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Induction of heme oxygenase-1 (HO-1) is protective in tissue injury in models of allograft rejection and vascular inflammation through either prevention of oxidative damage or via immunomodulatory effects. To examine the specific role of HO-1 in modulating the immune response, we examined the differences in immune phenotype between HO-1 knockout (HO-1−/−) and wild-type (HO-1+/+) mice. Consistent with previous findings, marked splenomegaly and fibrosis were observed in HO-1−/− mice. The lymph nodes of HO-1-deficient mice demonstrated a relative paucity of CD3- and B220-positive cells, but no such abnormalities were observed in the thymus. Flow cytometric analysis of isolated splenocytes demonstrated no differences in the proportions of T lymphocytes, B lymphocytes or monocytes/macrophages between the HO-1−/− and HO-1+/+ mice. Significantly higher baseline serum IgM levels were observed in HO-1−/− versus HO-1+/+ mice. Under mitogen stimulation with either lipopolysaccharide or anti-CD3/anti-CD28, HO-1−/− splenocytes secreted disproportionately higher levels of pro-inflammatory Th1 cytokines as compared to those from HO-1+/+ mice. These findings demonstrate significant differences in the immune phenotype between the HO-1−/− and the HO-1+/+ mice. The absence of HO-1 correlates with a Th1-weighted shift in cytokine responses suggesting a general pro-inflammatory tendency associated with HO-1 deficiency.

OBJECTIVE—The incidence of pediatric type 2 diabetes has recently seen an alarming increase. To improve our understanding of pediatric type 2 diabetes and identify markers that discriminate these subjects from those with type 1 diabetes, we performed a multivariant analysis associating serum adiponectin and leptin levels with anthropometrical parameters and disease state. RESEARCH DESIGN AND METHODS—Samples from children and adolescents with type 1 diabetes (n = 41) and type 2 diabetes (n = 17) and from nondiabetic individuals of similar age from the general population (n = 43) were investigated. An analysis included the parameters of matching for BMI and Tanner stage. Receiver-operator characteristic (ROC) curves were established to assess these analytes’ association with disease. RESULTS—Contrary to studies of adult type 1 diabetes, adiponectin levels in our pediatric type 1 diabetic subjects (10.2 μg/ml [95% CI 8.6–11.7]) did not differ from those of healthy control subjects (10.6 μg/ml [9.2–12.0]; P = NS). Children with type 2 diabetes (5.5 μg/ml [4.8–6.2]) had significantly lower adiponectin levels than both of those groups. Conversely, type 2 diabetic subjects showed marked elevations in serum leptin concentrations (24.3 ng/ml [17.1–31.5]) compared with healthy control subjects (2.7 ng/ml [1.3–4.1]; P < 0.001) and type 1 diabetic subjects (5.1 ng/ml [3.5–6.7]; P < 0.001). Importantly, each of the properties ascribed to pediatric type 2 diabetes was present when the comparison was restricted to healthy children or type 1 diabetic patients whose BMI was >85th percentile or who had Tanner stage 4 and 5. The evaluation of adiponectin-to-leptin ratios revealed a striking difference between children with type 1 diabetes (6.3 [3.8–8.8]) and type 2 diabetes (0.3 [0.2–0.5]; P < 0.001). CONCLUSIONS—In pediatric diabetes, where diagnosis of disease is often difficult, these studies suggest that the adiponectin-to-leptin ratio may provide additional help in the discrimination between type 1 and type 2 diabetes.

Large-scale programs designed to assess risk for type 1 diabetes through serologic assessment of autoantibodies to recombinant beta-cell autoantigens are hampered by several limitations, including the methods for sample collection and assay performance, as well as the volume required for autoantibody determinations. The present study was designed to develop a low sample-volume, primary screening method for autoantibody detection of high specificity and sensitivity, and to determine the feasibility of dried blood spots collected on filter paper in serving as vehicles for such determinations. Autoantibodies to glutamic acid decarboxylase (GAD) and ICA512bdc (IA-2), both individually and in combination, were determined in persons with type 1 diabetes, healthy controls, or individuals with other autoimmune disorders. Autoantibody results for serum, plasma, and dried blood spots were compared. GAD, IA-2, and combined GAD/IA-2 autoantibodies were concordant in their measurement from minimal volumes of serum, plasma, and whole blood extracted from dried filter paper. The autoantibody levels from the dried blood spots were, however, lower than corresponding serum samples, and, as currently designed, failed to detect low-titer autoantibodies. Despite this limitation, screening for diabetes risk can be performed using small volumes of whole blood, serum, or plasma collected onto filter paper. These methodological improvements should simplify matters, reduce costs, and increase the efficacy of screening programs for type 1 diabetes. Further development of better substrates/methods for blood-specimen collection seems necessary to exploit the full potential of this and other autoantibody measurement strategies for screening large populations.

