The fixation of complement and the activated first component (C1) of complement by complexes formed between antibody and divalent hapten.

Hapten-antibody complexes prepared at equivalence with the bivalent hapten bis-DNP-octamethylene-diamine and purified rabbit anti-DNP antibody were fractionated by Sepharose gel-filtration and the fractions examined by electron microscopy. Individual fractions were tested for whole-complement fixation and C1 fixation. Dimer forms did not show this type of biological activity, while fractions containing tetramers and larger polymers exhibited both C and C1 fixation, which could be inhibited by prior exposure of the complexes to the univalent hapten epsilon-DNP-caproic acid. The dose-response result indicated that the C-fixation observed was not due to interpolymeric cooperative effects. It was concluded that in the generation of biological activity by soluble antigen-antibody complexes made with complement-fixing antibody, quaternary structural changes following specific combination with antigen may be as important as any tertiary structural alterations that occur in the individual immunoglobulin molecule.

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.

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.

Complement lesions in cell membranes from joint effusions of various types of arthritis.

Electron microscopy of differentially centrifuged synovial fluid from patients with differing types of arthritis revealed a few membranes bearing complement lesions. It was shown that this is due to activation of the alternate pathway of complement by leukocytic enzymes. It is not clear where the site of action is in inflammatory exudates; it may be on internal membranes of the cells, as no detectable cytotoxic effect could be found using Rb(86) as a marker for specific release. In addition, lysosomal enzymes have the net effect of inhibiting the hemolysis of indicator cells caused by activation of the alternate pathway by inulin.

The structural events associated with the attachment of complement components to cell membranes in reactive lysis.

Electron microscopic study of the events occurring at the cell membrane during reactive lysis by complement, showed that a foliaceous particle was formed at the C5b-7 stage, that enlarged to a particle with a variable number of arms at the C5b-8 stage. Up to this point, no typical complement lesions were found. At the C5b-9 stages, the particles were completely converted to typical complement lesions, i.e. hollow cylinders projecting from the cell membrane and partly penetrating it. C5b-9 complexes assembled in the fluid phase did not show the typical structure of the lesions, but were amorphous masses of fibres.

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.

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.

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.

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.

Lymphocytic infiltration of the brain in sleeping sickness.

Cerebrospinal fluid mononuclear cells from 40 patients with advanced Gambian sleeping sickness were examined for intracytoplasmic immunoglobulin and for B- and T-lymphocyte markers. About 5% of mononuclear cells were plasma cells. Most of the lymphocytes present were B cells. These findings suggest that the considerable lymphocytic infiltration of the nervous system seen in advanced sleeping sickness is not a cell-mediated immune reaction to trypanosomes. Immune complexes may play a part in producing the brain damage characteristic of this disease.