wounds were produced on both sides of the back in 6 male minipigs (8 wounds in each animal). Then these wounds were randomly divided into 4 groups, which were saline control group, fat autografting group, basic fibroblast growth factor (bFGF) treatment group and epidermal growth factor (EGF) treatment group. At day 3, 7, 14 and 21 after wounding, the area and the volume of wounds were measured and the histological examination was performed to evaluate the velocity and quality of wounds healing in different groups.At day 3 and 7, the amount of granulation tissues and vessel density in fat treatment group were significantly more than that in other groups. Wound areas and volume in fat treatment wounds were markedly decreased in compared with those in other groups (P 〈 0.01). Regenerated epidermis in fat treatment group was thicker than that in other groups.These results confirmed that the wound healing velocity and quality in wounds treated with fat autografting were enhanced. It indicated that fat has a potential to accelerate velocity and improve the quality of wound healing after skin injury.

Notes: During wound healing, myofibroblasts play a major role in wound contraction and matrix formation. At the end of healing, myofibroblasts disappear via apoptotic pathways. Hypertrophic scars are a fibroproliferative disorder that leads to considerable morbidity. Although no evidence exists, it has been postulated that a defect in myofibroblast apoptosis could be responsible for the pathological scar formation. We have isolated and cultured human normal wound (Wmyo) and hypertrophic scar (Hmyo) myofibroblasts and compared their basal apoptotic rates and their sensitivity to serum starvation and Fas antibody-induced apoptosis to that obtained for dermal fibroblasts (Fb). A higher rate of apoptosis as evidenced by morphological criteria and a propidium iodide assay was observed for Wmyo in comparison to Fb and Hmyo. These results came along with a low level of the anti-apoptotic proteins Bcl-2 and BclxL in Wmyo, whereas there was an increase in the level of the pro-apoptotic molecule Bax when compared to the results obtained for Fb and Hmyo. Hmyo showed a higher level of Bcl-2 compared to Fb but no difference in the Bax or BclxL level. After serum starvation, Wmyo revealed an increased apoptotic rate whereas Hmyo and Fb did not show any difference. Anti-Fas treatment did not modify the levels of apoptosis but strongly increased the cell growth of Hmyo as compared to Wmyo. This is the first study presenting a broad vision of the apoptotic sensitivity of normal and pathological myofibroblasts. These results confirmed the hypothesis of defects in apoptosis and growth during pathological scar formation impeding myofibroblast disappearance at the end of healing.

Notes: Vacuum-assisted closure (VAC) therapy has been shown to facilitate wound healing. Data on the mechanisms are scarce, although beneficial effects on blood flow and granulation tissue formation have been presented. In the current study, laser Doppler was used to measure microvascular blood flow to an inguinal wound in pigs during VAC therapy (− 50 to − 200 mmHg), including consideration of the different tissue types and the distance from the wound edge. VAC treatment induced an increase in microvascular blood flow a few centimeters from the wound edge. The increase in blood flow occurred closer to the wound edge in muscular as compared to subcutaneous tissue (1.5 cm and 3 cm, at − 75 mmHg). In the immediate proximity to the wound edge, blood flow was decreased. This hypoperfused zone was increased with decreasing pressure and was especially prominent in subcutaneous as compared to muscular tissue (0–1.9 cm vs. 0–1.0 cm, at − 100 mmHg). When VAC therapy was terminated, blood flow increased multifold, which may be due to reactive hyperemia. In conclusion, VAC therapy affects microvascular blood flow to the wound edge and may thereby promote wound healing. A low negative pressure during treatment may be beneficial, especially in soft tissue, to minimize possible ischemic effects. Intermittent VAC therapy may further increase blood flow.