CD40 ligand (CD40L) regulates multiple phases of the humoral and cellular immune response through binding to CD40. Previous investigations have suggested that insulin-dependent diabetes (IDDM) in both humans and nonobese diabetic mice may be strongly influenced by similar immunoregulatory molecules. As persons with or at increased risk for the disease are characterized by a number of immunological abnormalities, including that of self-reactive autoantibody production (e.g. islet cell cytoplasmic autoantibodies), we analyzed the expression of CD40L on T lymphocytes (CD3+ cells) in a series of individuals with newly diagnosed IDDM (n = 11), nondiabetic relatives of IDDM probands at increased risk for the disease (n = 21; islet cell cytoplasmic autoantibodies positive; Juvenile Diabetes Foundation titer, ≥20), and healthy controls (n = 13). Both phorbol myristate acetate (PMA)-stimulated and unstimulated peripheral blood mononuclear cells from study subjects were analyzed by flow cytometry with a series of p...

Autoantibodies to the neuroendocrine protein insulinoma-associated protein 2 (IA-2), a member of the tyrosine phosphatase family, have been observed in individuals with or at increased risk for IDDM. Because this disease is thought to result from a T-cell-mediated autoimmune destruction of the insulin-producing pancreatic beta-cells, we analyzed humoral and cellular immune reactivity to this autoantigen to further define its role in the pathogenesis of IDDM. Peripheral blood mononuclear cells (PBMC) from individuals with newly diagnosed IDDM or at varying levels of risk for the disease were stimulated in vitro with the entire 42-kDa internal domain of IA-2 (amino acids 603-979), a series of control antigens (glutathionine-S-transferase, tetanus toxoid, Candida albicans, mumps, bovine serum albumin), and a mitogen (phytohemagglutinin). The frequency and mean stimulation index of PBMC proliferation against IA-2 was significantly higher in newly diagnosed IDDM subjects (14 of 33 [42%]; 3.8+/-4.5 at 10 microg/ml) and autoantibody-positive relatives at increased risk for IDDM (6 of 9 [66%]; 3.9+/-3.2) compared with autoantibody-negative relatives (1 of 15 [7%]; 1.8+/-1.0) or healthy control subjects (1 of 12 [8%]; 1.5+/-1.0). The frequencies of cellular immune reactivities to all other antigens were remarkably similar between each subject group. Sera from 58% of the newly diagnosed IDDM patients tested were IA-2 autoantibody positive. Despite investigations suggesting an inverse association between humoral and cellular immune reactivities against islet-cell-associated autoantigens, no such relationship was observed (rs=0.18, P=0.39) with respect to IA-2. These studies support the autoantigenic nature of IA-2 in IDDM and suggest the inclusion of cellular immune responses as an adjunct marker for the disease.

Previous studies suggest that therapeutic expression of interleukin (IL)-4 by islet cells improves their efficacy in transplantation models directed at reversing type 1 diabetes. We investigated the effects of introducing IL-4 into islets with recombinant adeno-associated virus (rAAV) on the reversal of hyperglycemia in a syngeneic marginal islet mass transplantation model. C57BL/6 islets were mock-transduced or transduced with rAAV expressing murine IL-4 (rAAV-IL-4) or rAAV expressing green fluorescent protein (rAAV-GFP) before transplantation of a marginal mass into diabetic mice. Normoglycemia was achieved in only 1/7 mice receiving rAAV-IL-4 transduced islets in comparison to 6/6 mock-transduced and 4/6 rAAV-GFP transduced animals. The failure of IL-4 expressing islets was not associated with cellular toxicity of rAAV or impairment of glucose-stimulated insulin release in vitro. Islet expression of IL-4 led to impaired metabolic function in mice receiving a marginal mass of syngeneic islets.

