Selective aflatoxin B1-induced sister chromatid exchanges and cytotoxicity in differentiating B and T lymphocytes in vivo

The purpose of this study was to assess the genotoxic and cytotoxic effects of the fungal metabolite aflatoxin B1 (AfB1) on the developing immune system of the chick embryo, a model in vivo system. Of particular interest was the assessment of AfB1 -mediated selective toxicity toward developing B lymphocytes as compared to T lymphocytes. In vivo bromodeoxyuridine (BrdU) labelling of DNA was used to detect the induction of sister chromatid exchanges (SCE) in lymphocytes and to assess the progression of these cells through successive cell cycles. Cytotoxicity was also assessed by studying the entrance and maintenance of cells in mitosis (mitotic index). Graded doses of AfB1 (1.09–17.4 μ/g embryo) were applied to chick embryos of 18 days of incubation (Dl). Embryos also received two doses of BrdU at 3 mg/200 μ (3 hr apart) to provide continuous labelling of B and T lymphocyte replicating DNA. B and T lymphocytes were harvested 20 hr post-AfB1/BrdU exposure from the bursa and thymus, respectively, and were processed for cytogenetic analyses. AfB1 induced dose-related increases in SCE in B lymphocytes; this induction was 6- to 8-fold that of controls at the higher doses tested, AfB1 -mediated induction of SCE in T cells was just 2-fold that of controls at the highest dose tested. AfB1 reduced the progression of B cells and to a lesser extent T ceels through successive rounds of replication. Furthermore, AfB1 dramatically reduced the mitotic index of B cells but not of T cells. These data indicate both selective genotoxicity and cytotoxicity of AfB1 toward B cells in the late stage embryo. © 1993 Wiley-Liss, Inc.     

In vivo and in vitro studies of the site of inhibitory action of omeprazole on adrenocortical steroidogenesis

Summary The site of omeprazole inhibition of adrenal steroidogenesis has been sought in vivo by analyzing the patterns of urinary steroid metabolite excretion after 6 days of treatment with placebo/omeprazole.Excretion rates of androsterone, aetiocholanolone, dehydroepiandrosterone, 11 hydroxyandrosterone, tetrahydrocortisone, tetrahydrocortisol and cortolone were reduced, indicating a block at an early step in steroidogenesis, possibly cholesterol side-chain cleavage. In vitro studies have confirmed this finding by measuring conversion of added precursors to cortisol in isolated bovine adrenocortical cells. Cortisol synthesis from added 20 hydroxycholesterol was inhibited by 83% in the presence of 100 µg omeprazole/ml. Conversion from pregnenolone and progesterone and their 17 hydroxylated derivatives was inhibited by 20–40% whereas cortisol production from added 11 deoxycortisol was not affected.These data suggest that omeprazole primarily inhibits cholesterol cleavage and does not inhibit 3 hydroxysteroid dehydrogenase, 17 hydroxylase or 11 hydroxylation; 21 hydroxylase activity may be marginally attenuated.     

Plädoyer für die primäre Resektion mit primärer Anastomose bei der komplizierten Sigmadiverticulitis

Zusammenfassung Von 1973 bis 1986 wurden 107 Patienten mit einer komplizierten Sigmadiverticulitis operiert. Es handelte sich um 47 Frauen und 60 Männer bei einem Durchschnittsalter von 62 Jahren. Von 107 Patienten wiesen 14 eine perforierte Diverticulitis mit diffuser eitriger/kotiger Peritonitis auf, 68 Patienten eine perforierte Diverticulitis mit lokalisierter eitriger Peritonitis/paracolischem Absceß und 25 Patienten eine akute phlegmonöse Diverticulitis ohne Perforation. Zusätzliche pathologische Befunde waren: innere Fisteln (13 Patienten), nekrotisierende Fasciitis (3 Patienten), Stenose mit Ileus (3 Patienten) und synchrone Carcinome (7 Patienten). Die Gesamtletalität der 107 Patienten betrug 9,3% (=10 Patienten), die Morbidität der 97 überlebenden Patienten 34,0% (= 33 Patienten). Die Letalität bei 14 Patienten mit perforierter Diverticulitis und diffuser eitriger/kotiger Peritonitis lag bei 50%, die der 68 Patienten mit perforierter Diverticulitis und lokaler eitriger Peritonitis/ paracolischem Absceß bei 4,4%, die der 25 Patienten mit akuter phlegmonöser Diverticulitis ohne Perforation bei 0%. Von den 10 Patienten verstarben 7 bei Operation ihrer perforierten Diverticulitis mit diffuser eitriger Peritonitis, 1 (5) nach primärer Resektion mit primärer Anastomose, 3 (5) nach Operation nach Hartmann, 3 (4) nach alleiniger Anlage einer Colostomie. Bei Operation der perforierten Diverticulitis mit lokaler Peritonitis verstarben 3 Patienten, 2 (6) nach Operation nach Hartman und 1 (5) nach alleiniger Anlage einer Colostomie. Trotz einer hohen Zunahme der Zahl primärer Resektionen mit primärer Anasto mose im Zeitraum 1980–1986 ergab sich im Vergleich zum 7-Jahresabschnitt 1973–1979 ein Rückgang der Letalität von 35,7% auf 0% bei dieser Operation. Ihre Anwendung ist auch gerechtfertigt bei der perforierten Diverticulitis mit lokaler wie diffus eitriger oder kotiger Peritonitis.

