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Notes: Abstract Islet autotransplantation offers the potential for preventing the surgically induced diabetes that is an inevitable consequence of total pancreatectomy. This paper describes the first islet autotransplant programme in the United Kingdom and the first series in the world to use the spleen as a site for the islet graft. Over an 11 month period, 7 patients underwent total pancreatectomy for chronic pancreatitis combined with a simultaneous islet autotransplant. All 7 patients had normal glucose-tolerance levels and normal C-peptide levels pre-operatively. In 6 patients, islets were embolized into the liver via the portal vein (median transplanted volume=8.5 ml). In addition, 3 patients received islets into the splenic sinusoids via a short gastric vein (median transplanted volume=4 ml). One patient received islets into the spleen alone. One patient died of a stroke 4 weeks post transplantation. Two patients have achieved insulin independence, with a further two patients achieving ”transient” insulin independence (〈1 month). The remaining 2 patients, although requiring reduced insulin doses, have not achieved insulin-independence. However, all patients have C-peptide levels within the normal range. In trying to explain these findings, split proinsulin levels were measured and found to be elevated. High levels of split proinsulin cross react with the C-peptide assay and this would explain the falsely elevated C-peptide levels. Indeed insulin levels in these patients were all below the normal range. These findings would suggest that the use of C-peptide levels as the ”gold standard” for monitoring islet autograft function, may require reappraisal.

Notes: Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptoticgenes, originally discovered in baculovirus, exists in animals ranging frominsects to humans. Here, we investigated the ability of IAPs to suppress celldeath in both a neuronal model of apoptosis and excitotoxicity. Cerebellargranule neurons undergo apoptosis when switched from 25 to 5 mMpotassium, and excitotoxic cell death in response to glutamate. We examinedthe endogenous expression of four members of the IAP family, Xchromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosisinhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblotanalysis in cultured cerebellar granule neurons. Cerebellar granule neuronsexpress significant levels of RIAP2 mRNA and protein, but expression of RIAP1,NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did notchange when cells were switched from 25 to 5 mM potassium. Todetermine whether ectopic expression of IAP influenced neuronal survival afterpotassium withdrawal or glutamate exposure, we used recombinant adenoviralvectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellargranule neurons. We demonstrate that forced expression of IAPs efficientlyblocked potassium withdrawal-inducedN-acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNAfragmentation. However, neurons were only protected from apoptosis up to 24 hafter potassium withdrawal, not at later time points suggesting that IAPSdelay but do not block apoptosis in cerebellar granule neurons. In contrast,treatment with 100 μM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.

Notes: We have recently demonstrated that specific neuroanatomical patterns of Fos-like immunoreactivity are predictive of atypical antipsychotic activity. However, the fact that neuroleptics must be administered chronically in order to generate both extrapyramidal side effects and an optimal therapeutic response calls into question the relevance of acute changes in Fos-like immunoreactivity for these slowly developing events. Fos-like immunoreactivity cannot be used to identify neurons activated by chronic neuroleptic administration because the increase in Fos-like immunoreactivity produced by an acute antipsychotic injection is dramatically reduced following repeated neuroleptic administration. In contrast, expression of the immediate-early gene product ΔFosB is persistently elevated in the striatum by chronic haloperidol administration. This suggests that ΔFosB-like immunoreactivity may be used to identify neurons activated by chronic antipsychotic administration. Since typical and atypical neuroleptics elevate Fos-like immunoreactivity in different regions of the forebrain acutely, the purpose of the present study was to determine whether typical (haloperidol) and atypical (clozapine, ICI 204,636) antipsychotics produce distinct patterns of elevated ΔFosB-like immunoreactivity in the forebrain after chronic administration. Administration of haloperidol (2 mg/kg/day) to rats for 19 days induced a homogeneous elevation of neurons which displayed ΔFosB-like immunoreactivity in the ventral, medial and dorsolateral aspects of the striatum. Chronic haloperidol administration did not enhance ΔFosB-like immunoreactivity in the prefrontal cortex and lateral septal nucleus. Repeated administration of clozapine (20 mg/kg/day) and ICI 204,636 (20 mg/kg/day) for 19 days elevated ΔFosB-like immunoreactivity not only in the ventral striatum but also in the prefrontal cortex and lateral septal nucleus. However, these compounds had weak effects on ΔFosB-like immunoreactivity in the dorsolateral striatum. These results suggest that a preferential action on limbic structures such as the prefrontal cortex, ventral striatum and lateral septal nucleus may account for the ability of chronic clozapine and ICI 204,636 administration to reduce the symptoms of schizophrenia without generating extrapyramidal side effects.

