Grateful Med on an institutional local area network.

Grateful Med version 6.0 provides new features very desirable to network users. These include: a single copy of the application resident on a server providing access to many users; a new communications architecture which provides access to Medline via the Internet or local network modems; additional scripting capabilities allowing local customization. These new features reduce the overhead in installing and maintaining Grateful Med (GM), allow much quicker downloading of citations and abstracts from Medline, and remove the requirement of a local modem for each PC accessing Medline.

Oral administration of cyclosporin does not prevent expansion of antigen-specific, gut-associated, and spleen lymphocyte populations during Chlamydia trachomatis proctitis in nonhuman primates.

To study the effects of oral cyclosporin (CsA) administration on immune responses in the gastrointestinal tract, humoral and cellular immune responses were studied in CsA-treated nonhuman primates having Chlamydia trachomatis proctitis (lymphogranuloma venereum, LGV). There was no apparent effect of CsA treatment on the gross or microscopic appearance of LGV proctitis, but CsA-treated animals, with or without LGV infection, had lymphoid hyperplasia of spleen and mesenteric lymph nodes. CsA treatment inhibited the primary antibody response to LGV, inhibited peripheral blood lymphocyte mitogen-induced proliferation and IL-2 production, and inhibited LGV-specific proliferation of peripheral blood lymphocytes. In contrast, mitogen-stimulated proliferation of spleen, mesenteric lymph node, and lamina propria lymphocytes was not significantly inhibited in CsA-treated animals. In addition, LGV-specific proliferation of spleen and mesenteric lymph node lymphocytes was not inhibited. High mitogen-stimulated IL-2 production of lamina propria lymphocytes was only partially inhibited in CsA-treated animals. In vitro CsA treatment had the expected inhibitory effects on mitogen- and antigen-induced proliferation of spleen and mesenteric lymph node lymphocytes. Thus, although oral cyclosporin inhibits the antibody and proliferative responses of peripheral blood lymphocytes to antigens and mitogens in animals having Chlamydia trachomatis proctitis, it does not prevent the expansion of antigen-specific, gut-associated, and spleen lymphocyte populations.

Mucosal T cells provide helper function but do not proliferate when stimulated by specific antigen in lymphogranuloma venereum proctitis in nonhuman primates.

To study antigen-specific immune responses of gut-associated T lymphocytes after gastrointestinal infection, Cynomolgus monkeys were inoculated rectally with Chlamydia trachomatis of the L2 [lymphogranuloma venereum (LGV)] strain. Infected monkeys developed a chronic proctitis with the appearance of LGV-specific immunoglobulin G-antibodies in the serum. Lymphocytes isolated from the peripheral blood, the spleen, and draining lymph nodes had a vigorous antigen-specific proliferative response to LGV in vitro. Both T and B cells proliferated in response to stimulation with LGV, but B-cell proliferation was T-cell-dependent, as shown by cell separation techniques and cell-cycle analysis with dual-laser flow cytometry. Lymphocytes isolated from both involved and uninvolved lamina propria did not proliferate in response to LGV stimulation, whereas mitogen-induced proliferation was not different in lamina propria lymphocytes and the other lymphocyte populations. This lack of antigen-specific proliferation was not caused by a suppressor effect of mucosal T cells or monocytes or the absence of antigen-presenting cells. In contrast, lamina propria T lymphocytes from infected animals were able to provide antigen-specific help for polyclonal immunoglobulin synthesis by immune B lymphocytes after stimulation with LGV. Thus, in LGV proctitis in monkeys, mucosal antigen-reactive T cells differ from lymphocytes in other sites in that they can provide helper function, but are not able to proliferate in response to LGV antigens.

Effect of IL-2 on immunoregulatory function of intestinal lamina propria T cells in normal non-human primates.

