Interactions of cultured rat synovial and ocular ciliary body cells with two strains of mycoplasma arthritidis

Summary Strains ofMycoplasma arthritidis differ in their ability to cause joint and ocular inflammations. Although the reasons for this difference are not fully understood, pathogenic myćoplasmas commonly require close associations with the cells they damage. Using³H-uridine labeled mycoplasma, we compared cellular interactions of in vitro cultivated rat synovial and ocular ciliary body epithelial cells with two American Type Culture Collection strains ofM. arthritidis shown to differ in their virulence. Radiolabeling assays gave evidence of á stronger retention capability on cultured cells by the more pathogenic strain, 14152. Scanning electron microscopy demonstrated cellular associations with the two strains of mycoplasma, with more of the 14152 adhering to both cell types. Examination by transmission electron microscopy showed evidence of contact between the more virulent 14152 strain and both cell types, but no similar evidence with the comparatively less virulent strain, 19611. The pathogenicity of different strains ofM. arthritidis may vary according to their ability to closely associate with specific target cells involved in the disease process. This work was supported by funding from the National Society for the Prevention of Blindness, Fight for Sight, Retinitis Pigmentosa International, grant EY05415 from the National Institutes of Health, Bethesda, MD, to Thirkill, and, in part, by an unrestricted grant from Research to Prevent Blindness.     

Rhamnose biosynthesis in mycoplasmas requires precursor glycans larger than monosaccharide

Despite the apparent absence of genes coding for the known pathways for biosynthesis, the monosaccharide rhamnose was detected in the d configuration in Mycoplasma pneumoniae and Mycoplasma pulmonis, and in both the d and l configurations in Mycoplasma arthritidis. Surprisingly, the monosaccharide glucose was not a precursor for rhamnose biosynthesis and was not incorporated at detectable levels in glucose‐containing polysaccharides or glycoconjugates. In contrast, carbon atoms from starch, a polymer of glucose, were incorporated into rhamnose in each of the three species examined. When grown in a serum‐free medium supplemented with starch, M. arthritidis synthesized higher levels of rhamnose, with a shift in the relative amounts of the d and l configurations. Our findings suggest the presence of a novel pathway for rhamnose synthesis that is widespread in the genus Mycoplasma.     

O‐linked protein glycosylation in mycoplasma

Although mycoplasmas have a paucity of glycosyltransferases and nucleotidyltransferases recognizable by bioinformatics, these bacteria are known to produce polysaccharides and glycolipids. We show here that mycoplasmas also produce glycoproteins and hence have glycomes more complex than previously realized. Proteins from several species of Mycoplasma reacted with a glycoprotein stain, and the murine pathogen Mycoplasma arthritidis was chosen for further study. The presence of M. arthritidis glycoproteins was confirmed by high‐resolution mass spectrometry. O‐linked glycosylation was clearly identified at both serine and threonine residues. No consensus amino acid sequence was evident for the glycosylation sites of the glycoproteins. A single hexose was identified as the O‐linked modification, and glucose was inferred by ¹³C‐labelling to be the hexose at several of the glycosylation sites. This is the first study to conclusively identify sites of protein glycosylation in any of the mollicutes.     

Intracellular Location and Survival of <Emphasis Type="Italic">Mycoplasma penetrans</Emphasis> Within HeLa Cells

Mycoplasma penetrans invades HeLa cells and survives within them for prolonged periods of time. The intracellular distribution of M. penetrans within HeLa cells was studied utilizing the acidotropic dye LysoTracker (green), which permeates cell membranes and upon protonation remains trapped in acidic compartments. The excitation and emission spectra of the green LysoTracker are suitable for colocalization studies with rabbit anti-M. penetrans antibodies and red Cy5 goat anti-rabbit IgG. The images collected by confocal laser scanning microscopy revealed that in the infected HeLa cells almost all Cy5 fluorescent foci (red) were located within the LysoTrack-labelled intracellular compartments, apparently endosomes. Viable mycoplasmas were detected within endosomes for prolonged periods of time, apparently due to a potent antioxidant activity detected in M. penetrans.     

