Further observations on serum viscosity changes in diabetes mellitus

The 8% increase in serum viscosity in diabetes mellitus, more striking in diabetics with evidence of microangiopathy, has been further investigated. Methodology has been developed to eliminate the effect of fluctuating levels of glucose, lipid, and protein. This was accomplished first by dialysis of fresh serum and later by ultracentrifugation to remove lipoprotein followed by dialysis. The distinction between diabetic and nondiabetic viscosity levels was improved by removing glucose and correcting for fluctuations in serum protein level, principally because variation between observations on the same subject was substantially reduced. Two methods were utilized to adjust for protein concentration differences. Either technique increased the statistical significance of the viscosity increase in diabetes. Without lipoprotein removal the regression of specific fluidity (1 - 1/relative viscosity) with concentration differed between the sexes. When lipoprotein was removed the sex difference disappeared and the regression line passed through the origin. Removal of lipoprotein also further increased the discrimination between diabetic and normal serum. Even without lipoprotein removal elevated serum viscosity was readily detected in advanced glucose intolerance. In both studies, viscosity was higher in diabetics with microangiopathic sequelae, increasing progressively with more evidence of microangiopathy. No effect of severity of hyperglycemia or duration of diabetes could be demonstrated. Increased serum viscosity's association with microvascular sequelae and its presence early in the disorder suggest that the protein changes responsible for its elevation may play a role in the development of diabetic microangiopathy.     

Further observations of urethan-induced thymic lymphoma in mice

Summary In mice of a low leukemic strain thymic lymphoma was induced by repeated application of urethan in early life. The thymus was grossly and microscopically examined to elucidate the appearance and development of the neoplastic changes at various ages during the latent period (see Table). The neoplastic transformation of the thymus occurs earlier and proceeds faster in females than in males. The sex difference may be due primarily to an inhibitory action of the testes in the male. In spite of the action of the testes, however, the thymus appeared finally to undergo neoplastic transformation also in males.

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Zusammenfassung Bekanntlich kann Urethan ein Thymuslymphom bei Mäusen hervorrufen. In dieser Mitteilung wird das Erscheinen und die Entwicklung der Lymphomentstehung im Thymus während der Latentzeit makroskopisch und mikroskopisch beschrieben (s. Tabelle). Die neoplastische Transformation des Thymus entwickelt sich und erscheint früher bei Weibchen als bei Männchen. Der Geschlechtsunterschied scheint auf einer hemmenden Wirkung vom männlichen Geschlechtshormon zu beruhen. Trotzdem erfährt aber der Thymus auch bei Männchen zuletzt eine neoplastische Transformation.

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With 7 Figures in the Text     

Prevention of low dose streptozotocin-induced diabetes by acetyl-homocysteine-thiolactone.

We used acetyl-homocysteine-thiolactone (citiolone) as an enhancer of superoxide dismutase (SOD), a free radical scavenger, in order to assay any possible prevention of the insulitis and subsequent B cell damage caused by streptozotocin (STZ) when given in multiple low doses. Mice were given citiolone (50 mg/kg b.wt.) as a long pretreatment or concomitantly with STZ for a shorter period. Ten days after the last STZ injection, pancreases were processed for SOD assay and morphological observations. Results demonstrate that citiolone increases SOD values, but to a variable degree, after the STZ administration. The highest SOD levels were found in animals treated for the longer period (P less than 0.001 vs saline-treated controls; P less than 0.0001 vs STZ-treated controls) but we did not observe a direct correspondence between high SOD values and morphological integrity of islet beta cells and/or low blood glucose levels. In conclusion, we hypothesize that the onset of type 1 diabetes in mice involves free radical generation but in addition some other factor may be responsible for the beta cell damage.     

Is Lipotoxicity presents in the early stages of an experimental model of autoimmune diabetes? Further studies in the multiple low dose of streptozotocin model