The role of T cell activation on the immunoregulatory function of lymphocytes in the lamina propria of the normal intestine was investigated. Lymphocytes were isolated from different sites in non-human primates, and their cell surface phenotypes, response to exogenous IL-2, and immunoregulatory function in pokeweed mitogen stimulated cultures were determined. The proportions of Leu-3+ (CD4) and Leu-2+ (CD8) lymphocytes in isolated lamina propria cells were similar to peripheral blood and spleen, but the proportion of Leu-3+ cells was significantly higher in mesenteric lymph node lymphocytes. The proportion of IL-2 receptor positive cells was significantly higher in lamina propria compared with peripheral blood, spleen and mesenteric lymph nodes. Increased IL-2 receptor expression was found on both Leu-3+ and Leu-2+ lamina propria T cells. In addition, although lamina propria T cells had a lower proliferative response to Con A, they had a significantly higher response when cultured with recombinant IL-2, indicating that they have increased expression of functional IL-2 receptors. The helper function of lamina propria T cells for pokeweed mitogen stimulated immunoglobulin synthesis was enhanced by recombinant IL-2. Although Leu-2+ lymphocytes in the lamina propria had increased IL-2 receptor expression, suppressor function of lamina propria T cells was similar to that of spleen cells, and was not enhanced by addition of exogenous IL-2. Thus, T cells in the lamina propria have increased expression of functional IL-2 receptors, and recombinant IL-2 enhances the helper, but not the suppressor function of lamina propria T cells for immunoglobulin synthesis.

Predominance of helper-inducer T cells in mesenteric lymph nodes and intestinal lamina propria of normal nonhuman primates.

To define the characteristics of T cells associated with the gastrointestinal tract, the phenotypes and immunoregulatory function of T cells from mesenteric lymph node (MLN) and lamina propria lymphocytes (LPL) were compared to peripheral blood (PBL) and spleen lymphocytes in normal nonhuman primates. Mesenteric lymph node lymphocytes were characterized by a higher proportion of Leu-3+(CD4+) and 9.3+(alpha-Tp44) lymphocytes and a lower proportion of Leu-2+(CD8) lymphocytes than lymphocytes in other sites. LPL and MLN lymphocytes were both characterized by a higher proportion of cells having the helper-inducer phenotypes (Leu-3+, Leu-8+, Leu-3+, 2H4+) compared to PBL. A lower proportion of cells with the suppressor-inducer phenotypes (Leu-3+, Leu-8+, Leu-3+, 2H4+) was found in LPL, but not in MLN lymphocytes compared to PBL. In studies of the Leu-2+ T cells, it was found that whereas PBL, spleen, and LPL contained approximately equal proportions of Leu-2+, Leu-15+ (suppressor phenotype) and Leu-2+, 9.3+ lymphocytes (cytolytic T-cell phenotype), the MLN T cells were predominantly Leu-2+, 9.3+. Furthermore, the Leu-3/Leu-2 ratio was significantly higher in MLN compared to other sites. In pokeweed mitogen-stimulated cultures, the highest helper function for Ig synthesis was found in MLN. Cells from none of the sites studied showed evidence of increased suppressor cell activity. These results show that MLN and LPL T cells in normal nonhuman primates differ from T cells in peripheral blood and spleen. While both MLN and LPL have a high proportion of T cells with the helper-inducer phenotype, cells with the suppressor-effector phenotype are infrequent in MLN, while cells with the suppressor-inducer phenotype are infrequent in LPL.

Cytotoxic and immunoregulatory function of intestinal lymphocytes in Chlamydia trachomatis proctitis of nonhuman primates.

To study the role of natural killer cells and immunoregulatory T cells in the pathogenesis of proctitis due to Chlamydia trachomatis (L2 serovar), lymphocytes were obtained from the rectal mucosa and other sites of nonhuman primates and studied by using phenotypic and functional assays. In animals with lymphogranuloma venereum (LGV) proctitis, the percentage of lymphocytes with the natural killer cell phenotype (Leu-11+) was not significantly higher at any site in LGV infection, and natural killer cell function of lymphocytes isolated from the rectum was lower during LGV infection. This was not due to the suppressive effect of factors in serum, rectal lymphocytes, or LGV elementary bodies. In studies of regulatory T cells, the Leu-3+/Leu-2+ ratio was lower in the peripheral blood and the spleen during LGV infection, but the ratio did not decrease in lamina propria T cells. Both peripheral blood and rectal lymphocytes had higher helper T-cell function for polyclonal immunoglobulin G (IgG) synthesis in pokeweed mitogen-stimulated cultures 2 weeks following LGV infection. Increased suppressor T-cell function for pokeweed mitogen-stimulated IgG synthesis was found only in the peripheral blood of animals 2 weeks after infection, but not in isolated rectal lymphocytes. These results indicate that in LGV proctitis natural killer cells are not an important component of the inflammatory infiltrate at the site of infection, and helper T-cell function increases in peripheral blood and rectal lymphocytes.

