一种引致人体原发性皮肤毛霉病的非高温根毛霉属真菌

由石家庄中国人民解放军白求恩国际和平医院皮肤科李成龙大夫提供的一株人体皮肤病菌经我们研究鉴定为毛霉目(Mucorales)毛霉科(Mucoraceae)根毛霉属(Rhizomucor)的一个种,并定名为多变根毛霉新种(Rhizomucor variabilis Zheng & G.-q.Chen sp. nov.)。据李大夫介绍,这株菌是从一名居住在江苏农村到该医院看病的女病人的手上病部分离的。这个病人没有一般真菌病病人所患有的其他疾病如糖尿病、白血病等等。她也没有患有其他毛霉病,因此她的皮肤毛霉病是原发性的而不是继发性的。我们查阅文献结果,国内由根毛霉属真菌引致的毛霉病过去仅有过一次肺部感染的报道;国外则有过较多次数的由根毛霉引致的人体毛霉病,主要为肺部疾病并可引致继发性的皮肤病,尚未见有由根毛霉属引起的原发性皮肤毛霉病的报道。无论国内、外引起人体毛霉病的根毛霉均为微小根毛霉[Rhizomucor pusillus(Lindt) Schipper,包括Mucor pusillus Lindt,Mucor parasiticus Lucet & Costanin等异名]一种。因此,本病例为我国第二例由根毛霉弓l起的人体毛霉病及第一例由根毛霉引起的人体皮肤毛霉病,同时又是全世界第一例由根毛霉属除微小根毛霉以外的另外一个种引起的人体毛霉病,很可能还是全世界第一例由根毛霉引起的人体原发性皮肤毛霉病。多变根毛霉与根毛霉属内所有过去已报道过的种都有显著差异。它的最适生长温度为24-30℃,最低9℃,最高38℃;其他种均为高温真菌,它们的最高生长温度可达55℃或更高。形态方面,多变根毛霉也与属内其他已知种明显不同。它的菌落高达4-8 mm并呈鲜明的浅黄色;其他已知种菌落低矮,除奈尼塔尔根毛霉(Rhizomucor nainitalensis Joshi)外全部为深暗灰色,奈尼塔尔根毛霉菌落色泽虽然较浅,但为浅灰或灰黄色,与多变根毛霉的鲜明黄色不同。它的假根异常发达并可从菌体的各个部位如菌丝、匍匐丝、孢子枝、孢子囊、囊轴上长出;其他已知种的假根一般都不发达且从未见有从孢子枝、孢子囊、囊轴等处长出的描述。它的孢子枝的分枝常常长于主枝;其他已知种则分枝长度一般不超过主枝。它的囊轴形状多变:球形、近球形、扁球形、卵形、椭圆形、梨形等等,两边对称或不对称,溢缩或不绕缩,纵向深裂或不作纵向深裂;其他已知种的囊轴形状通常为倒卵形至梨形的规则形状。它的囊领明显;其他已知种的囊领均很不明显至缺如。它的孢囊孢子形状和大小变化都较大,卵形、椭圆形、矩圆形、近球形、近三角形或其他各种不规则形状,长度范围2.5-16.5 μm;其他已知种除上面已经提到过的奈尼塔尔根毛霉外,它们的孢囊孢子形状仅限于卵形、椭圆形、近球形等较规则的形状,长度范围总是在3-6 μm范围内,奈尼塔尔根毛霉的孢囊孢子形状虽然多变,但其决度亦在3-6 μm的范围内。此外,多变根毛霉的孢子囊、囊轴、孢囊孢子等各种构造均较大;其他已知种则较小,其中肿梗根毛霉[Rhizomucor tauricus (Milko & Schkurenko) Schipper]虽亦较大,但除此之外与多变根毛霉迥异。多变根毛霉未见有接合孢子,将我们保存的全部微小根毛霉菌株与它分别配对时,或将我们的几对别的属的(+)(-)测试菌株与它分别配对时,均未见形成接合孢子或有任何反应。     

Transforming growth factor-beta does not alter interleukin-1 expression in cultured human macrophages

Transforming growth factor-beta (TGF beta) is a growth modulator that stimulates the growth of fibroblastic cells but inhibits the growth of cells of epithelial origin. TGF beta also influences the production of extracellular matrix proteins, and of proteases and the type 1 plasminogen activator inhibitor (PAI-1) by cultured cells. TGF beta appears also to have various immunoregulatory effects, suppressing both T- and B-cell activities. It has been proposed that it might increase the expression of interleukin-1 (IL-1) mRNA in cultured human monocytes, thus potentiating immune functions. To analyze the role of TGF beta in IL-1 production we have now quantitated the effect of this factor on the production of biologically active IL-1 as well as IL-1 beta mRNA expression. The effect of TGF beta on IL-1 production optimally activated with bacterial lipopolysaccharide (LPS) was also studied. It was found that IL-1 activity and mRNA levels were rapidly elevated by LPS but not by TGF beta. Culture fluids from monocytes treated with TGF beta alone or with TGF beta plus LPS inhibited the proliferation of the test thymocytes. After gel filtration, the media from TGF beta-treated cultures showed no activity in the molecular weight area of IL-1 (approx. 15 kD), while the supernatants from TGF beta plus LPS-induced cells contained IL-1 activity in these fractions, the magnitude of which was, however, at the same level as in the culture fluids derived from cells stimulated with LPS alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.Abbreviations ABZ albendazole - B[a]P benzo[a]pyrene - HPLC high-performance liquid chromatography - MC 3-methylcholanthrene - MFO mixed-function oxidase - UDPGT UDP-glucuronyltransferase     

Inhibition of hexose transport in the human erythrocyte by 5, 5′-dithiobis(2-nitrobenzoic acid): Role of an exofacial carrier sulfhydryl group

Summary The sulfhydryl reagent 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1mm DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10mm cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120mm accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 m cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfydryl, but reaction of this group may not always lock the carrier in an outward-facing conformation.     

