Reduced pteridine derivatives induce apoptosis in human neuronal NT2/HNT cells

Elevated concentrations of the pteridine compound neopterin, usually accompanied by 7,8-dihydroneopterin were found in cerebrospinal fluids of patients with neurodegenerative diseases and central nervous system infections. Here, the potential of pteridines to induce apoptosis of the human neuronal cell line (NT2) was investigated. Reduced neopterin, biopterin- and folate derivatives led to a time-dependent increase of apoptosis of cells. In contrast, non-reduced pteridines did not significantly alter cell survival. After differentiation of neuronal precursor cells to neurons and astrocyte-like cells, similar effects were detected. Antioxidants partly protected NT2 from pteridines-induced apoptosis, suggesting the involvement of reactive oxygen intermediates. In vitro experiments using dichlorofluorescin-diacetate further indicated a direct formation of reactive oxygen species in cells. Results implicate that high concentrations of reduced pteridines, might contribute to the loss of neuronal cells in neurodegenerative diseases.     

Legionella species of different human prevalence induce different rates of apoptosis in human monocytic cells

Legionella species of different human prevalence were examined with respect to induction of apoptosis in the human monocytic cell line Mono Mac 6 (MM6). L. pneumophila serogroup 1 (Pontiac), L. pneumophila serogroup 1 (Philadelphia-1), L. longbeachae serogroup 1, L. gormanii, L. micdadei and L. steigerwaltii were used to infect MM6 cells. Subsequent induction of apoptosis was investigated by enzyme-linked immunosorbent assay (ELISA), gel electrophoresis of cellular DNA extracts, and staining of cells with the DNA dye 4', 6-diamidino-2-phenylindole (DAPI). Additionally, the concomitant occurrence of infection and apoptosis was demonstrated by a combination of immunohistochemistry with nuclear DAPI counterstaining. Induction of apoptosis in MM6 cells by a given species of the genus Legionella correlates with their human prevalence rather than with their ability to multiply within this human monocytic cell line. Furthermore, we found that initiation of apoptosis of Mono Mac 6 cells was dependent on direct adherence of the pathogenic bacteria to the host cell and was triggered by extracellular bacteria.     

Reduced susceptibility to Fas-mediated apoptosis in B-1 cells

Elimination of activated T and B cells by Fas-dependent apoptosis may contribute to the maintenance of peripheral tolerance. CD40 ligation was recently shown to up-regulate Fas expression and enhance susceptibility to Fas-mediated apoptosis in mouse splenic B cells. In the present study, we have investigated the regulation of Fas expression and Fas-triggered apoptotis in mouse peritoneal B-1 cells. B-1 cells expressed a similar level of CD40 as that on B-2 cells, and proliferated in response to a soluble CD40 ligand (CD40L)-CD8α chimeric protein, suggesting that CD40 on B-1 cells is functional. In contrast to B-2 cells, B-1 cells expressed Fas at only low levels in response to CD40L-CD8α alone or CD40L-CD8α+interleukin-4, and were resistant to Fas-mediated apoptosis following these treatments. While Fas expression could be induced in B-1 cells to a comparable level as that in B-2 cells by cross-linking CD40L-CD8α with an anti-CD8α antibody, the sensitivity to Fas-mediated apoptosis in B-1 cells was significantly reduced compared with B2 cells. These results suggest that peritoneal B-1 cells from normal mice have a lower susceptibility to Fas-mediated apoptosis and may distinguish B-1 from B-2 cells. Similarly, B-1 cells from the peritoneal cavity and spleen of autoimmune-prone NZB mice exhibited reduced susceptibility to Fas-mediated apoptosis relative to their B-2 counterparts. NZB splenic B-1 cells, however, were more susceptible to Fas-mediated apoptosis than NZB peritoneal B-1 cells. The results presented here raise the possibility that the reduced susceptibility to Fas-triggered apoptosis in B-1 cells might be an accelerating factor for the autoantibody production in NZB mice.     