Microparticles stimulate angiogenesis by inducing ELR+ CXC‐chemokines in synovial fibroblasts

Microparticles (MPs) are small membrane‐vesicles that accumulate in the synovial fluids of patients with rheumatoid arthritis (RA). In the arthritic joints, MPs induce a pro‐inflammatory and invasive phenotype in synovial fibroblasts (SFs). The present study investigated whether activation of SFs by MPs stimulates angiogenesis in the inflamed joints of patients with RA. MPs were isolated from Jurkat cells and U937 cells by differential centrifugation. SFs were co‐cultured with increasing numbers of MPs. The effects of supernatants from co‐cultures on endothelial cells were studied in vitro and in vivo using MTT assays, annexin V and propidium iodide staining, trans‐well migration assays and modified matrigel pouch assays. MPs strongly induced the expression of the pro‐angiogenic ELR⁺ chemokines CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6 in RASFs. Other vascular growth factors were not induced. Supernatants from co‐cultures enhanced the migration of endothelial cells, which could be blocked by neutralizing antibodies against ELR⁺ chemokines. Consistent with the specific induction of ELR⁺ chemokines, proliferation and viability of endothelial cells were not affected by the supernatants. In the in vivo bio‐chamber assay, supernatants from RASFs co‐cultured with MPs stimulated angiogenesis with a significant increase of vessels infiltrating into the matrigel chamber. We demonstrated that MPs activate RASFs to release pro‐angiogenic ELR⁺ chemokines. These pro‐angiogenic mediators enhance migration of endothelial cells and stimulate the formation of new vessels. Our data suggest that MPs may contribute to the hypervascularization of inflamed joints in patients with rheumatoid arthritis.     

Development of competitive ELISA for neosporosis by employing immunoproteomics

In this study, proteomics was used to explore the antigenic proteins that are involved in cross-reactivity during serodiagnosis between Neospora caninum (N. caninum) and Toxoplasma gondii (T. gondii). Competitive enzyme-linked immunosorbent assay (C-ELISA) developed by proteomics shed a new light on the infection of N. caninum. Cross-reactivity of antigenic proteins between N. caninum and T. gondii tachyzoites was explored by using the conventional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (1-DE) and two-dimensional gel electrophoresis (2-DE) immunoblot. The proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The protein expression patterns in the immunoblot profiles of N. caninum were similar to bovine, chicken, and rabbit anti-N. caninum serum, but they were not similar to rabbit anti-T. gondii serum. Band at 79 kDa, HSP70, and actin on immunoblot profiles reacted, in general, with bovine, chicken, and rabbit anti-N. caninum serum and also with rabbit anti-T. gondii serum, respectively. Whereas the band at 144 kDa, and NCDG-1 were detected on bovine, chicken, and rabbit anti-N. caninum immunoblot profiles, they were not observed on rabbit anti-T. gondii immunoblot profile. These specific antigenic proteins were recorded as species-specific proteins of N. caninum against T. gondii. Based on the proteome analysis, C-ELISA was developed to screen the cattle infected with N. caninum by using N. caninum tachyzoite lysate as a coating antigen and chicken anti-N. caninum immunoglobulin (Ig)Y as a competitor. C-ELISA was able to detect the antibody of N. caninum without cross-reactivity with T. gondii. Furthermore, it achieved a fine diagnostic performance in the cases of 162 bovine sera.     