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Primary resection with primary anastomosis in complicated diverticulitis of the sigma

Summary Of the 107 patients with complicated diverticulitis operated from 1973–1986 47 were females and 60 males. In 14 of the 107 patients a perforated diverticulitis with diffuse purulent/faecal peritonitis was found, a perforated diverticulitis with localized purulent peritonitis/paracolic abscess in 68 patients and an acute phlegmonous diverticulitis without perforation in 25 patients. Additional pathologic findings were internal fistulae (13 patients), necrotizing fasciitis (3 patients), obstruction (3 patients) and synchronous carcinoma (7 patients). The overall mortality of the 107 patients was 9.3 % (=10 patients) and the morbidity of the 97 survivors 34% (=33 patients). The mortality of the 1.4 patients with perforated diverticulitis and diffuse purulent peritonitis was 50%, of the 68 patients with perforated diverticulitis and localized purulent peritonitis 4.4% and of the 25 patients with acute phlegmonous diverticulitis 0%. Seven of the 10 patients died after operation of the perforated diverticulitis with diffuse purulent peritonitis — 1 (5) after primary resection with primary anastomosis, 3 (5) after Hartmann procedure, 3 (4) after loop colostomy alone. Three patients died after operation of the perforated diverticulitis with localized purulent peritonitis — 2 (6) after Hartmann procedure, 1 (5) after loop colostomy alone. In spite of forcing the primary resection with primary anastomosis in the years from 1980–1986 the mortality decreased for these operations from 35.7% in 1973–1979 to 0% in 1980–1986. The indication of primary resection with primary anas tomosis is justified also for perforated diverticulitis with localized and diffuse peritonitis.

Auszugsweise vorgetragen auf dem Symposium Entzündliche Darmerkrankungen der Medizinischen Akademie Carl Gustav Carus, Dresden, 20. November 1987     

Autoimmune hepatitis type 1 and primary biliary cirrhosis have distinct bone marrow cytokine production

We have recently reported differences in the hematopoiesis between autoimmune hepatitis type 1 (AIH-1) and primary biliary cirrhosis (PBC). In view of the notion that cytokines are regulators of hematopoiesis, we investigated in our tertiary center the cytokine production in the bone marrow (BM) of the same consecutive cohort of patients (13 AIH-1, 13 PBC, 10 healthy and 7 patients with cirrhosis due to chronic hepatitis B). Interferon-gamma (IFN-gamma), interleukin-4 (IL-4), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) were determined in the supernatants of long-term BM cultures by ELISAs. IL-4, TNF-alpha and TGF-beta were found significantly increased in the BM of PBC patients compared to AIH-1 and both control groups. AIH-1 patients had significantly higher BM IL-10 compared to PBC patients and higher IL-10, IL-4 and TNF-alpha compared to controls. BM IFN-gamma was significantly higher in PBC and AIH-1 patients compared to controls. In AIH-1 patients, IL-10 was positively correlated with CD34+, CD34+/CD38- and CD34+/CD38+ cell proportions. In conclusion, the BM cytokine microenvironment of PBC and AIH-1 patients differs significantly compared to that of healthy individuals and cirrhotic patients of non-autoimmune etiology. Differences were also found between patients with PBC and AH-1. The implication of BM in the pathogenesis of autoimmune liver diseases is possible and needs further investigation.     

Cytogenetic diagnosis using midtrimester fetal blood samples: Application to suspected mosaicism and other diagnostic problems

Cytogenetic studies on fetal blood cells obtained at 18–25 weeks gestation have provided information for decision making in 25 cases identified as being at high risk of having an abnormal fetus. In particular, in the 21 cases studied to consider the possibility of true mosaicism, confirmation in fetal blood was obtained in three, one of which presented as a pseudomosaic on the original amniotic fluid cell study. Fetal blood was also informative in two cases (one positive and the other negative) in which a diagnosis of the fragile X syndrome was being considered. Furthermore, when high risk pregnancies presented late in gestation (21–24 weeks), these methods allowed for a rapid cytogenetic diagnosis. The procedure has proved useful in most of these cases since the couples involved had indicated that they would probably have terminated the pregnancy without the reassurance of normal fetal lymphocyte studies. Since the technique carries a much higher risk of pregnancy loss than does amniocentesis, its use should only be considered when there are compelling indications.     