Epidermolysis bullosa simplex: Expression of gelatinase activity in cultured human skin fibroblasts

We have measured the baseline level of gelatinase in fibroblast-conditioned medium from 41 Scandinavian individuals. They comprised 12 healthy persons, 11 individuals with the skin disorder dominant epidermolysis bullosa simplex (DEBS), 16 patients with other types of epidermolysis bullosa (EB) and 2 siblings with prolidase deficiency. These results divide the cell strains into those with low and those with high activity levels. Although this dual biochemical trait occurred in all the groups of individuals, the high-activity trait was more frequent among the DEBS patients. The localized DEBS forms showed an elevated activity level, in contrast to the previously reported generalized DEBS Köbner forms. Although a high level was found in some individuals with other EB forms, the high incidence in four families with localized DEBS Weber-Cockayne (eight of eight) and a single family with generalized DEBS—mottled pigmentation (two of two) may result from a close linkage between an EB gene and a gene responsible for the biochemical trait. In addition, in the single complete family tested, the level of gelatinase activity in cultured fibroblasts seemed to be regulated by codominant alleles or genes. A raised baseline level of gelatinase activity in cultured skin fibroblasts may be the result of either an altered expression of gelatinase or an allelic variant of this enzyme with increased specific activity. Further studies of gelatinase in cultured fibroblasts may provide insight into the regulatory mechanism and genetics behind this activity and allow formal linkage studies versus DEBS.This work was supported in part by the Norwegian Research Council for Science and the Humanities (NAVF) and the Concerted Action on Hereditary Connective Tissue Diseases of the European Community (1990–1992, project leader, M. Matton).Part of this work was performed at the Department of Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo.     

Interaction of free fatty acids with the erythrocyte membrane as affected by hyperthermia and ionizing radiation

The interference of hyperthermia and ionizing radiation, respectively, with the effects of capric (100), lauric (120), myristic (140), oleic (cis-181) and elaidic (trans-181) acids on the osmotic resistance of human erythrocytes was investigated. The results are summarized as follows: (A) not only at 37°, but also at 42° and 47°C lauric acid (120) represents the minimum chain length for the biphasic behaviour of protecting against hypotonic hemolysis at a certain lower concentration range and hemolysis promotion at subsequent higher concentrations; (B) with increasing temperatures the protecting as well as the hemolytic effects occur at lower concentrations of the fatty acids; (C) the increase of temperature promotes the extent of hemolysis and reduces the extent of protection against hypotonic hemolysis; (D) Gamma-irradiation of erythrocytes selectively affects the concentration of oleic acid at which maximum protection against hypotonic hemolysis occurs, without altering the minimum concentration for 100% hemolysis.     

Determination of mercury in human serum and packed blood cells by neutron activation analysis

A method is described for the determination of mercury in human blood serum and packed blood cells employing neutron activation analysis. Great attention was devoted to the collection and manipulation of the samples. The accuracy and precision of the method were tested by analyzing biological reference materials and by comparing the concentrations measured in a number of serum samples to those obtained by another, independent technique (cold vapor atomic absorption spectrometry) in the same samples. The article reports the levels measured in blood serum and packed blood cells samples from 15 adult volunteers, as well as the figures determined in a “second-generation” biological reference material (freeze-dried human serum), prepared and conditioned at the University of Ghent.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

Characterization of an in vitro system of human renal papillary collecting duct cells

Summary We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10⁴ live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins. Part of this work was presented at a Symposium of the Center for Alternatives to Animal Testing, April 4–5, 1989, Johns Hopkins Medical Institutions, Baltimore, MD 21205. This work was supported in part by grants R01-AI24179, PO1-A804393 for the Public Health Service, U.S. Department of Health and Human Services, and by a grant from the National Kidney Foundation, Baltimore, MD affiliate.     

Cytogenetic characterization of 20 lymphoblastoid lines derived from human individuals differing in bleomycin sensitivity

Summary Forty lymphoblastoid (lymphoid) lines were established from 42 volunteer blood donors, including healthy individuals and patients with head and neck carcinomas. Each peripheral blood sample was split into two portions, one for the establishment of a lymphoid line and the other for short-term culture, which was used to estimate bleomycin sensitivity by cytogenetic procedures. Twenty lymphoid lines were selected at random to compare bleomycin sensitivity with data obtained from short-term lymphocyte cultures. In each set, bleomycin sensitivity of lymphoid cells was similar to that of the lymphocytes. The lymphoid lines, which can be propagated for an unlimited supply of relatively homogeneous cellular material, will be useful for a variety of future investigations. This investigation was supported by grants from the John S. Dunn Foundation, Houston, TX, the Esther Knispel Fund administered by The University of Texas M. D. Anderson Cancer Center, Houston, TX, and Department of Health and Human Services PHS grant DE 07007.