One-year follow-up of anti-Leishmania antibody concentrations in serum and saliva from experimentally infected dogs

The quantification of anti-Leishmania antibodies in serum and saliva by a time-resolved immunofluorometric assay is useful for the diagnosis and treatment monitoring of dogs with clinical leishmaniasis. We compared the kinetics of anti-Leishmania IgG2 and IgA antibodies in serum and saliva from 11 Beagle dogs experimentally infected with Leishmania infantum. Most dogs showed detectable concentrations of anti-Leishmania IgG2 earlier in serum (between 3 and 4 months p.i.) than in saliva (between 4 and 6 months p.i.). Overall, a high correlation between concentrations of anti-Leishmania IgG2 in serum and saliva (r = 0.853; P < 0.0001) was observed. The quantification of anti-Leishmania IgA showed less diagnostic value than IgG2, since detectable amounts of IgA were not observed in the saliva of four dogs and in the serum of one dog. In addition, a very low correlation between anti-Leishmania IgA in serum and saliva (r = 0.289; P < 0.001) was observed. Our results indicate that the antibodies against L. infantum in saliva appear approximately 1 month later than in serum, and suggest that there is a threshold for the passing of immunoglobulins from serum to saliva in dogs. These facts should be taken into consideration for a proper interpretation of saliva assays for quantification of antibodies.     

Macrophage procoagulant-inducing activity of influenza-specific effector T cells

Three different types of immune mouse T cells raised against influenza virus were tested for their ability to induce the formation of macrophage procoagulant activity (MPCA) by a macrophage cell line PU5-1.8. They were primary spleen cells, taken 6 days after iv injection of virus, spleen cells from sensitized mice challenged with virus and cultured in vitro for 5 days (secondary cultured cells), and cloned T cells. With the last two preparations, some samples were K,D region restricted, Lyt 2⁺, and had cytotoxic activity; other samples were I region restricted, Lyt 2^?, and were not cytotoxic. Samples of a concanavalin A-activated T-cell supernatant which regularly induced MPCA with PU5-1.8 cells were included as controls in all assays. A few batches of T-cell preparations failed to induce MPCA production, however, most batches were active. Two sources of variation were detected: first, the number of cells (5-to 150-fold) needed to induce a certain level of MPCA, as measured by the decrease in clotting time; and second, the value of the gradient of the cell dose response. Both K,D- and I-region-restricted cells, either as cloned or secondary cultured cells, could induce MPCA but with the latter preparation, I-region-restricted cells were the better inducers by about eightfold. T cells tested in this way were also injected into mouse hind footpads and their ability to mediate delayed-type hypersensitivity (DTH) reactions was measured. A positive but not proportional correlation between the abilities to induce MPCA and mediate DTH activity for primary spleen cells was found, but this was not generally observed with cultured or cloned T cells.     

Inhibition of nitric oxide and caspase-3 mediated apoptosis by a tetrapeptide derivative (PEP1261) in cultured synovial fibroblasts from collagen-induced arthritis

In this study, the effect of (Boc-Lys (Boc)-Arg-Asp-Ser (tBu)-OtBu), a tetrapeptide derivative (PEP1261) was examined for antiproliferative potency and apoptotic induction. Synovial fibroblasts were isolated from collagen-induced arthritic (CIA) rats and exposed to peptides viz., PEP1261, and parental peptides (KRDS and RGDS). Viability of the cells decreased in the presence of PEP1261 at a lower concentration (0.1 mM) when compared to RGDS and KRDS (1 mM). The treatment of cells with peptides showed induction of apoptosis, resulting in the cleavage of caspase-3 as well as its substrate poly-(ADP-ribose) polymerase (PARP). Pretreatment of cells with caspase-3 inhibitor prevented inhibition of [³H] thymidine incorporation, DNA fragmentation, and cleavage of caspase-3 and PARP as confirmed by western blotting as well as annexin-V/PI-staining using flow cytometry. However, caspase-1 and caspase-2 inhibitors did not prevent the peptides from inducing apoptosis indicating that caspase-3 might have a role in the process of apoptosis induced by peptides. Treatment of synovial fibroblasts with nitric oxide donor, S-nitroso-N-acetyl-dl-penicillamine (SNAP) (500 μM) showed significant elevation of nitric oxide levels and resulted in absence of apoptosis by preventing the inhibition of [³H] thymidine incorporation. This was further evidenced by annexin V/propidium iodide (PI) staining and absence of DNA fragmentation, intra cellular caspase-3 activity and PARP cleavage. In contrast, SNAP followed by PEP1261 and parental peptides-induced apoptosis by lowering the levels of nitric oxide. These results suggested that PEP1261 suppressed the proliferation and induced apoptosis in cultured synovial fibroblasts from CIA rats. This study also confirmed that PEP1261 inhibited nitric oxide level in cultured synovial fibroblasts.     