Phenotypic analysis of lamina propria lymphocytes. Predominance of helper-inducer and cytolytic T-cell phenotypes and deficiency of suppressor-inducer phenotypes in Crohn's disease and control patients.

To better define the nature of intestinal T cells, the phenotypes of isolated lamina propria lymphocytes (LPL) were determined in both Crohn's disease patients and control patients using combinations of monoclonal antibodies that have been found to correlate with particular immunoregulatory functions. Isolated LPL and autologous peripheral blood lymphocytes (PBL) were stained with multiple combinations of monoclonal antibodies and studied by dual immunofluorescence flow cytometry. In LPL, compared with PBL, there was a significant increase in the proportion of T cells having the Leu-3+, Leu-8- and Leu-3+, 2H4- phenotypes (associated with helper-inducer function) and a corresponding decrease in the proportion of T cells having the Leu-3+, Leu-8+ and Leu-3+, 2H4+ phenotypes (associated with suppressor-inducer function). It was also found that in LPL, compared with PBL, the percentage of cells with the Leu-2+, Leu-15+ phenotype (associated with suppressor-effector function) was significantly lower. However, the percentage of T cells with the Leu-2+, 9.3+ phenotype (associated with cytolytic function) was similar in PBL and LPL in control patients. There were no major differences comparing Crohn's disease patients with control patients, except that the proportion of Leu-2+, 9.3+ lymphocytes was higher in PBL in Crohn's disease patients. These results show that the lymphocyte subpopulations in the lamina propria differ from those in peripheral blood in having predominantly the phenotypes of helper-inducer and cytolytic T cells, whereas the phenotypes of suppressor-inducer cells and activated suppressor cells are less frequently observed.

Spontaneous and lymphokine-induced cytotoxic activity of monkey intestinal mucosal lymphocytes.

We determined the capacity of primate (macaque monkey) intestinal mucosal lymphocytes to mediate natural killer cytotoxicity, and characterized the nature of cells mediating this form of cytotoxicity in the intestine. Isolated macaque monkey intestinal mucosal lymphocytes were found to have intermediate levels of spontaneous cytotoxicity against K562 target cells compared to higher values found in lymphocytes of peripheral blood (PBL) and spleen and low values for mesenteric lymph node (MLN) lymphocytes. Intestinal lymphocytes were similar to PBL in having the same range of target cell specificites, in having augmentation of activity by interferon or interleukin 2, and in demonstrating specificity in cold target inhibition studies. Both intestinal and PBL spontaneous cytotoxic function in primates was mediated predominantly by cells bearing antigens cross-reactive with the anti-human monoclonal antibody Leu-11. The percentage of Leu-11+ lymphocytes was significantly lower in isolated intestinal, spleen, and MLN lymphocytes compared to peripheral blood. Furthermore, isolated intestinal lymphocytes differed from PBL in that intestinal Leu-11+ were predominantly Leu-15-, while Leu-11+ PBL were predominantly Leu-15+. These studies demonstrate that the lower spontaneous cytotoxic function of intestinal mucosal lymphocytes compared to PBL is associated with a lower number of effector cells and with effector cells which differ qualitatively in expression of the Leu-15 antigen.

Immunoregulatory function of lamina propria T cells in Crohn's disease.