An increased availability of plasma free fatty acids (FFA) seems to play a role in the early stages of experimental type 1 diabetes mellitus induced in C57BL/6J mice by multiple low doses of streptozotoxin (mld-STZ). We analyzed the temporal changes of: (1) plasma and skeletal muscle lipids and their relationship with glucose metabolism; (2) triglyceride (Tg) concentration in isolated islets; (3) intraperitoneal glucose tolerance test; and (4) insulin secretion patterns when the three mutually interactive glucose signaling pathways were activated. Animals were killed by cervical dislocation at days 4, 6, 7, 8, 9 and 12 post first injection of mld-STZ. Compared with control mice, we observed: (1) at day 6, a significant increase of plasma FFA and both muscle and islet Tg content and a significant decrease of muscle pyruvate dehydrogenase activity. These parameters further deteriorated with time. (2) plasma Tg, glucose and insulin levels and glucose tolerance test were significantly different only after day 8. (3) an increase in both phases of the glucose plus palmitate-stimulated insulin secretion was observed at day 4. This effect progressively decreased since day 7 up to day 9. Moreover, an inhibitory action of cerulenin over glucose plus palmitate-stimulated insulin secretion was observed between days 6 and 9. Taken together these results suggest that early alteration in carbohydrate and lipid metabolism could represent a “metabolic window” which would develop between days 6 and 8. Afterwards, subsequent immunological alterations, apoptosis and necrosis induced the destruction of β cells and would mask the results mentioned above.     

Immunohistochemical study of insulitis induced by multiple low doses of streptozocin in CD-1 mice

In an attempt to characterize insulitis induced by multiple low doses of streptozocin (SZ), we immunohistochemically examined sequential changes of the subsets of lymphocytes infiltrating the pancreatic islets in CD-1 mice. Daily intraperitoneal injections of 30 mg/kg body wt of SZ for five consecutive days led to lymphocytic infiltration around the islets. Most of the infiltrated cells were initially CD4 positive (helper/inducer) T-lymphocytes and no immunoglobulin-bearing cells were detected. The number of helper/inducer T-lymphocytes increased with progression of the insulitis, then surface immunoglobulin-positive cells (B-lymphocytes) accumulated around the area of CD4 positive T-lymphocytes. CD8 positive (suppressor/cytotoxic) T-lymphocytes were seen scattered throughout the study and only a few asialo GM1 positive (natural killer) cells existed in the area of insulitis. Subsequently, the pancreatic insulin contents were considerably decreased and diabetes occurred on day 21. These observations suggest that CD4 positive T-lymphocyte-dependent B-lymphocyte infiltration in and around islet cells may be associated with islet cell destruction and the development of diabetes in CD-1 mice treated with multiple low doses of streptozocin.     

Further observations on tuberculin reactions in active tuberculosis.

One hundred patients with active tuberculosis were tested for tuberculin reactivity within 24 hours of their admission to the hospital. Commercial intermediate tuberculin, Tween stabilized intermediate tuberculin and the Tine test as well as a mumps antigen were applied simultaneously. False negative reactions were obtained in 28 per cent with Tine testing and in 21 per cent with Tween stabilized as well as plain tuberculin. These nonreactors were clinically identifiable as seriously ill with manifestations primarily attributable to protein depletion as a result of their illness. This is not specifically related to the effects of tuberculosis itself, but can be demonstrated in patients suffering the same sequelae of other debilitating illness. After two weeks of protein supplementation via a high calorie, high protein, hospital diet, skin reactivity was restored in the vast majority of these nonreactors. It is concluded that the lack of tuberculin reactivity on hospital admission probably results from impaired lymphocyte function in patients suffering serious protein depletion as a result of their illness; it is not attributable to deficiencies in the tuberculin test itself.     

Morphological and biochemical observations on hepatic glycogen metabolism in mice on a controlled feeding schedule

Changes in hepatocyte morphology were correlated with chemically measured liver glycogen, blood glucose, and plasma insulin levels in control-fed mice (6 hr fed, 18 hr fasted) sacrificed at various time intervals after initiation of a 6-hr meal. At initiation of feeding hepatic glycogen was low (0.05%) but deposition proceeded rapidly, reaching a maximum of 6.99±0.13% by the sixth hour. Glycogen was depleted during the subsequent fasting period, reaching the prefeeding levels by 24 hr. A relative hyperglycemia (140–192 mg/100 ml) predominated during all stages of glycogen deposition and depletion until the 21 st hour. Plasma insulin levels were maximum during feeding (63±7 U/ml, 3 hr) with mild hyperinsulinemia (insulin>16 U/ml) occurring during glycogen depletion (9–21 hr). Histochemical determinations (PAS) showed lobular patterns of hepatic glycogen which correlated with chemically measured glycogen levels. Six hours after initiation of feeding, periportal cells showed intensely stained masses of glycogen while centrilobular cells showed relatively diffuse staining. At 24 hours after initiation of feeding (18 hr of fasting), no significant staining was observed in the hepatocytes. Ultrastructurally, during all stages of glycogen deposition and depletion, centrilobular cells were characterized by the presence of dispersed glycogen particles with elements of smooth endoplasmic reticulum (SER) between the particles, while periportal cells showed dense glycogen deposits with SER restricted to the periphery of the glycogen masses.Supported by grants AM-06013, AM-27097 and in part by The University of Virginia Diabetes Research Training Center (AM-22125) from USPHS.     