Reduced pregnancy rates following the transfer of human embryos frozen or thawed in culture media supplemented with normal serum albumin

Over a 26 month period 17% of couples having treatment in our clinical programmes selected a commercially available protein (normal serum albumin, NSA) prepared from pooled human sera instead of using their own serum as a supplement for their embryo culture media. In a retrospective analysis of >2000 gonadotrophin-stimulated cycles and 1000 cycles where frozen/thawed embryos were transferred, fertilization, embryo quality and pregnancy rates following in-vitro fertilization (IVF), gamete intra-Fallopian transfer (GIFT) or intracytoplasmic sperm injection (ICSI) were unaffected by the type of protein used to supplement the culture medium. When embryos were thawed in medium containing NSA, both pregnancy (PR) and implantation rates (IR) were significantly lower (P <0.05) than if the medium was supplemented with serum (PR 8.3% and 17.5%; IR 4.6% and 10.5%). Inclusion of NSA before freezing reduced the IR of thawed embryos. To further test this observation all cycles where embryos were cultured and frozen in medium containing NSA (173 cycles) were matched to cycles where serum was used and the outcome was compared. At the end of 1995 just over half of the embryos in both groups had been thawed. No statistical difference was noted in the pregnancy rates (NSA, 5.6% versus serum, 11.3%) but the IR per embryo was significantly lower when embryos were cultured and frozen in medium supplemented with NSA (2.2%) than when serum was used as the supplement (6.6%).      

Reduced cold-induced thermogenesis in familial human obesity

Summary It is well known that cold and diet-induced thermogenesis, which is mediated in small rodents by the hypothalamic-noradrenergic fibersbrown adipose tissue axis, is impaired in genetically obese mice. To test whether these adaptive mechanisms are also impaired in obese humans, 12 young males who were otherwise healthy (6 lean and 6 obese) were examined. The obese subjects had an early-onset type of obesity with a strong family history of it as well. Deep body temperature was measured by using a deep body thermometer furnished with three thermocouples. They were respectively placed on the sternum, on the interscapular area immediately under the neck (HIS), and on the 4th intercostal space (LIS) in order to study core temperature as well as heat production where brown adipose tissue could also be present in adults. Both lean and obese subjects were kept in a thermoneutral environment (28° C) until they reached a steady-state body temperature and then rapidly transferred into a cold room (6–8° C) where they remained up to 60 min. Body temperature decreased in both groups, but the decrease was more marked in the obese individuals on the sternum (P<0.01), on HIS (P<0.05) and on LIS (P<0.05) when compared to lean individuals. In conclusion, cold-induced thermogenesis is impaired in familial early-onset human obesity and in genetically obese mice.Abbreviations BAT brown adipose tissue - BMI body mass index - HIS high interscapular area - LIS low interscapular area     

Activation of ERK,AKT and JNK signalling pathways in human schwannomas in situ

Aims: Schwannomas are common tumours that may be multiple in neurofibromatosis type 2, when they may be difficult to treat without significant morbidity using surgery and radiosurgery. Previous in vitro work has suggested that merlin loss is associated with activation of the JNK/JUN, PI3K/AKT and MEK/ERK pathways and that these pathways may be susceptible to pharmacological inhibition. The aim was to investigate the expression of proteins involved in these pathways in human schwannomas in situ. Methods and results: Immunohistochemistry using antibodies to AKT, pAKT, MEK, pMEK, ERK, pERK, JUN and pJUN was applied to 16 schwannomas (sporadic and NF2), and the results were compared with those seen in traumatic neuromas. Increased expression of pMEK, pERK and pJUN was seen in the schwannomas samples and of pAKT in schwannomas and controls. Conclusions: These findings provide further direct evidence for activation of the JNK/JUN, PI3K/AKT and MEK/ERK signalling pathways in schwannomas and support the development of therapeutic agents directed against these pathways for the treatment of this group of tumours.     

