AEB-071 has minimal impact on onset of autoimmune diabetes in NOD mice.

Protein kinase C (PKC) is an important signaling enzyme in the activation and regulation of T lymphocytes. T-cell-mediated destruction of beta-cells is a characteristic feature of autoimmune (Type 1) diabetes. Here we explore the ability of PKC inhibition, using the PKC inhibitor AEB-071 (AEB), to reduce disease in two animal models of spontaneous autoimmune diabetes (non-obese diabetic (NOD) mouse and biobreeding rat (BB)). NOD mice were treated with AEB for 4 weeks, starting at either 4 weeks of age (prior to the development of insulitis) or at 8 weeks of age, once insulitis is present. Animals treated with AEB during the effector phase of the disease (treatment onset at 8 weeks of age), showed a 2-week delay in diabetes onset (p < 0.05). In these animals, the extent of insulitis was lower than in vehicle-treated controls; however, neither serum autoimmune anti-GAD65 antibody levels nor pancreatic insulin content were different between experimental groups. Overall, inhibition of PKC can mildly reduce lymphocytic infiltrate of pancreatic islets and modestly delay onset of autoimmune diabetes in NOD mice. AEB, a T-cell-targeted immunosuppressive strategy, is only sufficient as a monothereapy to modestly delay onset of autoimmune disease in the NOD mouse.

Optimal implantation site for pancreatic islet transplantation.

BACKGROUND: Since the first report of successful pancreatic islet transplantation to reverse hyperglycaemia in diabetic rodents, there has been great interest in determining the optimal site for implantation. Although the portal vein remains the most frequently used site clinically, it is not ideal. About half of the islets introduced into the liver die during or shortly after transplantation. Although many patients achieve insulin independence after portal vein infusion of islets, in the long term most resume insulin injections. METHODS: This review considers possible sites and techniques of islet transplantation in small and large animal models, and in humans. Metabolic, immunological and technical aspects are discussed. RESULTS AND CONCLUSION: Many groups have sought an alternative site that might offer improved engraftment and long-term survival, together with reduced procedure-related complications. The spleen, pancreas, kidney capsule, peritoneum and omental pouch have been explored. The advantages and disadvantages of various sites are discussed in order to define the most suitable for clinical use and to direct future research.

The role of macrophage migration inhibitory factor on glucose metabolism and diabetes.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in many inflammatory reactions and disorders, and it has become evident that it also affects glucose homeostasis. The protein is produced by pancreatic beta cells and can promote the release of insulin. It also modulates glucose uptake, glycolysis and insulin resistance in insulin target cells such as the adipocyte, myocyte and cardiomyocyte. Possessing both immunological and endocrinological properties, MIF has been associated with the development of type 1 and type 2 diabetes, and it may be important in the setting of islet transplantation. The present review summarises our current knowledge, based on clinical and research data, on the impact of MIF on both physiological and pathological aspects of glucose metabolism.

Liraglutide, a long-acting human glucagon-like peptide 1 analog, improves glucose homeostasis in marginal mass islet transplantation in mice.

The current scarcity of high-quality deceased pancreas donors prevents widespread application of islet transplantation for treatment of labile type 1 diabetes mellitus. Opportunities for the improvement of current techniques include optimization of islet isolation and purification, use of culture with pharmacological insulinotropic agents, strategies to reduce graft rejection and inflammation, and the search for alternative insulin producing tissue. Here, we report our findings on the efficacy of the long-acting human glucagon-like peptide 1 analog, liraglutide, in a mouse model of marginal mass islet transplantation. Liraglutide was administered (200 microg/kg sc twice daily) after a marginal mass syngeneic islet transplant in streptozotocin-induced diabetic BALB/c mice. Time-to-normoglycemia was significantly shorter in liraglutide-treated animals (median 1 vs. 7 d; P = 0.0003), even in recipients receiving sirolimus (median 1 vs. 72.5 d; P < 0.0001). Liraglutide-treated animals also demonstrated improved glucose tolerance as assessed by an ip glucose tolerance test. Liraglutide discontinuation at postoperative d 90 resulted in diminished glucose tolerance during the ip glucose tolerance test, whereas a late-start liraglutide therapy 90 d after transplant resulted in no improvement. These findings suggest that liraglutide therapy mediates early and late insulinotropic effects. In accord with this hypothesis, insulin/terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling fluorescence microscopy showed reduced transplanted beta-cell apoptosis in liraglutide-treated recipients 48 h after transplant. In addition, liraglutide resulted in improved glucose-dependent insulin secretion. Overall, our data show that liraglutide has a beneficial impact on the engraftment and function of syngeneic islet transplants in mice, when administered continuously starting on the day of transplant.

