Phase I dose-escalation trial of tucatinib in combination with trastuzumab in patients with HER2-positive breast cancer brain metastases.

BACKGROUND: Brain metastases are frequent in HER2-positive breast cancer. ONT-380 (tucatinib) is a potent selective inhibitor of HER2 with intracranial activity in preclinical models. PATIENTS AND METHODS: This was a phase I study of tucatinib with trastuzumab, without chemotherapy, in patients with progressive, measurable HER2-positive brain metastases. The study tested two schedules of tucatinib: cohort A was twice daily and cohort B was once daily. The primary objective was determination of the maximum tolerated dose (MTD). Secondary end points included objective response (intracranial and extracranial) using modified RECIST and clinical benefit rate (CBR). RESULTS: Overall, 41 patients were enrolled (cohort A, n = 22; cohort B, n = 19). Patients had a median of two prior treatments for metastatic breast cancer and 83% had progressed after prior brain radiation. The MTD of tucatinib for cohort A was 300 mg twice daily and for cohort B was 750 mg once daily. The most common dose-limiting toxicities included thrombocytopenia and aspartate transaminase/alanine aminotransferase elevation. Grade 3/4 aspartate transaminase/alanine aminotransferase elevation occurred in nine of 41 patients (22%). Intracranial responses were observed in two of 17 (12%) patients in cohort A and one of 17 (6%) patients in cohort B treated at the MTD. In cohort A, CBR at 16 weeks was 35% (n = 6). In cohort B, CBR at 16 weeks was 53% (n = 9). Of 15 patients overall who experienced clinical benefit, 12 (80%) had received prior neratinib and/or lapatinib. Median progression-free survival for cohorts A and B was 3.4 and 4.1 months, respectively. CONCLUSION: The combination of tucatinib and trastuzumab is tolerable and demonstrated preliminary evidence of efficacy in patients with HER2-positive brain metastases. CLINICAL TRIAL REGISTRATION: NCT01921335.

Achilles tendinitis in systemic lupus erythematosus: search for an associated inflammatory disease.

OBJECTIVES: Except for traumatic and iatrogenic causes, Achilles tendinitis (AT) is mostly encountered in the context of inflammatory rheumatic diseases. This study aimed to describe AT in systemic lupus erythematosus (SLE). PATIENTS AND METHODS: Among 158 SLE patients who fulfilled the SLE criteria of the ACR classification followed between 1980 and 2013, we selected those who experienced at least one episode of AT not caused by traumatic or toxicity factors. RESULTS: Eight patients (one male, seven females), median age 52 years (range: 35-68), presented with 11 episodes of AT within an average of 10.5 (0-21) years after SLE diagnosis. Clinical presentation of SLE was mainly cutaneous (eight of eight), and articular (seven of eight). Axial symptoms were reported in six patients, two of whom had HLA-B27-positive status, and fulfilled the Amor and European Spondylarthropathy Study Group criteria. Resolution of AT was good with nonsteroidal anti-inflammatory topical or systemic drug therapies, which kept SLE quiescent and avoided any increase of specific treatment. CONCLUSION: Although the association is rare, when AT occurs in SLE patients, physicians should look for associated spondylarthritis.

Efficacy of rituximab in primary Sjogren's syndrome with peripheral nervous system involvement: results from the AIR registry.

OBJECTIVE: To evaluate rituximab (RTX) in primary Sjögren's syndrome (pSS) with peripheral nervous system (PNS) involvement. METHODS: Patients with pSS and PNS involvement who were included in the French AIR registry were analysed. RESULTS: 17 patients (age 60 years (44-78 years); 14 were female) were analysed. Neurological improvement was noted in 11 patients (65%) at 3 months. Rankin scale decreased from 3 (1-5) to 2 (1-5), 2 (1-5) and 2 (1-6) after 3, 6 and 9 months (p=0.02). European Sjögren's Syndrome Disease Activity Index decreased from 18 (10-44) to 11 (5-20), 11 (5-29) and 12 (5-30) after 3, 6 and 9 months (p<0.05). RTX was effective in neurological involvement in 9/10 patients with vasculitis or cryoglobulinaemia (90%) (group 1) at 3 months and in 2/7 cases (29%) without cryoglobulinaemia and vasculitis (p=0.03). Rankin and European Sjögren's Syndrome Disease Activity Index scales decreased significantly in group 1. CONCLUSION: RTX seems effective in cryoglobulinaemia or vasculitis-related PNS involvement in pSS.

