Cell Division Index Research Articles

Effect of sewage wastewater on the mitotic index of the root tips of Allium cepa in koya city /Kurdistan region of Iraq

The purpose of the experiment was to investigate onion (Allium cepa) root growth, cell division, and mitotic index. The onions were placed into four different water samples (Control water, Azadi bridge, Sar Razan, and Sar Baskan) for 72 hours in beakers. The results showed that the number of divided cells decreased significantly in the sewage water compared with the control water. The control water had a high percentage of divided cells (18%), whereas the Sar Razan sewage water had the least percentage of divided cells (1.3%). The percentage of divided cells in Sar Baskan sewage water was 2%, and the percentage of divided cells in Azadi Bridge was 2.6%. It was found that the mitotic index and the number of divided cells increased in the control water compared to the sewage water. The results showed that sewage water has a greater impact on root growth, cell division, and mitotic index of root tips of Allium cepa.

Cytological assessments and a methodological approach to observe the apoptotic effect of nickel sulfate (NiSO4) on Allium cepa L. root germination using EB/AO fluorescence staining

Ethidium bromide–acridine orange (EB/AO) is one of the fastest, most economically valid methods for separating living and dead cells in plant root tips. The present study aimed to investigate the apoptotic effect, nuclear abnormalities and cell division index using an Allium cepa test assay with EB/AO staining and an ImageJ program. Nickel sulphate (NiSO4) concentrations (1.75, 3.5, 7 and 14 ppm) were exposed to the root germination of A. cepa for 48 and 72 h to observe mitotic abnormalities, cytotoxic effects and 5-day exposure to apoptotic effects. It was found that amorphous nuclei, vacuolisation and C-mitosis were the most observed abnormalities, which increased at 14 ppm NiSO4 exposure after 48 and 72 h. The total number of nuclear abnormalities significantly increased at all concentrations and exposure periods. The ImageJ program was used to determine apoptosis rates. The data obtained showed that high concentrations of NiSO4 caused significantly more cell death in root tips than in the control group, resulting in root growth inhibition and increased apoptosis with increased concentration and exposure period. NiSO4 caused toxic activity on root growth, identified as apoptosis, especially at the cortex and vascular region in the root tips. The study found affected damaged and apoptotic areas on the root tips using an EB/AO staining method, which can be used as a marker of damaged tissue areas.

Clinical Characteristics and Local Histopathological Modulators of Endometriosis and Its Progression.

This retrospective study included 243 patients from whom tissue with E/A or normal control uterine tissue was harvested and stained by histochemical and classical immunohistochemical staining. We assessed the symptomatology of the patients, the structure of the ectopic epithelium and the presence of neovascularization, hormone receptors, inflammatory cells and oncoproteins involved in lesion development. Atypical areas were analyzed using multiple immunolabeling techniques. The cytokeratin (CK) CK7+/CK20- expression profile was present in E foci and differentiated them from digestive metastases. The neovascularization marker cluster of differentiation (CD) 34+ was increased, especially in areas with malignant transformation of E or A foci. T:CD3+ lymphocytes, B:CD20+ lymphocytes, CD68+ macrophages and tryptase+ mast cells were abundant, especially in cases associated with malignant transformation, being markers of the proinflammatory microenvironment. In addition, we found a significantly increased cell division index (Ki67+), with transformation and inactivation of tumor suppressor genes p53, B-cell lymphoma 2 (BCL-2) and Phosphatase and tensin homolog (PTEN) in areas with E/A-transformed malignancy. Proinflammatory/vascular/hormonal changes trigger E/A progression and the onset of cellular atypia and malignant transformation, exacerbating symptoms, especially local pain and vaginal bleeding. These triggers may represent future therapeutic targets.

