Library

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: Introduction: Ischemia-reperfusion (IR) injury is commonly associated with numerous pathologies including pressure sores, venous stasis ulcers, and lower extremity diabetic ulcers. The purpose of this study is to further investigate the relationship between age and ischemia-reperfusion skin injury in a rat model utilizing magnets for the purpose of injury creation.
Methods: Magnets were designed for subcutaneous placement and calibrated such that a second magnet placed externally over them would cause compression that exceeds capillary perfusion pressure (ischemia). Removing the external magnet results in reperfusion of the skin. After placing subcutaneous magnets in aged and young Fisher 344 rats, repeated cycles of external magnet placement and removal were performed. 
Results: Visual analysis of the skin revealed statistically significant greater areas of injury in the aged rats relative to their younger counterparts (37.4 ± 13.3% vs. 24.1 ± 14.8%, P 〈 .02)
Conclusions: Aged rats demonstrate an increased degree of injury relative to their younger counterparts in response to ischemia-reperfusion injury. Future studies will attempt to delineate differences in the markers of IR injury (such as myeloperoxidase and vitamin E levels) in aged versus young rats, giving insight to the mechanisms responsible for the impaired wound healing seen in the elderly.

Notes: Introduction: Telomeres are nucleoprotein structures at the ends of each chromosome. Due to the inability of DNA polymerase to replicate the full length of the chromosome, up to 50–200 base pairs of the telomere are lost during each successive round of cell division. In adult human somatic cells, telomerase is not active resulting in progressive loss of telomere length and entry into replicative senescence as observed in cell culture. hTERT is the catalytic subunit of telomerase, an enzyme which maintains telomere length. Transfection of human dermal fibroblasts (HDFs) by hTERT has been shown to reverse the senescent phenotype seen in aging HDFs in vitro. ERK (p44/42) is a MAP kinase which functions as a critical intermediary in the determination of cell growth and differentiation. Activation of ERK occurs through phosphorylation of threonine and tyrosine residues. 
Methods: In order to delineate some of the cellular mechanisms by which hTERT functions, we treated adenoviral hTERT (Ad-hTERT) transfected HDFs with TGFB1, and assayed phosphorylated ERK activity by Western blotting.
Results: Ad-hTERT treated HDFs demonstrated a 2–3 fold increase in phospho-ERK activity. In addition, our preliminary findings show that Ad-hTERT transfected HDFs have increased TGFB1, TGFB1-Receptor I and II, and COL1A1 gene expression by real-time rtPCR. 
Conclusions: Increased phosphor-ERK activity as well as increased TGFB1, TGFB1-Receptor I and II, and COL1A1 gene expression is seen in hTERT transfected HDF’s. Further studies will focus on defining other intermediary changes resulting from Ad-hTERT tranfection.Funding source: Geron Corporation

Notes: A critical limitation of using retroviral vectors for gene therapy is their inability to infect non-dividing cells. Although, the adenoviral vectors have the advantage of being able to infect both dividing and non-dividing cells, they elicit inflammatory response, thus making the interpretation of in vivo experiments harder. Adeno-associated virus (AAV) and Lentiviral vectors do not have those limitations, however, scant information is available about their transfection efficiency under low-oxygen tension. To determine if low-oxygen microenvironment affects viral vector-mediated gene transfection, we have used two other viral vectors, Adeno-associated virus (AAV) and Lentiviral constructs in vitro and in vivo to express foreign genes in hypoxic cultured human dermal fibroblasts and ischemic rat wounds. Both cultured normoxic and hypoxic (1% O2) human dermal fibroblasts were identically transfected by the AAV vector. A lenti6-LacZ construct was injected onto the periphery of rat ischemic and non-ischemic wound (106 pfu/wound) at the time of wounding. Wounds were harvested at post-operative day 7. Frozen sections of the wounds were fixed in cold acetone and stained with a in situ β-gal staining kit. Intense expression of β-gal was observed without any inflammatory response. No significant difference of transfection efficiency was observed between the ischemic and non-ischemic wounds. Thus our data indicates that both AAV and Lentiviral vectors are suitable to use in gene-therapy experiments in both ischemic and non-ischemic cells and tissues in vitro and in vivo.