Reduced rates of membrane turnover in lymphocytes from chronic lymphatic leukaemic patients

Lymphocytes in chronic lymphatic leukaemia (CLL) were found to release labelled antibodies against surface antigens at a slower rate than healthy cells and `escape from sensitization' was similarly prolonged in the abnormal lymphocytes. This suggests that membrane turnover in CLL lymphocytes is abnormally slow. The leukaemic cells can be provoked to turn over antisera more quickly by prior exposure to mitogenic stimulation and if nuclear material is used as a mitogen, membrane turnover will increase to a rate approaching that observed in normal lymphocytes. Once this acceleration has been achieved the leukaemic lymphocytes no longer react to an antiserum with specificity for leukaemic antigens and this result suggests that the leukaemic state of the CLL cell may be controlled by the surface membrane.     

Reduced Fhit protein expression in human malignant mesothelioma

Human malignant mesothelioma (MM) is an aggressive neoplasm related to occupational exposure to asbestos and characterised by a long latency time. Multiple chromosomal deletions and DNA losses have been revealed in MM by studies performed with karyotypic, comparative genomic hybridisation and loss of heterozygosity (LOH) analyses. Among frequently deleted chromosomal sites, LOH at chromosome 3p has been detected in MM, suggesting the presence of one or several tumour suppressor genes that have an important role in development of the disease. The FHIT (fragile histidine triad) tumour suppressor gene, located at 3p14.2, has been proposed to be a target to major human lung carcinogens, such as tobacco smoke and asbestos. Although many studies have indicated decreased Fhit protein expression in a variety of malignancies, there is no report of FHIT gene aberrations or Fhit protein abnormalities in MM. We examined expression of the Fhit protein and LOH at the FHIT gene in malignant mesothelioma. Altogether, 13 paraffin embedded MM tumours were analysed for Fhit protein expression, and 21 fresh tumours and 10 cell cultures for LOH at the FHIT gene with two intragenic microsatellite markers. All tumours showed less intense immunostaining than normal bronchial epithelium or mesothelium. Fhit expression was absent or reduced in 54% (7 of 13) of the tumours, with the weakest staining observed in poorly differentiated areas. Allele loss was seen in 3 of 10 (30%) of the MM cell lines, but only in 1 of the 21 fresh tumours studied, suggesting concealment of LOH by normal cells present in MM tumours. In conclusion, our present data indicate a frequent decrease of Fhit protein expression, thus supporting the significance of FHIT inactivation in development of MM.     

Reduced Morg1 expression in ischemic human brain

The mitogen-activated protein kinase organizer 1 (Morg1) has been recently identified as modular scaffold regulating ERK signaling. Morg1 also attenuates expression of the hypoxia-inducible factor-1α (HIF-1α) by activating or stabilizing of prolyl-hydroxylase 3 (PHD3). Here we demonstrate for the first time that Morg1 is expressed in the human brain in neurons, glial cells, and blood vessel walls. Immunohistochemistry, RT real-time PCR and western blotting indicated that Morg1 expression is reduced in human brain tissue with ischemic damage. Moreover, reactive astrocytes in the surrounding brain tissue showed strong Morg1 expression. Since hypoxic adaptation with enhancing HIF-1α expression can engage a genetic program leading to profound sparing of brain tissue and enhanced recovery of function, down-regulation of Morg1 expression in the ischemic brain may be viewed as an intrinsic mechanism to stimulate this response. On the other hand, upregulation of Morg1 in astrocytes surrounding the penumbra may counteract this hypoxic adaptation.     

Reduced apoptosis correlates with enhanced autophagy in synovial tissues of rheumatoid arthritis

Objective

Defective apoptosis contributes to the massive synovial hyperplasia in rheumatoid arthritis (RA), but the mechanism is largely unknown. To investigate the reasons for the reduced apoptosis in RA synovium, we analyzed autophagy and its relationship to apoptosis in synovial tissues from RA and osteoarthritis (OA) patients.

Methods

Synovial tissues were obtained from seven RA and 12 OA patients undergoing knee replacement surgery. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and staining for p85 fragment of PolyADP-ribose polymerase (PARP). Autophagy was determined by immunoblotting for the autophagic markers Beclin-1 and LC3. MicroRNA-30a (miR-30a), which targets Beclin-1, was measured by real-time RT-PCR. The interplay between autophagy and apoptosis was determined via Spearman’s correlation analysis.