Notes: The pattern of neurons which display haloperidol-induced Fos-like immunoreactivity closely matches the distribution of striatal D2 dopamine receptors, whereas clozapine-induced Fos-like immunoreactivity occurs primarily in regions that contain high levels of the D3 dopamine receptor. These neuroanatomical correlations suggest that haloperidol and clozapine may elevate Fos-like immunoreactivity by blocking D2 and D3 receptors respectively. In order to test this hypothesis, the abilities of prior administration of the D3 receptor-preferring agonist 7–hydroxy-N, N′-di-n-propyl-2-aminotetraline (7–OH-DPAT) to competitively reverse haloperidol- and clozapine-induced increases in Fos-like immunoreactivity were compared. Administration of 7–OH-DPAT (0.05 mg/kg, s.c.) 30 min before clozapine (20 mg/kg, s.c.) produced a 60% reduction in the number of neurons that displayed clozapine-induced Fos-like immunoreactivity in the major island of Calleja, nucleus accumbens and medial aspect of the striatum, while prior administration of 0.5 mg/kg (s.c.) of 7-OH-DPAT completely reversed these increases in Fos-like immunoreactivity. In contrast, the increases in Fos-like immunoreactivity in the major island of Calleja, nucleus accumbens and striatum (medial and dorsal aspects) induced by haloperidol (0.1 mg/kg, s.c.) were only reduced by the high dose of 7-OH-DPAT (0.5 mg/kg, s.c.). Hence, clozapine-induced increases in Fos-like immunoreactivity were more readily reversed by 7-OH-DPAT than elevations in Fos-like immunoreactivity produced by haloperidol. These results suggest that D3 receptor blockade plays a larger role in mediating clozapine- than haloperidol-induced increases in Fos-like immunoreactivity.

Notes: Using an antibody that recognizes the products of all known members of the fos family of immediate early genes, it was demonstrated that destruction of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle produces a prolonged (〉3 months) elevation of Fos-like immunoreactivity in the striatum. Using retrograde tract tracing techniques, we have previously shown that this increase in Foslike immunoreactivity is located predominantly in striatal neurons that project to the globus pallidus. In the present study, Western blots were performed on nuclear extracts from the intact and denervated striatum of 6-OHDA-lesioned rats to determine the nature of Fos-immunoreactive protein(s) responsible for this increase. Approximately 6 weeks after the 6-OHDA lesion, expression of two Fos-related antigens with apparent molecular masses of 43 and 45 kDa was enhanced in the denervated striatum. Chronic haloperidol administration also selectively elevated expression of these Fos-related antigens, suggesting that their induction after dopaminergic denervation is mediated by reduced activation of D2-like dopamine receptors. Western blot immunostaining using an antibody which recognizes the N-terminus of FosB indicated that the 43 and 45 kDa Fos-related antigens induced by dopaminergic denervation and chronic haloperidol administration may be related to a truncated from of FosB known as ΔFosB. Consistent with this proposal, retrograde tracing experiments confirmed that ΔFosB-like immunoreactivity in the deafferented striatum was located predominantly in striatopallidal neurons. Gel shift experiments demonstrated that elevated AP-1 binding activity in denervated striata contained FosB-like protein(s), suggesting that enhanced ΔFosB levels may mediate some of the effects of prolonged dopamine depletion on AP-1-regulated genes in striatopallidal neurons. In contrast, chronic administration of the D1-like receptor agonist CY 208–243 to 6-OHDA-lesioned rats dramatically enhanced ΔFosB-like immunoreactivity in striatal neurons projecting to the substantia nigra. Western blot immunostaining revealed that ΔFosB and, to a lesser extent, FosB are elevated by chronic D1-like agonist administration. Both the quantitative reverse transcriptase-polymerase chain reaction and the ribonuclease protection assay demonstrated that Δfos B mRNA levels were substantially enhanced in the denervated striatum by chronic D1-like agonist administration. Lastly, we examined the effects of chronic administration of D1-like and D2-like dopamine receptor agonists on striatal ΔFosB expression in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) primate model of Parkinson's disease. In monkeys rendered Parkinsonian by MPTP, there was a modest increase in ΔFosB-like protein(s), while the development of dyskinesia produced by chronic D1-like agonist administration was accompanied by large increases in ΔFosB-like protein(s). In contrast, administration of the long-acting D2-like agonist cabergoline, which alleviated Parkinsonian symptoms without producing dyskinesia reduced ΔFosB levels to near normal. Taken together, these results demonstrate that chronic alterations in dopaminergic neurotransmission produce a persistent elevation of ΔFosB-like protein(s) in both the rodent and primate striatum.