Notes: Accumulating data strongly suggests that under hypoxic conditions the adaptive physiological response of the cells involves co-operation between oxygen sensing and growth factor signals that cause HIF-1 mediated gene expression. The HIF-1 has been identified as a central and critical molecule in oxygen sensing and possibly a master switch. Of the many cytokines that have been shown to modulate collagen production, TGFβ1 appears to be crucial as it can sustain stimulation of collagen production as well as autoinduction of its own synthesis. Several genes that are known to be hypoxia inducible are also up-regulated by TGFβs, and the promoter of TGFβ3, a member of the family, has a hypoxia-response element (HRE). Recent reports indicate that HIF-1α physically interacts with Smad3 and that the TGFβ and hypoxia signaling pathways synergize at the transcriptional level to regulate gene expression. To determine if this interaction upregulates gene expression through the HRE element, we have co-transfected cultured human dermal fibroblasts with the mammalian expression plasmid for HIF-1α(pCEP1-HIF-1α) with a reporter construct 5HRE-luc, containing a concatemer of five copies of HRE derived from human vascular endothelial growth factor (VEGF), a minimal cytomegalovirus (CMV) promoter and a reporter gene,luciferase.). When treated with 2 ng/ml of TGFβ1 protein, co- transfected hypoxic aged and young human dermal fibroblasts showed significant synergistic upregulation of the reporter gene expression. To further confirm the TGFβ-HIF-1α interaction we have created a TGFβ1 -RNAi construct by subcloning a 19-nucleotide sequence derived from rabbit in a mammalian expression vector (p-SUPER) that directs the synthesis of small interfering RNAs (siRNAs). Hypoxic rabbit cultured dermal fibroblasts co-transfected with TGFβ1-RNAi and 5HRE-luc showed down-regulation of reporter gene expression.As a prerequisite to understanding the biology of physiological and pathological ischemic tissue repair process it is important to delineate the molecular basis of regulation of collagen gene expression in fibroblasts. Each step in the TGFβ signaling cascade is a potential target for highly specific therapy and information about how hypoxic low-oxygen microenvironments affect the TGFβ cascade could be useful to develop novel therapeutic strategies that will involve agonist and antagonist agents that directly interfere with these steps.

Notes: Hydration of keratinocytes modifies the levels of cytokines they secrete, which in turn impacts the secretory behaviour of dermal fibroblasts. In an in vitro coculture model, conditioned media (CM) collagen content was decreased 44% when keratinocytes were hydrated. We hypothesized that this is partly due to increased MMP-activity. We used the same coculture model to study changes in MMP-activity and TIMP secreted by keratinocytes as well as by fibroblasts in monoculture and in coculture in relation to air-treatment or hydration of keratinocytes. Stratified human epidermal keratinocytes (HEK) and confluent human dermal fibroblasts (HDF) were cocultured for 72 h under serum-free conditions. HEK were either kept at the air-interface or hydrated. CM was assayed for MMP-1, −2, −9, TIMP-1 and −2 were assayed using zymograms, western blotting, and ELISA. MMP-1, secreted by both cell types, increased significantly in cocultures compared to monocultures (4-fold in the air-treated group, 26-fold in the hydrated group). MMP-2, secreted mainly by HDFs, was significantly increased by coculture (hydration: 2.4-fold, air: 2.8-fold). MMP-9, predominantly secreted by air-treated HEKs and was significantly decreased in hydrated monoculture (76%) and coculture. HEK-monoculture hydration also significant decreased MMP-1 (86%) and MMP-2 (81%) activity. HDF-secreted TIMP-1 expression was significantly increased by coculture and was unaffected by hydration. Our findings demonstrate that paracrine interactions between HEK and HDF modify MMP activity and that HEK hydration significantly effects on MMP activity. The findings provide insight into the role of hydration on HEK and HDF ctivity during the wound healing process.