Sorafenib regulates vascular endothelial growth factor by runt-related transcription factor-3 to inhibit angiogenesis in hepatocellular carcinoma

Objective: To investigate the molecular mechanism of sorafenib against hepatocellular carcinoma. Methods: Sorafenib efficacy was screened and verified by the hepatocellular carcinoma patient-derived tumor xenograft (PDX) model. Veterinary B-mode ultrasonography and in vivo confocal laser scanning microscopy were used to observe PDX angiogenesis. Immunohistochemistry was used to observe the expression of proliferation and angiogenesis-related proteins in PDX tissue. Real-time quantitative PCR technology was used to observe the RUNX3 gene in PDX tissues. SPSS 17.0 statistical software was used for statistical analysis. Results: Four cases of PDX were used to screen the efficacy of sorafenib. PDX1 had a significant response to sorafenib, with an inhibition rate of 68.07%. Compared with the control group, sorafenib had significantly inhibited PDX1 relative tumor volume (5.76±2.14 vs. 11.71±2.87, P<0.05). Cell division index (39.50±7.72 vs. 67.10±9.14, P<0.05) and Ki67 expression (288.6±43.40 vs. 531.70±55.60, P<0.05) were significantly decreased. Veterinary B-mode ultrasonography showed evident blood flow signals in PDX1 tumors. In vivo confocal laser scanning microscopy results showed that sorafenib had significantly reduced the total vessel length (1573.00±236.21 vs. 2675.03±162.00, P<0.05) and area (11 145.33±1931.97 vs. 20 105.37±885.93, P<0.05)) of PDX1 tumors. Immunohistochemical results showed that sorafenib had significantly down-regulated the protein expressions of CD34 (27.55±3.76 vs. 45.47±5.57, P<0.05), VEGF (16.33±2.86 vs. 22.77±3.20, P<0.05) and MVD (38.75±6.01 vs. 55.50±8.61, P<0.05). Real-time PCR results showed that sorafenib had significantly up-regulated RUNX3 gene expression (2.14±0.71 vs. 1.00±0.36, P<0.05). However, there was a negative correlation between the expression of RUNX3 gene and the ratio of VEGF-positive cells in sorafenib group (R2=0.509 7). Conclusion: Sorafenib may inhibit the PDX angiogenesis and the growth of hepatocellular carcinoma by regulating the RUNX3-VEGF pathway.

FEATURES OF SPIRULINA PLATENSIS CULTIVATED UNDER AUTOTROPHIC AND MIXOTROPHIC CONDITIONS

Cultivation of Spirulina platensis in Zarrouk media containing 0–20 g l-1 glucose was studied in a photobioreactor for 30 days using a light intensity of 3 klux. Various parameters were measured to evaluate the enhancement of cell performance with glucose such as cell number, osmolarity, membrane stability, biomass productivity, doubling time, stress intensity, stress tolerance, chlorophyll, protein, carbohydrates, and lipid contents. Based on the results, we concluded that S. platensis is able to grow and produce some ingredients in Zarrouk media containing up to 20 g l-1 of glucose which is the first to be reported. The cell concentration of the mixotrophic cultures (80 cells per mm2) corresponded well to the sum of the autotrophic cell concentrations (50 cells per mm2), showing that the addition of carbohydrate positively effects on the microalgae growth. The continuous operation supplemented with 0.5 g l-1 of glucose (G0.5) led to the maximum cell concentration about 9.06 g l-1 wet and 1.32 g l-1 dry weights. The highest tolerance index, specific growth rate, biomass productivity, cell division, osmolarity and membrane stability index were respectively 102.5%, 0.15 d-1, 0.04 g l-1d-1, 0.26 div d-1, 0.87 osmol kg-1 and 93.8%, obtained in the same treatment. Chlorophyll (6.7 % in G0; 0.046 g l-1 in G0.5), protein (79.9 % and 0.884 g l-1 in G0.5), carbohydrates (55.5% in G20; 0.492 g l-1 in G6) and lipid (53.3% in G10; 0.636 g l-1 in G0) percentages and yields were mostly enhanced in the mixotrophic condition. This study indicated that mixotrophic growth of S. platensis is useful for commercial biomass production.

