Cytophotometric Evidence of Non-S-Phase Extra-Dna In Human Neuronal Nuclei

After Feulgen staining with acriflavine-Schiff, the DNA content of glial and neuronal nuclei from various sites of the human CNS (pre- and post-central gyrus, cerebellar cortex and spinal cord) were determined by fluorescence cytophotometry. the specimens were obtained from twelve adult human autopsy cases. Glial cell nuclei always revealed a biomodal DNA distribution pattern with a large 2c and a smaller 4c peak. the 4c peak was most prominent in the cerebellum. A few 8c glial nuclei were found. Neuronal cell nuclei disclosed unimodal DNA histograms with hyperdiploid means in the range 2.2–2.5c (1.8–2.9c for the individual populations). Tetraploid 4c DNA values were not observed, neither in Purkinje cells, nor in pyramidal cells. In eleven out of a total of forty-four slides the higher DNA means of neuronal nuclei were found to be statistically significant (P < 0.05) when compared with a population of 2c hepatocytes on the same slide. The results indicate the existence of some ‘extra DNA’ in human neuronal cell nuclei, the biological significance of which has still to be elucidated. It is however, suggested that it may play an important role in the functional activity of the CNS.     

Cell cycle analysis and DNA aneuploidy in autoimmune mice homozygous for the lpr and gld mutations.

Homozygosity for either of the mutations lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) in mice results in lymphoproliferation and autoimmune disease. To investigate the site and time of excessive lymphocyte proliferation in these mice, cell nuclei of normal and mutant mice of various ages were stained with propidium iodide and DNA profiles were analyzed by flow cytometry. Two major results were obtained. First, DNA aneuploidy was observed in the lymph nodes and spleen of these autoimmune mice and the cells involved in DNA aneuploidy were predominantly of a CD4-CD8- Thy-1- surface phenotype. Second, although DNA aneuploidy became apparent in mutant mice at 2 mo of age, the numbers of cycling cells were only minimally increased over control levels at all ages tested. Thus, the massive cellular accumulation in the lymph nodes of lpr and gld mice does not seem ascribable solely to excess cell proliferation in these tissues. Moreover, a previously unrecognized cell compartment (CD4-CD8-Thy-1-) characterized by apparent DNA aneuploidy appears in the same tissues and at the same times that the predominant "double negative" (CD4-CD8-Thy-1+) T cell subset accumulates.     

Destruction of pancreatic islet cells by cytotoxic T lymphocytes in nonobese diabetic mice

Proliferation of islet-associated leukocytes occurred when isolated islets from 20-wk-old female nonobese diabetic (NOD) mice were cultured with 10 U/ml rIL-2 for 7 days. Co-culture of these leukocytes with freshly isolated islets from 6- to 8-wk-old NOD donors in the presence of 1 U/ml rIL-2 produced islet structural deformation within 24 h and islet cytolysis within 48 h. Three lines of evidence suggest that these leukocytes were composed mainly of CTL specific for islet cells. First, morphologically, these proliferating cells adhered to NOD islets at 6 h and killed islets within 48 h of culture, but these phenomena could not be observed in the other tissues from NOD mice. These islet-derived cells were cytotoxic to NOD islet cells in a 51Cr-release assay, whereas no appreciable cytotoxicity was observed when NOD Con A-induced splenic blasts or fibroblasts were used as targets. Second, a flow cytometric analysis showed that these cells consisted of 97% Thy-1.2, 69% Lyt-2, 8% L3T4, and 4% asialo-GM1-positive cells, whereas Mac-1-positive cells could not be seen in these assays. After treatment with anti-Thy-1.2 or Lyt-2 mAb and C, these cells lost their activity to lyse NOD islet cells. However, these cells still had a full killing activity after the depletion of L3T4 or asialo GM1-positive cells. Third, islet cells from BALB/c, DBA/2, and B10.GD mice which share the same H-2K Ag with NOD mice were susceptible to cytolytic activity of these cells, whereas islet cells from NON, C57BL/6, C57BL/10, and C3H mice remained intact. Furthermore, anti-Kd antibody was capable of blocking this cytolysis. These results suggest that CTL expressing Thy-1.2 and Lyt-2 phenotypes appear to recognize the islet cell Ag with the restriction of MHC class I Kd, and then destroy NOD islet cells.     

