Lactate inhibits germ cell apoptosis in the human testis

Dysregulation of male germ cell apoptosis has been associated with the pathogenesis of male infertility. Therefore, factors involved in the regulation of germ cell death are being actively investigated. Here, we studied the effects of lactate on human male germ cell death, using as a model a testis tissue culture in which physiological contacts are maintained between the germ cells and the supportive somatic Sertoli cells. Apoptosis of spermatocytes, spermatids and a few spermatogonia was induced by culturing segments of seminiferous tubules under serum-free conditions. This germ cell death was inhibited effectively and dose-dependently by lactate, indicating that it plays a crucial role in controlling cell death cascades of male germ cells. Interestingly, the anti-apoptotic role of lactate was not associated with changes in testicular adenine nucleotide (ATP, ADP and AMP) levels. In the seminiferous tubules, the final site of the death-suppressing action of lactate appeared to be downstream along the cell death pathway activated by the Fas receptor of the germ cells. In conclusion, testicular cell death was effectively regulated by lactate, which may be regarded as a potential compound for optimizing in-vitro methods involving male germ cells for assisted reproduction.     

鞘氨醇-1-磷酸及其受体对免疫细胞功能影响的研究进展

自然杀伤性T细胞（NKT）和树突状细胞（DC）等免疫细胞作为免疫系统的重要组成成份在维持机体内环境的稳态和免疫功能的正常发挥中起着至关重要的作用.近年的研究发现,鞘氨醇-1-磷酸（SIP）及其受体相互作用不仅可以影响免疫细胞的迁移,而且还能改变多种免疫细胞如NKT细胞、DC、CD4^＋ CD25^＋调节性T细胞（Treg）等的功能,从而引起机体免疫反应的变化.与此信号通路相关的多种动物实验和临床试验研究均证明了免疫调节剂FTY720即鞘氨醇-1-磷酸受体1（S1PR1）的拮抗剂在疾病的诊断和治疗中具有广泛的应用前景.     

Environmental toxicant-induced germ cell apoptosis in the human fetal testis

BACKGROUND: Disorders of the male reproductive system are increasing in prevalence. The term testicular dysgenesis syndrome emphasizes the importance of developmental influences on the aetiology of conditions including cryptorchidism, testicular germ cell cancer and reduced spermatogenesis. Men whose mothers smoked during pregnancy have lower sperm production. Cigarette smoke contains agents acting on the aryl hydrocarbon receptor (AHR). We have investigated the presence of AHR in the developing human testis and the effects of functional activation. METHODS AND RESULTS: Immunohistochemistry determined AHR to be expressed by germ cells in the human testis between 7 and 19 week gestation, but not by other cells. Treatment of cultured fetal testis with an AHR ligand present in tobacco smoke increased markers of cell apoptosis, and this was prevented by an AHR receptor antagonist. Immunohistochemistry indicated that apoptosis was restricted to germ cells. CONCLUSIONS: Germ cells in the developing human testis are a target for regulation by AHR ligands. Activation of AHR by environmental toxicants and AHR-induced apoptotic pathways may be the mechanism of action underlying the epidemiological findings of reduced spermatogenesis in men exposed to cigarette smoke before birth, and may also be of importance in other conditions comprising the testicular dysgenesis syndrome.     

Interleukin-15 inhibits spontaneous apoptosis in human eosinophils via autocrine production of granulocyte macrophage-colony stimulating factor and nuclear factor-kappaB activation

Prolonged eosinophil survival, i.e., reduced apoptosis, is implicated in the pathogenesis of chronic allergic inflammation. Here we demonstrate that interleukin (IL)-15, in the presence or absence of tumor necrosis factor (TNF)-alpha, reduces spontaneous apoptosis in freshly isolated human eosinophils. The prosurvival effect of IL-15 was abrogated by neutralizing antibody to granulocyte macrophage-colony stimulating factor (GM-CSF), although GM-CSF was not detected in conditioned media by ELISA. Additionally, the effect of IL-15 on spontaneous eosinophil apoptosis appeared to require nuclear factor-kappaB (NF-kappaB) activation based on evidence for NF-kappaB nuclear translocation and abrogation of the effect by the NF-kappaB inhibitor, Bay 11- 7082. Finally, the data demonstrate that IL-15 expression is higher in the submucosa of endobronchial tissues from subjects with moderate to severe asthma when compared with control subjects. Thus, our results suggest that IL-15, either alone or in combination with TNF-alpha, may perpetuate allergic inflammation by reduction of spontaneous eosinophil apoptosis through autocrine production of GM-CSF and NF-kappaB activation.     