The concept of insulin production afforded by hepatic gene therapy retains promise as a potential therapy for type 1 diabetes, but the approach has been limited by the need for strict transgene regulation in response to fluctuating levels of both glucose and insulin. Furthermore, while hepatocytes contain various glucose-responsive elements, they lack the appropriate regulated secretory system necessary for insulin release, thereby necessitating the requirement for transcriptional regulation of hepatic insulin production under the direction of a glucose-responsive promoter. To address this, we have evaluated several glucose-responsive promoters that may be used successfully for hepatic insulin production via recombinant adeno-associated virus (rAAV) therapy. Our results suggest that the human insulin promoter represents a strong candidate as a robust, glucose-responsive promoter for regulated hepatic insulin production.

Investigations of humans and nonobese diabetic mice suggest that proinsulin and/or a fragment of the region spanning C-peptide and the B-chain of insulin (i.e., proinsulin peptide) may serve as key autoantigens in IDDM. Therefore, we analyzed cellular immune reactivities against these molecules in people with or at varying risks for the disease to clarify their role in the pathogenesis of IDDM. In vitro peripheral blood mononuclear cell (PBMC) responses against these antigens, a control antigen (tetanus toxoid), and phytohemaglutinin were determined in 60 individuals with newly diagnosed IDDM (< or = 1 day from diagnosis) in 34 islet cell cytoplasmic autoantibody- and/or insulin autoantibody-negative first-degree relatives of the IDDM subjects, and in 28 autoantibody-negative control subjects. Unlike previous reports suggesting diabetes-associated elevations in cellular immunity to other beta-cell antigens (e.g., GAD, IA-2, etc.), we observed equivalent levels of phytohemaglutinin stimulation and cellular proliferation in all groups against these antigens (all P values were not significant). The mean stimulation index +/- SD and frequency of reactivity to proinsulin for healthy control subjects and IDDM patients, respectively, were as follows: 1 microg/ml (1.5 +/- 1.0, 1 out of 17 [6%]; 1.9 +/- 1.4, 4 out of 33 [12%]); 10 microg/ml (1.7 +/- 1.3, 1 out of 17 [6%]; 1.2 +/- 0.6, 0 out of 28 [0%]); and 50 microg/ml (1.2 +/- 0.6, 1 out of 16 [6%]; 1.1 +/- 0.6, 1 out of 27 [4%]). The response in healthy control subjects, autoantibody-negative relatives, and IDDM patients, respectively, against the proinsulin peptide fragment were as follows: 1 microg/ml (0.9 +/- 0.4, 1 out of 12 [8%]; 1.3 +/- 1.1, 4 out of 34 [11%]; 1.1 +/- 0.3, 2 out of 28 [7%]); 10 microg/ml (0.9 +/- 0.6, 1 out of 12 [8%]; 1.2 +/- 0.6, 3 out of 34 [9%] 1.4 +/- 1.7, 2 out of 28 [7%]); and 50 microg/ml (1.0 +/- 0.7, 1 out of 12 [8%]; 1.2 +/- 0.5, 2 out of 34 [6%]; 1.3 +/- 0.5, 2 out of 28 [7%]). Taken together with previous studies reporting relatively infrequent occurrences of autoantibodies to proinsulin, the role of immunity to this molecule in the pathogenesis of IDDM in humans remains unclear.

Prophylactic insulin therapy prevents IDDM in spontaneous animal models of the disease and has shown promise in preventing the disease in humans. Although large clinical trials have been formed to use this therapy, a comparative analysis of the efficiency of different pharmaceutical forms and doses of insulin in preventing IDDM has not been performed, and the mechanism underlying the observed prevention of disease is unknown. In the NOD- scid/scid adoptive transfer model of IDDM (10(7) new-onset NOD splenocytes injected intravenously into 6- to 8-week NOD/ scid-scid recipients; insulitis develops at 6–9 days post-transfer and 100% IDDM by 32 days post-transfer), life-table (log-rank) analyses revealed that IDDM can be delayed (compared with insulin-free diluent, once daily, n = 8) with equivalent efficiency by prophylactic administration (–9–50 days post-transfer) of high (metabolism-altering) doses of short-acting (0.5 U, once daily, regular, n = 13) or long-acting (0.5 U, once daily, ultralente, n = 9) insulin as well as non-metabolism-altering low-dose insulin (0.02 U, once daily, regular, n = 8). Furthermore, IDDM was delayed with somatostatin (0.2 μg, twice daily, n = 11), an agent that suppresses endogenous insulin production. No significant difference was seen between the preventative effects of these agents. In an assessment of when therapies can be initiated and still maintain clinical efficiency, only prophylactic somatostatin therapy delayed IDDM ( n = 10, P = 0.02) when initiated at 14 days post-transfer, whereas the short-acting insulin regimen did not retard the onset of IDDM ( n = 8, P = 0.25) compared with diluent-treated controls. The 24-h urinary C-peptide levels were significantly reduced with short-acting (−56%, P = 0.01) and long-acting (–67%, P = 0.02) insulin products and somatostatin (–59%, P = 0.02) compared with diluent-treated controls. These results indicate that both immunological and metabolic (i.e., β-cell rest) factors may contribute to the beneficial effects of prophylactic insulin therapy.