The pathogenesis of Crohn's disease may involve altered function of immunoregulatory T cells in the intestine. To investigate this hypothesis, lamina propria lymphocytes were isolated from intestinal specimens resected from patients with active Crohn's disease and control subjects (colon carcinoma and diverticular disease) using an enzymatic technique. The T-cell phenotypes and function of these lymphocytes were compared with that of peripheral blood lymphocytes. The proportion of Leu-2-positive (suppressor/cytotoxic) cells was similar in peripheral blood and isolated lamina propria lymphocytes, both in Crohn's disease and control patients. Although the proportion of Leu-3-positive (helper/inducer) lymphocytes was lower in lamina propria lymphocytes than peripheral blood lymphocytes, there was no difference comparing Crohn's disease and control patients. Helper T-cell function, as determined by measuring the ability of T cells to increase immunoglobulin synthesis by pokeweed mitogen-stimulated normal peripheral blood B cells, was similar in peripheral blood lymphocytes and lamina propria lymphocytes, and comparing Crohn's disease with control patients. Suppressor T-cell function, as determined by measuring the ability of T cells to inhibit immunoglobulin production by cultures containing pokeweed mitogen-stimulated normal peripheral blood T and B cells, was also similar comparing peripheral blood lymphocytes and lamina propria lymphocytes, and comparing Crohn's disease with control patients: neither peripheral blood lymphocytes nor lamina propria lymphocytes significantly suppressed immunoglobulin synthesis. OKT8 (suppressor/cytotoxic)-enriched lamina propria lymphocytes mediated only marginal suppression, whereas concanavalin A-activated intestinal T cells did mediate significant suppression, in both Crohn's disease and control patients. Thus, patients with active Crohn's disease have no alteration of immunoregulatory T-cell function for polyclonal mitogen-induced immunoglobulin synthesis at the gut mucosal level, despite the presence of an inflammatory process in the intestine.

Suppression of immunoglobulin synthesis by lymphocyte subpopulations in patients with Crohn's disease.

In previous studies, patients with mild or inactive Crohn's disease were found to have increased suppressor T-cell activity. To further characterize suppressor T cells in Crohn's disease, studies were carried out with the use of monoclonal antibodies. Excessive suppressor activity was eliminated by removal of OKT 8+ lymphocytes by complement-mediated lysis. However, the percentage of OKT 8+ (or Leu 2a+) cells and the ratio of OKT 4+ to OKT 8+ (or Leu 3a+ to Leu 2a+) cells were not significantly different from normal. Although the subgroup of patients with increased suppression of immunoglobulin synthesis had a significantly lower mean Leu 3a to Leu 2a ratio than that of normal subjects, in the whole group of Crohn's patients studied, neither the percentage of Leu 2a+ cells nor the ratio of Leu 3a+ to Leu 2a+ cells correlated with excessive suppression of immunoglobulin synthesis. A subpopulation of Leu 2a+ lymphocytes reactive with the monoclonal antibody HNK-1 (Leu 2a+ HNK-1+) was increased in patients with Crohn's disease. Furthermore, elimination of HNK-1-reactive lymphocytes by complement-mediated lysis diminished the excessive suppressor cell function in patients with Crohn's disease. The percentage of Leu 2a+ HNK-1+ lymphocytes correlated significantly with the suppression of pokeweed mitogen-stimulated immunoglobulin synthesis in vitro. Thus, patients with mild Crohn's disease have an increased suppressor cell activity in vitro which correlates with the presence of a subset of lymphocytes that have an HNK-1+ Leu-2a+ phenotype.

Hypogammaglobulinemia due to abnormal suppressor T-cell activity in Crohn's disease.

Two patients are described who initially developed Crohn's disease followed by hypogammaglobulinemia. The immunologic profile of both was typical of acquired common variable hypogammaglobulinemia. The peripheral blood lymphocytes of neither patient were able to synthesize immunoglobulin when cultured with pokeweed mitogen in vitro nor were cultures containing mixtures of purified B cells and T cells. A potent suppressor T cell was present in the peripheral blood of both patients that was able to completely suppress immunoglobulin synthesis in cultures of normal B cells and T cells. When the patients' B cells were highly purified and appropriately stimulated in vitro, they were able to synthesize immunoglobulin, providing strong evidence that the circulating suppressor T cell was mediating the hypogammaglobulinemia. We have previously shown that many patients with Crohn's disease have a circulating "covert" suppressor T cell that is not expressed in cultures of unfractionated peripheral blood lymphocytes, but which becomes apparent after isolation of their T cells on antiimmunoglobulin columns. We conclude that these 2 patients represent instances in which the "covert" suppressor T cell of Crohn's disease has become overtly active in the systemic circulation and has resulted in hypogammaglobulinemia.