Development of binuclearity and DNA-polyploidization in the growing mouse liver

Summary Suspensions of intact liver cells were prepared from 36 male NMRI mice of different age after perfusion of the liver with ice-cold calcium- and magnesium-free phosphate buffer (CMF). The suspensions of the isolated hepatocytes were smeared on slides, fixed, hydrolized and stained by fluorescent acriflavine-Schiff-Feulgen reaction. The number of nuclei per cell was determined in a phase-contrast microscope. Quantitative fluorescent cytophotometric measurements of nuclear Feulgen-DNA were performed on individual nuclei. At the age of 0.5 month, 55% of the hepatocytes were found to be mononuclear, 45% binuclear. In the animal groups aged 1 month, 1.5 months, 3 months, 6 months and 12 months, the percentage of binuclear hepatocytes remained constant at about 80%. Very few liver cells with 3 or 4 nuclei were detected. Feulgen-DNA-measurements revealed a predominance of 2c and 4c nuclei at ages 1 month and 1.5 months with logarithmic increase of 8c nuclei and a decrease of the 2c nuclei. From 1.5 months on 16c nuclei were found, which never exceeded 8%. When total DNA-ploidy of the hepatocytes was calculated similar kinetics at a higher ploidy level were observed. 2c hepatocytes existed in small percentages at very young ages up to 1.5 months, but were also occasionally found in older animals. With increasing age the number of 16c hepatocytes increased logarithmically with a concomitant decrease of the 4c hepatocytes. The percentage of 8c liver cells remained more or less constant. Few hepatocytes with a 32c total DNA content were found in mice aged 3 months and older. In one-year-old mice the mean DNA-ploidy was calculated to be 5.8c per liver nucleus and 10.0c per whole hepatocyte.Supported by Deutsche Forschungsgemeinschaft, Grant No Bo 395/5     

Magnetic Resonance Imaging of Mouse Islet Grafts Labeled with Novel Chitosan-Coated Superparamagnetic Iron Oxide Nanoparticles

Object

To better understand the fate of islet isografts and allografts, we utilized a magnetic resonance (MR) imaging technique to monitor mouse islets labeled with a novel MR contrast agent, chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles.

Materials and Methods

After being incubated with and without CSPIO (10 µg/ml), C57BL/6 mouse islets were examined under transmission electron microscope (TEM) and their insulin secretion was measured. Cytotoxicity was examined in α (αTC1) and β (NIT-1 and βTC) cell lines as well as islets. C57BL/6 mice were used as donors and inbred C57BL/6 and Balb/c mice were used as recipients of islet transplantation. Three hundred islets were transplanted under the left kidney capsule of each mouse and then MR was performed in the recipients periodically. At the end of study, the islet graft was removed for histology and TEM studies.

Results

After incubation of mouse islets with CSPIO (10 µg/mL), TEM showed CSPIO in endocytotic vesicles of α- and β-cells at 8 h. Incubation with CSPIO did not affect insulin secretion from islets and death rates of αTC1, NIT-1 and βTC cell lines as well as islets. After syngeneic and allogeneic transplantation, grafts of CSPIO-labeled islets were visualized on MR scans as persistent hypointense areas. At 8 weeks after syngeneic transplantation and 31 days after allogeneic transplantation, histology of CSPIO-labeled islet grafts showed colocalized insulin and iron staining in the same areas but the size of allografts decreased with time. TEM with elementary iron mapping demonstrated CSPIO distributed in the cytoplasm of islet cells, which maintained intact ultrastructure.

Conclusion

Our results indicate that after syngeneic and allogeneic transplantation, islets labeled with CSPIO nanoparticles can be effectively and safely imaged by MR.     

