Endothelins urinary excretion is increased in spontaneously diabetic rats: BB/BB

Previously we reported (1) an increase of endothelin-1,2 (ET) content, in urine of rats made diabetic with streptozotocin (STZ), starting three days and up to 20 weeks from diabetes induction. The increased ET excretion was considered as an early marker of endothelial damage. To ascertain if this phenomenon was present also in a strain of spontaneously diabetic rats, endothelin-1,2 urinary excretion was determined in BB/BB diabetic rats, and their control (BB/WB), at different times after the onset of diabetes, (two, four, six and twelve weeks). BB/BB diabetic rats showed elevated urinary excretion of endothelins as compared to BB/WB control rats, starting two weeks after diabetes onset, and up to twelve weeks. In the same animals, Nerve Conduction Velocity (NCV), was monitored at the same time as an index of the occurrence of a diabetes complication (peripheral neuropathy). NCV resulted to be impaired in the BB/BB diabetic rats as compared to control rats; however the increase of ET in urine, is earlier in comparison to peripheral neuropathy. These data suggest the hypothesis that endothelial damages preceed the overt manifestations of peripheral neuropathy associated to diabetes.     

Genetic analysis of the BB/W diabetic rat

A large colony of BB/W diabetic rats has been developed as a research model for insulin dependent, type 1 diabetes mellitus. The foundation stock had 8% diabetics which appeared in a sporadic manner. The Worcester (W) colony was inbred by brother X sister matings for 11 generations and the proportion of diabetics increased to over 50%. The age of detection varies from 46 to 250 days. For selection purposes, classification was made at 120 days, which means that 15 to 20% potential diabetics were classified as normal. Evidence from different analyses indicates that the inheritance of diabetes is by a recessive gene or gene cluster with 50% penetrance at 120 days. The selection of breeding stock from diabetic parents raised the proportion of diabetics produced by two normal parents from 12 to 43%. Diallel tests show that diabetic and normal offspring of two diabetic parents have the same diabetic genotype. Outcrosses to other strains of rat indicate that the trait is transferred as a recessive with only 3% diabetics recovered in the F2 where noninbred BB stock was used as the diabetic source, and 36% where partially inbred BB/W was used as the diabetic parent. Since the proportion of diabetics produced by all types of crosses has changed, and may continue to change with changes in the genetic background, we have used the operational term penetrance to describe the frequency of diabetes in individuals homozygous for the diabetes gene cluster. At present the penetrance at 120 days is 59% in the BB/W colony.     

Engagement of 4-1BB inhibits the development of experimental allergic conjunctivitis in mice

The 4-1BB receptor acts as a costimulator in CD8(+) T cell activation. Agonistic stimulation through this molecule by treatment with anti-4-1BB Abs has been demonstrated to inhibit various experimentally induced diseases in animals. However, the effect of anti-4-1BB Abs on experimental allergic diseases has not been reported. We investigated the effect of anti-4-1BB Abs on the development and progression of experimental allergic conjunctivitis in mice. To examine the effects of Abs during the induction or effector phase, actively immunized mice or passively immunized mice by splenocyte transfer were treated with agonistic anti-4-1BB Abs, blocking anti-4-1BB ligand Abs, or normal rat IgG. Eosinophil infiltration into the conjunctiva was significantly reduced in wild-type mice by the anti-4-1BB Ab treatment during either induction or effector phase. Th2 cytokine production by splenocytes and total serum IgE were significantly reduced by the anti-4-1BB Ab treatment, while IFN-gamma production was increased. The anti-4-1BB Ab treatment induced a relative increase of CD8-positive cell numbers in the spleens. Moreover, inhibition of eosinophil infiltration by the treatment with anti-4-1BB Abs was also noted in actively immunized IFN-gamma knockout mice. Taken altogether, in vivo treatment with agonistic anti-4-1BB Abs in either induction or effector phase inhibits the development of experimental allergic conjunctivitis, and this inhibition is likely to be mediated by suppression of Th2 immune responses rather than up-regulation of IFN-gamma.     

Congenic diabetes-prone BB.Sa and BB.Xs rats differ from their progenitor strain BB/OK in frequency and severity of insulin-dependent diabetes mellitus.