Notes: To date, most shear-velocity heterogeneity models in the lower mantle have been derived using long-period data. Comparatively little use has been made of the vast International Seismological Centre (ISC) data base of shear-wave arrival times. the aim of this study is to use the ISC P and S arrival times to construct global models of P and S heterogeneity in the lower mantle, and then to compare them in order to investigate whether, within the limitations of the data distributions, they might be proportional. the advantages of constructing both compressional- and shear-wave models from the one data set is that they share similar resolution properties, and hence are most reliable in the same areas.We use data from over 21000 events to derive a data set of P and S summary rays whose residuals we invert jointly along with hypocentral parameters of over 600 summary events. Particular attention is paid to data weighting so that outliers are not given undue influence. Furthermore, summary rays with high internal variances are downweighted.In order to diminish the effect of model parametrization on our conclusion, we derive three sets of P and S models expanded in terms of Legendre polynomials for their depth variation, and spherical harmonics for their lateral dependence. Comprehensive resolution and error analysis is performed.Correlation coefficients between our P and S models are highly significant, averaging approximately 0.7 for our lowest parametrization (245 model coefficients), and 0.5 for our more highly parametrized models. Visual comparisons show strong similarities in areas where resolution is high and error is small.We also conduct an experiment in which we derive compressional- and shear-wave heterogeneity models from data sets which contain P and S arrivals from the same seismograms. These data sets sample the mantle almost identically. the resultant models compare well but correlation drops towards the core-mantle boundary, indicating that there are genuine physical differences in the lowermost mantle.Our models indicate that the ratio of relative S to P heterogeneities is close to 2. This value is based on both our complete and restricted data set models and hence is not highly dependent on data weighting.

Notes: Abstract Euro-Ficoll (EF) and bovine serum albumin (BSA) are the two most commonly used media for the density gradient purification of human pancreatic islets. The aim of this study was to compare these two media with respect to the efficiency of human islet isolation. Ten human pancreata were collagenase-digested, and samples of digest were separated on either a continuous linear density gradient of BSA or a discontinuous gradient of EF (1.108/1.096/1.037/Euro-Collins). Efficiency of islet purification was assessed by insulin and amylase assay of aliquots aspirated from the BSA gradients, and from the interfaces of the EF gradients. Islets were obtained from two interfaces in the EF gradients. Islet yield from the upper interface was generally poor (median 28% of total insulin; range 2–71%), but purity was better than for an equivalent yield using BSA [1% (0–3%) amylase contamination for EF versus 6% (0–37%) for BSA;P=0.013]. Pooling both EF interfaces increased yield to 66% (17–81%) but markedly reduced purity [46% (0–50%) amylase for EF versus 31% (0–52%) for BSA]. In conclusion, the efficiency of human islet purification is similar, though disappointingly low, with BSA and with EF. Considerable scope exists, therefore, for improvement in the density gradient purification of human islets.

Notes: Abstract The use of the COBE 2991 cell processor (COBE Laboratories, Colorado) for large-scale islet purification using discontinuous density gradients has been widely adopted. It minimizes many of the problems such as wall effects, normally encountered during centrifugation, and avoids the vortexing at interfaces that occurs during acceleration and deceleration by allowing the gradient to be formed and the islet-containing interface to be collected while continuing to spin. We have produced cross-sectional profiles of the 2991 bag during spinning which allow the area of interfaces in such step gradients to be calculated. This allows the volumes of the gradient media layers loaded on the machine to be adjusted in order to mazimize the area of the gradient interfaces. However, even using the maximal areas possible (144.5 cm2), clogging of tissue at such interfaces limits the volume of digest which can be separated on one gradient to 15 ml. We have shown that a linear continuous density gradient can be produced within the 2991 bag, that allows as much as 40 ml of digest to be successfully purified. Such a system combines the intrinsic advantages of the 2991 with those of continuous density gradients and provides the optimal method for density-dependent islet purification.

Notes: Abstract The purification of large numbers of human pancreatic islets remains one of the limiting factors in islet transplantation. This paper describes and validates a method for accurately and reproducibly determining the density of islets and exocrine tissue in pancreatic digest on the basis of their isopycnic distribution on linear continuous density gradients. The use of this data to analyse and compare the purity of a standard 60% islet yield is described. The results obtained using such gradients will enable factors responsible for the variation in yield between pancreases to be determined and optimized, improving the results and reliability of islet purification.