RESULTS OF INVESTIGATION OF THE EFFECTS OF BOVINE PLACENTAL PREPARATION ON IMMUNE FUNCTION

The present study aimed to investigate the effects of bovine placental preparation under in vitro and in vivo conditions. Cell Proliferation Kit I (MTT) was used for in vitro study of placental preparation effect to proliferate lymphocytes. Lymphocytes were isolated from spleen of Balb C mice, 100 μl cell was added to each well of 96 well culture plate, followed by addition of 10 μl placental preparation and mitogen (concanavalin-A) separately and in combination and cell culture only as control was used. Results were obtained by measuring and comparing the absorbance reading of the wells of the samples against the standards with ELISA microplate reader. Effect of placental preparation to proliferate lymphocytes in vivo condition was investigated in mice, which were divided into 4 groups and 4 subgroups. The results were estimtaed with spleen weight, spleen index and splenocyte counts. Results of in vitro study demonstrated that stimulation index increased by 1.19 or 19% for cell division in wells to which no mitogen was added, but the preparation was added as compared to control wells, cell division index increased with 1.38 or 38% for cell division in wells to which mitogen was added as compared to control wells and stimulation index was higher by 1.68 or 68% for cell division in wells with cells to which both mitogen and preparation were added than control wells. For in vivo experiments, spleen index and splenocyte count for animals of positive control subgroup-1 treated once by 0.2 ml sheep red blood cells were greater by 1.38 and 1.5 times respectively than relevant negative control animals, whereas spleen index and splenocyte count for animals of experimental subgroup -1 were greater by 3.09 and 2.2 times respectively. For animals of positive control subgroup -2, both spleen index and splenocyte count decreased by 1.35 and 1.3 times respectively than negative control animals, whereas they dropped by 1.1 and 1.17 times respectively in mice of experimental subgroup -2. Spleen index and splenocyte count in mice treated with the preprartion only increased by 1.2 times or 20% as compared to negative control animals. From above results, it is shown that bovine placental preparation is able to exert immunomodulatory effect regardless of antigen under both in vitro and in vivo conditions.

Nanodiamond-chymotrypsin and nanodiamond-papain conjugates, their synthesis and activity and visualization of their interaction with cells using optical and electron microscopy.

Two novel conjugates of detonation nanodiamonds (dNDs) with the proteolytic enzymes chymotrypsin and papain were synthesized. The synthesis was performed via functionalization of the dNDs' surface with acidic/alkali treatment followed by carbodiimide-mediated protein binding. Covalent binding of the enzymes was confirmed by Fourier transform infrared spectrographic analysis and high-performance liquid chromatography (HPLC) amino acid analysis. HPLC also proved the preservation of the enzymes' composition during synthesis. The same assay was used to determine the binding ratios. The ratios were 12% (mass to mass) for chymotrypsin and 7.4% for papain. The enzymatic activity of the conjugates was measured using chromogenic substrates and appeared to be approximately 40% of that of the native enzymes. The optimum pH values and stability under various conditions were determined. The sizes of resulting particles were measured using dynamic light scattering and direct electron microscopic observation. The enzyme conjugates were shown to be prone to aggregation, resulting in micrometer-sized particles. The ζ-potentials were measured and found to be positive for the conjugates. The conjugated enzymes were tested for biological activity using an in vitro model of cultured transformed human epithelial cells (HeLa cell line). It was shown that dND-conjugated enzymes effectively bind to the surface of the cells and that enzymes attack exposed proteins on the plasma membrane, including cell adhesion molecules. Incubation with conjugated enzymes results in morphological changes of the cells but does not affect cell viability, as judged by monitoring the cell division index and conducting ultrastructural studies. dNDs are internalized by the cells via endocytosis, being enclosed in forming coated vesicles by chance, and they accumulate in single membrane-bound vacuoles, presumably late endosomes/phagosomes, along with multimembranous onionlike structures. The authors propose a model of a stepwise conjugate binding to the cell membrane and gradual release of the enzymes.

Quantification of epithelial cell proliferation, cell dynamics, and cell kinetics in vivo.

The measurement of cell proliferation in vivo is usually carried out by the examination of static measures. These comprise the mitotic index or labeling indices using incorporation of DNA synthesis markers such as bromodeoxyuridine or tritiated thymidine, or intrinsic markers, such as Ki67 and proliferative cell nuclear antigen (PCNA). But static measures only provide a 'snapshot' of cell proliferation. Rate measures, including double labeling methods and the metaphase arrest method, can actually measure cell production rates but they are far less utilized at present. Transit times and migration rates can also be measured using pulse and chase labeling or by following the transit of labeled cells through the tissue. Simple indices of cell division can easily be confounded by concomitant changes in the compartment size and many alleged markers of proliferation have serious shortcomings, as the markers may be involved in multiple aspects of cell regulation. The complexities of studying proliferation in vivo are illustrated here with a focus on the gastrointestinal tract. Some of these methods can help elucidate the role of the stem cells and their relationship to label retaining cells. WIREs Dev Biol 2017, 6:e274. doi: 10.1002/wdev.274 For further resources related to this article, please visit the WIREs website.