Antibodies specific to acetylated histones document the existence of deposition- and transcription-related histone acetylation in Tetrahymena.

In this study, we have constructed synthetic peptides which are identical to hyperacetylated amino termini of two Tetrahymena core histones (tetra-acetylated H4 and penta-acetylated hv1) and used them to generate polyclonal antibodies specific for acetylated forms (mono-, di-, tri-, etc.) of these histones. Neither of these antisera recognizes histone that is unacetylated. Immunoblotting analyses demonstrate that both transcription-related and deposition-related acetate groups on H4 are recognized by both antisera. In addition, the antiserum raised against penta-acetylated hv1 also recognizes acetylated forms of this variant. Immunofluorescent analyses with both antisera demonstrate that, as expected, histone acetylation is specific to macronuclei (or new macronuclei) at all stages of the life cycle except when micronuclei undergo periods of rapid replication and chromatin assembly. During this time micronuclear staining is also detected. Our results also suggest that transcription-related acetylation begins selectively in new macronuclei immediately after the second postzygotic division. Acetylated histone is not observed in new micronuclei during stages corresponding to anlagen development and, therefore, histone acetylation can be distributed asymmetrically in development. Equally striking is the rapid turnover of acetylated histone in parental macronuclei during the time of their inactivation and elimination from the cell. Taken together, these data lend strong support to the idea that modulation of histone acetylation plays an important role in gene activation and in chromatin assembly.

Large scale refolding and purification of the catalytic domain of human BACE-2 produced in E. coli.

Evidence for a key role of beta-amyloid (Abeta) in Alzheimer's disease has led to considerable interest in potential therapeutic strategies targeting enzymes involved in processing the amyloid precursor protein (APP). Beta-site APP Cleaving Enzyme (BACE or beta-secretase) is a membrane bound aspartyl protease that has been shown to be directly involved in Abeta production and, therefore, is at the forefront of therapeutic targets in the treatment of Alzheimer's disease. BACE-2, an enzyme closely related to BACE, regulates Abeta production in a manner antagonistic to BACE, suggesting that non-selective inhibition of BACE-2 by BACE inhibitors might impair the lowering of Abeta. The design of BACE inhibitors that do not inhibit BACE-2 would be enhanced by structural and kinetic studies, efforts that typically demand considerable amounts of both enzymes. A BACE-2 construct containing 19 residues of the BACE prosegment followed by the BACE-2 catalytic domain sequence, Asp36-Trp447, was produced in E. coli inclusion bodies (IB) at 110-140 mg/L cell culture. Exploration of a variety of refolding conditions resulted in an efficient method for refolding the resulting pro-BACE-2 construct, and this protein undergoes facile autocatalytic cleavage, optimal at pH 4, at the Leu40- downward arrow-Ala41 bond. Refolded BACE-2 was purified by anion exchange, molecular sieve, and affinity chromatographies, yielding 105 mg of homogeneous enzyme (kcat/ Km = 1.2 x 10(4) x M(-1) x sec(-1)) from 8 liters of E. coli cell culture.

Large-scale purification of human BACE expressed in mammalian cells and removal of the prosegment with HIV-1 protease to improve crystal diffraction.

BACE, or beta-secretase, is an attractive target in the treatment of Alzheimer's Disease because of its involvement in the generation of amyloid beta peptides. BACE is a type I transmembrane aspartyl protease composed of pre-, pro-, catalytic, transmembrane and cytoplasmic domains. For the present study, the coding sequence was truncated just before the transmembrane domain and the resulting construct was extended with the C-terminal addition of a (His)(6) and expressed in several mammalian host cells. The enzyme expressed in CHO cells had the best crystallographic behavior and was purified in large quantities in a three step procedure. The purified BACE was comprised of two forms, namely the full length proBACE construct beginning with Thr(1), and a derivative missing the first 24 amino acids beginning with E(25). These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 A resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F(39)-V(40) bond, leaving the V(40)EM...ES(432) (His)(6) derivative that was purified yielding an enzyme that was no more active than untreated BACE but co-crystallized with inhibitors producing well shaped, bipyramidal co-crystals diffracting to 2.6 A resolution.

High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution diffracting crystal forms.