Notes: Celosia argentea (CA) is used in traditional medicine for sores, ulcers, and skin eruptions. The present study was aimed at investigating the healing efficacy of CA extract in an ointment formulated (10 % w/w) as an alcohol extract of CA using a rat burn wound model. Wound closure occurred earlier in the treated rats (15 days vs. 30 in the untreated group; p 〈 0.05). Granulation tissue collected on every fifth day of healing showed an increase in collagen and hexosamine content at a faster rate in the treated wounds. This correlated with the accelerated wound closure observed in the treated groups. To probe the cellular basis of this effect, we investigated the effect of this extract on two major cellular responses; cell proliferation and cell motility, in two key cell lineages, fibroblasts and keratinocytes. CA was not toxic at concentrations of 〈 3 µg/ml in fibroblasts and 〈 30 µg/ml in keratinocytes. The alcohol extract promoted cell motility and proliferation of primary dermal fibroblasts at 0.1–1.0 µg/ml but did not alter these responses in primary keratinocytes. In an initial examination of molecular mechanisms, we found that the CA extract did not alter fibroblast and keratinocyte responses to the wound repair-associated epidermal growth factor receptor ligands. In short, we demonstrate a salutary action of the CA extract on wound healing, and suggest that this may be due to mitogenic and motogenic promotion of dermal fibroblasts.

Notes: Keratinocytes are frequently used to examine efficacy of wound healing products and dermatological agents in vitro. Cultured keratinocyte sheets are also used as autologous or allogenic grafts to promote wound closure. Because it is well known that the expression patterns of keratin genes change when cell cultures reach confluence, we investigated the expression pattern of wound healing-related genes, including growth factors and cytokines. Of additional particular interest is a novel wound healing related factor, secretory leukocyte protease inhibitor (SLPI), which appears to enhance tissue repair. We found that the expression pattern varied for specific genes expressed by keratinocytes as confluence was reached. Specifically, SLPI expression peaked in the early postconfluent state and vascular endothelial growth factor and amphiregulin in the late postconfluent state. Some gene products exhibit autocrine activity, whereas others exert paracrine regulation of growth. These findings indicate that it is critical to define the growth and differentiation state of human keratinocyte cultures to better determine responses and efficacy in vitro to various dermatological/wound care agents tested.

Notes: Induction of bone tissue requires three elements: osteoprogenitor cells, osteoinductive factors, and a supporting extracellular matrix. In this study, we report on an experimental model in dogs of heterotopic bone tissue production, based on the integration of these osteo-inductive factors into abdominal implants. The implants consist of either a type I collagen sponge wrapped with periosteum and omentum or a type I collagen sponge embedded with demineralized bone powder, platelet-rich plasma, thrombin, and calcium chloride wrapped with omentum, with or without periosteum. Automated histomorphometric analysis showed an efficient production of trabecular bone, which corresponded to 50–70 % of the total tissue composition 4 months after implant formation. High expression of the osteoinductive cytokines transforming growth factor-β and bone morphogenetic proteins-2 and -4 was shown by immunohistochemistry in macrophages, endothelial cells from neoformed capillaries, osteoblasts, osteoclasts, and the mesenchymal tissue around the bone trabeculae. These approaches are novel and efficient surgical procedures to produce mature trabecular bone that could be used as a potential source of bone tissue for autotransplantation.

Notes: Tissue shortage complicates the surgery of cleft lip and palate anomalies and the healing of defects on the palate impairs growth of the dento-alveolar complex due to scar tissue formation. Implantation of substitutes into the wound area might overcome this adverse effect. The aim of this study was to compare the tissue response to three collagen-based (collagen type I substrate alone, or collagen coated with elastin or chondroitin-6-sulfate) and two skin-derived substrates (unprocessed dermis and AlloDerm) after implantation into 12 dogs. Histology was performed at 3, 10, and 20 days postsurgery. We showed that all substrates were well tolerated. However, it is unclear whether AlloDerm was rapidly degraded or if it was sequestrated. There was no elastin or collagen present in these wounds. All collagen-based substrates showed good epithelial regeneration, although heparan sulfate (JM 403) was absent. Wounds treated with the collagen-based substrates contained fewer myofibroblasts at 20 days postsurgery and the type III collagen fibers in the immature scar tissue were more randomly oriented than in an untreated wound. In conclusion, palatal wounds with a dermal substrate heal with fewer indications of scar tissue formation and evoke only a mild inflammatory reaction, which is preferred over the tissue reaction in an untreated wound.