CD4+ T-Cell Reactivity to Orexin/Hypocretin in Patients With Narcolepsy Type 1.

Introduction:Narcolepsy type 1 is accompanied by a selective loss of orexin/hypocretin (hcrt) neurons in the lateral hypothalamus caused by yet unknown mechanisms. Epidemiologic and genetic associations strongly suggest an immune-mediated pathogenesis of the disease.Methods:We compared specific T-cell reactivity to orexin/hcrt peptides in peripheral blood mononuclear cells of narcolepsy type 1 patients to healthy controls by a carboxyfluorescein succinimidyl ester proliferation assay. Orexin/hcrt-specific T-cell reactivity was also determined by cytokine (interferon gamma and granulocyte-macrophage colony-stimulating factor) analysis. Individuals were considered as responders if the cell division index of CD3+CD4+ T cells and both stimulation indices of cytokine secretion exceeded the cutoff 3. Additionally, T-cell reactivity to orexin/hcrt had to be confirmed by showing reactivity to single peptides present in different peptide pools.Results:Using these criteria, 3/15 patients (20%) and 0/13 controls (0%) showed orexin/hcrt-specific CD4+ T-cell proliferation (p = .2262). The heterogeneous reactivity pattern did not allow the identification of a preferential target epitope.Conclusions:A significant role of orexin/hcrt-specific T cells in narcolepsy type 1 patients could not be confirmed in this study. Further studies are needed to assess the exact role of CD4+ T cells and possible target antigens in narcolepsy type 1 patients.

Effect of low temperature on cell synchronization division in suspension culture cells of Pinellia ternata

To study the effect of low temperature on cell synchronization division in suspension culture cells of Pinellia ternata, in order to find a proper condition for cell synchronization. The loosened calli of P. ternata were used as materials. The effect of low temperature on cell synchronization division was studied and researched in suspension culture cells by the method of orthogonal experiment, the obtained experimental data was compared and analyzed through the analysis of range and analysis of variance. After the low temperature treatment, the cell division index was increased in various degrees, among the total results, when the temperature was set at 8 degrees C for 24 hours and then recovered for 36 hours, the effect was the best, the mitotic index was significantly increased to 11.3%. The low temperature could obviously influence the cell synchronization division in suspension culture cells of P. ternata, and the mitotic index was efficiently increased.

Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1exhibits slower rates of cell division and shoot induction

BackgroundPlant growth depends on both cell division and cell expansion. Plant hormones, including brassinosteroids (BRs), are central to the control of these two cellular processes. Despite clear evidence that BRs regulate cell elongation, their roles in cell division have remained elusive.ResultsHere, we report results emphasizing the importance of BRs in cell division. An Arabidopsis BR biosynthetic mutant, dwarf7-1, displayed various characteristics attributable to slower cell division rates. We found that the DWARF4 gene which encodes for an enzyme catalyzing a rate-determining step in the BR biosynthetic pathways, is highly expressed in the actively dividing callus, suggesting that BR biosynthesis is necessary for dividing cells. Furthermore, dwf7-1 showed noticeably slower rates of callus growth and shoot induction relative to wild-type control. Flow cytometric analyses of the nuclei derived from either calli or intact roots revealed that the cell division index, which was represented as the ratio of cells at the G2/M vs. G1 phases, was smaller in dwf7-1 plants. Finally, we found that the expression levels of the genes involved in cell division and shoot induction, such as PROLIFERATING CELL NUCLEAR ANTIGEN2 (PCNA2) and ENHANCER OF SHOOT REGENERATION2 (ESR2), were also lower in dwf7-1 as compared with wild type.ConclusionsTaken together, results of callus induction, shoot regeneration, flow cytometry, and semi-quantitative RT-PCR analysis suggest that BRs play important roles in both cell division and cell differentiation in Arabidopsis.