BACE (beta-site APP cleaving enzyme) or beta-secretase, the enzyme responsible for processing APP to give the N-terminal portion of the Abeta peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a C-terminal transmembrane segment and a cytoplasmic domain. Three BACE constructs, pET11a-BACE, pQE80L-BACE, and pQE70-BACE were designed to terminate at a position just before the transmembrane domain (Ser(432)) and are described schematically below. (1) pET11a-T7.Tag-G-S-M-(A-8GV......QTDES(432)), (2) pQE80L-Met-R-G-S-(His)(6)-G-S-I-E-T-D-(T(1)QH...QTDES(432)), and (3) pQE70-Met-BACE (R(36)GSFVEMG....PQTDES(432) (His) (6)) Each construct was over-expressed in Escherichia coli as inclusion bodies. The inclusion body proteins were solubilized in urea and refolded by dilution in water to yield active enzyme. Maximal activity for pET11a-BACE and pQE80L-BACE was usually reached at day 3 to 4, while construct pQE70-BACE required about 21 days. Active BACE was purified to homogeneity by anion-exchange chromatography and affinity chromatography over a column of immobilized peptide inhibitor. The process, easily scalable to 60 liters of cell culture, yielded in excess of 400 mg of active enzyme for crystallographic analysis. Highly purified pET11a-BACE and pQE70-BACE formed complexes with various inhibitors, the latter protein giving crystals diffracting up to 1.45 A resolution. In addition, a crystal form that does not require the presence of an inhibitor has been obtained for pQE70-BACE. This ligand-free crystal form has proven useful for the preparation of BACE-inhibitor complexes in soaking experiments.

ABRF ESRG 2006 study: Edman sequencing as a method for polypeptide quantitation.

The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for protein and peptide analysis. These studies provide a means for participating laboratories to compare their analyses against a benchmark of those from other laboratories that provide this valuable service. The main purpose of the 2006 study was to determine how accurate Edman sequencing is for quantitative analysis of polypeptides. Secondarily, participants were asked to identify a modified amino acid residue, N-epsilon-acetyl lysine [Lys(Ac)], present within one of the peptides. The ESRG 2006 peptide mixture consisted of three synthetic peptides. The Peptide Standards Research Group (PSRG) provided two peptides, with the following sequences: KAQYARSVLLEKDAEPDILELATGYR (peptide B), and RQAKVLLYSGR (peptide C). The third peptide, peptide C*, synthesized and characterized by ESRG, was identical to peptide C but with acetyl lysine in position 4. The mixture consisted of 20% peptide B and 40% each of peptide C and its acetylated form, peptide C*. Participating laboratories were provided with two tubes, each containing 100 picomoles of the peptide mixture (as determined by quantitative amino acid analysis) and were asked to provide amino acid assignments, peak areas, retention times at each cycle, as well as initial and repetitive yield estimates for each peptide in the mixture. Details about instruments and parameters used in the analysis were also collected. Participants in the study with access to a mass spectrometer (MALDI-TOF or ESI) were asked to provide information about the relative peak areas of the peptides in the mixture as a comparison with the peptide quantitation results from Edman sequencing. Positive amino acid assignments were 88% correct for peptide C and 93% correct for peptide B. The absolute initial sequencing yields were an average of 67% for peptide (C+C*) and 65.6 % for peptide B. The relative molar ratios determined by Edman sequencing were an average of 4.27 (expected ratio of 4) for peptides (C+C*)/B, and 1.49 for peptide C*/C (expected ratio of 1); the seemingly high 49% error in quantification of Lys(Ac) in peptide C* can be attributed to commercial unavailability of its PTH standard. These values compare very favorably with the values obtained by mass spectrometry.

Intrasteric inhibition of ATP binding is not required to prevent unregulated autophosphorylation or signaling by the insulin receptor.

Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in k(cat) and a 15-fold-lower K(m ATP) although K(m peptide) was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.

ABRF ESRG 2005 study: identification of seven modified amino acids by Edman sequencing.