Sphingosine-1-phosphate induces apoptosis of cultured hippocampal neurons that requires protein phosphatases and activator protein-1 complexes

A novel three-dimensional magnetometer-spatial filter system was developed to study human brain activity with high spatiotemporal resolution. The system combines the high temporal resolution of magnetoencephalography with the high spatial resolution achieved by using three-dimensional magnetometers and spatial filters to measure the direction and intensity of magnetic fields generated during brain activity. Simulation and phantom studies indicate that the system is capable of mapping current sources of magnetic fields with a spatial resolution comparable to that of any other brain functional imaging technique while maintaining millisecond temporal resolution. Application of this system to the human brain resolved magnetoencephalographic responses evoked by motion stimuli on a millisecond scale into responses occurring in visual cortical areas V1, V2/3 and V5. It also revealed signals related to contextual modulation in V1 and V2/3. This system provides a new way of studying the dynamics of human brain function.     

Stereological estimates of nuclear volume in normal germ cells and carcinoma in situ of the human testis

Carcinoma in situ of the testis may appear many years prior to the development of an invasive tumour. Using point-sampled intercepts, base-line data concerning unbiased stereological estimates of the volume-weighted mean nuclear volume (nuclear vV) were obtained in 50 retrospective serial testicular biopsies from 10 patients with carcinoma in situ. All but two patients eventually developed an invasive growth. Testicular biopsies from 10 normal adult individuals and five prepubertal boys were included as controls. Nuclear vV in testicular carcinoma in situ was significantly larger than that of morphologically normal spermatogonia (2P = 1.0 x 10(-19)), with only minor overlap. Normal spermatogonia from controls had, on average, smaller nuclear vV than morphologically normal spermatogonia in biopsies with ipsi- or contra-lateral carcinoma in situ (2P = 5.2 x 10(-3)). No difference in nuclear vV was found in carcinoma in situ with or without co-existing invasion, and no characteristic pattern of nuclear vV was disclosed by following the lesion in serial biopsies over time from individual patients. Estimation of nuclear vV may represent an adjuvant tool in morphologically puzzling cases of testicular carcinoma in situ, but the prognostic value requires further evaluation in larger series of patients.     

Inhibition of the 53BP2S-mediated apoptosis by nuclear factor kappaB and Bcl-2 family proteins

The p53 binding protein 2 (53BP2) has been identified independently as the interacting protein to p53, Bcl-2, and p65 subunit of nuclear factor kappaB (NF-kappaB). It was demonstrated that over-expression of 53BP2 (renamed as 53BP2S) induces apoptotic cell death. In this study we explored the effect of NF-kappaB activation elicited by a physiological NF-kappaB inducer, interleukin-1beta (IL-1beta), and anti-apoptotic Bcl-2 family proteins on the 53BP2S-mediated apoptosis. We found that both NF-kappaB activation and Bcl-2 family proteins could prevent the 53BP2S-mediated depression of mitochondrial transmembrane potential, activation of caspase-9, cleavage of poly ADP ribose polymerase (PARP), and cell death. These observations suggested that 53BP2S/Bbp and its directly or indirectly interacting proteins might play crucial roles in the regulation of apoptosis and contribute to carcinogenesis. It is also suggested that 53BP2S/Bbp induces apoptosis through the mitochondrial death pathway presumably by counteracting the actions of anti-apoptotic Bcl-2 family proteins. The regulatory network of the 53BP2S-mediated apoptosis cascade including its interacting proteins is discussed.     

Expression of transforming growth factor beta1 and its receptors in normal human urothelium and human transitional cell carcinomas

Previous studies indicated that transforming growth factor beta1 (TGFbeta1) is expressed by normal urothelial cells and exerts regulatory autocrine functions in urothelial maintenance and wound healing. However, little is known about the expression patterns of TGFbeta1 and its receptors in bladder tumors. Therefore, we studied the protein and mRNA localization of TGFbeta1 and TGFbeta receptor types I and II (TGFbetaRI and TGFbetaRII) in normal human urothelium and transitional cell carcinomas (TCCs) of different grades and stages. Expression of TGFbeta1 and its receptors was examined by immunocytochemistry and mRNA in situ hybridization in normal urothelium and TCCs using a semiquantitative method. By immunocytochemistry, the expression of TGFbeta1 and TGFbetaRII was higher in superficial and basal cell layers of normal urothelium than in the intermediate layer. A similar localization was seen in superficial TCCs. TGFbetaRI was mainly present in basal and intermediate cell layers of normal urothelium and superficial TCCs. In contrast, in muscle invasive TCCs, all tumor cells stained intensely for all three proteins. No correlation was found between immunostaining and TCC grade. In situ hybridization pointed out that all cell layers in normal urothelium exhibit similar TGFbeta1 mRNA levels. Elevated TGFbeta1 mRNA levels were noted in TCCs irrespective of grade or stage. In conclusion, these data indicate that in normal urothelium TGFbeta1, TGFbetaRI, and TGFbetaRII expression depend on maturation and differentiation. This pattern is particularly lost in muscle invasive TCCs, in which the expression of the three proteins is enhanced. These data suggest autocrine TGFbeta1 mechanisms in human TCC cells that may be more pronounced in muscle invasive TCC cells.     

