Interferon-gamma (IFN-gamma) and IL-4 expressed during mercury-induced membranous nephropathy are toxic for cultured podocytes.

The subepithelial immune deposits of Dorus Zadel Black (DZB) rats with mercury-induced membranous nephropathy consist of autoantibodies directed to laminin P1 and of complement. The animals develop massive proteinuria within 10-14 days which is associated with obliteration of foot processes of glomerular visceral epithelial cells (GVEC), or podocytes. Previous studies indicate that these autoantibodies are probably not the sole mediator of proteinuria and GVEC damage. In this study we investigated whether circulating or macrophage-derived cytokines can contribute to the GVEC changes as detected in vivo. In vivo at the height of the proteinuria, increased intraglomerular IFN-gamma immunoreactivity was found. In diseased rats a five-fold increase in intraglomerular macrophages was found, but we could not detect intraglomerular IFN-alpha, IFN-beta, IL-1 beta or tumour necrosis factor-alpha (TNF-alpha) by using immunohistology. Subsequently, we exposed cultured GVEC to these cytokines to investigate their cytotoxic effects on several physiological and structural parameters. IFN-gamma and IL-4 were the only cytokines that exerted toxic effects, resulting in a rapidly decreased transepithelial resistance of confluent monolayers, which was closely associated with altered immunoreactivity of the tight junction protein ZO-1. IL-4 also affected vimentin and laminin immunoreactivity. IFN-gamma and IL-4 only interfered with monolayer integrity when added to the basolateral side of the GVEC, indicating specific (receptor-mediated) effects. Only IL-4 decreased the viability of the cells, and treated monolayers demonstrated an increased passage of the 44-kD protein horseradish peroxidase. From our experiments we concluded that IFN-gamma subtly affected monolayer integrity at the level of the tight junctions, and that IL-4 additionally induced cell death. We hypothesize that the toxic effects of the cytokines IFN-gamma and IL-4 as seen with cultured podocytes are necessary together with the autoantibodies, for the ultimate induction of proteinuria in mercury nephropathy in the DZB rat.     

脐带血清对骨髓粒—巨噬细胞集落形成的影响

在骨髓细胞半固体琼脂培养体系中，研究脐带血清（ＣＢｓ）对骨髓粒—巨噬细胞集落形成单位（ＣＦＵ－ＧＭ）的影响，单纯应用ＣＢｓ培养可使骨髓ＣＦＵ－ＧＭ（ｘ±ｓ为２６．０３７５±１０．２３２７）显著的高于正常成人外周血清（ＰＢｓ）组（ｘ±ｓ为１１．５９１７±３．６０４７；Ｐ＜０．００１）。ＣＢｓ加粒－巨噬细胞刺激因子，ＧＭ－ＣＳＦ组ＣＦＵ－ＧＭ（ｘ±ｓ为１１９．０７９２±３５．０９９１）是单纯ＣＢｓ组的４．５７倍；而ＰＢｓ加ＧＭＣ－ＳＦ组（ｘ±ｓ为９７．１２５±２６．７５５９）是单纯ＰＢｓ组的８．３８倍。有白细胞介素－６（ＩＬ－６）存在的ＣＢｓ组，ＣＦＵ－ＧＭ（ｘ±ｓ为３１．９３３３±１０．９１４４）明显的高于单纯ＣＢｓ组（Ｐ＜０．０２）。提示ＣＢｓ中含有较高水平的内源性ＧＭ－ＣＳＦ。     

Sequential respiratory syncytial virus cytomegalovirus pneumonia following bone marrow transplantation

A 6-month-old child with familial hemophagocytic lymphohistiocytosis (FHL) experienced early sequential pneumonia due to respiratory syncytial virus (RSV) and cytomegalovirus (CMV) following bone marrow transplantation (BMT). The patient was deficient in natural killer (NK) cell activity (as found frequently in patients with FHL), and this risk factor may have played a major role in the concomitant infection by the two viral pathogens. Rapid diagnostic methods for both viruses are essential and early specific treatment may serve to ameliorate RSV- and CMV-induced lung injury in these life-threatening infections. © 1995 Wiley-Liss, Inc.     

Longitudinally orientated smooth muscle cells in rabbit arteries

Intima formation in vessels, spontaneous or experimentally induced, is generally characterized by the presence of longitudinally orientated smooth muscle cells (LSMC). During an experiment of neo-intima induction in carotid arteries in rabbits, by application of a nonconstrictive silastic cuff, a study was performed to investigate the presence of LSMC in the systemic and pulmonary circulations, in both elastic and muscular arteries. Three patterns could be distinguished: intimai cushions in muscular arteries, single or small groups of LSMC in the intima in elastic and larger muscular arteries, and intra-medially located layers or columns of LSMC in the aorta, the pulmonary artery, at the bifurcation of the aorta and around orifices of branches. In order to understand this peculiar orientation a biomechanical approach was used: this showed that near the lumen the circumferential stress is 4.5 times higher than the longitudinal. Because the cell surface of the smooth muscle cells exposed to this stress per unit vessel length is much less in the longitudinal than in the circular direction we conclude that the LSMC align in the direction which allows them to cope most effectively with the mechanical stresses.     

