Mitochondrial ultracondensation, but not swelling, is involved in TNF alpha-induced apoptosis in human T-lymphoblastic leukaemic cells.

Mitochondrial permeability transition (PT) pore opening and mitochondrial swelling have been reported in association with apoptosis. Conformational alterations of mitochondria induced by tumour necrosis factor-alpha (TNF alpha), and the association with TNF alpha-induced apoptosis, were, therefore, studied in the human acute T-lymphoblastic leukaemia (T-ALL) cell line, CCRF-CEM and its vinblastine-resistant CEM/VLB100 cell line by transmission electron microscopy (TEM). The CEM/VLB100 cell line possessed more condensed (C phase) mitochondria in the resting state compared with its parental cell line, consistent with increased activity of the mitochondrial electron transport chain (ETC). Following exposure to TNF alpha, conformational alterations of mitochondria occurred in both apoptotic and non-apoptotic cells. Orthodox (O phase) mitochondria in non-apoptotic cells underwent C-phase, transitional O-phase and slightly swollen (S-phase) conformational changes. TNF alpha-induced mitochondrial swelling was a late event and was found to a far lesser extent than mitochondrial condensation. No swollen mitochondria were observed in apoptotic cells. Ultracondensed (UC phase) mitochondria were observed in cells undergoing both TNF alpha-induced and spontaneous apoptosis and were seen when TNF alpha-induced apoptosis was inhibited by 3-methyladenine (3MA). The structural integrity of UC phase mitochondria persisted through the apoptotic process. We conclude that TNF alpha-induced mitochondrial swelling and apoptosis are separate events. Mitochondrial ultracondensation is associated with the processes signalling apoptosis and is not a result of TNF alpha-induced apoptotic shrinkage.

Inhibition of autophagy abrogates tumour necrosis factor alpha induced apoptosis in human T-lymphoblastic leukaemic cells.

The pattern and the sequence of tumour necrosis factor-alpha (TNF alpha) induced cell death in the acute T-lymphoblastic leukaemic cell line CCRF-CEM and its vinblastine-resistant subline CEM/VLB100 have been studied. Previously, we found that the CEM/VLB100 cell line was more sensitive to TNF alpha-induced killing than its parental CCRF-CEM cell line. TNF alpha-induced cell death showed an apoptotic pattern, as detected by agarose electrophoresis, flow cytometry and transmission electron microscopy (TEM). TEM images revealed that autophagy and codensed mitochondria occurred earlier than nuclear fragmentation. The specific inhibitor of autophagy, 3-methyladenine (3MA), inhibited the formation of autophagosomes. TNF alpha-induced DNA fragmentation and cytolysis were completely inhibited by 10 mM 3MA. Inhibition of the fusion of lysosomes with autophagosomes by asparagine did not block TNF alpha-induced apoptosis. In addition, amino acid and protein deprivation enhanced TNF alpha-induced autophagy but not apoptosis. We propose that the early stages of autophagy are required for, but do not necessarily result in, TNF alpha-induced apoptosis.

Small virus-like particles bud from the cell membranes of normal as well as HIV-infected human lymphoid cells.

Electron microscopy (EM) of cell sections showed cell associated virus-like particles (VLP), 50-60 nm in diameter, budding from the membrane of human lymphoid cells in culture. The particles had an envelope continuous with the cell membrane and a dense core that almost filled the particle. Particles 70-80 nm in diameter with prominent external spikes were found in the culture medium by negative staining (medium-associated VLP). Cell-associated VLP were also present in cord lymphocytes, both on initial separation and after culture with or without foetal calf serum, and therefore were considered to be endogenous to the cells and were not bovine diarrhoea virus. VLP were observed in most of the lymphoid cell lines examined. VLP were also found less frequently in established human tumour and nontumour cell lines. Both cell-associated and medium-associated VLP were also present in HIV infected cell cultures, and they could be distinguished from HIV by their characteristic morphology and smaller size. It was not determined whether the 2 types of particle represented the same entity, or whether they were defective virus particles or a cellular secretory product.