Identification of modified amino acids can be a challenging part for Edman degradation sequence analysis, largely because they are not included among the commonly used phenylthiohydantion amino acid standards. Yet many can have unique retention times and can be assigned by an experienced researcher or through the use of a guide showing their typical chromatography characteristics. The Edman Sequencing Research Group (ESRG) 2005 study is a continuation of the 2004 study, in which the participating laboratories were provided a synthetic peptide and asked to identify the modified amino acids present in the sequence. The study sample provided an opportunity to sequence a peptide containing a variety of modified amino acids and note their retention times relative to the common amino acids. It also allowed the ESRG to compile the chromatographic properties and intensities from multiple instruments and tabulate an average elution position for these modified amino acids on commonly used instruments. Participating laboratories were given 2000 pmoles of a synthetic peptide, 18 amino acids long, containing the following modified amino acids: dimethyl- and trimethyl-lysine, 3-methyl-histidine, N-carbamyl-lysine, cystine, N-methyl-alanine, and isoaspartic acid. The modified amino acids were interspersed with standard amino acids to help in the assessment of initial and repetitive yields. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the ESRG Web site. The ABRF ESRG 2005 sample is the seventeenth in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.

Immunochemical studies of an insulin-like material in the parotid gland of diabetic BB rats.

Immunocytochemical and radioimmunoassay studies were performed on pancreatic and parotid tissues from diabetic BB and control Wistar rats. Compared with those of normoglycemic controls, the pancreata of diabetic BB rats generally lacked insulin-containing B-cells. Extracts from the parotid glands of diabetic rats contained less immunoassayable insulin-like material than was present in parotid extracts of controls. However, the parotid glands of both groups of animals contained numerous cells displaying insulin-like immunoreactivity. These insulin-immunoreactive cells, located mainly in the intercalated portion of the duct system, were comparable to those we reported recently in the parotid glands of normal and streptozocin-diabetic Sprague-Dawley rats. The presence of an insulin-like material in the parotid salivary gland of two types of diabetic animals suggests that such cells may be spared, in part, from the effects of both chemical and hereditary diabetogenic factors.

Successful islet autotransplantation in humans: functional insulin secretory reserve as an estimate of surviving islet cell mass.

Islet autotransplantation for treatment of chronic painful pancreatitis in nondiabetic patients reliably establishes normoglycemia and phasic insulin secretion and can achieve prolonged insulin independence. Whether functional transplanted beta-cell reserve is normal after intrahepatic islet transplantation is not known, nor is it known whether conventional measures of insulin secretion accurately reflect the functional beta-cell mass. To determine insulin secretory reserve after islet transplant, we performed studies of glucose potentiation of arginine-induced insulin secretion (GPAIS) in eight recipients of intrahepatic islet autotransplants. All eight subjects (and matched, healthy controls) were studied cross-sectionally 49 +/- 12 months posttransplant, and four subjects were studied pre- and posttransplant. Subjects had received a mean +/- SE of 479,000 +/- 79,000 islets, and all were insulin independent and normoglycemic at the time of study. Acute insulin responses to arginine, glucose, and GPAIS were significantly reduced after islet transplantation in both study groups. Importantly, the magnitudes of these three responses were highly correlated to the mass of islets transplanted (response to glucose: r = 0.84, P < 0.01; response to arginine: r = 0.69, P < 0.05; response to GPAIS = 0.81, P < 0.01). Data from hemipancreatectomized and normal control subjects generally agreed with the regression lines. These findings demonstrate that despite normoglycemia and insulin independence, recipients of intrahepatic islet transplantation have significantly reduced functional beta-secretory reserve and that after islet transplantation, functional beta-cell mass can be estimated by measurements of glucose and arginine-induced insulin responses. Thus, these measurements can be used to estimate the mass and functional capacity of islets surviving intrahepatic transplantation in humans.

Lipids increase after solitary pancreas transplantation.

OBJECTIVE: The aims of this study were to determine 1) changes in lipids after solitary pancreas transplantation (SPTX) in patients with type 1 diabetes and 2) factors that influence those changes. RESEARCH DESIGN AND METHODS: Lipids were evaluated prospectively in 24 patients who underwent SPTX. Three were excluded because of early graft failure. The remaining patients (n = 21; 13 men, 8 women) were studied for changes in lipids over time (pre-SPTX, 0-2, 3-6, 7-12, and > 12 months). Glycohemoglobin, serum creatinine, BMI, and medications were also analyzed for their effects on lipid changes. RESULTS: Cholesterol, HDL, and LDL decreased in the immediate postoperative period (0-2 months), whereas triglycerides (TGs) increased (P < 0.05). At 3-6 months, cholesterol, HDL, and TG were higher than before the SPTX, whereas LDL returned to pre-SPTX levels. After 12 months, HDL and TG remained higher than their pre-SPTX levels (P < 0.05). During the study, systolic and diastolic blood pressure increased, renal function decreased, glyco-hemoglobin improved, and weight was unchanged. Changes in cholesterol/HDL ratio, HDL, and TG correlated with changes in prednisone dose (P < 0.05), and changes in TG correlated with changes in creatinine (P < 0.05). The same pattern of lipids occurred in patients prescribed or not prescribed hypolipidemic agents. CONCLUSIONS: Lipids do not improve within the 1st year after SPTX, despite improved glycemic control and blood pressure control, and renal function is worse. These results are in contrast to those reported for combined kidney-pancreas transplantation, where lipids, blood pressure, and renal function improved immediately after transplant. Further studies are needed to determine whether lipids continue to change with time after SPTX. The impact of these changes after SPTX on overall cardiovascular risk is unknown.

