Using a modification of the basic principles of pancreatic intraductal collagenase digestion and density gradient purification to isolate canine islets, in conjunction with simultaneous fluorogenic and dithizone islet staining, we quantified the yield, purity, and viability of the isolated islets. We then determined the combined influences of total and weight-corrected islet counts and implantation site on immediate and long-term functional outcome of purified canine islet autografts. Weight-corrected islet counts were 100% sensitive and specific in differentiating successful and unsuccessful islet autografts implanted to the liver (n = 10) and spleen (n = 10) of pancreatectomized dogs. The threshold number of islets required to achieve normoglycemia in the liver (4400 islets/kg) and spleen (4650 islets/kg) were nearly identical. Islet autografts failed to ameliorate hyperglycemia when implanted to the renal subcapsular space (n = 5) at counts of 4400 to 5500 islets/kg. The mean one- and three-month intravenous glucose tolerance test K-values of dogs with purified islet autografts to the liver (-1.43 +/- 0.27 and -1.69 +/- 0.27, respectively) and spleen (-1.78 +/- 0.36 and -1.64 +/- 0.3, respectively) were also similar. Time needed to achieve normoglycemia , however, was significantly (P less than 0.02) shorter for intrahepatic islets (1.0 +/- 0.0 days posttransplant) than intrasplenic islets (6.8 +/- 2.3 days posttransplant). The long-term durability of islet autograft function was not unlimited. Overall, thirteen canine islet autograft recipients have been followed for greater than or equal to 12 months posttransplant (range 12-18 months), seven canine islet autograft recipients (five intrahepatic and two intrasplenic) have had spontaneous recurrence of hyperglycemia at 2, 6, 11, 13, 14, 8, and 16 months, respectively. The phenomenon depended only on the number of islets implanted. The data underscore the significance of quantitatively defined islet preparations and the importance of islet number and implantation site on immediate and long-term functional outcome of canine islet autografts.

Animals↗

PubMed1990

The alloimmune response to murine islets occurs via indirect antigen presentation to LYT2+ and L3T4+ lymphocytes.

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Animals↗

PubMed1989

Comparison of the effects of four cholinomimetic agents on cognition in primates following disruption by scopolamine or by lists of objects.

The ability of four central cholinomimetics to reverse a scopolamine-induced spatial memory impairment or to improve visual recognition memory in primates was examined. Physostigmine (0.04-0.08 mg/kg IM) fully reversed the effects of scopolamine (0.03 mg/kg). Coadministration of pilocarpine (3.0-5.0 mg/kg) caused partial reversal of the scopolamine impairment after intermediate or long retention intervals (10 or 20 s). Treatment with arecoline (0.1-1.8 mg/kg) or nicotine (1.0-2.0 mg/kg) generally did not reverse the effects of scopolamine. A task in which memory could be taxed by increasing the number of visual stimuli presented appeared more sensitive to the effects of cholinomimetics on cognition than the scopolamine reversal model. In this paradigm treatment with physostigmine (0.001, 0.01 or 0.03 mg/kg) increased choice accuracy from about 55 to 70% correct. Arecoline improved performance at one dose only (0.1 mg/kg) which also induced marked adverse side-effects (salivation and tremor). Pilocarpine improved performance in the dose range 0.125-0.35 mg/kg, but not at higher doses which also induced marked salivation. Treatment with nicotine (0.001-2.0 mg/kg tended to improve performance but this did not reach statistical significance. The relevance of these findings for studies in man and for animal models of dementia is discussed.

Animals↗

PubMed1989

Acute cisplatin nephrotoxicity in the rat. Evidence for impaired entry of sodium into proximal tubule cells.