The regulatory role of FSH in steroid formation by luteinized human granulosa cells in culture

Granulosa cells were aspirated from human pre-ovulatory folliclesfollowing a combined clomiphene-gonadotrophin stimulation inan in-vitro fertilization (IVF) programme. The cells were culturedfor 8 days in medium M199 containing 10% bovine fetal calf serumunder 5% CO₂ in air. Pure human FSH and human LH were addedalone or in combination to the culture in various concentrationsand the progesterone (P) and oestradiol-17 (E₂) levels in themedium were measured every second day by a conventional RIAtechnique. In the presence of FSH or LH the formation of P increased2- to 3-fold with the pronounced effect after 4 to 6 days inculture. Addition of testosterone (T) (3 ? 10^–7 M) tothe culture medium affected neither basal nor gonadotrophinstimulated P formation. In this system, only minute amountsof E₂ were formed and neither FSH nor LH stimulated its formation.When the medium was fortified with T, basal E₂ formation increased50- to 100-fold. FSH further stimulated this conversion significantlyafter 6 and 8 days of culture, while LH had no significant influence.     

Separation of tumor-infiltrating lymphocytes from tumor cells in human solid tumors A comparison between velocity sedimentation and discontinuous density gradients

The separation of viable tumor-infiltrating lymphocytes (TIL) from surgical biopsies of human solid tumors was achieved by velocity sedimentation at unit gravity or by discontinuous density gradients. The two methods were adapted to small volumes and cell numbers not exceeding 1 × 10⁸. The recovery, purity and composition of the TIL-enriched fractions were comparable in the two methods. Density gradients were more rapid, simpler and more practical for preparation under sterile conditions of TIL from clinical material than velocity sedimentation. Lymphocytes in the TIL-enriched fractions obtained by either of the methods were poorly responsive to mitogens. This poor responsiveness is a characteristic of the human TIL and seems to be related to effects exerted by tumor cells.     

Macrophage and perisinusoidal cell kinetics in acute liver injury.

Perisinusoidal cells (PSCs) are currently regarded as the major source of extracellular matrix proteins during hepatic fibrogenesis in response to liver injury. However, the cellular mechanisms underlying their response to injury are not fully understood. One hypothesis is that the PSCs are stimulated by peptide growth factors produced by hepatic macrophages (Kupffer cells) in response to parenchymal cell damage. In this study we have investigated the kinetics of the PSC and macrophage populations in acute carbon tetrachloride-induced hepatic injury in rats. PSCs were identified immunohistochemically by detection of cytoplasmic desmin; monocytes and macrophages were detected using the monoclonal antibodies ED1 and ED2; cells in S phase were identified by immunohistochemical detection of nuclear-incorporated bromodeoxyuridine. The results showed an expansion of the desmin-positive PSC population, predominantly within the damaged perivenular zones, which reached a peak on days 3 and 4 following administration of carbon tetrachloride; this was contributed to by local PSC proliferation. The PSC response was preceded by an expansion of the macrophage population resulting from both local macrophage proliferation and influx of blood monocytes. These results are in keeping with the hypothesis that the PSC response to acute liver injury is mediated, at least in part, by hepatic macrophages.     

大鼠颌下腺、舌下腺副交感节前神经元胞体及树突的研究

将类霍乱毒素B原结合的辣根过氧化物酶(CB-HRP)注入大鼠一侧颌、舌下腺,标记了脑干内支配该腺副交感节前神经元的胞体及其轴、树突。在光学显微镜下,对所标神经元的位置、胞体形态、大小、树突的分布以及轴突在脑干内的行程做了较为系统的观察和测量,并结合有关参数的相关分析,对泌涎神经元的某些生理学特性做了初步探讨。     

Further analysis of ATP-mediated activation of K+ channels in renal epitheloid Madin Darby canine kidney (MDCK) cells

ATP activates K⁺ channels by increasing intracellular calcium activity in Madin Darby canine kidney (MDCK) cells. The present study has been performed to test for the involvement of G-proteins and of protein kinase C in the intracellular transmission of these effects. To this end, the effect of ATP on intracellular calcium and K⁺ channel activity has been studied in cells pretreated with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and/or pertussis toxin. The ATP-induced increase of intracellular calcium is not significantly affected by pretreatment with pertussis toxin, is significantly blunted by pretreatment with TPA and is abolished by pretreatment with both pertussis toxin and the phorbol ester. The ATP activation of K⁺ channels is similarly blunted by pretreatment with TPA, but is not abolished by pretreatment with both the phorbol ester and pertussis toxin. Furthermore, the ATP-induced hyperpolarization is not abolished in cells pretreated with both pertussis toxin and TPA. In those cells, ATP may activate K⁺ channels by calcium-independent mechanisms or lead to localized increases of intracellular calcium sufficient to activate the K⁺ channels but escaping detection with fura-2 fluorescence.