Kidney transplantation: a brief review.

Since the first successful kidney transplant in 1954, results of these transplants have dramatically improved. Given refinements in surgical techniques and perioperative care, combined with superior immunosuppression, the procedure is now the treatment of choice for patients of all ages with ESRD. Acute rejection no longer represents a significant threat to graft loss, and the newer immunosuppressive drugs will likely diminish this problem further. Complications such as sepsis are fewer and more reliably managed with current therapies. Chronic rejection remains a major problem whose incidence has not been significantly altered. This along with a better understanding of the processes that may ultimately lead to graft tolerance will be the major challenges facing the field of renal transplantation as it enters the 21st century.

Pancreas transplantation: indications, clinical management, and outcomes.

Although many advances have been made, pancreas transplantation still poses several challenges to the surgeon, internist and patient. With success rates now above 80% and improving yearly, diabetic patients must make a major life-style decision when considering a pancreas transplant. The main concerns are will the benefits of insulin-independence off-set the risks of surgery and immunosuppression. For diabetics near dialysis and considering a kidney transplant, the decision may not be as difficult. However, for those patients who are failing insulin therapy (brittle control) and remain with good renal function, the options are limited. As the success of pancreas transplantation improves, the procedure may become routine at more centers and become accepted by more third-party carriers. However, as with other solid organs, the availability of pancreases is limited and the supply soon to be exhausted. Thus, further advances are required for the prevention and treatment of Type 1 diabetes. Hopefully, the new frontiers of the next century will allow physicians to identify and preventively treat those at risk for the development of diabetes. Thus, the population of patients suffering from the consequences of this dreadful disease will be greatly reduced. With new developments in immunosuppression and islet transplantation, diabetic patients of the future may be offered the option of a procedure with reduced risks, less morbidity, and improved long-term cure rates.

Clopidogrel-associated thrombotic thrombocytopenic purpura/hemolytic uremic syndrome in a kidney/pancreas transplant recipient.

BACKGROUND: We present a case report of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) developing in a kidney/pancreas transplant recipient after the initiation of treatment with clopidogrel for symptomatic coronary artery disease. METHODS: A 35-year-old male kidney/pancreas recipient developed unstable angina 5 years after transplantation. The patient was treated with clopidogrel as adjunct therapy. A TTP/HUS condition developed, was diagnosed early, and successfully reversed with the implementation of plasmapheresis and cessation of clopidogrel and cyclosporine A. RESULTS: The patient continues taking cyclosporine A with good renal function 6 months after the incident, and successfully underwent coronary artery by-pass grafting 3 months after the event. DISCUSSION: This case demonstrates that early identification and treatment can reverse the TTP/HUS process associated with thienopyridine-derived agents. We strongly recommend that drugs of the thienopyridine class be used cautiously in transplant recipients, especially those taking calcineurin-inhibitors.

Reproductive hormones after pancreas transplantation.