用rAAV8-1.3HBV制备两种品系小鼠乙型肝炎病毒感染模型的比较研究

我们先前用rAAV8-1.3HBV静脉注射C57BL/6小鼠成功地制备了慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染模型。为了探讨不同品系的小鼠对rAAV8-1.3HBV静脉注射是否具有不同反应,本研究比较了C57BL/6和BALB/c小鼠静脉注射重组病毒后外周血中HBV抗原和抗体水平、病毒载量和肝脏组织HBcAg表达情况,以及不同剂量重组病毒注射与这些指标的关系。将低(4×109 Viral genome,vg)、中(4×1010vg)和高(4×1011vg)三种剂量的rAAV8-1.3HBV通过尾静脉注射至C57BL/6和BALB/c小鼠,分别利用ELISA和荧光定量PCR方法检测血清中的HBV抗原、抗体水平以及HBV DNA,利用免疫组化检测肝脏组织HBcAg的表达。结果发现,对于C57BL/6小鼠,三种不同剂量rAAV8-1.3HBV注射均可造成100%小鼠出现HBV持续感染;血清HBsAg、HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,其表达水平随重组病毒注射剂量的增加而升高,高剂量注射时可造成超过40%的肝细胞感染HBV,血清中HBV DNA可达105 IU/mL以上;未检测到针对HBV的抗体。对于BALB/c小鼠,三种不同剂量rAAV8-1.3HBV注射也可造成100%小鼠出现HBV持续感染;血清HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,但是血清HBsAg在重组病毒注射2周之后显著下降甚至消失;在中剂量注射组的BALB/c小鼠血清中检测到低水平的Anti-HBs;血清HBeAg和肝组织HBcAg的表达水平随重组病毒注射剂量的增加而增高,并且各剂量组表达水平均高于C57BL/6小鼠,高剂量注射时可造成超过50%的肝细胞感染HBV。本研究表明,低至4×109 vg剂量的rAAV8-1.3HBV注射即可造成C57BL/6和BALB/c两种品系小鼠出现HBV持续感染,并且HBV复制水平随重组病毒注射剂量增加而增高;BALB/c小鼠对HBV的免疫反应强于C57BL/6小鼠,可以产生针对HBsAg的体液免疫反应而使血清HBsAg转阴,但无法清除携带HBV的肝细胞。     

Influence of section thickness, mean nuclear diameter and nuclear crowding on DNA ploidy in histologic sections of melanocytic skin lesions

OBJECTIVE: To determine the influence of section thickness, nuclear diameter (MND) and area percentage of nuclei (a measure of nuclear crowding) on histologic DNA ploidy assessed by image cytometry (ICM) of primary melanocytic skin neoplasms (MSNs). STUDY DESIGN: Initially a feasibility study was performed to determine if comparable DNA ploidy histograms could be obtained from cell disaggregates and tissue sections. Following this, DNA ICM was performed on Feulgen-stained tissue sections (4, 6, 8 and 10 microns thick) from 30 primary MSNs (20 benign, 10 malignant) with nuclear diameters from 5.6 to 8.6 microns. Area percentage of nuclei was assessed in all cases at all section thicknesses. RESULTS: The feasibility study produced comparable results for cytocentrifuge and tissue section preparations. For sectioned MSNs, DNA ploidy histograms from 4-micron sections had a higher coefficient of variation of the 2c peak than those from 6-, 8- and 10-micron sections. Ten-micrometer sections had marked overlapping of nuclei, and only small numbers of cells could be measured, giving inadequate results. MND and area percentage of nuclei did not have an important influence on the results. CONCLUSION: Adequate DNA ploidy profiles can be obtained by DNA ICM on 6- and 8-micron-thick histologic sections of MSNs, provided that a strict measurement protocol is followed.     

Specific nuclear elimination in polyploid plasmodia of the slime mold Physarum polycephalum

In growing plasmodia of the myxomycete Physarum polycephalum (G2-phase), three distinct classes of nuclei with a relative DNA content of 1x, 2x, and 4x are observed in the presumed haploid strain CL. The 2x and 4x species comprise up to 35% and 5% of the nuclei. Quantitative cytofluorometric studies of nuclei isolated in either G2- or S-phase or after FUDR treatment (G1 arrest) show that the three nuclear populations undergo a synchronous mitotic cycle and that the relative DNA content of the nuclear fractions in G-2 phase reflects the 2c, 4c, and 8c state. The heterogeneity of the nuclear population does, however, seem to be restricted to the growth phase. During a starvation period of 4 days that always preceeds sporulation (and also meiosis), the 4c nuclear population is reduced to 7%, 8c nuclei are no longer detected. These results suggest that a mechanism exists in Physarum for the selective detection and elimination of polyploid nuclei.     