Glutamic acid decarboxylase is attracting much interest because of its putative involvement in two clinical disorders: stiffman syndrome and insulin-dependent diabetes. Here we discuss the clinical significance of an autoimmune response against GAD and consider how such information may help identify the disease mechanisms of these disorders.

The present studies were designed to test the hypothesis that arginine vasopressin (AVP) can interact with hydrocortisone and 3,5,3 ′-triiodothyronine (T3) to induce maturation of lung liquid reabsorptive processes in fetal sheep < 130 days gestation. Lung liquid production rates were measured in chronically catheterized thyroidectomized fetal sheep during eight different experimental treatments. Each experiment consisted of a 2-h control period followed by a 5-h treatment period. Net secretion or reabsorption of lung liquid was measured by using impermeant marker dilution techniques. AVP alone (50 mU/kg bolus plus 5.0 mU.kg-1.min-1 i.v. infusion) does not alter lung liquid secretion in fetal sheep 125 +/- 0.72 (SE) days gestation. In contrast, AVP (same dose as above) with T3 (30 micrograms) and hydrocortisone (6.94 mg/min) depressed lung liquid secretion and caused reabsorption of fluid. T3 alone, T3 and hydrocortisone, T3 and AVP, hydrocortisone alone, hydrocortisone and AVP, and saline did not result in net lung liquid reabsorption over a 5-h treatment period. These investigations demonstrate that AVP, T3, and hydrocortisone interact to cause lung liquid reabsorption in immature fetal lungs.

1. Lingual salt glands, secreting hyperosmotic Na/K solutions in response to methacholine, are present inCrocodylus acutus andC. johnstoni but apparently absent from the alligatorids,Alligator mississipiensis andCaiman crocodilus. 2. Both secretory rates (6–20 μmol/100 g·h) and concentrations (450–600 mM Na) of glandular secretions are essentially identical in the marine/estuarineC. acutus andC. porosus and significantly higher than in the freshwaterC. johnstoni (1–2 μmol/100 g·h; 320–420 mM Na). 3. Lingual glands inAlligator secrete isosmotic Na/K at low rates (1–2 μmol/100 g·h) while those ofCaiman show no response to methacholine. 4. The physiological contrast between alligatorids and crocodylids is reflected in distinct differences in the superficial appearance of the tongue and lingual pores. 5. It is postulated that the alligatorid condition of low secretory capacity and isosmotic secretion reflects the primitive salivary function of lingual glands from which the salt-secreting capability in crocodylids was derived.

Alligators in fresh water regulate their plasma and cloacal fluid electrolytes at concentrations similar to those of other crocodilians. They have an exchangeable Na pool of 60.8 μmol/gm and a unidirectional Na efflux of 3.9 μmol/100 gm·hr. Of this 11% is excreted from the cloaca, 43% presumably diffuses across the body integument, and the remaining 46% is lost from the head region. The integumental Na efflux is estimated to be 0.01 μmol/cm2·hr. In fresh water the net flux is 0.5 μmol/100 gm·hr, which suggests that nondietary Na uptake can compensate for 86% of unidirectional Na loss.