Non-pathogenic Entamoeba histolytica: functional and biochemical characterization of a monoxenic strain

We have cultured under monoxenic conditions and characterized an Entamoeba histolytica clone, MAV-I CINVESTAV (MAV-I), obtained from feces from an asymptomatic carrier. The clone shows the non-pathogenic E. histolytica zymodeme type I, which did not change through the process of monoxenization. Clone MAV-I was non-pathogenic in both in vivo and in vitro tests, and it did not have a functional 112-kDa adhesin. As far as we know, this is the first non-pathogenic monoxenic strain reported. Clone A (strain HM1:IMSS), a highly virulent clone with pathogenic zymodeme type II, and which has the 112-kDa adhesin, was used as a control. Protein patterns from both clones were almost identical in one-dimensional gels. In two-dimensional gels, differences in high-molecular-weight proteins were detected. Clone MAV-I adhered and phagocytosed only 12% of the red blood cells adhered and phagocytosed by clone A. MAV-I trophozoites did not destroy cell culture monolayers and did not produce hepatic abscesses in hamsters. They also showed deficiency in protease activity. The absence of virulence in clone MAV-I correlated directly with the absence of a functional 112-kDa adhesin, supporting the role that this protein plays in virulence.     

大鼠脑皮质微血管内皮细胞培养和形态学观察

为获取较纯净脑微血管内皮细胞进行血脑屏障的病理生理研究，我们采用脑组织匀浆、过滤和酶消化技术分离大鼠脑微血管，对分离的脑微血管内皮细胞进行了体外培养和形态学观察。倒置显微镜下，细胞具有单层“卵石样”排列的典型特征、电镜观察可见细胞间连接，免疫酶技术显示，95％以上的细胞为第Ⅷ因子相关抗原反应阳性，进一步证实为血管内皮细胞。     

Preparation of primary monolayer cultures of mouse pancreatic epithelial cells

Summary We describe a technique using normal and diabetic (dbdb) mice to establish primary pancreatic cultures that spread and assume a characteristic epithelial morphology. These cultures contain 4 to 7% beta cells, secrete insulin in response to stimuli for 10 to 14 d, contain few fibroblasts, and have a cell viability that is greater than 95%. The cells attach firmly to glass cover slips and are ideal for the study of insulin secretory granules or contractile proteins using indirect immunofluorescence.     

The major histocompatibility complex (CyLA) of the cynomolgus monkey. I. Serologic definition of 21 specificities

Thirty-seven lymphocytotoxic antisera, 27 of which were raised by immunization with skin grafts and blood from partially matched donors, were tested against cells obtained from 218 unrelated animals and 205 offspring from a colony of cynomolgus monkey (Macaca fascicularis). Evidence was obtained for the presence of at least 21 specificities defined by cluster analysis and segregation within families. Allelic relationships between 16 specificities was suggested by segregation patterns, the absence of triplets and statistical analysis of association in the unrelated population sample. The data support a two-locus model, with tentative assignment of seven specificities to the A locus and six to the B locus. That these lymphocyte alloantigens constitute the major histocompatibility complex (MHC) of the cynomolgus monkey is suggested by analogy with other known MHCs and by the increased survival times of skin grafts between paternally matched half sibs compared to haplodistinct full sibs.     

Cell-Growth Kinetics and Karyotype Analysis of Human Memory Lymphocytes Responding to Alloantigen and Mitogen

Peripheral-blood lymphocytes were primed in vitro with the mitogen phytohemagglutinin (PHA) or with allogeneic cells and their memory responses studied following sequential restimulation with either mitogen or alloantigen. Chromosome preparations were made every 12 hours following exposure to the stimulating agents. Cultures were labeled with BUdR for sister-chromatid staining of the chromosomes which provided information about the kinetics of cell growth and rates of sister chromatid exchange. Cultures containing no BUdR were used for the investigation of cell karyotypes after chromosome-banding.Following PHA as well as alloantigen restimulation, an earlier reaction of the responding cells was observed. The peak response after the first stimulation was found at 120 h with allogeneic stimulation and at 60 h with mitogen stimulation. In the second round of stimulation, the peak occurred after 48 h (allogeneic) and 36 h (PHA) and following the third stimulation after 36 h (allogeneic) and 24 h (PHA). The speed of cell growth was decreased following restimulation with either alloantigen and mitogen. In contrast to the allogeneic restimulation, the number of cells responding after PHA restimulation was decreased.No systematic numerical or structural aberration of the karyotype was detected following repeated stimulation with either alloantigen or mitogen. In this sense, the lymphocyte subpopulations selected by repeated stimulation did not differ from the starting material. On the other hand, the sister-chromatid exchange (SCE) frequency was increased following allogeneic restimulation, whereas it remained constant with PHA restimulation.