Notes: Abdominal wall fascial wound healing failure is a common clinical problem for general surgeons, manifesting in early postoperative fascial dehiscence as well as delayed development of incisional hernias. We previously reported that abdominal wall fascial incisions normally recover breaking strength faster than simultaneous dermal incisions in a rodent model. The accelerated fascial repair was associated with greater fibroblast cellularity within fascial wounds and increased wound collagen deposition. The current study was designed to determine whether accelerated fascial healing is the result of increased fascial fibroblast kinetic activity as measured by a more efficient fibroblast phenotype for binding to and remodeling a collagen matrix. Using a new model of abdominal wall repair, fibroblast cell cultures were developed from uninjured and wounded fascia and compared to dermal fibroblasts in order to define the fibroproliferative kinetic properties of abdominal wall fibroblasts. Fascial wound fibroblasts produced a more efficient and greater overall collagen lattice compaction compared to dermal fibroblasts. Acute fascial wound fibroblasts also showed enhanced cell proliferation compared to dermal fibroblasts but no significant differences in collagen production when normalized to cell number. These results suggest that fascial fibroblasts express distinct acute repair phenotypes and therefore a specific mechanism for fascial repair following injury.

Notes: We have previously shown that fibroblasts obtained from adhesions produce greater amounts of transforming growth factor-beta 1 (TGF-β1) and extracellular matrix (ECM) molecules than normal fibroblasts isolated from normal peritoneum. The purpose of the current studies was to examine the effect of Tisseel (Baxter Healthcare Corporation, Glendale, CA), a fibrin sealant containing fibrinogen, aprotinin (a protease inhibitor), thrombin, and CaC12, on TGF-β1 and ECM production by human peritoneal mesothelial cells, normal peritoneal fibroblasts, and adhesion fibroblasts. Multiplex reverse transcription–polymerase chain reaction using β-actin as a housekeeping gene was used to determine mRNA levels of TGF-β1 and ECM in these cells at 6, 12, 24, and 48 hours under normoxic conditions in the following treatment groups :  fibrin sealant (Tisseel) alone; fibrin sealant with the two components diluted 1 : 2; fibrin sealant with the sealer protein component reconstituted without aprotinin (a protease inhibitor); fibrin sealant with the sealer protein component reconstituted without aprotinin (and both components diluted 1 : 2); fibrin sealant components diluted to physiologic concentrations; and control (culture media). The test compositions had little effect on TGF-β1 mRNA expression in mesothelial cells and normal peritoneal fibroblasts, but resulted in a marked reduction of TGF-β1 from adhesion fibroblasts. Expression of type I collagen by human peritoneal mesothelial cells was not detected; the compositions reduced type I collagen mRNA expression by both types of fibroblasts. Type III collagen was detected at six hours, and increased approximately 50 percent by culturing for 48 hours. Tisseel at full strength and with both components diluted 1 : 2 initially increased type III collagen mRNA levels; in contrast, type III collagen mRNA levels were reduced in mesothelial cells by the fibrin sealant without aprotinin at both concentrations and at physiologic concentrations. In both types of fibroblasts, the Tisseel compositions reduced type III collagen mRNA expression. Fibronectin mRNA were transiently reduced at six hours by approximately 50 percent in the presence of the Tisseel components, but then returned to control levels. Fibronectin mRNA levels were not altered in normal peritoneal fibroblasts, but were reduced by all but the physiologic concentration in adhesion fibroblasts. Tisseel may modulate human peritoneal mesothelial cell, normal peritoneal fibroblast, and adhesion fibroblast function. These results suggest that fibrin sealant prepared from the Tisseel kit without aprotinin has the ability to reduce ECM and TGF-β1 mRNA levels, especially from adhesion fibroblasts, which may indicate a role in reduction of postoperative adhesion development.

