Detection of human herpesviruses 6 and 7 in heart transplant recipients by a multiplex polymerase chain reaction method

Cystic lesions of the pancreas are relatively uncommon. We describe the case of a young man with a complex cystic mass located within the head of the pancreas. The patient underwent exploration with resection of the mass. Pathology revealed a ciliated epithelial cyst, a rare cystic lesion of the pancreas.     

In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase C mu

To investigate whether thromboxane and/or platelet activating factor (PAF) mediate the pulmonary vasoconstrictive response to antigen in vivo, we intra-arterially injected human erythrocytes as antigen into sensitized rabbits after administration of putative inhibitors: a cyclooxygenase synthetase inhibitor (indomethacin, 5 mg.kg-1), a thromboxane synthetase inhibitor (OKY 046, 10 mg.kg-1 + 100 micrograms.kg-1.min-1), and a PAF blocker (CV6209, .1 mg.kg-1). Pulmonary artery and airway pressures significantly increased after the antigen challenge in sensitized rabbits, but did not in nonsensitized rabbits. Both indomethacin and OKY046 significantly inhibited the increase in pulmonary artery pressure after the antigen challenge, while CV6209 did not. CV6209 significantly attenuated the decrease in femoral artery pressure after the antigen challenge, while neither indomethacin nor OKY046 did. There were no significant differences in the increase in airway pressure among the groups. We conclude that thromboxane rather than PAF mediates the pulmonary vasoconstriction after the antigen challenge and that mediators other than thromboxane and PAF mediate bronchoconstriction after the antigen challenge in sensitized rabbits.     

In vitro mutagenesis of PH-20 hyaluronidase from human sperm

The cDNA encoding PH-20 hyaluronidase from human sperm has been mutated at five positions by in vitro mutagenesis. We have changed three acidic amino acids and two arginine residues that are conserved in the sequence of mammalian PH-20 polypeptides as well as in the hyaluronidases from bee and hornet venom. Of the former, the mutants [Gln113]PH-20 and [Gln249]PH-20 had no detectable enzymatic activity; the mutant [Asn111]PH-20 had about 3% activity. The mutant [Thr252]PH-20 was also inactive, while [Gly176]PH-20 had only about 1% activity. This indicates that the PH-20 hyaluronidases, like numerous enzymes that hydrolyze glycosidic bonds, have acidic amino acids in their active site. Moreover, for the binding of the substrate, the polyanion hyaluronan, arginine residues appear to be essential.     

In vivo and in vitro macrophage activation by systemic adjuvants

Six systemic adjuvants of immunity were tested for their ability to induce macrophage activation. Four of them: living BCG, hydrosoluble extracts from BCG (HIU II) and from M.smegmatis (IPM), and lipopolysaccharide from E.coli (LPS), when administered to normal mice render macrophages non-specifically cytotoxic for tumor cells in vitro. The intensity of this phenomenon varied according to the route and time of adjuvant administration. In contrast, lentinan extracted from Lentinus edodes, and levamisole which is a synthetic chemical compound, depressed macrophage cytotoxic potential. BCG, IPM and LPS were shown to have a direct action on macrophages. After in vitro exposure to these agents, the cytotoxic potential of normal macrophages was greatly increased. Levamisole was unable to stimulate this macrophage function directly in vitro. On the other hand, such a macrophage activation has been induced in vitro when normal macrophages were cultivated in the presence of MIF coming from the supernatant of human lymphoblastoid cell lines.     

In vitro growth and drug sensitivity of tumor colony-forming units from human tumor xenografts