Blocking LFA-1 Activation with Lovastatin Prevents Graft-versus-Host Disease in Mouse Bone Marrow Transplantation

Graft-versus-host disease (GVHD) following bone marrow transplantation (BMT) is mediated by alloreactive donor T lymphocytes. Migration and activation of donor-derived T lymphocytes play critical roles in the development of GVHD. Leukocyte function-associated antigen-1 (LFA-1) regulates T cell adhesion and activation. We previously demonstrated that the I-domain, the ligand-binding site of LFA-1, changes from the low-affinity state to the high-affinity state on LFA-1 activation. Therapeutic antagonists, such as statins, inhibit LFA-1 activation and immune responses by modulating the affinity state of the LFA-1 I-domain. In the present study, we report that lovastatin blocked mouse T cell adhesion, proliferation, and cytokine production in vitro. Furthermore, blocking LFA-1 in the low-affinity state with lovastatin reduced the mortality and morbidity associated with GVHD in a murine BMT model. Specifically, lovastatin prevented T lymphocytes from homing to lymph nodes and Peyer's patches during the GVHD initiation phase and after donor lymphocyte infusion (DLI) after the establishment of GVHD. In addition, treatment with lovastatin impaired donor-derived T cell proliferation in vivo. Taken together, these results indicate the important role of lovastatin in the treatment of GVHD.

Effects of Maternal Exposure to Diethylstilbestrol on Epididymal Development in Rat Offspring

In our previous study, prenatal diethylstilbestrol (DES) exposure (days 7-21 of gestation) suppressed plasma testosterone levels and histological development in the epididymis of rat offspring. In this study, we measured cell proliferation in epididymal ductules and the expression of steroid hormone receptors and 5alpha-reductase 1 in the epididymis to assess the effect of DES on epididymal development in the offspring. Prenatal DES exposure did not alter the cell division index, but suppressed the expression of androgen receptor mRNA at 15 weeks after birth, and stimulated estrogen receptor alpha mRNA at 6 weeks. These results suggest that prenatal DES exposure results in the retardation of epididymal tissue maturation by disruption of the postnatal expression of steroid hormone receptors.

Reduced proliferative capacity of highly purified T cells from patients with Autism Spectrum Disorders (ASD)

A pilot study assessing the proliferation of peripheral blood T cells from ASD patients as compared to controls was performed. Peripheral blood mononuclear cells (PBMC) were purified via ficoll. T cells were isolated from PBMC by negative selection (&gt;95% CD3+), labeled with cytoplasmic dye, activated in vitro, and assessed for proliferation by flow cytometry on day 5. Results indicate that the percent of T cells entering the cell cycle upon activation with anti‐CD3/anti‐CD28 coated beads from ASD patients (73%) and controls (76.4%) were not statistically different. Likewise the division index, defined as the number of divisions on average that every T cell in the culture undergoes, was also not statistically different between ASD patients (1.8) and controls (2.1) activated with the antibody beads. However, the average number of divisions that a T cell that has divided undergoes, the proliferative index, was significantly less in ASD patients (2.45) activated with the beads than in controls (2.67), p‐value = 0.03. Proliferation of T cells to lectins were not different between ASD patients and controls as measured by the percent of cells entering the cell cycle, division index, or proliferative index. These results indicate that while ASD patient T cells are not profoundly deficient in proliferative capacity, they may demonstrate subtle defects in activation via antibody‐coated beads.

Stem Cells, Hormones and Pituitary Adenomas

We still do not understand the pathogenesis of the majority of pituitary adenomas or why, once formed, their behaviour tends to be so benign. Understanding trophic activity in the normal pituitary may be the key. Despite the fact that changes in indices of cell division and programmed cell death that are too small to measure can produce highly significant fluxes in cell populations, little by little, the fascinating patterns of integrated responses of pituitary cells to hormonal stimuli are now being revealed.

T cell proliferation and cytokine responses to ovalbumin and ovomucoid detected in children with and without egg allergy