Interactions of a nonneutralizing IgM antibody and complement in parainfluenza virus neutralization.

While many viruses activate the complement cascade directly, this is generally not a neutralizing event in the absence of antibody. We used a nonneutralizing IgM monoclonal antibody to parainfluenza virus type 3 (PIV3) hemagglutinin-neuraminidase (HN) to explore the role of antibody in complement-dependent neutralization of PIV3. Neither the antibody nor nonimmune guinea pig serum (GPS) neutralized PIV3 significantly, but a more than 100-fold reduction in titer was found when antibody and GPS were combined. Heat-inactivated GPS or GPS lacking either of two different complement proteins were all inactive with or without antibody. Specific repletion of the deficient sera with highly purified complement proteins restored neutralizing activity, indicating an absolute requirement for the classical pathway of complement activation and lytic terminal complement components, and viral lysis was confirmed by electron microscopy. The presence of antibody before complement activation was essential; later addition had no effect. Spontaneous complement activation by PIV3 occurred via the classical pathway in the absence of antibody. Addition of antibody did not alter the overall rate or extent of complement component C3 binding to PIV3 in these experiments. We conclude that certain nonneutralizing antibodies may support complement-dependent PIV3 neutralization by facilitating viral lysis. This process does not, however, involve enhanced activation through the C3 step. Lysis may require antibody-dependent localization of the membrane attack complex or reorganization of the viral envelope structures to facilitate attack complex insertion and lysis.

Epithelial patchy necrosis in Crohn's disease.

Electron and light microscopic studies of the intestinal epithelium in Crohn's disease demonstrated localized areas of damage to the superficial epithelium, occurring without accompanying acute inflammation. In a blind study of rectal biopsy specimens from seven patients with Crohn's disease, four with ulcerative colitis, and four normal controls, this finding of patchy necrosis without acute inflammation was observed exclusively in four of the seven cases of Crohn's disease.

Chronic malabsorption due to cryptosporidiosis in a child with immunoglobulin deficiency.

A case of fatal cryptosporidiosis in a child with primary immunoglobulin deficiency is described. This is always a serious complication in immunodeficient patients because there is no known effective therapy.

Ultrastructure of a hyaluronic acid matrix.

Freeze-etch replicas of a hylauronic acid matrix were visualized by electron microscopy. In water a coarse branching fibrillar network of hyaluronic acid aggregates was seen. The high solvent permeability of this matrix suggests that the spaces observed are relatively devoid of unaggregated polymer. Addition of calcium disordered the matrix, resulting in a more dispersed felt of polymer.

Ribonucleoprotein of avian infectious bronchitis virus.

The ribonucleoprotein (RNP) of avian infectious bronchitis virus (IBV) was examined by electron microscopy after shadowing with carbon/platinum. Linear RNP strands up to 6.7 microns in length, from three IVB strains, were sensitive to both pancreatic RNase and to proteases. These strands were obtained from spontaneously disrupted complete particles but not from disrupted incomplete particles that lacked RNP. They were also released from Nonidet P40-disrupted particles and could be isolated on sucrose density gradients at a density of 1.27 g/ml. In some cases, helical RNP complexes associated with virus particles were observed that were similar to RNPs of human coronavirus strain 229E and mouse hepatitis virus strain 3.

Electron microscopic demonstration of lesions in target cell membranes associated with antibody-dependent cellular cytotoxicity.

To test the hypothesis that complement-mediated cell lysis and cell-mediated cytotoxicity operate by analogous mechanisms, cell membranes from two antibody-dependent cytotoxicity systems were examined by electron microscopy after negative staining. Ring-shaped membrane lesions generally similar to, but larger than, those previously described for complement lysis were observed. These findings are in agreement with recent measurements of larger functional pores for ADCC than complement.

Interaction of Mycoplasma pneumoniae with human lung fibroblasts: characterization of the in vitro model.