To investigate the feasibility of using tissue obtained from human tumor xenografts for in vitro screening of antineoplastic agents, we grew human tumor colony-forming units (CFU) in semisold agar from xenografts serially passaged in nude mice. Growth of human tumor CFU was accomplished from nine xenografts representing five different histological tumor types (ovarian carcinoma, adenocarcinoma of the colon, malignant melanoma, epidermoid carcinoma of the lung, and malignant astrocytoma). Cloning efficiency ranged from 0.04 to 0.1% and showed significant variability both between tumor types and between individual animals bearing the same type of xenograft. A high percentage of tumor CFU was in S phase [47 +/- 20% (S.D.)] as determined by the thymidine "suicide" technique. The number of tumor CFU observed increased linearly with increasing numbers of cells plated. In vitro drug sensitivity of the tumor CFU was assessed to Adriamycin, cis-platinum, and melphalan. The patterns of drug sensitivity were found to be reproducible and stable over a period of 9 months. Drug sensitivity curves to Adriamycin for five xenografts representing four tumor types showed complex patterns with plateau portions similar to those described for tumor CFU from primary tumors. The rank order of sensitivity of the tumors was compared to that of normal granulocyte-macrophage progenitors and, with the exception of the melanomas, was found to correlate well with clinical experience (order of sensitivity = colon less than ovary less than bone marrow). Growth of human tumor CFU from xenografts represents a reproducible and stable means for the study of the biology of tumor CFU and has potential applications as a means for screening new anticancer agents.     

In vitro activation of mouse macrophages by rat lymphocyte mediators

Macrophage-activating factors (MAF)3 were released by presensitized rat lymphocytes stimulated in vitro with the appropriate antigens. Different supernatants of presensitized rat lymphocytes specifically stimulated in vitro with several different mouse, dog, and rat tumor or normal cells were capable of rendering normal rat and mouse macrophages nonspecifically cytotoxic in vitro to their respective syngeneic tumor cells. The release of active mediators by rat lymphocytes sensitized in vivo was dependent upon immunologically specific recognition of an antigen in vitro. When rat lymphocytes were incubated in vitro with antigens unrelated to the in vivo sensitizing antigens, no release of MAF occurred. Once rat MAF was released, it activated both syngeneic (rat) and xenogeneic (mouse) macrophages to kill tumor cells in vitro. These activated marcophages destroyed all syngeneic tumor targets. Such cytotoxicity was obtained even when the cells used to elicit release of MAF were totally unrelated to the target tumor cells. The data thus demonstrated that MAF can cross strain and even species specificities and can activate macrophages to kill tumors in a nonspecific manner. The cytotoxicity mediated by in vitro activated mouse macrophages decreased with time once the macrophages were removed from MAF; and by 7 days postactivation, the macrophages were not cytotoxic. However, when incubated again with MAF, significant reactivation was observed. This suggested that activation of macrophages in vivo may be a continuous process of lymphocyte-macrophage interaction.     

In vitro stimulation of lymphocytes by neutral proteinases from human polymorphonuclear leukocyte granules

Two neutral proteinases from human polymorphonuclear leukocytes (PMN), an elastase and the chymotrypsin-like cathepsin G, were purified, and their actions on lymphocytes in culture were studied. Both PMN proteinases stimulate lymphocytes from human peripheral blood and from mouse spleen in vitro, but do not affect thymic cells from either normal or hydrocortisone-treated mice. In stimulated mouse spleen cell cultures, most of the developing blast cells bear surface immunoglobulins, and subsequently appear to engage in antibody synthesis. In their stimulatory action, the two PMN proteinases thus resemble the classic B-cell mitogen LPS and neutral pancreatic proteinases such as trypsin, chymotrypsin, and elastase. The effects of proteinase inhibitors indicate that lymphocyte stimulation is dependent on the proteolytic activity of the enzymes. This work suggests that PMN proteinases, which are released at sites of inflammation, may modulate the function of lymphocytes.     

Primary human herpesvirus 7 infection: a comparison of human herpesvirus 7 and human herpesvirus 6 infections in children

Atrial fibrillation is an important and independent risk factor for cerebrovascular disease and vascular dementia. There is increasing evidence that atrial fibrillation is associated with an increased risk of asymptomatic or silent cerebral infarction and as a result may confer an increased risk of progressive cognitive impairment on a person. In this study we sought to determine whether this hypothesis could be explored in a prospective case controlled design. Twenty seven patients with non-valvular atrial fibrillation (NVAF) and no history of stroke, transient ischaemic attack, dementia, and thyrotoxicosis were compared with 54 age and sex matched controls in sinus rhythm. All cases underwent clinical examination, ECG, and psychological assessment using a battery of nine neuropsychological tests. Between group analysis and a comparison of mean test scores of paired controls with cases were undertaken. The presence of atrial fibrillation was consistently associated with poorer performances on all the subtests of the neuropsychological battery. There was no association between duration of atrial fibrillation and performance. These results provide evidence to justify further examination of the hypothesis in a larger prospective study to determine whether antithrombotic therapy may protect against cognitive decline in patients at maximal risk of silent cerebral ischaemia and associated cognitive decline.     

