The functions of some CLC Cl(-) channels are evident from human diseases that result from their mutations, but the role of the broadly expressed ClC-2 Cl(-) channel is less clear. Several important functions have been attributed to ClC-2, but contrary to these expectations ClC-2-deficient mice lacked overt abnormalities except for a severe degeneration of the retina and the testes, which led to selective male infertility. Seminiferous tubules did not develop lumina and germ cells failed to complete meiosis. Beginning around puberty there was a massive death of primary spermatocytes and later also of spermatogonia. Tubules were filled with abnormal Sertoli cells, which normally express ClC-2 in patches adjacent to germ cells. In the retina, photoreceptors lacked normal outer segments and degenerated between days P10 and P30. The current across the retinal pigment epithelium was severely reduced at P36. Thus, ClC-2 disruption entails the death of two cell types which depend on supporting cells that form the blood-testes and blood-retina barriers. We propose that ClC-2 is crucial for controlling the ionic environment of these cells.

The cells responsible for production of the male sex hormone testosterone, the Leydig cells of the testis, are post-mitotic cells with neuroendocrine characteristics. Their origin during ontogeny and regeneration processes is still a matter of debate. Here, we show that cells of testicular blood vessels, namely vascular smooth muscle cells and pericytes, are the progenitors of Leydig cells. Resembling stem cells of the nervous system, the Leydig cell progenitors are characterized by the expression of nestin. Using an in vivo model to induce and monitor the synchronized generation of a completely new Leydig cell population in adult rats, we demonstrate specific proliferation of vascular progenitors and their subsequent transdifferentiation into steroidogenic Leydig cells which, in addition, rapidly acquire neuronal and glial properties. These findings, shown to be representative also for ontogenetic Leydig cell formation and for the human testis, provide further evidence that cellular components of blood vessels can act as progenitor cells for organogenesis and repair.

The transcription factor Pax6 has been implicated in neocortical neurogenesis in vertebrates, including humans. Analyses of the role of Pax6 in layer formation and cognitive abilities have been hampered by perinatal lethality of Pax6 mutants. Here, we generated viable mutants exhibiting timed, restricted inactivation of Pax6 during early and late cortical neurogenesis using Emx1-Cre and hGFAP-Cre lines, respectively. The disruption of Pax6 at the onset of neurogenesis using Emx1-Cre line resulted in premature cell cycle exit of early progenitors, increase of early born neuronal subsets located in the marginal zone and lower layers, and a nearly complete absence of upper layer neurons, especially in the rostral cortex. Furthermore, progenitors, which accumulated in the enlarged germinal neuroepithelium at the pallial/subpallial border in the Pax6 mutants, produced an excess of oligodendrocytes. The inactivation of Pax6 after generation of the lower neuronal layers using hGFAP-Cre line did not affect specification or numbers of late-born neurons, indicating that the severe reduction of upper layer neurons in Pax6 deficiency is mostly attributable to a depletion of the progenitor pool, available for late neurogenesis. We further show that Pax6 fl/fl ;Emx1-Cre mutants exhibited deficiencies in sensorimotor information integration, and both hippocampus-dependent short-term and neocortex-dependent long-term memory recall. Because a majority of the morphological and behavior disabilities of the Pax6 mutant mice parallel abnormalities reported for aniridia patients, a condition caused by PAX6 haploinsufficiency, the Pax6 conditional mutant mice generated here represent a valuable genetic tool to understand how the developmental cortical disruption can lead to a human behavior abnormality.