Combined coinhibitory and costimulatory modulation with anti-BTLA and CTLA4Ig facilitates tolerance in murine islet allografts.

Complex interactions between positive and negative cosignaling receptors ultimately determine the fate of the immune response. The recently identified coinhibitory receptor, B and T lymphocyte attenuator (BTLA), contributes to regulation of autoimmune and potentially alloimmune responses. We investigated the role of BTLA in a fully major histocompatibility complex-mismatched mouse islet transplant model. We report that anti-BTLA mAb (6F7) alone does not accelerate graft rejection. Rather, while CTLA4Ig alone improved allograft survival, the addition of anti-BTLA mAb to CTLA4Ig led to indefinite (>100 days) allograft survival. Immediately after treatment with anti-BTLA mAb and CTLA4Ig, islet allografts showed intact islets and insulin production despite a host cellular response, with local accumulation of Foxp3+ cells. We clearly demonstrate that combined therapy with anti-BTLA mAb and CTLA4Ig mice induced donor-specific tolerance, since mice accepted a second donor-specific islet graft without further treatment and rejected third party grafts. CTLA4Ig and anti-BTLA mAb limited the initial in vivo proliferation of CFSE-labeled allogeneic lymphocytes, and anti-BTLA mAb enhanced the proportion of PD-1 expressing T cells while depleting pathogenic BTLA+ lymphocytes. We conclude that targeting the BTLA pathway in conjunction with CTLA4Ig costimulatory blockade may be a useful strategy for promoting immunological tolerance in murine islet allografts.

Human islet function is not impaired by the sphingosine-1-phosphate receptor modulator FTY720.

Clinical islet transplantation for type 1 diabetes mellitus currently requires potent immunosuppressive drugs, which limits the procedure to the most severe forms of the disease, and many of the drugs are directly beta-cell toxic. A class of compounds called sphingosine-1-phosphate receptor modulators has been explored in transplantation and shown to be highly effective in multiple sclerosis and other autoimmune conditions. While FTY720, the first drug in this class, may not move forward initially in transplantation, this class requires detailed investigation to assess direct impact upon human beta-cell function and survival. We set out to evaluate the effects of FTY720 on human islets in vitro by investigating glucose-stimulated insulin and apoptosis; and in vivo, after transplantation into immunodeficient mice with chemically induced diabetes, by examining blood glucose levels, oral glucose tolerance tests and stimulated human C-peptide over a 50-day follow-up period. Our data showed that neither in vitro, nor in vivo human islet function was impaired by FTY720 exposure. Since FTY720 demonstrated no detrimental effects on human islet function in vitro or in vivo, emerging S1PR modulators may prove to be useful adjuncts in clinical islet transplantation through lack of diabetogenicity and potent immunological protection.

Genome sizes in afrotheria, xenarthra, euarchontoglires, and laurasiatheria.

Topical literature and Web site databases provide genome sizes for approximately 4,000 animal species, invertebrates and vertebrates, 330 of which are mammals. We provide the genome size for 67 mammalian species, including 51 never reported before. Knowledge of genome size facilitates sequencing projects. The data presented here encompassed 5 Metatheria (order Didelphimorphia) and 62 Eutheria: 15 Xenarthra, 24 Euarchontoglires (Rodentia), as well as 23 Laurasiatheria (22 Chiroptera and 1 species from Perissodactyla). Already available karyotypes supplement the haploid nuclear DNA contents of the respective species. Thus, we established the first comprehensive set of genome size measurements for 15 Xenarthra species (armadillos) and for 12 house-mouse species; each group was previously represented by only one species. The Xenarthra exhibited much larger genomes than the modal 3 pg DNA known for mammals. Within the genus Mus, genome sizes varied between 2.98 pg and 3.68 pg. The 22 bat species we measured support the low 2.63 pg modal value for Chiroptera. In general, the genomes of Euarchontoglires and Laurasiatheria were found being smaller than those of (Afrotheria and) Xenarthra. Interspecific variation in genome sizes is discussed with particular attention to repetitive elements, which probably promoted the adaptation of extant mammals to their environment.