Two newly established congenic diabetes-prone BB rat strains designated BB.Sa and BB.Xs carrying a region of chromosome 1 (Sa-Lsn-Secr-Igf2-Tnt, 16 cM) and a region of chromosome X (DXMgh3-Mycs/Pfkb1-Ar, 36 cM) of the SHR rats, respectively, were studied to determine whether the transferred chromosomal regions influence diabetes frequency, age at onset, and clinical picture. Therefore, 4 complete litters of BB/OK (n = 43), BB.Sa (n = 45), and BB.Xs (n = 41) were observed for diabetes occurrence up to the age of 30 weeks. From these litters 6 diabetic males of each strain manifesting in an interval of 1 week were chosen to study body weight, blood glucose, insulin requirement to survive, and several diabetes-related serum constituents at onset of diabetes and after a diabetes duration of 150 days. The diabetes frequency was significantly lower in BB.Xs than in rats of the parental strain BB/OK, whereas comparable frequencies were found between BB/OK and BB.Sa rats. Obvious differences were observed 150 days after diabetes onset between BB/OK and both BB.Sa and BB.Xs rats. BB/OK rats were significantly heavier and needed significantly more insulin/100 g body weight than BB.Sa and BB.Xs rats. Comparisons of the serum constituents as lipids, proteins, and minerals revealed significant differences between diabetic BB/OK rats and their diabetic congenic derivatives in several traits studied at onset and after 150 days of insulin treatment. These results not only show the power of congenic lines in diabetes research, but indicate for the first time that there are genetic factors on chromosomes 1 and X influencing frequency and severity of diabetes in the BB/OK rat.     

Increased plasma and tissue levels of vitamin E in the spontaneously diabetic BB rat

Increased plasma and tissue levels of vitamin E were found in spontaneously diabetic BB rats (D) as well as asymptomatic/diabetes-prone BB rats (AD) in comparison to levels in non-diabetic control rats (ND). Treatment of D rats with insulin for 30 days returned plasma and tissue values of vitamin E to control levels. The changes reported here could not be explained solely on the basis of variations in total lipid content of plasma. These data suggest the metabolism of vitamin E is altered in asymptomatic and spontaneously diabetic BB rats and this alteration returns to control values following insulin treatment. Furthermore, it might be speculated that these data indicate a relationship between vitamin E and insulin.     

4-1BB: Still in the Midst of Darkness

4-1BB is a member of the tumor necrosis factor receptor superfamily. The receptor functions mainly as a costimulatory molecule in T lymphocytes. In addition, several lines of evidence have shown that interactions between 4-1BB and its ligand are involved in the antigen presentation process and the generation of cytotoxic T cells. Recent studies, however, have demonstrated that 4-1BB plays more diverse roles: Signals through 4-1BB are important for long-term survival of CD8⁺ T cells and the induction of helper T cell anergy. Clinically, there is great interest in 4-1BB, because T-cell activation induced by anti-4-1BB monoclonal antibodies is highly efficient in the eradication of established tumor cells in mice. Now, since mice deficient in 4-1BB or the 4-1BB ligand are available, subtle roles played by 4-1BB may be revealed in the near future.     

Persistence of betanodavirus in Barramundi brain (BB) cell line involves the induction of Interferon response

The BB cell line derived from the brain tissue of a barramundi (Lates calcarifer) that survived nervous necrosis virus (NNV) infection is persistently infected with NNV. To elucidate whether interferon (IFN) plays a role in the mechanism of NNV-persistent infection in BB cell line, a virus-negative control cell line was obtained by treating BB cells with NNV-specific rabbit antiserum for 5 subcultures. After the treatment, NNV titer or RNA or capsid protein was no longer detected in the cured BB (cBB) cells. Expression of Mx gene, encoding a type I IFN-inducible antiviral protein, was found in BB cells and cBB cells following NNV infection, but not in NNV-free cBB cells. Moreover, expression of Mx gene and antiviral activity against NNV were induced in cBB cells by the treatment with MAb-neutralized BB cell supernatant. Furthermore, NNV persistent infection was induced again in cBB cell culture if multiplicity of infection (MOI) was low (< or = 1). These experimental results indicated that IFN-like cytokines existed in the culture supernatant of BB cells, and IFN-induced response played an important role in protecting the majority of cells from virus lytic infection and regulating NNV persistence in the BB cell line.     