Notes: Relatively intense burning has been suggested as a possible alternative to the restoration of pre-European settlement forest conditions and fire regime in mixed conifer forests, in contrast to thinning of trees and light prescribed burning. In 1993 a management-ignited fire in a dense, never-harvested forest in Grand Canyon National Park escaped prescription and burned with greater intensity and severity than anticipated. We sampled the burned site and an adjacent unburned site (270 ha each) 6 years after the fire to assess burn effects on tree structure (species composition, size and age distributions, regeneration, and snags), forest floor fuels, and coarse woody debris. Tree structure before fire-regime disruption (1879 CE) was reconstructed with dendroecological techniques. By 6 years after burn the fire reduced average tree density (331 trees/ha) and basal area (28.5 m2/ha) to levels similar to pre-European reference conditions (approximately 246 trees/ha and 28.5 m2/ha). Mortality was concentrated in fire-susceptible species, especially white fir, restoring dominance by fire-resistant ponderosa pine. Forest floor fuels were reduced, and regeneration by aspen and understory plants was vigorous. Densities of large snags and logs were high. However the fire also killed a high proportion of old-growth trees, especially aspen. Burning created more spatial variability in forest structure than was present before fire-regime disruption by killing many trees in some areas of the site but few in other areas. The intentional use of severe burning would be challenging to managers because of the increased risk of escaped fires, but the ecological outcome of this particular wildfire was not inconsistent with ecological restoration goals for this ecosystem type.

Notes: Twenty-four-hour recall data from the Continuing Survey of Food Intake by Individuals (CSFII) are frequently used to estimate dietary exposure for risk assessment. Food frequency questionnaires are traditional instruments of epidemiological research; however, their application in dietary exposure and risk assessment has been limited. This article presents a probabilistic method of bridging the National Health and Nutrition Examination Survey (NHANES) food frequency and the CSFII data to estimate longitudinal (usual) intake, using a case study of seafood mercury exposures for two population subgroups (females 16 to 49 years and children 1 to 5 years). Two hundred forty-nine CSFII food codes were mapped into 28 NHANES fish/shellfish categories. FDA and state/local seafood mercury data were used. A uniform distribution with minimum and maximum blood-diet ratios of 0.66 to 1.07 was assumed. A probabilistic assessment was conducted to estimate distributions of individual 30-day average daily fish/shellfish intakes, methyl mercury exposure, and blood levels. The upper percentile estimates of fish and shellfish intakes based on the 30-day daily averages were lower than those based on two- and three-day daily averages. These results support previous findings that distributions of “usual” intakes based on a small number of consumption days provide overestimates in the upper percentiles. About 10% of the females (16 to 49 years) and children (1 to 5 years) may be exposed to mercury levels above the EPA's RfD. The predicted 75th and 90th percentile blood mercury levels for the females in the 16-to-49-year group were similar to those reported by NHANES. The predicted 90th percentile blood mercury levels for children in the 1-to-5-year subgroup was similar to NHANES and the 75th percentile estimates were slightly above the NHANES.

Notes: In recent years, there has been a renewed interest in the criminal victimization of elderly persons. Given that homicides of the elderly are thought to occur primarily in the context of robbery/burglary situations, this study seeks to identify situational as well as structural and cultural factors that influence the murder of older persons. In particular, it has been noted that Southern culture promotes a notion that one should ‘respect their elders.’ Using conservative Protestant affiliation as a proxy for Southern culture, this study also examines the relationship between Southern culture and homicide of the elderly. Findings show that Southern culture as reflected in conservative Protestant affiliation does not insulate the elderly from victimization.

Notes: (J Interven Cardiol 2004;17:93–94)