Notes: Transforming growth factor-β1 is a potent mediator of the differentiation of fibroblasts into myofibroblasts, which is characterized by the appearance of the cytoskeletal protein α-smooth muscle actin. The aim of this study was to investigate the role of integrin extracellular matrix receptors in transforming growth factor-β1–induced myofibroblast differentiation. We show that blockade of the αv and/or β1 integrins prevents the transforming growth factor-β1–induced myofibroblast differentiation, seen by the increased expression of α-smooth muscle actin and enhanced collagen gel contraction in three human fibroblast cell lines (from the mouth, skin, and kidney). Further, blockade of αv specific integrins αvβ5 and αvβ3 suppressed myofibroblast differentiation in fibroblasts from the mouth and skin; however, in the kidney cells, the prevention of differentiation was seen only with blockade of αvβ5 integrin but not αvβ3. A possible reason for this result may be different degrees of responsiveness to transforming growth factor-β1 treatment seen from different anatomical origins of the cell lines. These data indicate a novel role for αv integrins in the differentiation of human fibroblasts from the mouth, skin, and kidney into myofibroblasts and suggest that there is a common differentiation pathway.

Notes: First identified in psoriatic epidermis and subsequently in other inflammatory cutaneous lesions, human β-defensin-2 (hβD-2) is one of two endogenous antimicrobial peptides related to defensins in plants and animals. Our objective was to determine the expression of hβD-2 after injury and in chronic wounds. Biopsies of normal ipsilateral thigh skin and wound edges were taken from nine consecutive patients with venous leg ulcers (day 1) and from the same biopsy sites 2 days later (day 3). Sequential samples were also obtained from intact or meshed bilayered bioengineered skin consisting of neonatal human keratinocytes and dermal fibroblasts in a collagen matrix. Specimens were processed and immunostained for hβD-2 using a polyclonal rabbit antibody. In both human tissues and bioengineered skin, staining for hβD-2 was confined to the upper epidermal layers, sparing the basal cells. Analysis of 26 tissue samples from patients showed that normal skin had no hβD-2 expression but that marked up-regulation occurred after wounding by day 3. Conversely, chronic ulcers showed moderate-to-strong immunostaining for hβD-2 at baseline on day 1, with little or no change in intensity after wounding by day 3. In vitro, bioengineered skin showed increased distribution of cytoplasmic hβD-2 immunostaining after meshing. We conclude that the expression of hβD-2 is up-regulated after injury. Chronic wounds uniformly show a constitutively high baseline expression of hβD-2, possibly due to ongoing tissue injury and bacterial colonization.

Notes: That a contractile actin isoform has been found in cells of other cartilage tissues in healing and disease states prompted this investigation of the presence of α-smooth muscle actin (α-SMA) in pathological human intervertebral disc tissue. The presence of this isoform has been reported in human intervertebral disc specimens obtained at autopsy from subjects for whom there were no reported symptoms. An objective of this study was to evaluate the cell density and percentage of α-SMA–containing cells in pathological nucleus pulposus tissue obtained from lumbar disc surgery from 17 patients. Additionally, explants of nucleus pulposus material were cultured to determine how α-SMA expression changed with time in vitro. Seventy-six 5-mm diameter explants (approximately 2 mm thick) pooled from six lumbar surgeries were cultured for 1, 2, 4, or 6 weeks. Microtomed sections of paraffin-embedded specimens were stained with hematoxylin and eosin or a monoclonal antibody to α-SMA. Histologically, cells were categorized as to α-SMA phenotype (positive or negative), and the areal cell density was determined. The evaluation of the cultured nucleus pulposus explants also included documentation of the percentage of cells that were round or elongated and the percentage of the cells that were part of a group (group: ≥ 2 cells). Every nucleus pulposus section exhibited the presence of α-SMA–containing cells, which accounted for approximately 24 percent of the cells in vivo. In vivo, the cell density was significantly higher in older individuals (p = 0.02). The average time for cell outgrowth from the explants was 8.6 days. Approximately 10–15 percent of the cells in the explants stained positive for α-SMA. The time in culture had no significant effect on any of the outcome measures except the percentage of α-SMA–containing cells that were round (p = 0.008), with values decreasing through 4 weeks and then slightly rising at 6 weeks. The role of α-SMA in intervertebral disc pathology warrants further investigation.