Identification of profound peripheral T lymphocyte immunodeficiencies in the spontaneously diabetic BB rat

The BB rat is presently the best available animal model for human insulin dependent diabetes (IDD). Because of the extreme susceptibility of the strain to opportunistic infections and because current studies suggest that they have an autoimmune diathesis, of which IDD is but one result, aspects of the immune system of the BB rat were studied. Severe T lymphopenia was observed in all BB rats, irrespective of sex or the presence of IDD, while numbers of B cells and serum immunoglobulin levels were normal. Both the helper T lymphocyte and cytotoxic/suppressor T lymphocyte subsets, defined by reactions with monoclonal antibodies, were depressed, and an inversion of the helper T cell subset to cytotoxic/suppressor T lymphocyte subset ratio occurred in all BB rats with increasing maturity. Concomitantly, severe impairments of T cell-mediated immune responses were noted. BB rats poorly rejected allografts across both major and minor histocompatibility barriers, and BB splenic or peripheral blood lymphocytes had markedly defective proliferative responses to mitogens and to allogeneic cells in MLC. Irradiated and nonirradiated BB spleen cells did not inhibit WF mitogenic or MLC responses, which suggests that the T cell defect in BB rats is not solely due to increased suppressor activity. Because irradiated WF cells and Con A supernatants did not restore BB proliferative responses, and BB lymphocytes were able to produce IL-2 normally, a reduced ability of BB lymphocytes to respond to helper factors such as IL-2 is suggested. In contrast to T lymphocytes from spleen or peripheral blood, BB thymocytes responded as well as did WF thymocytes to Con A or Con A supernatants. Percentages of T lymphocyte subsets and histology of BB thymuses were also normal when compared to WF thymuses. However, spleens and lymph nodes from BB rats were severely depleted of T lymphocytes, and thymocytotoxic autoantibodies were detected in many BB rat sera. The above findings indicate that BB rats have T lymphocyte immunoincompetence, which appears to be a post-thymic or peripherally acquired maturational defect.     

Antigenic properties of BB creatine kinases from various tissues in hens

Studies have been made on interaction between rabbit antiserum to BB creatine kinase from hen gizzard and BB creatine kinases from the brain, heart and gizzard of hen. The degree of interaction was evaluated by the intensity of fluorescence of the immune complexes labeled by a fluorescent dye. It was shown that the intensity of fluorescence of the immune complexes formed by BB creatine kinases from the brain and gizzard of hen is approximately the same, whereas that formed by BB creatine kinase from the heart is twice lower. The data obtained indicate that structural differences in the region of antigenic determinants exist between BB creatine kinase from the heart and those from the brain and gizzard of hen.     

Blocking 4-1BB/4-1BB ligand interactions prevents herpetic stromal keratitis

Herpetic stromal keratitis (HSK) is a chronic inflammatory process in corneal stroma that results from recurrent HSV type 1 infection. We used the murine model of HSK to demonstrate the importance of the interaction between an inducible T cell costimulatory receptor, 4-1BB, and its ligand, 4-1BB ligand (4-1BBL), in the development of this disease. In BALB/c mice, HSK ordinarily induced by infection with the RE strain of herpes was prevented by blocking 4-1BB/4-1BBL interaction, either by deleting 4-1BB (in mutant 4-1BB(-/-) mice) or by introducing mAbs against 4-1BBL. The majority of T cells infiltrating the infected corneas were 4-1BB(+) activated effector cells that expressed cell surface markers CD44, CD25, and/or CD62L, as well as chemokine receptors CCR1, CCR2, and CCR5, and a limited number of TCR Vbeta chains (Vbeta8.1/8.2, Vbeta8.3, Vbeta10b, and Vbeta5.1/5.2, in order of abundance). Analysis of cell surface phenotypes showed that the failure to develop HSK in the 4-1BB(-/-) mice was associated with a reduced expression of CD62L at the time of T cell migration into the corneal stroma.     