The earliest phase of cisplatin nephrotoxicity involves natriuresis due to impaired sodium reabsorption by the proximal tubule. To define the cell mechanism of this transport lesion, electron microprobe X-ray analysis was used to determine changes in the electrolyte composition of proximal tubule cells in kidneys taken from rats treated acutely with cisplatin (1 mg/100 g body weight). Compared to control animals injected with vehicle, cisplatin treated rats developed significant natriuresis, the fractional excretion of sodium rising over sevenfold. In kidneys removed 90 min following cisplatin, sodium concentration in proximal tubule cells was reduced by 4.2 mmol/kg wet weight, or 19%, compared to control values. When allowance was made for cell shrinkage in cisplatin-treated kidneys by deriving the cell content of sodium (mmol/kg dry weight), the reduction was even greater (28%). These data suggest that cisplatin reduces proximal tubule sodium reabsorption by inhibiting the entry of sodium into the cells across the apical membrane.

Animals↗

PubMed1989

Cold storage preservation of pancreatic tissue prior to and after islet preparation in a dog autotransplantation model.

Utilizing an intrasplenic canine islet autotransplant model, the effects of cold storage preservation on pancreatic tissue prior to and after collagenase dispersion were examined. A control series, in which freshly retrieved and prepared tissue was transplanted, yielded a 75% success rate (6/8). In contrast, when the pancreas was stored in modified silica gel filtered plasma (SGF I) for 24 hr, no autotransplant was successful (0/6). However, when the islet tissue was prepared following pancreatectomy and then stored in a mannitol-containing modification of SGF (SGF III), autotransplantation was successful in 83% (5/6) after 24 hr of preservation and in 60% (3/5) after 48 hr of preservation. Similarly, the islet tissue was stored in a hyperkalemic hydroxyethyl starch solution (HES) and this was successful in 20% (1/5) after 24 hr of preservation and in 50% (1/2) after 48 hr of preservation. Cold storage preservation techniques for the pancreas prior to islet isolation need to be refined, but dispersed islet-enriched pancreatic tissue can be successfully maintained at 4 degrees C for up to 48 hr prior to transplantation in dogs using established pancreas preservation solutions.

Animals↗

PubMed1989

Experimental pancreatic preservation prior to islet isolation and transplantation.

Preservation of the Lewis rat pancreas prior to islet isolation was accomplished by initial intraductal distension with the University of Wisconsin (UW) hydroxyethyl starch-lactobionate solution to which collagenase had been added, followed by simple cold storage at 4 degrees C for 0, 3, 12, 24, and 48 hr (n = 16-21 at each interval). The pancreases were then processed by digestion and mechanical dispersion to produce free islets of Langerhans. The mean islet yields (+/- standard errors of the means) were controls = 819 +/- 58 (n = 21), 3 hr = 867 +/- 51 (n = 20), 12 hr = 770 +/- 71 (n = 16), 24 hr = 805 +/- 62 (n = 18), and 48 hr = 722 +/- 55 (n = 16). None of these means differed significantly. The islets from pairs of donor pancreases (mean dose of islets = 1586 +/- 72) were transplanted intraportally into single isogeneic recipients with streptozotocin-induced diabetes (plasma glucose greater than 400). The preservation interval directly influenced the outcome of these islet isografts in the following manner: (i) Rates of functional success (nonfasting glucose less than 200 mg/dl) were 100% after storage times of 0 hr (n = 10), 3 hr (n = 8), and 12 hr (n = 8); 86% with a storage time of 24 hr (n = 7); and 0% after 48 hr (n = 8). (ii) Return of euglycemia was increasingly delayed with increasing preservation intervals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Modulation of MHC class I antigen decreases pancreatic islet immunogenicity.