Notes: Antimicrobial peptides are essential to innate host defense as effectors of pathogen clearance and can modify host cell behaviors to promote wound repair. While these two functions appear interrelated, it is unclear whether the ability to aid in wound repair requires inherent antimicrobial function. We hypothesized that the influence of antimicrobial peptides on wound repair is not dependent on antimicrobial function. To explore this, we analyzed the microbial killing activity of peptide fragments and correlated this with the ability to influence wound repair in mice. HB-107, a peptide lacking antimicrobial activity and originally derived from the antimicrobial cecropin B, showed up to 64 percent improvement in wound repair compared to scrambled peptide and vehicle controls, an effect comparable to treatment with recombinant human platelet-derived growth factor-BB (formulated as RegranexTM). Wounds treated with HB-107 showed keratinocyte hyperplasia and increased leukocyte infiltration. Furthermore, HB-107 stimulated interleukin-8 secretion from cultured endothelial cells, an effect that may explain the increase in leukocyte migration. These findings confirm that antimicrobial peptides can function as effectors of cutaneous wound repair. Moreover, this study furthers our understanding of antimicrobial peptides by showing that their wound repair properties can be independent of antimicrobial function.

Notes: We examined the structural characteristics of repair tissue induced by recombinant human bone morphogenetic protein-2 in a rabbit model of laryngotracheal reconstruction. Twenty-four New Zealand White rabbits were randomly divided into four groups of six rabbits. Two groups were treated with recombinant human bone morphogenetic protein-2 delivered on an absorbable collagen sponge, while two groups were used as controls. Rabbits were euthanized at 1 and 4 weeks after surgery. The larynx was removed, fixed, and sectioned. The sections were stained with hematoxylin-eosin, safranine O/fast green, and immunostained with an antibody for tissue inhibitor of metalloproteinases-1. In rabbits treated with bone morphogenetic protein-2, the defects were filled with new cartilage and bone at 4 weeks after surgery. There were no discontinuities or gaps at the margins of the cartilage defects. Proteoglycans were synthesized in new cartilage in rabbits treated with bone morphogenetic protein-2, and were present 4 weeks after surgery. The general aspects of the vascular pattern and the pattern of tissue inhibitor of metalloproteinases-1 expression were similar in control and treated rabbits, both 1 week and 4 weeks after surgery. The repair tissue induced by recombinant human bone morphogenetic protein-2 consisted of new cartilage and bone perfectly integrated with host tissue at the site of the cricoid cartilage defects. This new cartilage was able to mature and produce proteoglycans.

Notes: This study examined dermal wound healing in juvenile red Duroc pigs and determined that these animals exhibit a unique healing phenotype at multiple levels. Gross and histologic analysis revealed that full-thickness and deep dermal (1.8 mm deep) wounds both heal via formation of hypercontracted, hyperpigmented scars. Molecular analysis using reverse-transcriptase polymerase chain reaction and porcine-specific primer sets revealed that types I and III collagen, heat shock protein 47, bone morphogenetic protein-1, several proteoglycans, and tissue inhibitor of metalloproteinases 1–3 all showed a unique biphasic pattern of mRNA expression compared to previous results with Yorkshire pigs. This pattern was characterized by an initial peak of expression early after wounding, followed by a return to near-normal levels by days 28–42, and then a second increase in mRNA levels at days 56–70. The second phase of increased gene expression correlated with an increased collagen deposition as observed by picrosirius red staining and polarizing light microscopy. Reverse-transcriptase polymerase chain reaction analysis also revealed a prolonged expression of matrix metalloproteinase-2 compared to previous findings in the Yorkshire strain. Further characterization of the genetics and molecular biology associated with the red Duroc phenotype may provide insight into aberrant human wound healing.