Morphological aspects on pancreatic islets of non-obese diabetic (NOD) mice

The pancreatic islets of female non-obese diabetic (NOD) mice (a model of insulin-dependent diabetes mellitus), have been examined by both light and electron microscopy. At about the age of 2 weeks, mononuclear cells began to infiltrate in or near the islets and some of these cells were in contact with the islet cells. Following this degeneration of islet B-cells took place, the process occurring in two ways. In many cells numerous secretory granules with extremely dense cores occupied the cytoplasm. Other cells, however, were filled with low-density secretory granules and the nuclei of these cells became pycnotic. After degeneration of B-cells, the islets were effaced by numerous mononuclear cells. With the onset of the diabetic state these mononuclear cells gradually disappeared, and thereafter small islets remained. By electron microscopy, retrovirus-like particles were observed in cisternae of the rough endoplasmic reticulum in islet B-cells at all stages. With an anti-retrovirus serum (goat anti-KiMSV-NIHxeno serum), positive immunofluorescence was observed in some pancreatic islet cells of NOD mice aged 1 day and 4, 6, 8, 9, 10 and 14 weeks. It is suggested that these virus particles may be intimately related to the inflammatory reaction occurring in the islets and to the development of diabetes mellitus.     

Effect of sonication on paraffin-embedded tissue preparation for DNA flow cytometry

Since the publication of paraffin block extraction procedures, flow cytometric analysis of DNA ploidy and S-phase of tumor specimens has been widely applied. DNA aneuploidy, DNA tetraploid (elevated G2/M), and elevated S-phase are clinically significant in some tumor systems. True DNA tetraploid cell lines will contain a large 4c population and perhaps an 8c population; samples with cell aggregates will also contain a 6c population. Microscopic examination of samples having a 6c peak revealed nuclei with adhering debris and doublets, triplets, and larger nuclear aggregates. After sonication, a uniform suspension of single nuclei without adherent debris was seen. In addition to reducing the percent of G2/M cells, sonication also reduced S-phase percent such that it was closer to the bromodeoxyuridine labeling index. The DNA ploidy classification of specimens was also compared pre- and post-sonication. Four of 96 breast cancer samples changed classification; all were specimens in which the histogram became cleaner and a small DNA aneuploid peak became apparent after sonication.     

重组恶性疟原虫DNA质粒免疫小鼠制备单克隆抗体

用恶性疟原虫MSP131基因片段的重组质粒DNA直接免疫BALB/c小鼠,诱导产生体液,免疫后取脾细胞与SP2/0小鼠骨髓瘤细胞在PEG1450作用下进行融合,获得了2株能分泌抗恶性疟原虫MSP131单克隆抗体的小鼠杂交瘤细胞株9H9和8A2。用酶联免疫吸附试验检测,小鼠腹水抗体滴度最高为1∶10 000。经免疫球蛋白类型和亚类鉴定,2株杂交瘤细胞株均为IgG\-1\.蛋白免疫印迹试验表明,此单克隆抗体与MSP1\|31蛋白抗原有特异免疫反应,证明通过质粒DNA直接免疫小鼠可制备特异性单克隆抗体。     

Effects of precryopreservation culture on survival of rat islets transplanted after slow cooling and rapid thawing

Despite the frequent use of in vitro tissue culture before islet cryopreservation, no study has evaluated the ability of this procedure to improve the recovery or in vivo function of frozen-thawed islets. To evaluate this, quantities of 2500 Wistar-Furth (WF) rat islets were allocated to each of four groups (n = 8 each): group 1, freshly isolated; group 2, 48 hr in vitro culture; group 3, cryopreservation; group 4, cryopreservation after 48 hr in culture. Islets were frozen slowly at 0.25 degrees C/min and thawed rapidly at 200 degrees C/min. The number of islets recovered after culture or cryopreservation was determined and viability was assessed by measuring weekly indices of plasma glucose (PG), urine glucose (UG), urine volume (UV), and weight after implantation into the portal vein of streptozotocin-diabetic WF recipients. Islet recovery was 97% after culture, 95% after cryopreservation, and 94% after culture-then cryopreservation. After implantation of group 1 and 2 islets, PG was less than 150 mg/dl at 1 week and UG and UV were normal by 1-2 weeks. Group 3 islets restored normoglycemia at 3 weeks and other indices of diabetes were reversed by 4 weeks; group 4 islets restored normoglycemia at 4 weeks and indices returned to basal after 4 weeks. At intravenous glucose tolerance testing (ivGTT), the K values (mean decline in glucose, %/min, +/- SE) were group 1, 1.6 +/- 0.3; group 2, 1.5 +/- 0.3; group 3, 0.6 +/- 0.1; and group 4, 0.7 +/- 0.2. These data show that cryopreservation preserves freshly isolated or cultured islets that reverse the indices of diabetes after implantation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation of monoclonal antibodies to nuclear DNA of mammalian cells by immunization with group A streptococcal polysaccharide conjugated with synthetic polyelectrolytes