Surface receptors and immune activity of purified human circulating mononuclear cells: IV. The demonstration of seven subclasses of T cells in the circulation of the normal individual: The cytotoxic activities of these cells

T lymphocytes were isolated from monocyte-depleted mononuclear cells of normal individuals by rosetting them with sheep erythrocytes. These purified T cells were preferentially depleted of cells with receptors for FcG (T_G cells), FcM (T_M cells), or C3 (T_C cells) by rosette formation with EA(G), EA(M), and EAC, respectively, before or after incubation for 24 hr in medium 199 fortified with fetal calf serum (20%). The unfractionated lymphocytes and the purified and the depleted T cells were analyzed for receptors to FcG, FcM, and C3 and for cytotoxic activity in the natural killer (NK), antibody dependent cell-mediated cytotoxicity (ADCC), and mitogen-induced cell-mediated cytotoxicity (MICC) assays. The T_G and T_C cells were detected among the freshly isolated T cells, whereas the T_M cells were detected only following 24 hr of incubation. Removal of T_C cells from the 24-hr-cultured T cells resulted in removal of all the T_C cells and in the concomitant removal of the majority of T_M cells. Similarly, removal of T_M cells from the 24-hr-cultured T cells resulted in the elimination of all T_M cells as well as the majority of T_C cells. These results demonstrate the in vitro generation of T cells with receptors for both FcM and C3 (T_M+C cells). Ten percent of the freshly isolated T_G cells possessed detectable receptors for C3 and/or FcM. These cells constitute the T_G+C and T_G+M lymphocytes. Support for consideration of these receptor-bearing cells as unique and stable cells is provided by the finding that T_M and T_C cells maintained in culture for up to 72 hr do not generate other receptors but retain the single receptor which characterizes each of these cells. Only a small percentage of cultured T_G cells generate receptors for C3 and FcM. It may therefore be concluded that the T_G, T_M, and T_C cells are stable unireceptor-bearing cells. The T_G, T_M, T_C, T_G+C, T_G+M, and T_M+C lymphocytes account for approximately 50% of the circulating lymphocytes. Whether the remaining cells, the T null or T_N cells, constitute the precursors for any or all of the receptor-bearing T cells remains to be determined. Unfractionated freshly isolated T cells were highly cytotoxic in the NK and PWM-mediated MICC assays but were relatively inactive in the ADCC, naturally occurring cell-mediated cytotoxicity (NOCC), and PHA- and Con-A-mediated MICC assays. In contradistinction, T cells incubated for 24 hr displayed marked cytotoxic activity in the ADCC and PHA-mediated MICC assays; they were inactive in the NOCC and Con-Amediated MICC assays. The T_G cells were the predominant cytotoxic cells in the ADCC, NK, and MICC cytotoxic assays since their selective elimination from either the freshly isolated or 24-hr-incubated T cells resulted in almost total loss of cytotoxic activity of the remaining cells. Removal of the T_G+C cells from the freshly isolated or 24-hr-incubated T cells resulted in a significant decrease in PHA- and PWM-mediated MICC cytotoxic activity. T cells depleted of T_M, T_M+C, and T_C cells exhibited the same cytotoxic activity as did the unfractionated T cells. These results suggest that the predominant cytotoxic T cells in all the assays investigated are the T_G cells, that limited cytotoxic activity is also displayed by the T_G+C cells, and that the T_M, T_M+C, T_C, and T_N cells display no cytotoxic activity in the assays utilized in this investigation.     

