Preliminary characterization of the procoagulant material in human ascites

Disseminated intravascular coagulation invariably accompanies placement of peritoneovenous (LeVeen) shunts, which suggests that ascitic fluid contains procoagulant material capable of activating blood coagulation. In this study, we identified thrombogenic activity in human ascites and the hemostatic pathway by which it acts. Peritoneal fluid was removed percutaneously from patients with ascites due to various causes. Four fractions were prepared by centrifugation: cells, a low-speed, cell-free fluid, a high-speed supernatant, and the precipitate from the high-speed centrifugation. Cellular fractions from all ascitic fluids shortened a one-stage clotting time of normal pooled plasma by 68% in comparison with saline solution and endotoxin controls. Similarly, the cell-free fluids also shortened the clotting time of normal pooled plasma by 41%. The cellular and cell-free fractions shortened the clotting time of factor VIII-deficient plasma but failed to demonstrate procoagulant activity in factor VII-deficient plasma. These fractions had no effect on platelet aggregation or the platelet release reaction. The high-speed precipitate was dissociated by ethylenediaminetetra-acetate (EDTA) into fluid phase and precipitate, both of which demonstrated procoagulant activity. Furthermore, high-speed precipitate contained protein, phospholipid, and sterol in proportions similar to those of plasma membranes and contained membrane-bound vesicles as identified by means of electron microscopy. This material could be rendered inactive by heating to 100 degrees C for 2 minutes or by incubation with phospholipase C for 15 minutes. Finally, the ability of the high-speed precipitate to shorten the clotting time was prevented by preincubation with a monoclonal antibody, which is known to inhibit the procoagulant activity of human tissue factor. We suggest that several entities contribute to the procoagulant properties of human ascites, with procoagulant material deriving at least in part from peritoneal cells. The sedimentable procoagulant factor appears to be associated with cellular membranes or membrane fragments and is thromboplastin-like in its chemical composition, immunoreactivity, and substrate specificity.     

Immunofluorescence-digital image processing system for the quantitation of secreted immunoglobulin by single cells

To quantitate the amount of secreted immunoglobulin (Ig) by a single cell, the immunofluorescence digital image processing (IDIP) system was adapted to the modified enzyme-linked immunospot (ELI-SPOT) assay. In this assay, an immunofluorescence (tetramethylrhodamine isothiocyanate) conjugated antibody was used for the detection of spots instead of the usual method of enzyme coupling. We have named this the immunofluorescence-linked immunospot (ILISPOT) assay. In addition to the quantitation of secreted Ig by single cells, this method allowed us to objectively determine the exact number of Ig producing spot forming cells (SFC). 96 well culture plates were pre-coated with goat anti-mouse Ig. The mouse IgM producing hybridoma (E-3-4) was incubated in the plates for 4 h at 37 degrees C. Cells were removed prior to the addition of biotinylated goat anti-mouse mu antibody. After overnight incubation, immunofluorescence conjugated avidin was added for the visualization of spots by the IDIP system. The IDIP system consists of a fluorescent microscope equipped with a video camera and computer. The gray scale of secreted IgM was initially established as a standard by the known amount of purified IgM. By using digital image processing, the number of spots and the gray scale of individual spots were computed. The shape and pattern of gray scale data were used to distinguish between the real spots and pseudo spots. This IDIP system could detect as little as 0.19 pg of secreted IgM (1.2 x 10(5) molecules) and an average of approximately 1.33 pg (8.3 x 10(5) molecules) produced by a single cell. Adaptation of the digital image processing system to the ILISPOT assay allowed the measurement of both the amount of Ig produced at the single cell level and also the exact numbers of SFC present in a totally objective fashion.     