Chromosome studies of murine T-cell lymphoid leukemia and derived cell lines.

Several cell lines were previously established from a spontaneous murine T-cell leukemia (LB). The aim of this study was to analyze the G- and C-banded karyotypes of the parental LB tumor cells and the derived cell lines. A sensitive cell line (LBL) from which two sublines originated, as well as Vincristine (LBR-V160) and Doxorubicin (LBR-D160) resistant cell lines, were used. Our results showed that LB cells had a pseudo-diploid karyotype with 40 acrocentric chromosomes in which trisomy of chromosome 14 was the most relevant alteration. The sensitive cell line showed this alteration in all metaphases studied; no changes in karyotypes were observed in either subline, despite their dissimilar morphology and growth patterns. In contrast, both resistant lines displayed a more heterogeneous karyotype with no common markers, except for the finding that chromosome 5 was involved in a trisomy in LBR-V160 and in a translocation with chromosome 12 in LBR-D160. Taking into account that the mdr genes are located in chromosome 5, these results suggest a possible association between such alterations and the acquisition of drug resistance.

Five novel hormone-responsive cell lines derived from murine mammary ductal carcinomas: in vivo and in vitro effects of estrogens and progestins.

We have developed an experimental model of mammary carcinogenesis in which the administration of medroxyprogesterone acetate (MPA) to female BALB/c mice induces progestin-dependent ductal metastatic mammary tumors with high levels of estrogen receptor (ER) and progesterone receptor (PR). Through selective transplants in untreated mice, we have obtained progestin-independent variants, still expressing high levels of ER and PR. Primary cultures of the MPA-induced carcinomas C4-HD and C7-HI were set up, and after 3-4 months, several different cell lines were obtained. Four of these, MC4-L1, MC4-L2, MC4-L3, and MC4-L5 were established from C4-HD and a fifth, MC7-L1, from C7-HI. All cells were of epithelial origin, as demonstrated by electron microscopy and by immunocytochemical identification of cytokeratin and cadherin. In vitro MC4-L1, MC4-L3, and MC4-L5 showed a typical epithelial morphology; when transplanted in vivo, they originated metastatic carcinomas with different degrees of differentiation. MC4-L2 and MC7-L1 deviated from the standard epithelial picture; they disclosed a spindle-shaped morphology in vitro and in vivo gave rise to a biphasic spindle cell/tubular carcinoma and an anaplastic carcinoma, respectively; both lines gave rise to metastases. This differential morphology correlated with a higher degree of aggressiveness, as compared with MC4-L1, MC4-L3, and MC4-L5. ERs and PRs were detected by binding, immunocytochemistry, and Western blot. In vitro, MC4-L2 and MC7-L1 were stimulated by MPA (nM to microM) and 17beta-estradiol (nM and 10 nM); no significant stimulation was observed in MC4-L1, MC4-L3, and MC4-L5 under the same experimental conditions. In vivo, MPA significantly stimulated tumor growth in all epithelioid lines but not in MC4-L2 and MC7-L1. A progestin-dependent growth pattern was confirmed for MC4-L1, MC4-L3, and MC4-L5 in successive transplants, whereas MC4-L2 and MC7-L1 behaved as progestin independent. This is the first description of mouse mammary carcinoma cell lines expressing ER and PR. The different in vitro hormone responses as compared with in vivo and the differential effects of 17beta-estradiol in the parental tumors and in cell lines render these lines useful tools for the in vitro and in vivo study of hormone regulation of tumor growth and metastases.

Sensitivity to CCK-4 in women with and without premenstrual dysphoric disorder (PMDD) during their follicular and luteal phases.