Monoclonal antibodies identifying a novel T-Cell antigen and Ia antigens of human lymphocytes

We describe here two new monoclonal antibodies that react with surface antigens of human lymphocytes. Antibody 7.2 identified a determinant on the framework region of the human Ia antigen. It was cytotoxic for all cultured B-cell lines, normal B cells, and monocytes. The antibody was not cytotoxic for normal T cells or for established T leukemic cell lines. In immune precipitation assays, the 7.2 antibody reacted with a bimolecular complex of two chains that resolved in polyacrylamide gels as polypeptides with molecular weights of 29000 and 34000 daltons. These precipitation results were analogous to those achieved with a rabbit antiserum prepared against human Ia antigens. Antibody 9.3 identified a determinant on the framework region of a T-cell antigen. It was cytotoxic for 50–80% of peripheral T cells and for 20–50% of thymocytes. It was not cytotoxic for cultured B-cell lines, normal B cells, or monocytes. In immune precipitation assays, the 9.3 antibody reacted with a single polypeptide with a molecular weight of 44000 daltons. Due to the expression of this antigen on a limited subpopulation of human T cells, we have designated the antigen HuLyt-1.     

Transfer of Protection to Murine Typhoid Conferred by L-Form Salmonella typhimurium in Dependence of Cooperation between L Form-Adopted Macrophages and L Form-Induced Lyt-2+ T Cells

The effector cells responsible for protection to Salmonella typhimurium in C3H/HeJ mice, conferred by L-form S. typhimurium, were determined by cell transfer test. Nonfractionated spleen cells from 6-week immune mice but not from 24-week immune animals transferred anti-S. typhimurium immunity. Treatment with anti-macrophage antiserum and complement most effectively abolished protective capacity in 6-week immune cells, while anti-T cell monoclonal antibody plus complement reduced it to a lesser extent. However, adoptive protection was achieved only by transfer of immune macrophages along with Lyt-2⁺ T cells selected from 6-week immune spleen cells. These Lyt-2⁺ T cells were cytotoxic to Kupffer cells from C3H/HeJ mice which had been infected 48 hr previously and from the mice which had been immunized 1 week previously, but not to the cells from 6-week immune mice and from normal animals. Moreover, protective capacity in immune macrophages seemed to be correlated to the degree of colonization by the L forms, and the inability to transfer immunity of 24-week immune spleen cells may be due to the decrease in the L form-colonization. These results suggest that cooperation between the L form-colonized macrophages and L form-induced cytotoxic Lyt-2⁺ T cells contributes to anti-S. typhimurium immunity, and might imply the immunological difference between the 6-week immune phagocytes and the cells at an early stage of infection or immunization.     

Quantitative immunocytochemical studies on the gonadotrophs isolated from the pituitary of the male rat

Summary Quantitative studies on the population of the gonadotropic cells in the pituitary of adult male rats were performed after trypsic dissociation of the pituitary glands and immunoenzymatic staining with anti--LH or anti--FSH antisera. Number, area and extinction of labelled cells were measured by use of an image analyser and a cytophotometer. The gonadotrophs represent approximately 14% of the pituitary cells. The mean area of gonadotrophs is significantly larger after staining with anti--LH serum than after staining with anti--FSH serum. Planimetric measurement of the gonadotrophs reveals their variability in size ranging between 30 and 160 m². Moreover, the size distribution depends on the staining serum used: more numerous small-sized cells (<75 m²) are stained with anti--FSH serum than with anti--LH serum, which conversely stains more numerous large-sized cells. Cytophotometric measurements indicate that immunostaining varies greatly among cells of the same size class and that the staining intensity appears to increase according to the cell size. These results emphasize the morphofunctional heterogeneity of the gonadotropic cell population.     