c-Kit in early onset of diabetes: a morphological and functional analysis of pancreatic beta-cells in c-KitW-v mutant mice

c-Kit tyrosine receptor kinase, a well-established stem cell marker, is expressed in a variety of tissues including the pancreas. The involvement of c-Kit in fetal rat and human endocrine pancreatic development, survival, and function has been well characterized but primarily using in vitro experimental approaches. Therefore, the aim of the current study was to examine whether deficiency of a functional c-Kit receptor would have physiological and functional implications in vivo. We characterized the c-Kit mutant mouse, c-Kit(W-v/+), to evaluate the in vivo role of c-Kit in beta-cell growth and function. Here we report that male c-Kit(W-v/+) mice, at 8 wk of age, showed high fasting blood glucose levels and impaired glucose tolerance, which was associated with low levels of insulin secretion after glucose stimulation in vivo and in isolated islets. Morphometric analysis revealed that beta-cell mass was significantly reduced (50%) in male c-Kit(W-v/+) mice when compared with controls (c-Kit(+/+)) (P < 0.05). In parallel, a reduction in pancreatic duodenal homeobox-1 and insulin gene expression in whole pancreas as well as isolated islets of c-Kit(W-v/+) male mice was noted along with a decrease in pancreatic insulin content. Furthermore, the reduction in beta-cell mass in male c-Kit(W-v/+) mice was associated with a decrease in beta-cell proliferation. Interestingly, these changes were not observed in female c-Kit(W-v/+) mice until 40 wk of age. Our results clearly demonstrate that the c-Kit receptor is involved in the regulation of glucose metabolism, likely through an important role in beta-cell development and function.     

Proliferative synovitis in hemophilia. biochemical and morphologic observations

The synovium removed from the knee of a 10-year-old with hemophilia A was characterized morphologically and biochemically. The specimen showed villous hypertrophy with hyperplasia of synovial lining cells which contained abundant intracytoplasmic granules of hemosiderin. Monolayer cultures prepared from enzymatically dispersed tissue were characterized by pigment-laden fibroblast-like cells and round cells. Both explants of synovium and adherent cells secreted a large amount of latent collagenase and neutral proteinase into the culture medium. The secretion of these enzymes dropped sharply and intracellular pigment decreased with passage of these cultures. Lysozyme was secreted by the explants but was not detected in the monolayer culture medium. These data establish the degradative potential of the synovitis found in hemophilia and support the concept that recurrent hemarthrosis without inflammation is sufficient in and of itself to produce proliferative synovitis.     

Further observations on the incidence and properties of lymphocytotoxins in leukaemia.

54 our of 112 patients with leukaemia have been shown to produce a lymphocytotoxin in their serum when the number of malignant cells present in the peripheral blood was considerably raised but the normal blood cells were low. This lymphocytotoxin reacts with all normal cells. It is a high molecular weight protein, though not an antibody. Partial purification was achieved by elution from DEAE-52-cellulose at about pH 6.9.     

Combination therapy with low dose sirolimus and tacrolimus is synergistic in preventing spontaneous and recurrent autoimmune diabetes in non-obese diabetic mice