The authors determined whether women with premenstrual dysphoric disorder (PMDD) exhibit a heightened sensitivity to the panicogenic effects of CCK-4 administration and whether this enhanced sensitivity to CCK-4 would vary with the phase of the menstrual cycle at the time of CCK-4 injection. Twenty-one normal controls and 18 PMDD women were randomly assigned to receive the first and second CCK-4 injection during the follicular phase and the luteal phase or vice versa. PMDD women showed a greater anxiety and panic response to CCK-4. These preliminary results suggest that the CCK-B system may play a role in the pathophysiology of PMDD.

Effect of oral ondansetron on total cholecystokinin plasma levels following CCK-4 panic challenge procedure in healthy men.

OBJECTIVE: To gain insight into whether ondansetron treatment induces changes in total cholecystokinin (CCKT) plasma levels before and after administration of the cholecystokinin tetrapeptide (CCK-4) panic challenge procedure in healthy men. METHODS: Thirty-eight volunteers received a 50-microgram bolus of CCK-4 60 minutes after a single oral dose (acute treatment) and multiple oral doses (chronic treatment) of ondansetron or placebo. RESULTS: Results showed no difference in CCKT plasma levels of CCKT elimination rate constant between the ondansetron and the placebo groups after either acute or chronic treatment. CONCLUSION: Results from this study suggest that total CCK plasma levels are not influenced by either acute or chronic treatment with ondansetron. However, the effect of ondansetron on the different CCK component fractions still needs exploration.

Development of a sensitive and specific assay system for cholecystokinin tetrapeptide.

Cholecystokinin is a gastrointestinal and neuropeptide which has been implicated in a wide range of physiological and behavioral processes. We have developed a sensitive and specific assay system to measure the various forms of cholecystokinin (CCK) in human plasma. This 3-step system involves i) extraction of CCK fragments from plasma using reverse phase chromatography; ii) separation of peptides by high performance liquid chromatography; and iii) detection and quantification of peptides with a double-antibody radioimmunoassay, using an antibody raised against cholecystokinin tetrapeptide (CCK-4) coupled to thyroglobulin and 125I Bolton-Hunter CCK-4 as tracer. The antibody detects CCK-4, sulfated CCK-8 (CCK-8S) and nonsulfated CCK-8 (CCK-8ns) with equal affinity. The lower limit of detection is 2.7 fmol, with an ED50 of 10.6 +/- 2.2 fmol. Mean CCK-like immunoreactivity (CCK-LI) in the plasma of 12 healthy subjects was determined to be 12.9 +/- 2.1 pM CCK-4 equivalents. Concentrations of each individual peptide in plasma were determined to be 1.0 +/- 0.2 pM, 3.4 +/- 0.8 pM and 1.9 +/- 0.4 pM for CCK-4, CCK-8s and CCK-8ns respectively.

The sex-determining zinc finger sequences in XY females of Akodon azarae (Rodentia, Cricetidae).

Wild populations of Akodon azarae comprise females with a karyotype indistinguishable from that of males. These individuals were formerly assumed to be Xx, the x being an X chromosome with a deletion of most of its long arm. By using a DNA probe derived from the testis-determining region of the human Y chromosome (comprising a candidate gene for the testis-determining factor, Y-linked zinc finger [ZFY]), we demonstrate that A. azarae gonosomally variant females are XY and not Xx. The ZFY sequences in A. azarae are amplified and located in two different families of EcoRI fragments derived from Y-chromosome DNA. No rearrangement or change in the state of methylation of ZFY or ZFX (X-linked zinc finger) sequences were found in XY females. We propose that sex reversal in A. azarae may be mediated by a gene or genes other than ZFX or ZFY.

Mortality induced by adoptive immunity in Junin virus-infected athymic mice.

The effect of normal or sensitized spleen cell transfer from syngeneic euthymic mice to Junin virus-infected suckling athymic mice was studied. Transfer was performed 1 or 7 days after infection. In both cases, an acute lethal disease developed 6-11 days after transfer. The mortality reached 100% in all infected groups receiving normal or sensitized splenocytes, while it was negligible for different control groups of athymic mice. Transfer of normal or sensitized splenocytes was unable to significantly modify brain viral titers, as compared with infected nontransferred athymic mice killed after a 25-day observation period. Brain lesions were demonstrated in about half of the infected athymic mice transferred with sensitized splenocytes and in all euthymic infected mice. These results show that splenocyte transfer from immunocompetent donors is able to change the normal course of persistent Junin virus infection in nude mice to a lethal acute disease, thus pointing to a main role for T cells in its pathogenesis.