Immunoregulatory function of T cells activated in the autologous mixed lymphocyte reaction.

This study was undertaken to define the functional properties of T cells stimulated in the autologous mixed lymphocyte reaction (MLR) by purified B cells or macrophages. In preliminary experiments, it was found that T cells that had been cultured with autologous non-T cells inhibited pokeweed mitogen- (PWM) stimulated immunoglobulin synthesis by autologous B cells. In addition, the T cell-mediated suppression was eliminated by x-irradiation and hydrocortisone treatment, was mediated by a mechanism that occurred early in the PWM-stimulated cultures, and did not involve killing of mature immunoglobulin-secreting cells. T cells were then cultured with either autologous B cells or macrophages in order to determine whether such autoreactive T cells had a similar capacity to regulate PWM-induced immunoglobulin synthesis. Although T cell populations stimulated either by B cells or by macrophages suppressed proliferative responses and immunoglobulin synthesis, both these populations of autoreactive T cells provided help for immunoglobulin synthesis that was not significantly different from that provided by fresh T cells. These results suggest that the predominant functional consequence of activation of T cells in the autologous MLR is the generation of suppressor T cells capable of inhibiting immunoglobulin synthesis. Thus, the autologous MLR may represent a negative feedback mechanism for the regulation of the immune response.

Covert suppressor T cells in Crohn's disease.

To determine whether patients with Crohn's disease have a defect in immune regulation, suppressor T-cell activity was assessed in 16 patients with mild or inactive Crohn's disease and was compared with that of an equal number of randomly selected normal controls. Pokeweed mitogen-stimulated cultures of peripheral blood lymphocytes from patients synthesized as much IgM as those from normal controls. In addition, cocultures of patient and normal peripheral blood lymphocytes did not result in either suppression or enhancement of immunoglobulin M synthesis. In contrast to these results with cultures of peripheral blood lymphocytes, cultures containing optimal ratios of purified B cells and T cells from patients synthesized significantly less immunoglobulin M (p less than 0.005) than those from normals; in fact, the latter cultures from 6 patients synthesized no detectable immunoglobulin M. Detailed studies of cells from these patients indicated that a suppressor T cell was revealed in vitro during the cell-purification procedure. Finally, in those patients in whom it could be measured, radiation-sensitive suppressor T-cell activity was found to be normal. We conclude that there is no deficiency of suppressor T cells regulating antibody synthesis in patients with Crohn's disease; on the contrary, at least one-half of these patients have suppressor T cells that markedly inhibit the synthesis of immunoglobulin M, but which are revealed only after purification of the T cells.

Simultaneous induction of antigen-specific IgA helper T cells and IgG suppressor T cells in the murine Peyer's patch after protein feeding.

The effects of feeding the dietary protein antigen, ovalbumin (OVA), on OVA-specific IgG and IgA immune responses involving Peyer's patches (PP) and mesenteric lymph nodes (MLN) were examined. Mice were administered soluble OVA by gastric intubation. One to 3 days later, PP, MLN, or spleen cells from these donor mice were adoptively transferred into normal syngeneic recipients. After two subsequent immunizations, spleens from the recipient mice were assayed for IgA and IgG anti-OVA plaque-forming cell (PFC) responses. None of the tissues from normal (unfed) mice had the inherent ability to alter recipients' IgG or IgA PFC responses. Within 1 day of OVA feeding, however, cells were generated in the PP that could augment recipients' IgA anti-OVA PFC responses and suppress IgG PFC responses. Three days after OVA feeding, these cells were present in MLN as well, and whereas the IgG suppressor cell also appeared to migrate to spleen, the IgA helper cell did not. The cells mediating antigen-specific IgG suppression and IgA help were both T cells but could be distinguished by surface phenotype. We therefore conclude that protein feeding induces differential, isotype-specific immunoregulation in gut-associated lymphoid tissues, part of which is mediated by an antigen-specific IgA helper T cell.