Notes: Lactate accumulation is a characteristic of wounds in which glycolysis, occurring both aerobically and anaerobically, contributes to its production. Cell proliferation is a critical component of healing wounds. Recently it has been shown that lactate can chelate iron and thus promotes production of hydroxyl radicals. We report here that exogenous lactate increases intracellular oxidants and that the oxidants promote cell growth in cultured dermal fibroblasts in a dose-dependent manner. The production of lactate-mediated oxidant requires iron and hydrogen peroxide and with increasing iron concentration oxidant production is raised as well. However, we found cell proliferation is retarded by 15 mM lactate in the presence of a high iron concentration (7.25 µM). The antioxidants catalase and mannitol abolish the inhibitory effect of high lactate. We conclude from these results that increased proliferation of cultured human fibroblasts by exogenous lactate is mediated by oxidant production.

Notes: The release of mast cell granules is commonly associated with inflammation and fibrosis. However, does direct communication between mast cells and fibroblasts through gap junction intercellular communication (GJIC) occur? Fibroblast populated collagen lattice (FPCL) cast with mast cells show enhanced lattice contraction. Do released granules or GJIC between mast cells and fibroblasts promote enhanced lattice contraction? Mast cells preloaded with a fluorescent dye that readily passes through gap junctions were cast in FPCL. Dye passed from mast cells into fibroblasts within these cocultured mast cell-FPCLs. Fatty acid amide hydrolase inhibitor blocks the breakdown of oleamide, which is a potent endogenous inhibitor of GJIC. GJIC was blocked for 3 days when mast cells were pulsed for 3 hours with fatty acid amide hydrolase inhibitor. Mast cells pretreated with fatty acid amide hydrolase inhibitor cast in cocultured mast cell-FPCLs failed to enhance cocultured lattice contraction. Mast cell-FPCLs made with mouse fibroblasts unable to generate GJIC failed to show enhanced lattice contraction. Degranulated mast cells were equal to intact mast cells at enhancing cocultured mast cell-FPCL contraction. The supernatant from degranulated mast cells had no effect upon FPCL contraction. Therefore, enhanced mast cell-FPCL contraction appears to be independent of mast cell granules, but dependent upon GJIC between fibroblasts and mast cells. We speculate that mast cell–fibroblast GJIC may play a role in fibrosis.

Notes: Proteolytic activity is required for the turnover of the extracellular matrix during wound healing. Matrix metalloproteinases can collectively cleave all components of the extracellular matrix, with the endogenous tissue inhibitor of metalloproteinase-1 regulating their activity. Breast tissue taken at varying postoperative times (n= 92) or during surgery (controls, n= 17), was used to investigate the temporal and spatial activity of matrix metalloproteinase-2 and -9 and tissue inhibitor of metalloproteinase-1 during human wound healing. Matrix metalloproteinase activity, determined using a quenched fluorescence substrate assay, increased during early healing (3–8 weeks) compared to controls, and then decreased between 24 and 36 weeks after surgery (p 〈 0.05 until 24 weeks, Mann-Whitney U-test). Immunohistochemistry scores for matrix metalloproteinase-9 expression were significantly elevated compared to controls in scar endothelial cells and fibroblasts from 2 until 12 and 20 weeks, respectively. Matrix metalloproteinase-2 staining was observed exclusively in fibroblasts, reaching maximum levels 8–12 weeks after surgery, decreasing by 1.5 years but remaining significantly increased. Tissue inhibitor of metalloproteinase-1 staining was relatively sparse but was significantly increased until 8 weeks after surgery. These results show that matrix metalloproteinases are present at elevated levels during early wound healing, when angiogenesis occurs, and suggest that matrix metalloproteinase-9 may play a significant role. The later expression of matrix metalloproteinase-2 and -9 in fibroblasts suggests a role in extracellular matrix remodeling.