Chromosome changes in germ cell tumors of the testis

Chromosome analysis was performed on short-term cultures established from samples of six tumors of the testis. Histologically, four tumors were embryonal cell carcinomas (three primary, one metastatic) and two of mixed histology with predominance of teratoma. The modal chromosome number was hypotriploid in four tumors, triploid in one, and hypertriploid in another. All tumors contained structurally abnormal chromosomes, ranging in number from 1 to 10 in different cases. A small metacentric marker chromosome, identified as an isochromosome of the short arm of chromosome #12 [i(12p)], was present in all tumors analyzed. Unlike other marker chromosomes, this one was invariably present in at least two copies per metaphase in all cases; all other chromosome markers were present in single copy in all tumors. Together with the previous reports on the presence of i(12p) in seminoma and teratoma of the testis, our findings suggest that this karyotypic abnormality is characteristic for all histologic varieties of germ cell tumors of the testis.     

SIRT1 promotes proliferation and inhibits apoptosis of human malignant glioma cell lines

In mammalian cells, SIRT1 decreases PTEN acetylation and inactivates the AKT pathway in a SIRT1 deacetylase-dependent manner. However, the function of SIRT1 in glioma was unknown. SIRT1 reexpression or knockdown was induced in human glioma cell lines. The cell synchronization, BrdU labeling and mitotic index were detected. Subsequently, cell cycle, cell viability, apoptosis, cell growth and proliferation were analyzed. Our work identified that SIRT1-knockdown significantly delayed mitotic entry of glioma cells, inhibited its growth and proliferation, and promoted its apoptosis. The apoptosis was related to PTEN/PI3K/AKT signaling pathway. The results showed that SIRT1 might be a promoter factor on tumorigenesis of glioma through PTEN/PI3K/AKT signaling pathway.     

Immunohistochemical localization of lactate dehydrogenase isoenzyme 1 in germ cell tumors of the testis

In this study, antiserum to lactate dehydrogenase isoenzyme 1 (LD 1) was used to determine immunohistochemical patterns of localization in a variety of germ cell neoplasms of the testis. All 29 seminomas and teratocarcinomas and four of seven embryonal carcinomas were positive for LD 1. These results suggest that elevated serum LD 1 levels noted in patients with germ cell neoplasms may be derived directly from neoplastic tissue and explain the relationship between serum LD 1 levels and tumor burden. A variety of neoplasms from other organs also were evaluated in order to determine the specificity of LD 1 staining for testicular tumors.     

Restricted 12p amplification and RAS mutation in human germ cell tumors of the adult testis

Human testicular germ-cell tumors of young adults (TGCTs), both seminomas and nonseminomas, are characterized by 12p overrepresentation, mostly as isochromosomes, of which the biological and clinical significance is still unclear. A limited number of TGCTs has been identified with an additional high-level amplification of a restricted region of 12p including the K-RAS proto-oncogene. Here we show that the incidence of these restricted 12p amplifications is approximately 8% in primary TGCTs. Within a single cell formation of i(12p) and restricted 12p amplification is mutually exclusive. The borders of the amplicons cluster in short regions, and the amplicon was never found in the adjacent carcinoma in situ cells. Seminomas with the restricted 12p amplification virtually lacked apoptosis and the tumor cells showed prolonged in vitro survival like seminoma cells with a mutated RAS gene. However, no differences in proliferation index between these different groups of seminomas were found. Although patients with a seminoma containing a homogeneous restricted 12p amplification presented at a significantly younger age than those lacking it, the presence of a restricted 12p amplification/RAS mutation did not predict the stage of the disease at clinical presentation and the treatment response of primary seminomas. In 55 primary and metastatic tumors from 44 different patients who failed cisplatinum-based chemotherapy, the restricted 12p amplification and RAS mutations had the same incidence as in the consecutive series of responding patients. These data support the model that gain of 12p in TGCTs is related to invasive growth. It allows tumor cells, in particular those showing characteristics of early germ cells (ie, the seminoma cells), to survive outside their specific microenvironment. Overexpression of certain genes on 12p probably inhibits apoptosis in these tumor cells. However, the copy numbers of the restricted amplification of 12p and K-RAS mutations do not predict response to therapy and survival of the patients.     