Pretreatment regimens directed at reducing the immunogenicity of pancreatic islets have emphasized the elimination or alteration of the major histocompatibility complex (MHC) class II-positive dendritic cells within the islet. Unfortunately, the efficacy of such pretreatment regimens has been extremely variable and the relative contribution of the dendritic cells to the overall immunogenicity of pancreatic islets has remained ambiguous. Recent evidence has suggested that the MHC class I antigen present on the endocrine cells within the islets may play an important role in the alloimmune response. This study utilized the in vitro mixed lymphocyte-islet co-culture system to determine if pretreatment of whole islets with an anti-MHC class I monoclonal antibody specific to the donor strain would block the generation of cytotoxic T lymphocytes (CTL) in the in vitro mixed lymphocyte-islet coculture. Pretreatment of B10.BR (H-2k) and DBA/2J (H-2d) islets with an allospecific anti-MHC class I monoclonal antibody blocked the generation of allospecific CTL when the pretreated islets were placed into coculture with C57Bl/6 (H-2b) splenocytes. If such a pretreatment regimen is similarly effective in vivo, it could potentially be used as an antirejection strategy in pancreatic islet allotransplantation.

Animals↗

PubMed1989

A simple method of canine pancreatic islet isolation and intrahepatic transplantation.

Clinical pancreatic islet transplantation has been impeded by the inability to isolate an adequate mass of functional tissue that will ameliorate diabetes. A simplified method of canine islet isolation was developed that allowed for either intrasplenic or intrahepatic transplantation. Following total pancreatectomy, parenchymal digestion was accomplished by intraductal collagenase perfusion and stationary incubation. The digested tissue was dispersed by filtration through a steel mesh (400 microns), washed, and separated on a discontinuous dextran density gradient. Enhanced islet tissue (2-4 ml) was recovered from the uppermost interface of the gradient and autotransplanted. The islet isolation procedure was tested in two series of dogs undergoing either intrasplenic or intrahepatic engraftment. Immediate and sustained normoglycemia (plasma glucose less than 200 mg%) was obtained in 5 of 8 dogs (63%) in the intrasplenic group and 6 of 8 dogs (75%) in the intrahepatic group. The mean fasting plasma glucose concentration 2 weeks after transplantation was 102.8 +/- 6.4 mg% in the intrasplenic group and 103.3 +/- 8.4 mg% in the intraportal group. The mean IVGTT K-values 2 weeks after transplantation were -1.41 +/- 0.35% and -1.21 +/- 0.13%, respectively. On the basis of insulin content, the islet yield was 33.0 +/- 3.7% of the total pancreas in the intrasplenic group and 33.0 +/- 3.1% in the intrahepatic group. Islet mass was enhanced 10.2 +/- 1.5 and 20.0 +/- 6.2 fold, respectively, on the basis of insulin/amylase ratios. The success rate in this canine model compared favorably with previously published results from other laboratories.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Cold-storage preservation of the canine and rat pancreas prior to islet isolation.

Cold-storage preservation of the canine pancreas prior to islet isolation has previously been noted to reduce the intrasplenic islet autograft success rate; but the mechanism of this deleterious effect has not been determined. We undertook a study in both outbred dogs and Lewis (RT1-1) rats to determine the influence of cold-storage preservation interval, preservation solution, and flushing technique on islet yield and islet viability. The preservation solutions used were those that had proved most efficacious in preserving segmental canine pancreases--namely, the modifications of silica gel fractionated plasma (SGF-III and SGF-IV) and an hydroxyethylstarch/lactobionate solution (UW-1). In the first set of experiments, the traditional vascular flush was used; this was followed by storage at 4 degrees C. After brief periods of preservation (3 hr in the rat, 12 hr in the dog) there was a significant (P less than 0.006) reduction in islet yield. The reduced yields were similar with each solution tested, were made worse with increasing intervals of storage, and resulted in a significant reduction in autograft success rate. The second set of experiments examined the effect of using an intraductal flush prior to preservation, along with the effect of adding collagenase to the preservation fluid. Islet yields were maintained at control values in both animal models using preservation intervals of up to 24 hr. These islet yields produced auto- or isograft success rates similar to those obtained by transplanting freshly obtained tissue; verifying adequate islet viability. We recommend that a pre-storage ductal flush technique be used for cold-storage preservation of the pancreas prior to islet isolation and transplantation.

Animals↗

PubMed1989

Micropuncture comparison of nephron function during acute and chronic ADH excess in the rat.