BACKGROUND: Reproductive hormone function after pancreas transplantation (PTX) is unknown as it has not been studied. METHODS: We prospectively studied PTX recipients to determine changes in reproductive hormones after PTX. Testosterone or estradiol, leutinizing hormone, follicle stimulating hormone, and prolactin were determined before and 1 year after PTX in 23 patients (10 women, 13 men) followed for more than 1 year after PTX. Of these, 11 received simultaneous kidney-PTX; 8 PTX only; and 4, PTX after kidney. Average age was 38.4+/-1.6 years and average duration of diabetes was 24.5+/-1.3 years. Nine (four women, five men) patients had been on dialysis pre-PTX. Sixteen of 23 patients were treated with cyclosporine and seven with FK-506 along with prednisone and azathioprine post-PTX. RESULTS: Mean testosterone in men was normal pre- and post-PTX. Two men had secondary hypogonadism pre-PTX with resolution in one and persistence in the other post-PTX. Five of the ten women had evidence of hypogonadism pre-PTX: three had primary hypogonadism and two had secondary hypogonadism. Post-PTX, 7 of 10 women had abnormal reproductive hormones: 4 had primary hypogonadism, 2 had secondary hypogonadism, and 1 developed hyperestrogenemia with elevated estradiol (482 pg/ml) and leutinizing hormone (41 IU/liter). Mean prednisone dose and cyclosporine trough level were higher in the women than the men (P<0.05). No cases of secondary hypogonadism that developed or resolved post-PTX were related to changes in prolactin, renal function, or hyperglycemia. CONCLUSIONS: Women are more likely than men to have reproductive hormone abnormalities pre- and post-PTX and the causes may be multiple.

Posttransplant nonfunction of canine islets in PVG rats deficient in complement component C6.

BACKGROUND: Discordant islet xenografts are immediately nonfunctional in nonimmunosuppressed recipients other than the mouse, a process called primary nonfunction. Although at present it is unknown whether complement is involved, complement might participate in the induction of primary nonfunction through a number of mechanisms. We investigated the potential role of the membrane attack complex of complement in primary nonfunction of transplanted xenoislets. METHODS: Canine islets were transplanted into both nonimmunosuppressed and immunosuppressed normocomplementemic and C6-deficient (C6D) PVG rats. Cyclosporine, rapamycin, deoxyspergualin, and mycophenolate mofetil were used for immunosuppression from day -3 to cessation of islet cell function. Serum glucose was measured at 6 hr after transplant and daily thereafter. Xenograft tissue sections were obtained at various times after transplant and stained for inflammatory cells and insulin. RESULTS: Canine islets grafted in nonimmunosuppressed C6D rats and normocomplementemic rats underwent primary nonfunction in all animals. The incidence of primary nonfunction in animals receiving a four-drug immunosuppressive regimen was 33% in the normocomplementemic rats but only 10% in the C6D rats. The mean functional islet survival time was 1.57+/-0.33 days in the normocomplementemic group and 2.70+/-0.67 days in the C6D group (P=0.38). The islet xenografts showed little difference in degree and composition of cell infiltration between normocomplementemic and C6D rats. CONCLUSION: The membrane attack complex does not appear to play a major role in primary nonfunction of canine islet xenografts in nonimmunosuppressed PVG rats. However, there was a lower incidence of primary nonfunction and a longer posttransplant survival time in immunosuppressed C6D rats, suggesting the membrane attack complex may play a minor role in recipients that are heavily immunosuppressed.

Effect of pancreatic warm ischemia on islet yield and viability in dogs.

BACKGROUND: Defining tolerable warm ischemia (WI) is mandatory before nonheartbeating cadavers can be used to enlarge the donor pool. No studies to date have precisely evaluated the effect of pancreatic WI on islet yield and viability in a large animal model. METHODS: We used mongrel dogs in our study at the University of Minnesota. Excised pancreases were left in situ for a designated period (0, 30, 45, and 60 min in groups 1 to 4, respectively) of WI. Then, they were digested by the automated collagenase digestion method of Ricordi, purified on Euro-Ficoll discontinuous gradients with the COBE cell processor, and autotransplanted into the liver via a mesenteric vein. We compared the four groups in terms of islet yield, expressed as islet equivalents (IE; diameter standardizing to 150 microm) per pancreas weight (IE/g pancreas), and viability, assessed by functional success (maintenance of normoglycemia for 2 weeks) after transplant. RESULTS: Mean islet yield (+/- SD) and the functional success rate after transplant were as follows: 6200+/-1800 IE/g pancreas and 4 of 4 (100%) in group 1; 6300+/-4400 and 4 of 4 (100%) in group 2; 3800+/-2600 and 2 of 4 (50%) in group 3; and 1400+/-1300 and 0 of 4 (0%) in group 4 (P=0.01 vs. group 1). CONCLUSIONS: With 30 min or less of WI, there are no deleterious effects on islet yield and viability. However, with periods of WI longer than 30 min, the loss in islet yield is severe, resulting in functional failure after autotransplantation. The limit of WI that is tolerable for islets is shorter than for a whole pancreas.