A corneal model for slit-scanning elevation topography

Computerised videokeratography is increasing in sophistication and accuracy. The Orbscan II Videokeratographer combines the advantages of Placido derived and slit-scanning derived topography, to acquire shape information on the posterior surface of the cornea. The purpose of this paper is to construct a model of slit-scanning elevation topography and highlight potential sources of variation in this methodology. Ray tracing calculations were performed on a defined, theoretical, spherical model to obtain a pachymetric measurement. These calculations were tested by comparing the pachymetry measurement derived from the model, over a refractive index shift from 1.376 to 1.400 with the pachymetry measurement obtained by Orbscan II, of a human eye, with an identical alteration in refractive index. The two methods of measurement differed by only 0.157 microm, suggesting that the model is a good representation of slit-scanning elevation topography, and that refractive index variations have only a minimal effect on Orbscan II pachymetry measurement.     

Intestinal intraepithelial lymphocyte T cells are resistant to lpr gene-induced T cell abnormalities.

The mucosal immune system of the gastrointestinal (GI) tract consists of Peyer's patches (PP), which are IgA inductive sites, and more diffuse effector regions which include cells in the intraepithelial lymphocyte (IEL) compartment. Since autoimmune MRL lpr/lpr (MRL/lpr) mice develop a proliferating CD3+, CD4-, CD8- (double negative; DN), B220+ T cell subset in systemic lymphoid tissue, we have initiated studies to determine the distribution of CD3+, DN, B220+ T cells (B220+ T cells or lpr/lpr T cells) in the GI immune system. Specifically, we examined T cell subsets separated according to expression of CD4, CD8, Thy-1, B220, alpha/beta T cell receptor (TcR) and gamma/delta TcR in PP and IEL of MRL/lpr mice at 6, 12 and 21 weeks of age. Increased numbers of CD3+ T cells were noted in both PP and spleen of 12- and 21-week-old mice in which the development of autoimmune disorders were also evident. However, normal numbers of CD3+ IEL T cells were seen in MRL/lpr mice in all three age groups tested. When the presence of T cell lymphadenopathy was examined in both IgA inductive and effector tissues, the PP followed the B220+ T cell pattern seen in the spleen, where approximately 30%-50% of CD3+ T cells in the PP of 12- and 21-week-old MRL/lpr mice expressed the phenotype of lpr/lpr T cells and greater than 90% were alpha/beta TcR+. On the other hand, B220+ T cells had not developed in PP or spleen of 6-week-old MRL/lpr mice. Of interest was the finding that IEL from lpr/lpr homozygous mice did not contain B220+ T cells in any age group tested. In this regard, the IEL of MRL/lpr mice comprised an identical pattern and frequency of CD4-/CD8+, CD4+/CD8-, DN and CD4+/CD8+ (double positive, DP) T cell subsets as their normal counterparts (i.e. MRL +/+, BALB/c and C3H/HeN mice) which consisted of approximately 75%, approximately 7.5%, approximately 7.5% and approximately 10%, respectively. Further, Thy-1, gamma/delta TcR and alpha/beta TcR expression in these four subsets of MRL/lpr IEL were very similar to normal mice. These results suggest that the intestinal IEL compartment is minimally affected by the lpr/lpr mutation which induces T cell abnormalities and indicate that B220+ T cells do not preferentially home to IEL. Further, our results support the concept that IEL T cells develop as a separate T cell lineage from thymus-derived cells.     

Virulence of Streptococcus mutans: comparison of the effects of a coupling sugar and sucrose on certain metabolic activities and cariogenicity.

A coupling sugar preparation (sucrose-free [CSSF]), which contains a mixture of sugars, oligosaccharides, and oligosaccharides terminated at the reducing end by sucrose, served as a substrate for growth and acid production by Streptococcus mutans 6715. However, CSSF was a poor substrate for cellular aggregation, glucosyltransferase activity, plaque formation, and adherence of cells to glass surfaces. In the presence of sucrose, CSSF inhibited glucosyltransfer activity and adherence of cells. The substitution of CSSF for sucrose in a rat diet significantly reduced caries score. Furthermore, rats fed diets containing sucrose and CSSF had significantly fewer carious lesions than did rats fed a sucrose diet.     