1. The aim of this study was to compare the effect of acute versus chronic ADH administration on the handling of sodium, potassium and water by the nephron. Simultaneous clearance and free-flow micropuncture experiments were performed on rats, infused with hypotonic Ringer solution, following either 1-3 h ('acute') or 10-12 days ('chronic') of continuous treatment with arginine vasopressin, 50 mU/h. A third group of animals receiving no exogenous ADH acted as controls. 2. Chronic ADH treatment led to more profound concentration of the urine and antidiuresis than acute treatment, due to enhanced extraction of water in the renal medulla. 3. Fractional sodium excretion during this protocol was increased after 'acute' but not 'chronic' ADH treatment. The acute natriuretic response was brought about principally along the length of the proximal tubule, probably due to volume expansion and increased arterial blood pressure. 4. Fractional excretion of potassium was also increased by 'acute' ADH, but this response was due to stimulation of potassium secretion in the late distal tubule. 5. It is concluded that acute and chronic exposure to ADH have different effects on nephron function, as a result of both direct and indirect actions.

Animals↗

PubMed1989

Should we regulate 'utilization management?'.

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Government↗

PubMed1989

Effect of immunodepletion of MHC class II-positive cells from pancreatic islets on generation of cytotoxic T-lymphocytes in mixed islet-lymphocyte coculture.

In vitro manipulation of pancreatic islets to decrease islet immunogenicity before transplantation has largely been directed at eliminating the major histocompatibility complex (MHC) class II-positive passenger leukocytes from the islets. The mixed islet-lymphocyte coculture (MILC) system was used to quantitate the efficacy of immunodepletion of MHC class II-positive cells from pancreatic islets in terms of reducing immunogenicity. With these experiments we compared the in vitro immunogenicity of MHC class II-depleted islets with untreated islets. B10.BR (H-2k) islets were treated with anti-Iak alloserum followed by complement. This treatment successfully eliminated MHC class II-positive cells from the islets, as demonstrated by indirect immunofluorescence techniques. Depleted islets generated slightly lower amounts of allospecific cytotoxic T-lymphocyte (CTL) activity when exposed to C57BL/6 (H-2b) splenocytes in the MILC than untreated control islets. Although the amount of CTL generated by the depleted islets was slightly less than that generated by untreated islets, there was significant stimulation of CTL by the MHC class II-depleted islets. Therefore, the presence or absence of MHC class II cells within the islet is unlikely to be the decisive factor contributing to islet immunogenicity.

Animals↗

PubMed1989

Successful transplantation of canine islets of Langerhans after 24 hours cold-storage.

Utilizing the intrasplenic canine islet autograft model, it was possible to examine the effect of cold-storage of pancreatic tissue, both prior to and following dispersion, on functional outcome. A control group of dogs receiving freshly prepared autografts (n = 8) obtained durable euglycaemia in 75% of cases; the mean K value at two weeks being -1.70 (Standard Deviation (sd) = 0.06). In the first experimental group, animals (n = 8) were transplanted following 24 hours of whole pancreas cold-storage in silica gel fractionated plasma (SGF) using intraductal perfusion prior to preservation. None of these animals obtained euglycaemia; the transplanted material having a significantly reduced insulin content compared with controls (p = 0.0006). In the second experimental group, animals (n = 6) were transplanted following 24 hours of dispersed pancreatic tissue storage in SGF. This resulted in an 83% incidence of durable euglycaemia; the mean K value at two weeks being -1.60 (sd = 0.3). The glucose decay curve improved with time in this group and at three months the mean K value was -2.00 (sd = 0.38); this value being significantly superior to that of the control animals (K = -1.34, sd = 0.34, p less than 0.05). We conclude that while storage of the whole pancreas prior to islet isolation remains problematic, it is possible to reliably preserve dispersed pancreatic tissue for 24 hours by simple cold-storage, as assessed by the functional outcome of intrasplenic autografting in the dog model. These findings have important clinical implications.

Amylases↗