Notes: Since pronounced differences exist between the fetal and adult repair processes, we studied the proliferative response of skin fibroblasts from these two stages to transforming growth factor-β (TGF-β), a cytokine with a broad range of activities in tissue repair. Here, we present evidence that TGF-β inhibits fetal human skin fibroblasts, while it is stimulatory for adult ones. This proliferative effect of TGF-β was found to be concentration- dependent, but isoform-independent. Furthermore, even a transient exposure of the cells to this growth factor was sufficient to exert its stimulatory or inhibitory action. Accordingly, we have studied the immediate responses provoked by TGF-β in major signaling pathways, and we have found that it induces a rapid activation of the SMAD pathway, i.e., phosphorylation and nuclear translocation of SMAD2, followed by dephosphorylation, most probably due to degradation by the proteasome. However, similar intensity and kinetics of this activation have been observed in both fetal and adult fibroblasts. On the other hand, curcumin, a natural product with wound healing properties that inhibits several intracellular signaling pathways, was found to completely abrogate the inhibitory effect of TGF-β1 on human fetal skin fibroblasts, without affecting the stimulatory action on fibroblasts from adult donors. In conclusion, there is a major radical in the proliferative response of fetal and adult human skin fibroblasts to TGF-β, possibly reflecting the different repair strategies followed in these two stages of development.

Notes: Previous studies in a pig model of skin wound healing showed a coordinate expression of transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF), and exposure of porcine skin fibroblasts in vitro to recombinant human CTGF significantly up-regulated mRNA levels for a number of molecules. Therefore, based on recent reports that small interfering RNA (siRNA; double-stranded RNA) can effect silencing of the expression of gene(s), this approach has now been used with CTGF-specific siRNA to better understand the function of this growth factor in regulating matrix homeostasis and repair. Normal skin fibroblasts from Yorkshire pigs were treated with 0.1–0.8 µM CTGF siRNA, TGF-β, or TGF-β plus CTGF siRNA for 12–48 hours. Total RNA was isolated and quantified, and then mRNA levels for specific molecules were analyzed by reverse transcription-polymerase chain reaction. Protein levels for CTGF and HSP47 were assessed by Western-blot analysis. CTGF siRNA transfection led to significant decreases in mRNA and protein levels for CTGF in both a dose- and time-dependent manner. mRNA levels for types I and III procollagen, decorin, HSP47, tissue inhibitor of metalloproteinase -1, -2, -3, and basic fibroblast growth factor were also significantly and uniquely decreased following exposure of cells to CTGF siRNA. Addition of TGF-β to the cells led to increases in CTGF mRNA levels that were blocked by CTGF siRNA. CTGF siRNA exposure also significantly and selectively down-regulated TGF-β–mediated increases in mRNA levels for types I and III procollagen. The results indicate that CTGF can regulate extracellular matrix molecule, growth factor, and proteinase inhibitor gene expression, and that some of the TGF-β effects on skin fibroblasts are via a CTGF-dependent pathway. (WOUND REP REG 2004;12:–)

Notes: This article represents a process paper describing the development, at our facility, of a wound care fellowship that was scheduled to begin in July of 2003. The proposed program is in no way a finished product or our statement of how the program must be. This article is presented as a call to wound care professionals for input, criticism, guidance, and—we hope—adoption and acceptance of wound care fellowships in some format in the future. After many years of work in this field, it has become apparent that without medical specialization wound care will never rise from its current status of part-time avocation to full-time occupation. After a brief background and description of the present status of wound care education, an initial curriculum, program objectives, and clinical rotation schedule are presented. We look forward to the day when this program will have been replaced with a fully accredited, readily accepted, board-certifiable fellowship program with all the rights and responsibilities afforded the other medical specialties.

Notes: Venous leg ulcers are among the most common chronic wounds. Treatment is commonly with a limb compression bandage. Previous small, often single-center, studies have shown that it is possible to predict which wounds are likely to respond to compression therapy. We designed this cohort study using a dataset of over 20,000 individuals with a venous leg ulcer to investigate the accuracy of several prognostic models. Creating complex models using logistic regression, as well as simply counting prognostic factors, we show that initial measures of wound size and duration accurately predict, as measured by area under the receiver operator curve and Brier score, who will heal by the 24th week of care. For example, a wound that is less than 10 cm2 and less than 12 months old at the first visit has a 29 percent chance of not healing by the 24th week of care, while a wound greater than 10 cm2 and greater than 12 months old has a 78 percent chance of not healing. Ultimately, these models can be applied by a clinician to help determine whom to continue to treat with standard care and perhaps whom to treat with adjuvant therapies. They may also aid in the design of clinical trials.