In vitro culture of human peritoneal fluid cells

The author has studied the behaviour of cells from human ascitic fluid in long-term culture (5-6 months). Three cellular types are described with different morphological features, namely the cellular shape, the fashion in which the cell spreads on the glass, the nucleocytoplasmic ratio, the chromatin appearance and the abundance of mitochondria. The three cellular types can phagocytose, but each one in a different way. The first one phagocytoses exclusively erythrocytes 'by contact' without emission of pseudopods; the second one phagocytoses degenerating nucleated cells in the same way as the first; the third type phagocytoses degenerating nucleated cells by emission of long pseudopods. The origin of these three cellular types is discussed; it is felt that they are transformed mesothelial cells. According to this study, it cannot be excluded, especially for the second and the third type, that they are histiocytes coming from serous membranes. The life in vitro of the three cellular types is depending upon the composition of nourishing medium. Cells can divide by mitosis only during the first 10 - 15 days of culture (mitotic index 0.1-3.0(0/00). Nuclear amitosis, nucleolus expulsion into cytoplasm and cytoplasmatic DNA synthesis can be observed in healthy cells.     

In vitro generation of human dendritic cells and cell therapy

HHV-7 growth on Sup-T1, an immature T-cell line, was studied using different HHV-7 isolates obtained in our laboratory. Titration of viral yields showed that all the virus isolates propagate on this cell line more efficiently than in cord blood lymphocytes, the cells usually recommended for HHV-7 growth. The permissivity of Sup-T1 to HHV-6, whose ability to replicate in these cells was still unknown, was also investigated using two virus isolates representative of variants A and B respectively. Both isolates were able to propagate on Sup-T1 and viral titres were similar to those obtained in cord blood lymphocytes. As the efficient propagation of both HHV-7 and HHV-6 isolates in Sup-T1 cultures, these cells may replace more time consuming and expensive cord blood lymphocyte preparations for the propagation of both the viruses.     

Cobalt-chromium-molybdenum but not titanium-6aluminium-4vanadium alloy discs inhibit human T cell activation in vitro

The Authors studied the morphological, biochemical, physico-chemical and biological characteristics of Vibrio anguillarum cultured on different growth conditions, characterized by low osmolarity and high temperature (37 degrees). One culture was subcultured for several days in tryptone soya agar with 0.5% Nacl at 37 degrees C incubation until the cell morphology was stabilized. The low osmolarity, through an osmotic shock, induced remarkable morphological modifications in the strain, evidenced by optic and electron microscopic studied; in addition SDS-PAGE analysis of saline extracts from the culture at 37 degrees C showed a specific new protein band of about 66KDa. This band was correlated with remarkable differences in outer membrane protein composition (OMPs) evidenced by Ag/Ah cross-reactions with rabbit hyperimmune sera against the modified and the reference V. anguillarum strains. Finally, the modified strain proved to be non pathogenic for trout and sea bass.     

In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain

Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Deltap6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Deltap6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Deltap6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Deltap6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Deltap6 and nucleic acid are not sufficient for the formation of normal-sized particles.     

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.     

In vitro percutaneous penetration of methyl-parathion from a commercial formulation through the human skin

OBJECTIVE: To compare in vitro percutaneous absorption of methyl-parathion dissolved in an acetone vehicle and in the form of a commercial formulation. METHODS: Penetration through the human skin was measured in Franz diffusion cells with full thickness skin from a human cadaver as the membrane. The two tailed non-parametric Mann-Whitney U test was used to compare the cumulative diffusion of methyl-parathion in the receptor fluid of the cells at various time intervals. RESULTS: In vitro skin penetration of methyl-parathion was significantly higher with the commercial formulation. The percentage of the applied dose absorbed after 24 hours was 5.20% v 1.35%. The mean lag time was < 8 hours. CONCLUSION: Assessments of uptake and internal dose after exposure to pesticides should be based on the commercial products rather than active ingredients, because of the crucial role of the vehicle, as shown in this study.     