Monoclonal antibodies (MAb) were obtained by hybridization of spleen cells from BALB/c mice immunized with streptococcal group A polysaccharide (A-PS) conjugated with synthetic polyelectrolytes (PEL). These MAb reacted with nuclei from human and mouse cells. MAb reacting with nuclei were obtained after prolonged immunization with conjugates and were not formed by hybridization of spleen cells from non-immunized mice or by the immunization with PEL. The investigation of Mab (B1/2 and A5/2) reacting with nuclei has shown that these Mab are directed against DNA and do not react with other tissue substances. No cross-reactions of Mab with A-PS used for immunization have been revealed. Mab B1/2 and A5/2 belong to autoantibodies.     

Regeneration of the liver of Chinese hamster Cricetulus griseus

Using cytofluorimetry and interferometry, hepatocyte DNA, dry mass and distribution of hepatocyte ploidy classes were measured in hamsters Cricetulus griseus in 1 month after partial hepatoctomy. Ploidy of normal liver hepatocyte was 2.35 +/- 0.03 (mean +/- SD) c. Modal ploidy class was presented by mononuclear hepatocytes with diploid nuclei (82.4 +/- 1.3 %). Hepatocyte dry mass was 605.2 +/- 4.8 pg. One month after partial hepatectomy the distribution of ploidy classes and dry mass of hepatocyte did not change. A similar hepatectomy in mice resulted in significant polyploidization of liver parenchyma: the middle level of hepatocyte ploidy increased by 32% and mononuclear octaploid cells, the number of which increased 5-fold, composed modal ploidy class in place of 4cx2-hepatocytes predominated in control mice. The number of 8cx2-hepatocytes in the liver of mice creased by more than 5-fold. Thus, in contrast with mice, in hamsters Cricetulus griseus an increase in the liver mass followed partial hepatectomy depended completely on hepatocyte proliferation.     

Immunohistochemical localization of tonin in rat salivary glands and kidney

Summary As a part of a microfluorometric investigation of the nucleoproteins of nuclei whose chromatin displays varying degrees of condensation, a comparison was made of mouse small thymocyte and hepatocyte nuclei stained with the acidic dye, brilliant sulfaflavine, at pH 2.8. These estimates of total protein content were compared with measurements obtained in similarly stained nuclei after extraction either with 0.4 N H₂SO₄ to remove all histones or with 0.35 M NaCl to remove nucleoplasmic proteins and some loosely bound non-histone chromosomal proteins. Treatment with 5% TCA at 60°C was used to remove nucleic acids and to reverse the effects of formaldehyde fixation. In all instances, the fluorescence of 2c hepatocyte nuclei greatly exceeded that of similarly treated thymocyte nuclei. While extraction with 0.4 N H₂SO₄ resulted in reductions of as much as 75% of the total fluorescence of small thymocyte nuclei, the losses of fluorescence in 2c hepatocyte nuclei amounted to only 20–30%. Nevertheless, the absolute values of fluorescence lost in both types of nuclei were very similar. After extraction in 0.35 M NaCl, thymocyte nuclei displayed slightly greater fluorescence than control thymocyte nuclei, while the total fluorescence of hepatocyte nuclei declined. In hepatocyte nuclei extracted with TCA, with and without treatment with 0.35 M NaCl, two populations of diploid nuclei were apparent: one corresponding to parenchymal cell nuclei and the other comprised of non-parenchymal cell nuclei. Only single diploid populations were visible in acid-extracted material. The ratios of 4c2c, 8c4c, and 8c2c hepatocyte nuclei in control, acid-extracted, and NaCl-extracted groups were generally lower than the expected 224 values. These results indicate that total nuclear histones may be estimated microfluorometrically by computing the difference between acid-extracted and unextracted preparations treated in otherwise equivalent ways. In addition, despite very similar absolute losses of fluorescence after removal of histones in thymocyte and 2c hepatocyte nuclei, the proportion of total protein ascribable to histones is much greater in thymocyte nuclei than in 2c hepatocyte nuclei — or, conversely, the percentage of total protein attributable to non-histone proteins is much greater in 2c hepatocyte nuclei than in thymocyte nuclei.     