Results

In comparison with OA, the synovial tissues from RA displayed decreased TUNEL-positive nuclei (P < 0.01). In contrast, Beclin-1 and LC3 were overexpressed in the synovial lining layers of RA, which was correlated with decreased levels of miR-30a. Moreover, there was a significant reverse relationship between apoptosis and autophagy in RA synovial tissues (P < 0.01 and r = ?0.8937).

Conclusion

The impaired apoptosis in RA synovium might result from increased autophagy, which in turn could be due to the deregulation of miRNA-30a.     

Immunoreactivity of bovine schwannomas

Thirty schwannomas from 22 cows were examined immunohistochemically. All were positive for vimentin and Ki-67 but negative for pancytokeratin, neurofilament, and desmin. S-100 immunolabelling varied between and within lesions. The numbers of tumours giving positive results for S-100, neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were 16, 30 and 25, respectively. It was concluded that vimentin-positive tumours suspected to be schwannomas should also be immunolabelled for NSE and GFAP to confirm the diagnosis.     

神经鞘瘤假腺性结构的本质

目的：认识神经鞘瘤假腺性结构的本质及其发生原因,决定是否采纳假腺性神经鞘瘤之命名。方法：运用免疫组化ABC法观察126例神经鞘瘤的病理形态表现。结果：颅内和椎管内神经鞘瘤除了具有躯体神经普通型神经鞘瘤的A、B区结构表现外,尚易发生出血、囊性变、核多形性,泡沫细胞和假腺样结构等变化。免疫组化证明这些假腺样或腔隙状结构由神经鞘瘤的瘤细胞组成。对S-100蛋白、MBP呈阳性表达,对EMA和CEA呈阴性表达。结论：神经鞘瘤的假腺样和腔隙状结构和颅内椎管内神经鞘瘤的出血,囊性变,核异形,泡沫细胞等改变一样,可能是在肿瘤的退行性改变中对囊液的反应性改变,以不采用“假腺性神经鞘瘤”为妥。     

Reduced and oxidized glutathione in human and monkey brain

Prior animal studies have indicated that levels of oxidized glutathione (GSSG) in brain and other organs are low, comprising several percent, or less, of the total glutathione. An exception is seen in reports for autopsy and biopsy specimens of human brain, where very high levels of GSSG have been indicated. The latter observations imply an unusual redox state for human brain. In the current study, GSSG and reduced glutathione (GSH) were measured in monkey brain and autopsy specimens of human brain. Levels of GSSG were 1.2% or less, of the total glutathione. These results are similar to those for rodent brain, but disagree with earlier reports concerning human brain.     

Histamine prevents apoptosis in human monocytes

BACKGROUND: As chronic atopic dermatitis (AD) is associated with activation of circulating and infiltrating monocytes, monocytes are considered to play a pivotal role in the establishment of chronic lesions in AD. Histamine is an important mediator of inflammatory and allergic responses. Although new immunomodulatory functions of histamine have recently become apparent, the effect of histamine on the life span of monocytes remains unclear. OBJECTIVE: In the present study, we investigated the effect of histamine on the life span of human monocytes from normal healthy donors and patients with AD. METHODS: Monocyte apoptosis was induced by serum deprivation, CD95/Fas ligation, or dexamethasone in the presence of histamine, and measured using annexin V-and propidium iodide-staining. Bcl-2 protein and activated caspase-3 were determined by flow cytometry. We also examined the effect of soluble, histamine-induced factors produced by monocytes on apoptosis. Furthermore, we examined whether monocytic apoptosis is dependent on the cAMP pathway. RESULTS: Histamine prevented monocytic apoptosis induced by serum deprivation, CD95/Fas ligation, or dexamethasone in a dose- and time-dependent fashion. The inhibitory effects of histamine on monocytic apoptosis were blocked by an H2R antagonist, and mimicked by an H2R agonist. Histamine also up-regulated the expression of Bcl-2 and Mcl-1, and inhibited the activation of caspase-3. The culture supernatants from histamine-treated monocytes inhibited monocytic apoptosis, which was partly reversed by the removal of IL-10. Monocytes cultured with anti-IL-10 mAb and histamine did not exhibit an inhibitory effect on apoptosis. The histamine-induced anti-apoptotic effect was attenuated when monocytes were cultured in the presence of a cAMP inhibitor. CONCLUSIONS: These results indicate that the H2R signals induced by histamine allow monocytes to prolong their life span and infiltrate to the site of inflammation. This process may contribute to the establishment of chronic allergic disorders, such as AD.     