In vitro effects of diazepam on human ciliary function

This study demonstrated the in vitro effect of diazepam on human ciliary beat frequency (CBF) from fifteen normal subjects using brush cytology. Tube A contained culture medium, tube B the diluent that diazepam intravenous injectate was carried in and culture medium (controls). Tube C, D and E contained, 0.4 mg/l, 4.0 mg/l and 40.0 mg/l of diazepam in culture medium respectively. The mean effective diazepam concentration in plasma is 0.4 mg/l. CBF was measured photometrically. The most vigorous cilia were measured in 5 areas taking 10 readings on each sample, 30 minutes and 1 hour after mixing. Standard deviation (SD) and confidence limits were calculated along with significance testing (p < 0.05) using the paired t-test and ANOVA. The mean of the CBF of tubes A and B were 13.44 (SD 2.65) and 13.67 (SD 2.48). There was a reduction of the CBF with increasing concentrations of diazepam at 30 minutes, 11.32 (SD 2.14), 10.29 (SD 1.58) and 4.14 (SD 1.57) tubes C. D and E respectively. There was a significant lowering in CBF of 17% (p < 0.01) of diazepam at the mean effective plasma level (tube C) when compared against the controls. CBF decreased over time and at 1 hour was 10.57 (SD, 1.36), 9.02 (SD, 1.39) and 3.58 (SD, 1.31) tubes C, D and E respectively. A proposed mechanism of altered intracellular calcium flux via the action diazepam on GABA receptors is described.     

In vitro study of pesticide hematotoxicity in human and rat progenitors

Some pesticides are hematotoxic and cause aplastic anemia, agranulocytosis, neutropenia, and thrombopenia. In order to evaluate hematopoietic progenitor cultures in the exploration of pesticide hematotoxicity, human and rat colony-forming unit-granulocyte and macrophage (CFU-GM) were cultured in the presence of different concentrations of pesticides, known to be either hematotoxic or innocuous for blood cells. The results were compared to the control culture of the same sample. Four insecticides (lindane, azinphos, mevinphos, parathion methyl), three herbicides, (2,4,5, T, bromacil, MCPA), and two fungicides (fosethyl-aluminum, DNOC) were tested and exhibited the following data: 1) Pesticides, known to be hematotoxic, inhibited the development of progenitors. Different phenomena were observed and suggested different mechanisms: cell destruction, block in mitosis, decrease or delay in mitosis. 2) Difference in sensitivity to molecules between human and rat progenitors was observed. Human progenitors were more sensitive to pesticides, except for 2,4,5 T.     

In vitro expansion and characterization of dendritic cells derived from human bone marrow CD34+ cells

Dendritic cells (DC), as professional antigen-presenting cells, play a major role in stimulating naive T cell responses in vivo and in vitro, and may exacerbate or modulate T lymphocyte-mediated reactions, such as interactions between a hematopoietic graft and the recipient, eg GVHD and graft-versus-leukemia. Here, we describe a two-stage cell culture system for expansion of functionally active human DC from CD34+ marrow precursors. Optimal outgrowth was achieved by initially culturing CD34+ cells for 5 days in medium containing GM-CSF, MGF and TNF-alpha. Substitution of CD40L and IL-4 for TNF-alpha during a subsequent 5-day subculture increased DC content, such that by 10 days the cultures contained approximately 40% DC as determined by immunophenotype and morphology. An increase in DC purity to 84% at 10 days was achieved by immunomagnetic separation for CD1a+ cells from 5-day cultures and subculturing these cells in medium with IL-4 and CD40L. Reversing the sequence of growth factors during culture and subculture decreased the yield and purity of DC. Expression of CD80 and CD86 was enhanced by adding CD40L and IL-4, and the DC showed stimulatory activity in MLC. In conclusion, we have described a simple two-stage culture system to generate functional DC from CD34+ marrow precursors.