质粒DNA单次脾内注射制备单克隆抗体

探索一种新的快捷有效DNA免疫制备单克隆抗体的方法,辅助实现构建高通量无蛋白纯化体系单克隆抗体制备和筛选。分别通过“重叠PCR”和“无模板PCR”在pVAX1真核载体中分别引入IL-2信号肽、IgG kappa链信号肽构建分泌型真核表达载体,将代表抗原基因的profilin1基因克隆到经改造带有信号肽基因的表达载体上,构建重组质粒pVAX-IL2-prof1和pVAX-Igκ-prof1,单次脾内注射重组质粒DNA免疫BALB/c小鼠。经过细胞融合、ELISA筛选,获得两株抗profilin1的单克隆抗体。单抗亚型分别为IgM和IgG3。单次脾内质粒DNA免疫便捷有效,是制备单克隆抗体的有效方法。     

Polyamines in pancreatic islets of obese-hyperglycemic (ob/ob) mice of different ages

To further evaluatethe role of polyamines in insulin production and cell replication indiabetic pancreatic islets, we have studied hyperplastic islets ofobese-hyperglycemic mice of different ages and normal islets of thesame strain. The aims of the study were to investigate the impact ofthe diabetic state and aging on polyamine contents and requirements inthese islets. Cultured islets from lean and obese animals containedsignificantly less polyamines than freshly isolated islets.Spermine-to-spermidine ratio was elevated in freshly isolated isletsfrom young obese mice compared with those from lean mice. In isletsfrom old obese animals, spermidine content was decreased, whereas thecontent of spermine was not different from that of young obese mice.The physiological significance of polyamines was investigated byexposing islets in tissue culture to inhibitors of polyamine synthesis. This treatment caused a partial polyamine depletion in whole islets butfailed to affect polyamine content of cell nuclei. Insulin content wasnot affected in polyamine-deficient islets of obese mice, irrespectiveof age, in contrast to decreased islet insulin content inpolyamine-depleted young lean animals. Polyamine depletion depressedDNA synthesis rate in obese mouse islets; in lean mice it actuallystimulated DNA synthesis. We concluded that important qualitative andquantitative differences exist between islets from obese-hyperglycemicand normal mice with respect to polyamine content and requirements ofpolyamines for regulation of insulin content and cell proliferation.The results suggest that spermine may be involved in mediating therapid islet cell proliferation noted early in obese-hyperglycemicsyndrome, but changes in spermine concentration do not seem to accountfor the decline in islet cell DNA synthesis in aged normoglycemic animals.

     

The effect of starvation on phosphodiesterase activity and the content of adenosine 3′:5′-cyclic monophosphate in isolated mouse pancreatic islets

1. The concentration of cyclic AMP and the activity of phosphodiesterase were measured in isolated pancreatic islets from fed or 48h-starved mice. 2. Two different phosphodiesterases were detected. Neither the maximum activity nor the K(m) values of these enzymes were changed by starvation. 3. The concentration of cyclic AMP in non-incubated islets was the same in islets from fed and starved mice. 4. Incubation with 3.3mm-glucose for 5-30min had no effect on the concentration of cyclic AMP, irrespective of the nutritional state of the mice. Incubation with 16.7mm-glucose for 5-30min raised the concentration of cyclic AMP by about 30% in islets from fed mice. This rise was prevented by addition of mannoheptulose (3mg/ml). Incubation with 16.7mm-glucose had no effect on the cyclic AMP content in islets from starved mice. 5. In islets from fed mice 10min incubation with 5mm-caffeine had no effect on the concentration of cyclic AMP in the presence of 3.3 or 16.7mm-glucose, whereas the cyclic AMP content was increased approx. 150% in islets from starved mice. 6. After 10min incubation with 1mm-3-isobutyl-1-methylxanthine in the presence of 3.3 or 16.7mm-glucose the concentration of cyclic AMP was raised by 250% in islets from fed mice and by 400% in islets from starved mice. 7. A threefold function of glucose in the insulin-secretory process is suggested, according to which the decreased islet glucose metabolism is the primary defect in the insulin-secretory mechanism during starvation.     