The interaction of pathogenic Mycoplasma pneumoniae and host cells was studied in cell cultures of MRC-5 human lung fibroblasts. A comparison of results obtained with fibroblasts in a monolayer format and with hamster tracheal explant cultures indicated that the former can bind significantly larger numbers of mycoplasmas. In addition, the attachment was 96% specific, that is, mediated through a neuraminidase-sensitive receptor on the host cell. Uptake of mycoplasmas was directly related to the number of mycoplasma cells present in the inoculum, and attachment was virtually complete within a 30-min period at 37 degrees C. High doses of M. pneumoniae induced a marked cytopathic effect, whereas doses of less than or equal to 10(6) colony-forming units per ml produced grossly observable cell damage that was moderate and variable. Transmission electron microscopy studies indicated that attachment of M. pneumoniae to the surface of lung fibroblasts occurred with the specialized terminal structure or binding site oriented closest to the epithelial cell surface. The filamentous mycoplasma cells were spatially arranged in several configurations and were not limited to a vertical orientation. The advantages and disadvantages of human lung fibroblast monolayer cultures, in reference to other in vitro models are discussed. A new mycoplasma agar medium (G-200 agar) with a defined tissue culture base and 10% horse serum is also described.

Growth and effect of chlamydiae in human and bovine oviduct organ cultures.

Organ cultures of 10 Fallopian tubes were inoculated with a genital strain of Chlamydia trachomatis and seven were infected. Infection was enhanced by centrifuging the organisms on to the tissues, larger numbers of organisms being reisolated from the tissues after this procedure. There was evidence of chlamydial multiplication because the number of organisms which were recovered from the tissues three to five days after inoculation had increased. Recovery was rare, however, after the sixth day, thus suggesting a self-limiting infection. Organ cultures of two bovine oviducts were infected with the bovine abortion strain of Chlamydia psittaci, but in these experiments centrifugation of the inocula did not enhance infection. The organisms were found in both the tissue and medium of cultures up to 18 days after inoculation and in much greater numbers than in the C. trachomatis-infected Fallopian cultures. Chlamydial infection was not entirely host-tissue specific, because C. trachomatis organisms were isolated from bovine oviduct cultures. Inclusions, however, were not detected histologically or electron microscopically in the epithelium of C. trachomatis-infected cultures, but they were detected by these means in C. psittaci-infected bovine cultures. All the elements of the chlamydial growth cycle were seen by electron microscopy, organisms being found in ciliated and possibly non-ciliated cells, and shedding of some infected epithelial cells was observed. No evidence of extensive epithelial cell damage was observed, however, and no loss of ciliary activity was detected in cultures infected with either C. trachomatis or C. psittaci when compared with uninoculated cultures. Thus acute salpingitis, when caused by chlamydial infection, is probably immunologically mediated.

A year's experience of the rotavirus syndrome and its association with respiratory illness.

In a hospital study rotavirus was identified in 51% of 152 children with diarrhoea. These patients showed a clinical pattern that was distinct from patients in whom the diarrhoea was associated with bacteria, other viruses, or no pathogens. A respiratory illness was described in 66% of rotavirus patients and usually preceded the gastrointestinal symptoms. Vomiting lasted between one and 3 days and was curtailed by substituting the normal diet with clear fluids. Watery diarrhoes continued for 4 or 5 days, even when rehydration was by the intravenous rather than the oral route. Prolonged diarrhoea was rare. Most children infected with rotavirus were under 2 years of age, but dehydration was most severe in infants aged between 12 and 18 months. A clinician can thus recognise the rotavirus syndrome and expect spontaneous recovery if adequate rehydration is maintained for a critical few days.

Studies on the mechanism of influenza virus entry into cells.

Inhibitors of glycolysis, oxidative phosphorylation, protein synthesis, membrane Na&-K& transport and microfilament and microtubule function have been employed to elucidate the mechanism of influenza virus uptake by CAM and CEF cells. Electron microscopy demonstrated uptake of virus by viropexis in the presence of all these inhibitors. Utilizing a pulse labelling technique, virus entering CEF cells in the presence of inhibitors was shown to initiate specific virus polypeptide synthesis after neutralization of remaining extracellular virus and removal of the inhibitors. As a consequence of these findings an energy independent mechanism of viropexis has been proposed.