BACKGROUNDTenascin‐C (TN‐C), a large extracellular matrix (ECM) glycoprotein with a molecular weight of 180–250 kilodaltons, is present in several normal adult tissues. TN‐C is up‐regulated during embryogenesis, in wound healing, and in tumor tissues. Glioblastoma multiforme (GBM) is the most frequent and malignant astrocytic tumor comprised of poorly differentiated, neoplastic astrocytes. Recently, TN‐C‐based radioimmunotherapy was administered to patients with GBM.METHODSIn the current study, the authors used immunohistochemistry to conduct a systematic investigation of TN‐C distribution patterns in normal human brain tissue and in a large variety of brain tumors (n = 485 tumors). Immunoreactivity for TN‐C was assessed with regard to its localization within tumor cells, blood vessels, and ECM using three different monoclonal antibodies (clones BC2, BC4, and TN2).RESULTSIn control human brains, a significant difference was noted in the expression of TN‐C when comparing gray with white matter using either Western blot analysis or immunohistochemistry. TN‐C was found in the white matter of the frontal, temporal, parietal, and occipital lobes and in the hippocampus, where the immunoreaction was especially strong in the hippocampal formation. In 181 astrocytomas of different grades (World Health Organization [WHO] Grade 2–4), TN‐C immunopositivity was seen to varying degrees in the cellular and stromal components of the tumor and in tumor‐associated vessels. Glioblastomas (n = 113 tumors) showed strong immunopositivity in the vessels and moderate immunopositivity of the ECM. A statistically significant reduction of TN‐C immunopositivity in tumor‐associated vessels or ECM was observed in anaplastic astrocytomas (WHO Grade 3) compared with GBM (WHO Grade 4). A Kaplan–Meier analysis showed that patients who had GBM lesions that lacked TN‐C immunopositivity in the ECM had a significantly longer survival (median, 28 months; standard error, 7.8 months) (n = 12 patients) compared with patients who had GBM lesions with TN‐C immunopositivity (median, 12 months; standard error, 1.6 months) (n = 87 patients). In meningiomas (n = 24 tumors), the neoplastic cells, the ECM of the tumor, and the vessels were TN‐C negative. In schwannomas (n = 31 tumors), the tumor cells were TN‐C negative; whereas, in > 50% of tumors, the vessels and the ECM of regressively altered tumor areas were positive. In metastatic carcinomas (n = 53 tumors), the tumor cells were negative; seldom were vessels stained positive for TN‐C. Focal areas of the ECM, often accompanied with fibrotic changes, were immunopositive for TN‐C.CONCLUSIONSThe most constant TN‐C immunopositivity was noted in the ECM of the fibrotic stroma in highly malignant brain tumors and along the tumor border, especially in high‐grade astrocytomas. The current results suggest that TN‐C expression may be correlated with the grade of malignancy in astrocytic tumors and that the presence or absence of TN‐C expression in the stroma of astrocytic tumors may play a not yet clearly understood role in shortening or prolonging, respectively, the survival of patients. Cancer 2003. © 2003 American Cancer Society.

A number of marker substances for neuronal and neuroendocrine cells have been demonstrated in the cytoplasm of the interstitial Leydig cells of human testes using basic immunocytochemical methods and some of their modifications. We were able to reveal immunoreactivity for enzymes involved in the synthesis of the catecholamines dopamine and noradrenaline (tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine-beta-hydroxylase), for the indolamine 5-hydroxytryptamine (serotonin), as well as for a number of well-known neuronal markers such as the neurofilament protein 200, synaptophysin, chromogranin A + B, the neural cell-adhesion molecule (N-CAM), the microtubule-associated protein (MAP-2), and the calcium-binding proteins: S-100, calbindin and parvalbumin. Immunoreactivity for these substances was found in the majority of the interstitial cells although differences in the staining intensity among the individual Leydig cells and among Leydig cells from different patients were observed. At the electron-microscopic level the Leydig cell cytoplasm was seen to contain microtubules, intermediate- and microfilaments as well as clear (40-60 nm) and dense-core (100-300 nm) vesicles, providing a morphological correlate for some of the immunocytochemical results. Although individual marker substances are not absolutely specific for nerve and neuroendocrine cells, the results obtained, together with the already established neuron-specific enolase-, substance P-, methionine-enkephalin- and proopiomelanocortin (POMC)-derived peptide-like immunoreactivity, provide strong evidence for the neuroendocrine (paraneuronal, APUD-like) nature of the Leydig cells of the human testis.