Spontaneous apoptosis in human thymocytes.

Apoptosis seems to be involved in different stages of immune cell development. In particular, experimental evidence suggests that it is a major form of cell death in the thymus. The present analysis of human thymocytes reveals that a fraction of these cells, cultured in vitro, undergoes spontaneous apoptosis. This observation is based both on molecular (DNA fragmentation) and morphological (electron microscopic) investigations of the cells. The apoptotic thymocytes are CD3- or CD3lo, CD4lo, and CD8lo and do not express Bcl-2 protein. Furthermore, thymocytes die by apoptosis when exposed to pharmacological stimuli, such as tumor necrosis factor-alpha, dexamethasone, ATP, or Ca++ ionophore. Thus the apoptotic machinery in thymocytes can be triggered by an imbalance in growth factors in the in vitro culture media and can be modulated by various biochemical signals. The process of spontaneous apoptosis is independent of mRNA or protein synthesis, as actinomycin D and cycloheximide fail to inhibit this phenomenon. Furthermore, apoptosis seems to require active oxidative phosphorylation, as it is prevented by incubation of the cells with inhibitors of the respiratory chain.     

Reduced cancer rates in agricultural workers: a benefit of environmental and occupational endotoxin exposure

Numerous epidemiological studies have suggested reduced cancer rates in workers employed in agricultural industries. Traditionally, these observations have been attributed to low tobacco consumption and the healthy worker effect(s). Recent investigations have suggested that endotoxin may be responsible for reducing lung cancer rates in various occupational groups. Endotoxin anticancer properties are believed to be mediated through immunological mechanisms. This paper provides evidence and suggests a hypothesis for endotoxin-mediated reduced cancer rates in agricultural workers.     

sDR5封闭TRAIL减少内毒素引起小鼠肝细胞凋亡

目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)在内毒素诱导小鼠肝细胞凋亡中的作用.方法 内毒素作用于可溶性死亡受体-5(sDR5)封闭TRAIL的小鼠,不同时间检测小鼠血清中的谷丙转氨酶(ALT)、谷草转氨酶(AST)和乳酸脱氧酶(LDH)的含量;HE染色及Annexin V、PI双染法流式细胞术检测小鼠肝细胞凋亡;免疫组织化学法及流式细胞术检测小鼠肝细胞和肝细胞膜表面DR5的表达.结果 内毒素可以上调肝细胞DR5的表达.DR5封闭TRAIL可显著抑制内毒素引起的小鼠肝细胞凋亡和改善肝细胞损伤.结论 内毒素引起疾病过程中TRAIL起重要作用.     

Reduced activity of the red-cell sodium-potassium pump in human obesity

Looking for evidence of reduced energy use in the cells of obese persons, we measured the numbers of sodium-potassium-pump units in erythrocytes from a group of 21 obese human subjects and found them to be reduced by 22 per cent as compared with those of nonobese controls (P <0.001). The cation-transport activity of the pump, as measured by 86rubidium uptake by the cells, we also reduced in parallel with decrease in pump units. An increased concentration of sodium in the red cells of obese subjectes was also found (9.6 +/- 0.7 vs. 7.1 +/- 0.6 mmol per liter of cells; P<0.01). This finding demonstrates independently the physiologic importance of reduced numbers of sodium-pump units and reduced pump activity as measured by ouabain binding and rubidium transport, respectively. The magnitude of the reduction in the number of pump units was found to be negatively correlated with the percentage of ideal body weight (r = 0.56, P<0.001); this observation suggests a possible role of abnormalities of the sodium pump in the pathophysiology of obesity.