Oxidative modification of creatine kinase BB in Alzheimer's disease brain

Creatine kinase (CK) BB, a member of the CK gene family, is a predominantly cytosolic CK isoform in the brain and plays a key role in regulation of the ATP level in neural cells. CK BB levels are reduced in brain regions affected by neurodegeneration in Alzheimer's disease (AD), Pick's disease, and Lewy body dementia, and this reduction is not a result of decreased mRNA levels. This study demonstrates that posttranslational modification of CK BB plays a role in the decrease of CK activity in AD brain. The specific CK BB activity and protein carbonyl content were determined in brain extracts of six AD and six age-matched control subjects. CK BB activity per microgram of immunoreactive CK BB protein was lower in AD than in control brain extracts, indicating the presence of inactive CK BB molecules. The analysis of specific protein carbonyl levels in CK BB, performed by two-dimensional fingerprinting of oxidatively modified proteins, identified CK BB as one of the targets of protein oxidation in the AD brain. The increase of protein carbonyl content in CK BB provides evidence that oxidative posttranslational modification of CK BB plays a role in the loss of CK BB activity in AD.     

Insulin-dependent diabetes and gut dysfunction: the BB rat model.

Accumulating data indicate that intestinal dysfunction and dysregulation of the gut immune system may play a role in the development of type 1 diabetes. This review deals with the occurrence of gut damage and dysfunction in BB rats, an animal model of spontaneous immune type 1 diabetes, placing special emphasis on the effect of diet on the incidence of diabetes in BB rats, the identification of a type 1 diabetes-related protein from wheat, and preliminary observations documenting anomalies in the inductive tissues of the gut immune system (Peyer's patch cells and mesenteric lymph node cells) and pancreatic lymph node cells of diabetes-prone BB rats. In addition to histological evidence of gut damage, the review will also draw attention to altered intestinal disaccharidase activity, changes in intestinal peroxidase activity, glucagon-like peptide 1 anomalies, and perturbation of both intestinal permeability and mucin content in BB rats. In all these cases, the findings in rats fed a diabetes-promoting diet are compared to those collected in animals receiving a protective diabetes-retardant diet.     

Neonatal Treatment With Beta-Cell Stimulatory Agents Reduces the Incidence of Diabetes in BB Rats

The aim of the study was to investigate whether various beta-cell stimulatory drugs, given neonatally, influence the incidence of diabetes in BB rats. Newborn BB rats were treated twice daily for 6 days and diabetes development was observed during the following 200-day study period. Compared to a diabetes incidence of 63.8% in 163 control BB rats which received saline or were untreated, the percentage of experimental BB rats that developed diabetes was as follows in the different subgroups: arginineglucose: 47% (n= 73, p < 0.02); glucagon: 37% (n = 93, p < 0.0001); tolbutamide-glucose: 36% (n = 58, p < 0.0005); and theophylline-glucose: 39% (n = 41, p < 0.005). A long-term arginine-glucose treatment was not superior to the shorter neonatal treatment. Histological examination revealed a higher degree of insulitis in diabetic than in non-diabetic animals but no difference according to the kind of treatment was observed. Finally, we found that the diabetes incidence in BB rats was higher in the first litter compared to subsequent litters (p = 0.04). Thus, neonatal treatment with various beta-cell stimulatory agents reduces diabetes incidence in BB rats. The theory behind the study, that the treatment accelerates beta-cell maturation leading to increased immunological tolerance towards beta cells, is discussed.     

Breeding soundness and libido examination of Belgian Blue and Holstein Friesian artificial insemination bulls in Belgium and The Netherlands

Data on breeding soundness and libido evaluations in Belgian Blue (BB) bulls are scarce. The present study compared results of breeding soundness and libido evaluations of young BB bulls to young Holstein Friesian (HF) bulls prior to acceptance into an AI program. Breed differences for breeding soundness exist between BB and HF bulls, as 93.7% of the young BB bulls failed the breeding soundness examination (BSE) compared to 59.3% of the HF bulls (P=0.0005). Within the BB breed, differences were present between bulls of different ages, and bull selection for better fertility with increasing age apparently influenced the results. The number of reasons for which bulls failed the test differed between the age groups in the BB breed, whereas a tendency for more failure reasons in the BB breed was noticed in the breed comparison. The most important reasons for failure were sperm morphology and scrotal circumference (SC), but far more BB bulls failed for these traits compared to the HF breed (82.8% versus 56.0% and 43.8% versus 17.6% in the BB and the HF breed for sperm morphology (P=0.0005) and SC (P<0.0001), respectively). The high proportion of BB bulls with a substandard SC and poor sperm morphology might suggest an increased prevalence of testicular hypoplasia or degeneration within this breed. Concerning libido, the reaction time did not differ either between breeds or between age groups within the BB breed, whereas mounting enthusiasm, although not different between the two breeds, did decline with increasing age, probably due to the greater mating experience of the older bulls. All in all, libido did not seem to be different between the breeds.     