The specific T cell responses in egg allergy and resolution have not been fully elucidated. To characterize egg allergen-specific T cells of children with active and resolved egg allergy, in comparison with non-allergic controls. We studied children with active (n=35) or resolved (n=20) egg allergy determined by oral challenge, and non-allergic controls (n=15). Peripheral blood mononuclear cells were labelled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with ovalbumin (OVA), ovomucoid (OM) or tetanus toxoid. Flow cytometry was used to detect divided CD3+ CFSE(lo) cells that expressed intra-cytoplasmic IL-4 or IFN-gamma. The cell division index (CDI) was calculated as a measure of allergen-specific proliferation. Peanut-specific T cells of a subgroup of children who also had peanut allergy were also studied. OVA-specific T cells were found in subjects with active (87%) or resolved (75%) egg allergy and in controls (67%), with a trend towards increased T cell proliferation in allergy. OM-induced weaker T cell responses than OVA, stimulating fewer responders (46% allergic, 50% resolved, 60% controls) and 10-fold less proliferation [CDI(OVA) 2.0 (median), 25.6 (maximum) vs. CDI(OM) 0.2 (median), 15.1 (maximum); P<0.01]. Both egg allergens induced significant IL-4+ (median 10%, range 1.4-58%) and IFN-gamma+ (median 28%, range 4.5-63%) cells in responders, including non-allergics. There were no significant differences in IFN-gamma+ or IL-4+ cells or in IFN-gamma/IL-4 ratios between groups. Peanut-specific T cell proliferation was significantly higher in peanut allergy [CDI(CPE) 16.5 (median), 24.8 (maximum)] compared with controls [CDI(CPE) 2.1 (median), 16.1 (maximum)] but cytokine profiles were not different. Tetanus-specific T cells were seen in 90% of the subjects, with no significant inter-group differences in responses. Egg allergen-specific T cells are readily detected in all groups and not restricted to egg allergy. In contrast, peanut-specific proliferation was significantly higher in peanut allergy. This suggests that T cell responses in peanut and egg allergy may differ. We did not find T helper type 2-deviated cytokine responses in egg or peanut allergy.

Development of Human Cytomegalovirus–Specific T Cell Immunity during Primary Infection of Pregnant Women and Its Correlation with Virus Transmission to the Fetus

We sought to study the development of human cytomegalovirus (HCMV)-specific T cell-mediated immune responses during primary HCMV infection in pregnancy. The HCMV-specific lymphoproliferative response (LPR) and intracellular cytokine (interferon [IFN]- gamma and interleukin [IL]-2) production were investigated during the first year after primary infection in 49 pregnant women and 9 nonpregnant control subjects. An HCMV-specific CD4(+) and CD8(+) T cell LPR was detected by the 5,6-carboxyfluorescein diacetate succinimidyl ester dilution method, and a cell-division index was calculated. The CD4(+) T cell LPR developed slightly earlier than the CD8(+) T cell LPR. However, CDI values for both T cell subpopulations were lower than those of seropositive control subjects in both pregnant and nonpregnant individuals. During the first month after infection, IFN- gamma -producing CD4(+) and CD8(+) T cells were consistently observed, whereas IL-2-producing T cells were very rarely detected in blood. A correlation between the development of HCMV-specific LPR and virus clearance from blood was observed. A significantly delayed development of the CD4(+) T cell LPR was observed in infected mothers who transmitted virus to the fetus, compared with those who did not. The development of adaptive T cell immunity after primary HCMV infection appears to be a complex and slow process until a memory T cell response develops. The T cell immune response appears to influence vertical HCMV transmission.

Immune reactivity to a homologue of wheat storage globulin, Glb1 in human type 1 diabetes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease dependent on gene-environment interaction. A wheat storage globulin with 80% amino acid homology to Glb1 (referred to as Glb1), was identified as the first candidate diabetes-related wheat protein in diabetes-prone rats. Glb1 antibodies were also found in pooled diabetic serum (MacFarlane et al., JBC, 2003). In this study, we evaluated the response of peripheral blood T cells to recombinant Glb1 protein in T1D patients and healthy controls. T cell response was measured using a CFSE proliferation assay and reported as cell division index (CDI). Th1 and Th2 cytokines were analyzed using cytometric bead arrays. Glb1 antibodies were evaluated by ELISA. T1D patients showed a higher mean CDI in response to Glb1. More than 1/3 of patients were responders (CDI > control mean + 3 SD). Cytokine analysis of the culture supernatant from Glb1-stimulated PBMC of patients showed a Th1 response, with IFN-γ most abundant. Approximately 25% of patients displayed Glb1 antibody levels higher than the control group mean + 2 SD. This study demonstrates high T cell reactivity with a Th1 response and antibodies to Glb1 in a subset of T1D patients. Further study is required to determine whether Glb1 plays a direct role in the pathogenesis of T1D or whether immune reactivity against this protein is a biomarker of a related process. (Supported by JDRF and CIHR)