Monocyte activation in patients with non-seminomatous germ cell tumour of the testis before and after tumour eradication.

AIMS: To investigate the kinetics of normalisation of monocyte oxidative activity following tumour eradication. METHODS: Whole blood lucigenin enhanced chemiluminescence was studied in patients with non-seminomatous germ cell tumours. Group 1 comprised 14 patients who had been "cured" of their cancer (the term "cured" as used in this report denotes a relapse free period of at least three years). Group 2 comprised 15 patients who were followed from diagnosis to up to two years after the start of treatment. RESULTS: Lucigenin enhanced chemiluminescence of whole blood in the "cured" patients was similar to that of controls and lower than that in patients who had not yet received chemotherapy (group 2). After treatment, chemiluminescence decreased slowly and did not normalise until 18 months after the start of treatment. Tumour necrosis factor alpha (TNF alpha) concentrations were normal in "cured" patients but were raised in those who had not yet received treatment. TNF alpha was normalised 12 months after start of treatment. Alpha-fetoprotein concentrations were raised in most patients but normalised rapidly after tumour eradication. CONCLUSIONS: The activity of blood monocytes, as measured by whole blood lucigenin enhanced chemiluminescence, is increased in cancer. This activity may be a consequence of the presence of tumour cells. Immunocompetent cells remain active for over a year after eradication of the tumour.     

Sphingosine-1-phosphate via activation of a G-protein-coupled receptor(s) enhances the excitability of rat sensory neurons

Sphingosine-1-phosphate (S1P) is released by immune cells and is thought to play a key role in chemotaxis and the onset of the inflammatory response. The question remains whether this lipid mediator also contributes to the enhanced sensitivity of nociceptive neurons that is associated with inflammation. Therefore we examined whether S1P alters the excitability of small diameter, capsaicin-sensitive sensory neurons by measuring action potential (AP) firing and two of the membrane currents critical in regulating the properties of the AP. External application of S1P augments the number of APs evoked by a depolarizing current ramp. The enhanced firing is associated with a decrease in the rheobase and an increase in the resistance at firing threshold although neither the firing threshold nor the resting membrane potential are changed. Treatment with S1P enhanced the tetrodotoxin-resistant sodium current and decreased the total outward potassium current (IK). When sensory neurons were internally perfused with GDP-beta-S, a blocker of G protein activation, the S1P-induced increase in APs was completely blocked and suggests the excitatory actions of S1P are mediated through G-protein-coupled receptors called endothelial differentiation gene or S1PR. In contrast, internal perfusion with GDP-beta-S and S1P increased the number of APs evoked by the current ramp. These results and our finding that the mRNAs for S1PRs are expressed in both the intact dorsal root ganglion and cultures of adult sensory neurons supports the notion that S1P acts on S1PRs linked to G proteins. Together these findings demonstrate that S1P can regulate the excitability of small diameter sensory neurons by acting as an external paracrine-type ligand through activation of G-protein-coupled receptors and thus may contribute to the hypersensitivity during inflammation.     

N-cadherin expression in malignant germ cell tumours of the testis

Background

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men aged 18–35 years. They are clinically and histologically subdivided into seminomas and non-seminomas. Cadherins are calcium-dependent transmembrane proteins of the group of adhesion proteins. They play a role in the stabilization of cell-cell contacts, the embryonic morphogenesis, in the maintenance of cell polarity and signal transduction. N-cadherin (CDH2), the neuronal cadherin, stimulates cell-cell contacts during migration and invasion of cells and is able to suppress tumour cell growth.

Methods

Tumour tissues were acquired from 113 male patients and investigated by immunohistochemistry, as were the three TGCT cell lines NCCIT, NTERA-2 and Tcam2. A monoclonal antibody against N-cadherin was used.

Results

Tumour-free testis and intratubular germ cell neoplasias (unclassified) (IGCNU) strongly expressed N-cadherin within the cytoplasm. In all seminomas investigated, N-cadherin expression displayed a membrane-bound location. In addition, the teratomas and yolk sac tumours investigated also differentially expressed N-cadherin. In contrast, no N-cadherin could be detected in any of the embryonal carcinomas and chorionic carcinomas examined. This expression pattern was also seen in the investigated mixed tumours consisting of seminomas, teratomas, and embryonal carcinoma.

Conclusions

N-cadherin expression can be used to differentiate embryonal carcinomas and chorionic carcinomas from other histological subtypes of TGCT.