Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T cell receptor α chain gene rearrangement

Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic β cells. Although both major histocompatibility complex class I-restricted CD8+ and class II-restricted CD4+ T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8+ T cells responsible, we isolated and propagated in vitro CD8+ T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2Kd, and showed a diverse T cell receptor β chain repertoire. In contrast, their α chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of Vα17 family members frequently joined to the Jα42 gene segment. These results suggest that a number of the CD8+ T cells participating in the initial phase of autoimmune β cell destruction recognize a common structural component of Kd/peptide complexes on pancreatic β cells, possibly a single peptide.

Apoptosis resistance of nonobese diabetic peripheral lymphocytes linked to the Idd5 diabetes susceptibility region

Defects in lymphocyte apoptosis may lead to autoimmune disorders and contribute to the pathogenesis of type 1 diabetes. Lymphocytes of nonobese diabetic (NOD) mice, an animal model of autoimmune diabetes, have been found resistant to various apoptosis signals, including the alkylating drug cyclophosphamide. Using an F2 intercross between the apoptosis-resistant NOD mouse and the apoptosis-susceptible C57BL/6 mouse, we define a major locus controlling the apoptosis-resistance phenotype and demonstrate its linkage (logarithm of odds score = 3.9) to a group of medial markers on chromosome 1. The newly defined gene cannot be dissociated from Ctla4 and Cd28 and in fact marks a 20-centimorgan region encompassing Idd5, a previously postulated diabetes susceptibility locus. Interestingly, we find that the CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and the CD28 costimulatory molecules are defectively expressed in NOD mice, suggesting that one or both of these molecules may be involved in the control of apoptosis resistance and, in turn, in diabetes susceptibility.

Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses

Hepatic fibrosis represents the generalized response of the liver to injury and is characterized by excessive deposition of extracellular matrix. The cellular basis of this process is complex and involves interplay of many factors, of which cytokines are prominent. We have identified divergent fibrosing responses to injury among mouse strains and taken advantage of these differences to examine and contrast T helper (Th)-derived cytokines during fibrogenesis. Liver injury was induced with carbon tetrachloride, fibrosis was quantitated, and Th1/Th2 cytokine mRNAs measured. Liver injury in BALB/c mice resulted in severe fibrosis, whereas C57BL/6 mice developed comparatively minimal fibrosis. Fibrogenesis was significantly modified in T and B cell-deficient BALB/c and C57BL/6 severe combined immunodeficient (SCID) mice compared with wild-type counterparts, suggesting a role of Th subsets. Fibrogenic BALB/c mice exhibited a Th2 response during the wounding response, whereas C57BL/6 mice displayed a Th1 response, suggesting that hepatic fibrosis is influenced by different T helper subsets. Moreover, mice lacking interferon γ, which default to the Th2 cytokine pathway, exhibited more pronounced fibrotic lesions than did wild-type animals. Finally, shifting of the Th2 response toward a Th1 response by treatment with neutralizing anti-interleukin 4 or with interferon γ itself ameliorated fibrosis in BALB/c mice. These data support a role for immune modulation of hepatic fibrosis and suggest that Th cytokine subsets can modulate the fibrotic response to injury.

Induction of resistance to diabetes in non-obese diabetic mice by targeting CD44 with a specific monoclonal antibody

Inflammatory destruction of insulin-producing β cells in the pancreatic islets is the hallmark of insulin-dependent diabetes mellitus, a spontaneous autoimmune disease of non-obese diabetic mice resembling human juvenile (type I) diabetes. Histochemical analysis of diabetic pancreata revealed that mononuclear cells infiltrating the islets and causing autoimmune insulitis, as well as local islet cells, express the CD44 receptor; hyaluronic acid, the principal ligand of CD44, is detected in the islet periphery and islet endothelium. Injection of anti-CD44 mAb 1 hr before cell transfer of diabetogenic splenocytes and subsequently on alternate days for 4 weeks induced considerable resistance to diabetes in recipient mice, reflected by reduced insulitis. Contact sensitivity to oxazolone was not influenced by this treatment. A similar antidiabetic effect was observed even when the anti-CD44 mAb administration was initiated at the time of disease onset: i.e., 4–7 weeks after cell transfer. Administration of the enzyme hyaluronidase also induced appreciable resistance to insulin-dependent diabetes mellitus, suggesting that the CD44–hyaluronic acid interaction is involved in the development of the disease. These findings demonstrate that CD44-positive inflammatory cells may be a potential therapeutic target in insulin-dependent diabetes.

Identification of a renal-specific oxido-reductase in newborn diabetic mice

Aldose reductase (ALR2), a NADPH-dependent aldo-keto reductase (AKR), is widely distributed in mammalian tissues and has been implicated in complications of diabetes, including diabetic nephropathy. To identify a renal-specific reductase belonging to the AKR family, representational difference analyses of cDNA from diabetic mouse kidney were performed. A full-length cDNA with an ORF of 855 nt and yielding a ≈1.5-kb mRNA transcript was isolated from a mouse kidney library. Human and rat homologues also were isolated, and they had ≈91% and ≈97% amino acid identity with mouse protein. In vitro translation of the cDNA yielded a protein product of ≈33 kDa. Northern and Western blot analyses, using the cDNA and antirecombinant protein antibody, revealed its expression exclusively confined to the kidney. Like ALR2, the expression was up-regulated in diabetic kidneys. Its mRNA and protein expression was restricted to renal proximal tubules. The gene neither codistributed with Tamm–Horsfall protein nor aquaporin-2. The deduced protein sequence revealed an AKR-3 motif located near the N terminus, unlike the other AKR family members where it is confined to the C terminus. Fluorescence quenching and reactive blue agarose chromatography studies revealed that it binds to NADPH with high affinity (KdNADPH = 66.9 ± 2.3 nM). This binding domain is a tetrapeptide (Met-Ala-Lys-Ser) located within the AKR-3 motif that is similar to the other AKR members. The identified protein is designated as RSOR because it is renal-specific with properties of an oxido-reductase, and like ALR2 it may be relevant in the renal complications of diabetes mellitus.