Serum and Egg Antibody Responses in Chickens to Escherichia coli

The effects of Freund’s adjuvants on antibody production in chickens against E. coli whole cells were examined. The levels of anti-E. coli IgG antibodies in serum were higher when Freund’s complete (FCA) or incomplete adjuvant (FIA) was administered than that without adjuvant. Production of antibodies recognizing E. coli cells and their lipopolysaccharide was enhanced by FIA, while both FIA and FCA enhanced production of antibodies recognizing outer membrane components. In contrast, serum IgM antibody levels were higher when no adjuvant was used. Anti-E. coli IgG antibodies in serum were efficiently transferred to egg yolk, giving antibody activity in egg yolk similar to that in serum. However, anti-E. coli IgM antibodies were not detected in the egg, suggesting that egg (white) IgM was not influenced by antigenic stimulation of the humoral immune system. Antimicrobial activity of the egg yolk IgG was highest when the bacteria antigen was injected with FIA.     

Alloantigens expressed on a subset of unstimulated human T lymphocytes that generate suppressor function

Human alloantisera were tested for antibodies reacting with T-cell subpopulations. T-cell subsets were separated using the monoclonal antibodies OKT4 and OKT8. Five sera reacting with the T4-T8+ subset and two sera reacting with T4+T8- lymphocytes were identified. Serum Z. G. reacted with T4-T8+ cells from 8 of a panel of 19 donors. T cells treated with Z. G. serum and rabbit complement lost the capacity to generate suppressor cells but showed no decrease in the development of cytotoxic effector cells. ZG antigens were demonstrated by absorption also on monocytes but not on B cells. Their reactions on T cells were blocked by chicken anti-human la serum, but not by turkey anti-₂-microglobulin or by a monoclonal anti-human DR (L227). Studies in four informative families suggested that the ZG determinants are inherited in linkage with HLA. Although the similarities between ZG antigens and mouse I-J products are striking, structural studies are needed to establish their homology.     

Effects of Bacillus sphaericus 1593 and 2362 spore/crystal toxin on cultured mosquito cells

An in vitro assay system for the toxin of Bacillus sphaericus strains 1593 and 2362 has been developed utilizing cultured Culex quinquefasciatus cells. The cytotoxic activity of extracts of B. sphaericus strain 1593 did not necessarily correlate with insecticidal activity. Cytotoxicity and larvicidal activity were neutralized by immune rabbit serum prepared against crude toxin extracts as well as by serum prepared against purified toxin from strain 2362. This purified toxin was also found to be cytotoxic. Activation with mosquito larval gut homogenates enhanced cytotoxicity of both 1593 extracts and purified toxin from 2362. The activity of cytotoxic preparations against three mosquito cell lines paralleled the activity of B. sphaericus spores against larvae of these mosquito species. The results suggest the presence of a protoxin and one or more cytotoxic proteins derived from it.     

In Vitro Proliferative Activity of Spleen Cells in Mice Infected with Attenuated Salmonella typhimurium

Proliferative activity of cultured spleen cells obtained from mice 1 to 5 weeks after infection with attenuated strains of Salmonella typhimurium was examined in the presence or absence of lipopolysaccharide (LPS) or concanavalin A (Con A). Spontaneous uptake of ³H-thymidine (TdR) by cells taken from infected mice at the 2nd and 3rd weeks was obviously lower than that by cells from uninfected, control mice. Cells from infected mice at the 4th and 5th weeks also showed a lower proliferative response to LPS than that of the controls. However, the responses of the cells to Con A remained virtually unchanged during the entire period. Furthermore, the reduction of spontaneous ³H-TdR uptake by the cells could be achieved also by the injection of heat-killed instead of living organisms. The T- and B-lymphocyte populations of these spleen cells were examined by the dye exclusion cytotoxic test using rabbit anti-mouse T- and anti-mouse B-lymphocyte sera, respectively. There was some alteration of the populations in the cells, but it did not correlate with the reduction in ³H-TdR uptake. Results of expriments with cultured cells reconstituted with lymphocytes and macrophages isolated from spleen cells suggested that the spontaneous reduction of proliferative activity observed in cells taken from the infected mice could be attributed to the dysfunction of macrophages.     

Modulation and regrowth of allotype on normal rabbit peripheral blood lymphocytes: allelic inclusion of b4 and b6 in single cells.