Potential Value of Changes in Cell Markers in Organotypic Hippocampal Cultures Associated With Chronic EtOH Exposure and Withdrawal: Comparison with NMDA‐Induced Changes

Previous studies have shown that withdrawal from ethanol (EtOH) exposure induces neuronal damage, as indicated by propidium iodide (PI) uptake, in organotypic hippocampal slice cultures. This is prevented by MK801, suggesting that damage is "excitotoxic," resulting from activation of N-methyl-d-aspartate (NMDA) receptors by endogenous glutamate. To avoid reliance on a single indicator, and to enable assessment of recovery from the EtOH withdrawal (EWD) insult, we assessed changes in cell markers for neurons and glia, as well as cell division, following either EWD or NMDA challenge (as a positive control). Organotypic cultures from postnatal day (PND) 8 rats were cultured for 5 days before exposure to EtOH (mean concentration approximately 65 mM) for 10 days before EWD. Cultures of the same "days in vitro" age (DIV16) were exposed to NMDA (200 microM) for 1 hour. Neuronal injury was visualized using PI and indices of neurons, glia, or cell division were measured at intervals up to 10 days following the neurotoxic insults. Each time point and measurement used separate slice cultures, and these were treated as separate experiments with paired controls. Regional neuronal content was assessed by neuronal nuclear protein (NeuN) and calbindin D28k (Calb), glial content by glial fibrillary acidic protein (GFAP), and cell division by bromodeoxyuridine (BrdU) incorporation, all measured immunohistochemically. Chronic exposure to EtOH was associated with a dramatic reduction in BrdU incorporation in all regions of cultures. Propidium iodide fluorescence in the CA1 region was elevated significantly after EWD and more so after NMDA challenge. Reduced immunoreactivity (IR) of NeuN and Calb suggested that loss of neurons resulted from the EWD insult. Bromodeoxyuridine incorporation was initially depressed even further by EWD, but had returned to control levels after 3 days. In contrast, following NMDA insult, BrdU incorporation was significantly and persistently elevated above control levels after 3 days. Glial fibrillary acidic protein was reduced immediately after both EWD and NMDA challenge. Several days after EWD, expression of neuronal and glial markers, although variable, had generally returned to control levels. In contrast, NeuN IR remained significantly reduced after NMDA challenge. In general, the use of additional markers supports data obtained with PI uptake alone and suggests that neurons (and glia) are lost from the culture following EWD or NMDA challenge. These cell markers recover several days after EWD, but it is unclear whether functional recovery accompanies these changes. If the dramatic effect of EtOH exposure and EWD on BrdU incorporation reflects reduced neuro- and gliogenesis, it is likely that this adversely affects long-term recovery from EWD. Finally, some markers showed significant and consistent changes after EWD, whereas others did not. This information may facilitate the use of this model in evaluation of potential medications that protect against and/or promote recovery from neurotoxicity.

Recovery in the fetal pancreatic islet following fetal administration of streptozotocin in the rat in vivo and in vitro

In our previous study, after direct administration of streptozotocin (STZ; 400 microg/g) to fetuses on day 19 of gestation, the B-cell volume in fetal pancreatic islets showed a marked decrease, but gradually recovered with electron microscopic confirmation of B-cell regeneration. However, STZ at this dose often caused fetal death. In this study, therefore, we determined whether B-cells are newly generated after treatment with STZ at a smaller dose in vivo and in vitro. For in vivo experiment, fetuses were administered STZ at 40 microg/g on day 19 of gestation. The B-cell volume in pancreatic islets decreased markedly 3 hr after the administration of STZ, but it began to increase after 6 hr. The fetal plasma insulin concentration decreased from 6 to 12 hr after the administration, but recovered after 48 hr. The cell division index in fetal pancreatic islets of the STZ-treated group began to be significantly larger after 6 hr. For in vitro experiment, fetal pancreases on day 18 of gestation were pretreated with 10 mM STZ for 6 hr and cultured for 98 hr. B-cells were completely destroyed with STZ treatment; however, as these pancreases were cultured in a medium free of STZ, B-cells began to appear and insulin secretion was detected after 48 hr. After 72 hr, the cell division index was significantly greater. These results suggest that the fetal pancreas treated with STZ has the ability to regenerate B-cells both in vivo and in vitro.