Hepatitis B immunoglobulin is used for prophylaxis against hepatitis B virus (HBV) and is thought to act by neutralization of virions and hepatitis B virus surface antigen (HBsAgHepatitis B immunoglobulin (HBIG) is used clinically as passive immunoprophylaxis for accidental exposure to hepatitis B virus (HBV) and long term to prevent HBV recurrence in the graft after liver transplantation (24). It contains high-titer antibodies against HBV surface antigen (HBsAg), which is the major component of the outer envelope of the 42-nm-diameter hepatitis B virion, as well as the 22-nm-diameter subviral particles. The therapeutic effect of HBIG is believed to be due to high-affinity binding with HBs-containing particles and neutralization of HBV in the circulation.Despite HBIG prophylaxis, HBV infection recurs in 30% of patients who receive transplants for HBsAg-positive cirrhosis (24). The failure of immunoprophylaxis is due either to a high HBV load and inadequate neutralization by HBIG or to the emergence of antibody-induced escape HBV mutants (3, 6, 25). These mutant HBV strains contain amino acid substitutions within the conserved a-determinant (a group-reactive region between amino acids 124 and 149 of HBsAg), which abrogate the binding affinity of anti-HBs (4, 5, 22, 27). The most frequent mutation occurs at codon 145 of the surface open reading frame, leading to a glycine (G)-to-arginine (R) substitution-G145R-which has been shown to emerge both in liver transplant patients receiving HBIG prophylaxis and in HBV vaccine recipients (2,6,22). The mechanism for the emergence of HBsAg mutations that escape antibody recognition has not been defined.Earlier studies have demonstrated the presence of membrane-bound and/or nuclear localization of immunoglobulin G (IgG) in hepatocytes of patients with chronic HBV infection, who express HBV core antigen or hepatitis delta virus antigen in the liver (17,19,23). Recently, a novel Fc receptor for IgG (FcRn) which mediates the transcytosis of IgG from serum to bile and protects the internalized IgG from catabolism has been identified on the plasma membranes of adult rat hepatocytes (1, 9). FcRn is a heterodimer of ␤ 2 -microglobulin light chain and a major histocompatibility complex class I-like heavy chain that binds IgG via Fc residues in a pH-dependent manner. IgG binding to FcRn is followed by endocytosis of the complex in the acidic endosome environment, trafficking through cellular conduits to bypass lysosomal activities and finally releasing IgG in the extracellular fluids (7). Whether hepatitis B immunoglobulin enters HBV-infected hepatocytes and whether an interaction with HBsAg occurs within cells, in addition to the interaction in serum, have not been investigated.In the present study, we investigated the hypothesis that HBIG is able to bind to hepatocytes and affect the secretion of HBsAg and HBV virions from the cells. For this purpose, we used a panel of human hepatocyte-derived cell lines cultured together with monoclonal and polyclonal HBs-specific antibodies. The...

The lamina propria of human seminiferous tubules is composed of 5 to 7 cellular layers separated by laminae of extracellular connective-tissue components. By means of immunocytochemical methods the different nature of the cellular layers could be defined for the first time. Based on the light-microscopic demonstration of both desmin-like and vimentin-like immuno-reactivity in the inner 3 to 4 layers of the lamina propria, these cells can be identified as myofibroblasts. The outermost one or two cellular layers, on the contrary, only show a vimentin-like immunoreactivity indicating the pure fibroblastic nature of these cells. Therefore, the outermost cellular layers are suggested to be derivatives of the interstitium. In cases of disturbed spermatogenesis, the lamina propria is frequently considerably thickened by an increase in the extracellular matrix components between the cellular layers. Whereas the ultrastructural localization of laminin-, collagen type-IV- and fibronectin-like immunoreactivity remains unaffected in the thickened lamina propria, the desmin-like immunoreactive cells of the inner layers strongly decrease in number and staining intensity. Most probably, the myofibroblasts lose their myoid characteristics to participate in the secretion of increased amounts of extracellular matrix components, which in turn presumably block the mediation of the lamina propria between the interstitium and the germinal epithelium. It is still unclear whether the thickened lamina propria provokes the disturbance of spermatogenesis or vice versa.

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