Immunity to melanoma mediated by 4-1BB is associated with enhanced activity of tumour-infiltrating lymphocytes

4-1BB costimulates T cells to carry out effector functions such as eradication of established tumours. 4-1BB (CD137) is a member of the TNF receptor family, and its triggering by either 4-1BB ligand or antibody ligation induces T-cell activation and growth. We analysed tumour-infiltrating lymphocytes (TIL) in the experimental B16F10 melanoma model to determine the mechanisms involved in 4-1BB-mediated tumour suppression. 4-1BB(+/+) mice survived longer than 4-1BB(-/-) mice, and survival was further prolonged by triggering 4-1BB with an agonistic mAb. The number of metastatic B16F10 colonies in the lung was much greater in 4-1BB(-/-) mice than in their 4-1BB(+/+) littermates. Administration of agonistic anti-4-1BB mAb increased the number of TIL in the tumour masses in the lungs of 4-1BB(+/+) mice. The numbers of CD4(+) T, CD8(+) T and CD11b(+) TIL increased in these mice. Anti-4-1BB mAb induced not only CD8(+) 4-1BB(+) T cells but also a CD8(+) IFN-gamma(+) T-cell population. B16F10 cells from the lungs of anti-4-1BB-treated mice showed enhanced expression of MHC class Iota and IotaIota antigens compared with the same cells from control IgG-treated mice. Thus, the increase in number of CD8(+) T cells and enhanced MHC Iota and IotaIota expression in B16F10 cells that result from augmented IFN-gamma production in response to anti-4-1BB mAb may lead to suppression of tumour growth and metastasis.     

4-1BB triggering ameliorates experimental autoimmune encephalomyelitis by modulating the balance between Th17 and regulatory T cells

Agonistic anti-4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8(+) T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8(+) T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB-triggered suppression of autoimmune responses using IFN-γ- or IFN-γR-deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3(+)CD4(+) regulatory T (Treg) cells, particularly among CD4(+) T cells. This was not due to a direct effect of 4-1BB signaling on CD4(+) T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8(+) T cells, but also did so in IFN-γ-deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.     

Enhanced CD4 T cell responsiveness in the absence of 4-1BB

The 4-1BB (CD137) is a member of the TNFR superfamily, and is expressed on several cell types, including activated T cells. Although 4-1BB ligation by agonistic Ab or 4-1BB ligand-expressing APCs can costimulate T cells, the physiological significance of 4-1BB expression in vivo during T cell responses is still being elucidated. In this study, we have addressed the impact on CD4 T cell priming when 4-1BB is absent after gene targeting. Surprisingly, 4-1BB(-/-) mice generated more enhanced effector CD4 T cell responses to OVA protein in adjuvant, even though Ab responses in 4-1BB(-/-) mice were normal. Using an adoptive transfer system with OT-II TCR transgenic CD4 T cells, we found that 4-1BB(-/-) CD4 cells responding in a 4-1BB-sufficient environment had enhanced cell division compared with wild-type cells and displayed augmented clonal expansion during the primary response. This was not due to a developmental defect as 4-1BB-deficient CD4 cells could respond normally to Ag in vitro. These results demonstrate that the absence of 4-1BB can make CD4 T cells hyperresponsive to protein Ag in vivo, suggesting a new unappreciated negative regulatory role of 4-1BB when expressed on a T cell.     

Creatine phosphokinase BB distribution in different structures of the human brain

Regional localization of creatine phosphokinase BB was studied in postmortem human brain and its stability was shown. The content of CPK BB in different brain structures was unequal: from 0.5 mcg/mg of protein in the occipital lobe and tuber cinereum to 4.5 mcg/mg in the frontal lobe. In the regional localization of CPK BB in the postmortem brain of schizophrenia patients, some changes in isoenzyme content were found as compared to the control group. The reduction of CPK BB concentration at schizophrenia was found in the frontal lobe (45%, P less than 0.001) and s. nigra (70%, P less than 0.001); the concentration was higher in the thalamus and occipital lobe (15%, P less than 0.001), as well as in the parietal lobe, cingulate gyrus, tuber cinereum, cerebellum cortex, inferior olive--50-80%, P less than 0.001.