Induction of Junin virus persistence in adult athymic mice.

To determine the role of T lymphocytes in adult mice infected with Junin virus, 60-day-old athymic (nu/nu) mice and their immunocompetent (nu/+) littermates were inoculated intracerebrally with 10(3) TCD50 of the XJ strain. None of them exhibited neurologic illness during a 6-month observation period, and mortality was 3% for nu/nu and 7% for nu/+ animals. The main features in infected nu/nu mice were: high viral titers in brain, reaching a late peak (6.5 log/ml) 32 days postinoculation and persisting at least 6 months; low, late viremia appearing simultaneously with the viral peak in the central nervous system (CNS) and persisting up to 3 months after infection; absence of signs of neurologic disease or histologic lesions in brain and almost no mortality; and lack of detectable circulating antibodies either in IgM or in other immunoglobulins (Igs). Circulating anti-Junin antibodies were demonstrated in IgM and other Igs in immunocompetent mice, although no infectious virus or histologic lesions could be detected. These results show an important role for T lymphocytes in the clearance of Junin virus in the CNS, as demonstrated by viral persistence induced in adult athymic mice.

Cytogenetics of South American akodont rodents (Cricetidae). V. Segregation of chromosome No. 1 polymorphism in Akodon molinae.

Akodon molinae is polymorphic with 2n=42, 43, 44, where the metacentric autosome No. 1 is homologous to 2 acrocentrics 1a and 1b. Matings between 2n=43 heterozygotes 1/1a, 1b gave a surplus of 1/1 offspring, a moderate reduction of heterozygous and a strong reduction of homozygous 1a, 1b/1a, 1b offspring. The latter type also has a highly reduced fertility.

The half-lives of angiotensin II, angiotensin II-amide, angiotensin III, Sar1-Ala8-angiotensin II and renin in the circulatory system of the rat.

1. Methods are described for estimating the half-life of angiotensin analogues and renin in the rat, from the time course of the blood pressure changes they evoke. 2. The following half-life values were measured: angiotensin II, 16 +/- 1 sec; angiotensin III, 14 +/- 1 sec; angiotensin II-amide, 15 +/- 1 sec; Sar1-Ala8-angiotensin II, 6.4 +/- 0.6 min; renin, 3.0 +/- 0.4 min. The distribution volume of angiotensin was found to be 18 ml./kg body wt. 3. It is inferred that the Asp1 residue does not reduce the rate of angiotensin II catabolism, but that substitution of this residue by sarcosine may inhibit catabolism while substitution by asparagine has no effect. 4. Five experimental criteria were identified which indicate that these methods give reliable estimates of the half-life. It is suggested that these results are more accurate than most previous half-life estimates. 5 When tachyphylaxis to angiotensin II-amide occurs, the pressor activity of the plasma is not reduced.

G- and C-banding patterns in the T2 murine leukemia.

The bone marrow cells of BALB/c mice with T2 murine leukemia were analyzed cytogenetically. Of 98 leukemia metaphases, 16.3% were hypodiploid, 4.1% hyperdiploid, and 79.5% diploid. The distribution of G bands in diploid metaphases indicated that almost half of them were pseudodiploid, with chromosome abnormalities such as trisomies, monosomies, nullisomies, unidentified chromosomes, translocations, deletions, or duplications. Since all mouse chromosomes are acrocentric and can be identified only tentatively most of the anomalies detected with G-banding procedures would have passed unnoticed with conventional cytogenetic techniques. The C-banding pattern of leukemia cells did not differ from that of normal controls. However, a considerable number of leukemia cell metaphases had bridges connecting the centromeric C bands of two or more chromosomes. This phenomenon probably indicates an increased stickiness of the heterochromatin, which may produce mitotic nondisjunction and the appearance of monosomies and trisomies.