Gingival mononuclear cells from chronic inflammatory periodontal tissues produce interleukin (IL)-5 and IL-6 but not IL-2 and IL-4.

A unique characteristic of the localized inflammatory tissue in the periodontium (e.g., adult periodontitis [AP]) is the accumulation of IgG (IgG1 > IgG2 > IgG3 > or = IgG4) followed by IgA plasma cells (IgA1 > IgA2). However, the exact molecular mechanisms contributing to these elevated B-cell responses at the local disease site are still unknown. Thus, this study has examined the production of cytokines of importance in B-cell responses, e.g., interleukin (IL)-2, IL-4, IL-5, and IL-6 by gingival mononuclear cells (GMC) isolated from patients in severe stages of AP. These cytokines were assessed at the protein and messenger (m)RNA levels to understand their importance for the observed increased B-cell responses present in these tissues. Among the four cytokines tested by respective cytokine-specific, polymerase chain reaction and dot-blot hybridization, high levels of IL-5- and IL-6-specific mRNA were noted in GMC freshly isolated from AP patients. On the other hand, specific message for IL-2 and IL-4 were not present. Further, the analysis of culture supernatants of GMC also revealed that cells from AP patients spontaneously produced IL-5 and IL-6 but not IL-2 and IL-4. In contrast, when peripheral blood mononuclear cells isolated from the same patients were examined for these cytokines, no detectable levels of mRNA or secreted cytokines were noted. These results showed that GMC from localized inflammatory tissues in severe stages of AP possess a distinct cytokine profile represented by high levels of IL-5 and IL-6 mRNA expression and protein synthesis, whereas IL-2 and IL-4 were not detected. Further, this study supports the concept that AP is a localized inflammatory disease, because GMC from the inflamed tissue actively produce IL-5 and IL-6, whereas peripheral blood mononuclear cells from the same patients do not.     

A T cell/B cell/epithelial cell internet for mucosal inflammation and immunity

Conclusions Mucosal immune responses are strongly regulated by CD4⁺ T cells and their derived cytokines. In this regard, IFN-^–/– mice (i.e., which lack Th1 and have elevated Th2 cells) showed strong mucosal Th2-type responses together with S-IgA production, while IL-4^–/– (e.g., dominant Th1 and lack of Th2 cells) mice had impaired mucosal Th2 and IgA responses following oral delivery of TT and CT. However, when rSalmonella or radenovirus were used for antigen delivery, significant levels of mucosal IgA responses were induced in both IFN-^–/^– and IL-4^–/^– mice. The choice of the antigen delivery system which leads to optimal Th and B cell interactions are important for the induction of effective IgA responses, even in situations where the immune system is compromised. It is clear that Th2-type cytokines are important in mucosal IgA responses; however, other cytokine combinations can compensate for mucosal immunity in situations in which Th2 cell responses are absent. Mucosally induced tolerance may be one approach to prevent several systemic immune disorders; however, the mechanism of this phenomenon still needs to be elucidated. Our recent findings have suggested that IFN- may play an important role in induction of systemic unresponsiveness since oral tolerance was not induced in IFN-^–/^– mice.Our studies as well as those of others indicated that at least two phases of a triad of cell interactions are important for the mucosal immune system. First, it has been shown that epithelial cell-produced IL-7 and SCF and T cell-derived IL-2 are essential activation and growth signals for intestinal T cells. Second, our studies with TCR knockout mice have suggested that mucosal T cells also play a critical role in the regulation of mucosal IgA responses. Thus, a mucosal internet among T cells, T cells, and IgA B cells appear critical for mucosal homeostasis and for regulation of specific mucosal immune responses.     

Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant.

Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 micrograms of TT given orally with 10 micrograms of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-TT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or IgM were found compared with the numbers in mice given TT alone. The results clearly indicate that serum and mucosal antibody responses develop with different kinetics and that protective TT-specific antibody responses are generated in the systemic compartment when TT is administered with CT via the oral route.