Chronic Resveratrol Treatment Protects Pancreatic Islets against Oxidative Stress in db/db Mice

Resveratrol (RSV) has anti-inflammatory and anti-oxidant actions which may contribute to its cardiovascular protective effects. We examined whether RSV has any beneficial effects on pancreatic islets in db/db mice, an animal model of type 2 diabetes. The db/db and db/dm mice (non-diabetic control) were treated with (db-RSV) or without RSV (db-control) (20 mg/kg daily) for 12 weeks. After performing an intraperitoneal glucose tolerance test and insulin tolerance test, mice were sacrificed, the pancreas was weighed, pancreatic β-cell mass was quantified by point count method, and the amount of islet fibrosis was determined. 8-Hydroxydeoxyguanosine (8-OHdG), an oxidative stress marker, was determined in 24 h urine and pancreatic islets. RSV treatment significantly improved glucose tolerance at 2 hrs in db/db mice (P = 0.036), but not in db/dm mice (P = 0.623). This was associated with a significant increase in both pancreas weight (P = 0.011) and β-cell mass (P = 0.016). Islet fibrosis was much less in RSV-treated mice (P = 0.048). RSV treatment also decreased urinary 8-OHdG levels (P = 0.03) and the percentage of islet nuclei that were positive for 8-OHdG immunostaining (P = 0.019). We conclude that RSV treatment improves glucose tolerance, attenuates β-cell loss, and reduces oxidative stress in type 2 diabetes. These findings suggest that RSV may have a therapeutic implication in the prevention and management of diabetes.     

Toward testing the hypothesis that group B coxsackieviruses (CVB) trigger insulin-dependent diabetes: inoculating nonobese diabetic mice with CVB markedly lowers diabetes incidence

Insulin-dependent (type 1) diabetes mellitus (T1D) onset is mediated by individual human genetics as well as undefined environmental influences such as viral infections. The group B coxsackieviruses (CVB) are commonly named as putative T1D-inducing agents. We studied CVB replication in nonobese diabetic (NOD) mice to assess how infection by diverse CVB strains affected T1D incidence in a model of human T1D. Inoculation of 4- or 8-week-old NOD mice with any of nine different CVB strains significantly reduced the incidence of T1D by 2- to 10-fold over a 10-month period relative to T1D incidences in mock-infected control mice. Greater protection was conferred by more-pathogenic CVB strains relative to less-virulent or avirulent strains. Two CVB3 strains were employed to further explore the relationship of CVB virulence phenotypes to T1D onset and incidence: a pathogenic strain (CVB3/M) and a nonvirulent strain (CVB3/GA). CVB3/M replicated to four- to fivefold-higher titers than CVB3/GA in the pancreas and induced widespread pancreatitis, whereas CVB3/GA induced no pancreatitis. Apoptotic nuclei were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay in CVB3/M-infected pancreata but not in CVB3/GA-infected pancreata. In situ hybridization detected CVB3 RNA in acinar tissue but not in pancreatic islets. Although islets demonstrated inflammatory infiltrates in CVB3-protected mice, insulin remained detectable by immunohistochemistry in these islets but not in those from diabetic mice. Enzyme-linked immunosorbent assay-based examination of murine sera for immunoglobulin G1 (IgG1) and IgG2a immunoreactivity against diabetic autoantigens insulin and HSP60 revealed no statistically significant relationship between CVB3-protected mice or diabetic mice and specific autoimmunity. However, when pooled sera from CVB3/M-protected mice were used to probe a Western blot of pancreatic proteins, numerous proteins were detected, whereas only one band was detected by sera from CVB3/GA-protected mice. No proteins were detected by sera from diabetic or normal mice. Cumulatively, these data do not support the hypothesis that CVB are causative agents of T1D. To the contrary, CVB infections provide significant protection from T1D onset in NOD mice. Possible mechanisms by which this virus-induced protection may occur are discussed.