Allelic inclusion at the b-locus by heterozygous peripheral blood rabbit lymphocytes was demonstrated by using the mixed antiglobulin techniques (6, 7, 19). Heterozygous cells (64, 6) were treated with monospecific antiallotype reagents at 4 °C and warmed at 37 °C. The removal of surface allotype determinants was studied and, both the b4-and b6-specificities co-modulated after sensitization with either anti-b4 or anti-b6. Experiments were undertaken in which cells stripped of one or both allotypes by antiallotype induced modulation were cultured overnight. Allotype was then regenerated. Double expression of allotype by cells before antiallotype treatment was recovered following overnight regrowth at levels equal to those seen before treatment. Such was not the case when b44 and b66 cells were cultured together. These results indicate that normal heterozygous peripheral blood lymphocytes may express both b-locus alleles and that these determinants are in some manner physically associated with one another in the cell membrane.     

Differential Responses of Single Cells and Aggregates of Mycoplasmas to Ultraviolet Irradiation

Except for Mycoplasma fermentans strain PG 18, single-cell suspensions of M. arthritidis, M. fermentans (ATCC 19989), M. hominis type 1, M. orale types 1 and 2, M. pneumoniae, and M. salivarium were inactivated exponentially by ultraviolet (UV) irradiation, in contrast to broth cultures containing clusters of elementary bodies. The susceptibility of the mycoplasmas was unaffected by storage at 2-4 C and at -70 C, by sonication, and by filtration. The rate of inactivation was dependent on the intensity of the radiations but independent of the concentration of the cells. Therefore, single-cell suspensions of these mycoplasmas could be differentiated from aggregates of cells by exponential inactivation of the colony-forming units (CFU). By this criterion, the CFU of M. arthritidis in the exponential phase of growth consisted of single cells, in contrast to the other species in which the CFU contained two or more elementary bodies. Even though the cultures of M. fermentans (PG 18) were grown from single cells, they were not homogeneous in their susceptibility to UV light. Neither were cultures of M. arthritidis and M. orale type 1 grown from single cells which had survived irradiation.     

Hypochlorous acid enhances immunogenicity and uptake of allogeneic ovarian tumor cells by dendritic cells to cross-prime tumor-specific T cells

Background: Ovarian cancer commonly relapses after remission and new strategies to target microscopic residual diseases are required. One approach is to activate tumor-specific cytotoxic T cells with dendritic cells loaded with tumor cells. In order to enhance their immunogenicity, ovarian tumor cells (SK-OV-3, which express two well-characterized antigens HER-2/neu and MUC-1) were killed by oxidation with hypochlorous acid (HOCl). Results: Treatment for 1 h with 60 μM HOCl was found to induce necrosis in all SK-OV-3 cells. Oxidized, but not live, SK-OV-3 was rapidly taken up by monocyte-derived dendritic cells, and induced partial dendritic cell maturation. Dendritic cells cultured from HLA-A2 healthy volunteers were loaded with oxidized SK-OV-3 (HLA-A2⁻) and co-cultured with autologous T cells. Responding T cells were tested for specificity after a further round of antigen stimulation. In ELISPOT assays, T cells produced interferon-gamma (IFN-γ) in response to the immunizing cellular antigen, and also to peptides coding for MUC-1 and HER-2/neu HLA-A2 restricted epitopes, demonstrating efficient cross-presentation of cell-associated antigens. In contrast, no responses were seen after priming with heat-killed or HCl-killed SK-OV-3, indicating that HOCl oxidation and not cell death/necrosis per se enhanced the immunogenicity of SK-OV-3. Finally, T cells stimulated with oxidized SK-OV-3 showed no cross-reaction to oxidized melanoma cells, nor vice versa, demonstrating that the response was tumor-type specific. Conclusions: Immunization with oxidized ovarian tumor cell lines may represent an improved therapeutic strategy to stimulate a polyclonal anti-tumor cellular immune response and hence extend remission in ovarian cancer.