Continuous amperometric monitoring of glucose in a brittle diabetic chimpanzee with a miniature subcutaneous electrode

The performance of an amperometric biosensor, consisting of a subcutaneously implanted miniature (0.29 mm diameter, 5 × 10−4 cm2 mass transporting area), 90 s 10–90% rise/decay time glucose electrode, and an on-the-skin electrocardiogram Ag/AgCl electrode was tested in an unconstrained, naturally diabetic, brittle, type I, insulin-dependent chimpanzee. The chimpanzee was trained to wear on her wrist a small electronic package and to present her heel for capillary blood samples. In five sets of measurements, averaging 5 h each, 82 capillary blood samples were assayed, their concentrations ranging from 35 to 400 mg/dl. The current readings were translated to blood glucose concentration by assaying, at t = 1 h, one blood sample for each implanted sensor. The rms error in the correlation between the sensor-measured glucose concentration and that in capillary blood was 17.2%, 4.9% above the intrinsic 12.3% rms error of the Accu-Chek II reference, through which the illness of the chimpanzee was routinely managed. Linear regression analysis of the data points taken at t>1 h yielded the relationship (Accu-Chek) = 0.98 × (implanted sensor) + 4.2 mg/dl, r2 = 0.94. The capillary blood and the subcutaneous glucose concentrations were statistically indistinguishable when the rate of change was less than 1 mg/(dl⋅min). However, when the rate of decline exceeded 1.8 mg/(dl⋅min) after insulin injection, the subcutaneous glucose concentration was transiently higher.

Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction

We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.

Localization and regulation of GLUTx1 glucose transporter in the hippocampus of streptozotocin diabetic rats

We describe the localization of the recently identified glucose transporter GLUTx1 and the regulation of GLUTx1 in the hippocampus of diabetic and control rats. GLUTx1 mRNA and protein exhibit a unique distribution when compared with other glucose transporter isoforms expressed in the rat hippocampus. In particular, GLUTx1 mRNA was detected in hippocampal pyramidal neurons and granule neurons of the dentate gyrus as well as in nonprincipal neurons. With immunohistochemistry, GLUTx1 protein expression is limited to neuronal cell bodies and the most proximal dendrites, unlike GLUT3 expression that is observed throughout the neuropil. Immunoblot analysis of hippocampal membrane fractions revealed that GLUTx1 protein expression is primarily localized to the intracellular compartment and exhibits limited association with the plasma membrane. In streptozotocin diabetic rats compared with vehicle-treated controls, quantitative autoradiography showed increased GLUTx1 mRNA levels in pyramidal neurons and granule neurons; up-regulation of GLUTx1 mRNA also was found in nonprincipal cells, as shown by single-cell emulsion autoradiography. In contrast, diabetic and control rats expressed similar levels of hippocampal GLUTx1 protein. These results indicate that GLUTx1 mRNA and protein have a unique expression pattern in rat hippocampus and suggest that streptozotocin diabetes increases steady-state mRNA levels in the absence of concomitant increases in GLUTx1 protein expression.

Unbalanced expression of the different subunits of elongation factor 1 in diabetic skeletal muscle

In studies using subtraction cloning to screen for alterations in mRNA expression in skeletal muscle from humans with Type 2 diabetes mellitus and control subjects, one of the most prominent differences was in the mRNA for elongation factor (EF)-1α. With Northern blot analysis, EF-1α expression was enhanced by 2- to 6-fold in both Types 1 and 2 human diabetics. In contrast, no changes in expression of EF-1β or -γ were noted. We observed similar results in animal models of Type 1 diabetes. EF-1α expression, but not EF-1β or -γ expression, was also enhanced in streptozotocin-induced diabetic rats, and this effect was reversed by insulin treatment. An increased level of EF-1α mRNA was also observed in nonobese diabetic mice. This unbalanced regulation of the expression of the different subunits of EF-1 may contribute to alterations not only in protein synthesis but also in other cellular events observed in the diabetic state.

Inhibition of Endothelial Wound Repair by Dominant Negative Connexin Inhibitors

Wounding of endothelial cells is associated with altered direct intercellular communication. To determine whether gap junctional communication participates to the wound repair process, we have compared connexin (Cx) expression, cell-to-cell coupling and kinetics of wound repair in monolayer cultures of PymT-transformed mouse endothelial cells (clone bEnd.3) and in bEnd.3 cells expressing different dominant negative Cx inhibitors. In parental bEnd.3 cells, mechanical wounding increased expression of Cx43 and decreased expression of Cx37 at the site of injury, whereas Cx40 expression was unaffected. These wound-induced changes in Cx expression were associated with functional changes in cell-to-cell coupling, as assessed with different fluorescent tracers. Stable transfection with cDNAs encoding for the chimeric connexin 3243H7 or the fusion protein Cx43-βGal resulted in perturbed gap junctional communication between bEnd.3 cells under both basal and wounded conditions. The time required for complete repair of a defined wound within a confluent monolayer was increased by ∼50% in cells expressing the dominant negative Cx inhibitors, whereas other cell properties, such as proliferation rate, migration of single cells, cyst formation and extracellular proteolytic activity, were unaltered. These findings demonstrate that proper Cx expression is required for coordinated migration during repair of an endothelial wound.