Thermogenic incubation has been documented in two large species of pythons, but the phenomenon has not been studied in small species with concomitantly large heat transfer coefficients. We describe behavior, metabolic rates, mass changes, and temperature relations for adult ball pythons (Python regius), the smallest member of the genus, during the reproductive cycle. Egg and hatchling metabolism and hatchling growth rates were also examined. Rates of oxygen consumption ( $$\dot V_{O_2 } $$ ) of both gravid and non-gravid snakes showed typical ectothermic responses to changing ambient temperature (T a). TheQ 10 forT a's of 20–35°C was 2.2–2.3. The $$\dot V_{O_2 } $$ of gravid females was significantly greater than that of non-gravid snakes at allT a. Maximum oxygen consumption ( $$\dot V_{O_2 } $$ max) during forced exercise was about 12 times resting $$\dot V_{O_2 } $$ atT a=30°C. Eggs (5–6 per female) were laid in April. Total clutch mass was approximately 32% of the females' pre-oviposition mass. After oviposition, mother snakes coiled tightly around their clutches and remained in close attendance until the eggs hatched in June. Sudden decreases inT a elicited abrupt but transient 2- to 4-fold increases in the $$\dot V_{O_2 } $$ of incubating females. Similar responses were not observed in non-incubating snakes. The steady-state $$\dot V_{O_2 } $$ of incubating females was independent ofT a. In no case was body temperature (T b) elevated more than a few tenths of a degree aboveT a in steady-state conditions. The $$\dot V_{O_2 } $$ of developing eggs increased sigmoidally through the 58–70 day incubation period. Total oxygen consumption during incubation atT a=29.2°C was about 3.61 per egg. Young snakes quadrupled their mass during their first year of growth. Compared to larger python species which are endothermic during incubation, ball pythons have similar aerobic scopes and higher mass-specific $$\dot V_{O_2 } $$ max. However, effective endothermy in ball pythons is precluded by high thermal conductance and limited energy stores.

Resting metabolic rates (RMR) of 34 species from 18 genera of boas and pythons (Serpentes: Boidae), with body masses ranging from 2 to 67,800 g, were determined as oxygen consumption ( $$\dot V_{O_2 }$$ ) and carbon dioxide production ( $$\dot V_{CO_2 }$$ ) at three ambient temperatures (T a). The temperature coefficient of metabolism (Q10) averaged 2.61 betweenT a of 20–30°C and 2.65 between 30 and 34°C. The respiratory exchange ratio RE (= $$\dot V_{CO_2 }$$ / $$\dot V_{O_2 }$$ ) increased slightly with increasingT a (0.795 at 20°C, 0.819 at 30°C, and 0.834 at 34°C). Interspecific differences in Q10 and RE were slight or insignificant. A multiple regression relating metabolism ( $$\dot V_{O_2 }$$ ) to mass andT a explained 97% of the variance in the pooled interspecific data. The mass exponent was 0.806, which is approximately the same as reported for squamates and for all reptilian taxa combined. The mean within-species slope (0.732) was significantly less than the slope for pooled data, but did not differ significantly from 0.75. In 40 of 42 cases (14 species at 3T a), within-species slopes did not differ from each other. Values of the adjusted mean Y, from covariance analysis, were significantly and positively correlated with mass, indicating that the mass coefficient increases with increasing mass. Considerable variation in metabolic rate is apparent both within and between ecological and taxonomic categories.

Early systemic treatment of nonobese diabetic mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 prevents type 1 diabetes. To determine the therapeutic parameters and immunological mechanisms underlying this observation, female nonobese diabetic mice at 4, 8, and 12 wk of age were given a single i.m. injection of rAAV-murine IL-10 (104, 106, 108, and 109 infectious units (IU)), rAAV-vector expressing truncated murine IL-10 fragment (109 IU), or saline. Transduction with rAAV-IL-10 at 109 IU completely prevented diabetes in all animals injected at all time points, including, surprisingly, 12-wk-old animals. Treatment with 108 IU provided no protection in the 12-wk-old injected mice, partial prevention in 8-wk-old mice, and full protection in all animals injected at 4 wk of age. All other treatment groups developed diabetes at a similar rate. The rAAV-IL-10 therapy attenuated pancreatic insulitis, decreased MHC II expression on CD11b+ cells, increased the population of CD11b+ cells, and modulated insulin autoantibody production. Interestingly, rAAV-IL-10 therapy dramatically increased the percentage of CD4+CD25+ regulatory T cells. Adoptive transfer studies suggest that rAAV-IL-10 treatment alters the capacity of splenocytes to impart type 1 diabetes in recipient animals. This study indicates the potential for immunomodulatory gene therapy to prevent autoimmune diseases, including type 1 diabetes, and implicates IL-10 as a molecule capable of increasing the percentages of regulatory cells in vivo.