Aims/hypothesis: Sirolimus and tacrolimus are immunosuppressive drugs that prevent rejection of pancreatic islet allografts transplanted into patients with Type I (insulin-dependent) diabetes mellitus. This study aimed to determine whether sirolimus and tacrolimus can prevent autoimmune beta-cell destruction, and if so, what the mechanisms of action are. Methods: Sirolimus and tacrolimus were given separately and together to female non-obese diabetic (NOD) mice from age 12 to 35 weeks. Diabetes incidence was determined and pancreatic insulitis and insulin content were measured. Sirolimus and tacrolimus were also given separately and together to diabetic NOD mice from the time of syngeneic islet transplantation until the reappearance of hyperglycaemia. Islet grafts were examined by RT-PCR assay for expression of interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-10 and transforming growth factor (TGF)-β1. Results: Low doses of sirolimus (0.1 mg/kg) and tacrolimus (0.1 mg/kg) were synergistic in reducing insulitis, preserving pancreatic insulin content and preventing diabetes in female NOD mice (8 % diabetes incidence at 35 weeks vs 66 % in vehicle-treated mice). Also, the combination of sirolimus and tacrolimus prolonged syngeneic islet graft survival (median 34 days vs 13 days for vehicle-treated mice). Islet grafts from sirolimus plus tacrolimus-treated mice expressed significantly decreased mRNA contents of Th1-type cytokines (IFN-γ and IL-2) and the highest ratio of TGF-β1/IFN-γ mRNA. Conclusion/interpretation: These findings suggest that combination therapy with sirolimus and tacrolimus prevent autoimmune beta-cell destruction by upregulating expression of the immunoregulatory cytokine, TGF-β1 and reducing Th1 cytokines (IFN-γ and IL-2) expressed in the islets. Low-dose sirolimus and tacrolimus combination therapy could warrant consideration for prevention or early treatment of human Type I diabetes. [Diabetologia (2002) 45: 224–230] Received: 20 August 2001 and in revised form: 29 October 2001     

BCG vaccine prevents insulitis in low dose streptozotocin-induced diabetic mice

Autoimmune type 1 diabetes mellitus is caused by the immunologic destruction of pancreatic beta-cells; therefore, there have been many attempts at immunologic modulation as a block or prevention of the underlying process. The aim of this study is to investigate the effect of BCG vaccination on low dose streptozotocin-induced diabetic (LDSD) mice. The mice were pretreated with BCG 7 days before starting low dose streptozotocin (STZ), observed body weight and blood glucose for 2 months, then analyzed the severity of the STZ-induced insulitis after the animals were sacrificed. In this experiment, the mean body weights in the BCG-STZ group on days 1, 19, 33, 47, and 61 of the experiment were 37.5 +/- 3.6, 37.3 +/- 3.6, 37.5 +/- 3.5, 39.4 +/- 3.9, and 39.3 +/- 4.5 (g), respectively. Those in the STZ group were 37.7 +/- 3.5, 38.3 +/- 4.5, 38.4 +/- 3.9, 36.2 +/- 4.5, and 36.3 +/- 4.0 (g), respectively (P < 0.05). The mean blood glucose levels in the BCG-STZ group on days 1, 19, 33, 47, and 61 were 106.5 +/- 8, 150 +/- 37, 147 +/- 54, 143 +/- 60, and 142 +/- 66 (mg/dl), respectively. Those in the STZ group were 103 +/- 12, 196 +/- 90, 261 +/- 236, 236 +/- 91, and 224 +/- 89 (mg/dl), respectively (P < 0.05). The numbers developing grade 0, I, II, III, and IV insulitis in the BCG-treated group were 63, 48, 33, 4, and 2, respectively, and in the control group were 16, 23, 31, 45, and 35, respectively. This study indicates that BCG vaccination reduces the development of insulitis and overt diabetes in LDSD mice.     

Importance of db gene for the development of low dose streptozotocin diabetes in C57BL/KsJ mice

The paper is concerned with some data on the effect of the diabetic gene (db) on mouse sensitivity to streptozotocin (SC). Male mice aged 2-3 mos. of mutant C57BL/KsJ strain (genotypes: m+/+m, db+/+m, db+/+db) were used for investigation. Insulin-dependent diabetes mellitus was induced by intraperitoneal injections of streptozotocin at a daily dose of 40 mg/kg for 5 days. The structure and function of the insular apparatus were histologically assessed as well as by the blood level of insulin and glucose within 15 days after the start of the experiment. The earliest hyperglycemic reaction to SC was typical of mice, homozygous by the diabetic gene; they had normoglycemia at the time of treatment. In mice, heterozygous by the diabetic gene, a hyperglycemic reaction developed later after treatment. However by the end of the investigation it reached values which were typical of mice, homozygous by the diabetic gene, with basal normoglycemia. Mice, not carrying the diabetic gene, as well as homozygotes by this gene with basal hyperglycemia, possessed lesser sensitivity to SC. The expression of hyperglycemic reactions showed correlation with a degree of dystrophic changes and the development of lymphocellular infiltration in the pancreatic islets of mice with basal normoglycemia in low dose streptozotocin diabetes. The development of spontaneous hyperglycemia in homozygotes by the diabetic gene lowered their sensitivity to SC diabetogenic effects.