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The frequency of cytotoxic NK cells has been directly measured. The cytotoxic assay is performed by preparing conjugates of effector cells bound to target cells and assessing under the microscope target cell lysis by the trypan blue dye exclusion test. With Ficoll-Hypaque purified cells, it was found that 11--13% of lymphocytes formed conjugates with K562 or Molt-4 target cells, but only 3--4% of the total lymphocytes actually killed. Thus, the NK cell's ability to bind targets cannot be equated to the ability to kill. The nature of the noncytotoxic binding lymphocytes is discussed. The kinetics of lysis by NK cells show a random distribution of killing in relationship to time and plateaus at 3--4 hours. The frequency of killing is the same for a wide variety of populations tested at different times. We conclude that some of the target binding cells are antigen specific NK cells that can be converted to cytotoxic natural killer cells.

It was recently reported that, using a T-cell receptor alpha-chain complementary deoxyribonucleic acid probe (pGA5) and the restriction enzyme Eco RV, a 10-kilobase restriction fragment length polymorphism was detected significantly more frequently in patients with ulcerative colitis than in patients with Crohn's disease and controls. This finding had great potential importance, as no gene marker had previously been found to be strongly associated with inflammatory bowel disease. Therefore, an attempt to confirm it in an independent laboratory and patient population has been made in this study. Thirty patients with ulcerative colitis, 30 with Crohn's disease, and 30 healthy control subjects were studied using the Eco RV restriction enzyme and the same T-cell receptor alpha-chain complementary deoxyribonucleic acid probe as was used in the prior report. No 10-kilobase fragment or any other polymorphism using this probe-enzyme combination was found in any of the individuals studied. Polymorphisms were observed with the restriction enzyme Bgl II, but their frequencies did not distinguish between cases and controls. Therefore, there is as yet no evidence for an association between polymorphisms of Eco RV-digested genomic DNA probed with the pGA5 T-cell receptor alpha-chain complementary deoxyribonucleic acid and the predisposition to inflammatory bowel disease.

A case of hyperamylasemia associated with gonococcal salpingitis and perihepatitis (Fitzhugh-Curtis syndrome) is reported. Other potential causes of hyperamylasemia such as pancreatic, parotic, biliary, or intestinal disease were carefully excluded. In addition, the amylase to creatinine clearance ratio was within normal limits, suggesting tubal rather than pancreatic source of hyperamylasemia. This finding is of clinical importance since acute salpingitis is a prevalent disease and its initial presentation may mimic acute pancreatitis or other causes of surgical abdomen. The amylase to creatinine clearance ratio is of value in differentiating such cases from those with hyperamylasemia due to pancreatitis.

The mechanism of lysis by cytotoxic T lymphocytes, K cells, and natural killer (NK) cells is imperfectly understood at this point. In this report, material (glycopeptide) isolated from the plasma membranes of K562 cells and fractionated on lectin affinity adsorbents which has been shown to inhibit NK lysis, was used in several specific NK assays to ascertain what stages of the NK-lytic sequence is inhibited by this substance. Results indicate that this glycopeptide (a) does not inhibit initial binding, but dissociates conjugates following initial effector target interactions; (b) inhibits NK lysis beyond Ca-dependent programming, and (c) inhibits lysis induced by NK cell-derived soluble cytotoxic factors (NKCF) in a soluble factor assay. These results suggest that this glycopeptide can effect the lethal hit stage of NK lysis and may represent structures which can associate directly with NKCF.

The intestine is a unique immunologic organ that comprises an afferent and efferent compartment and provides the host with the ability to respond through several different effector mechanisms against environmental factors. We discuss mechanisms in three intestinal diseases in this overview of the mucosal immune system. Genetic and immunologic factors are important in the pathogenesis of celiac disease, which is characterized by damage to the mucosa of the small intestine with resultant malabsorption. Pathogenic microbes are important environmental agents that interact with the intestinal mucosa and initiate local immune responses. Advances in the understanding of the mucosal immune response to these pathogenic microbes have produced a clear picture of the way in which this specialized immune system works in concert with systemic immunity. As to the autoimmune nature of inflammatory bowel disease, no specific antigen has been shown to incite the inflammatory reactions and neither the target cells nor the effector mechanism involved have been identified. Several factors exist, however, to suggest an autoimmune mechanism and the role of mucosal immunologic factors in this disease.

The role of anaerobic or microaerophillic bacteria in spontaneous peritonitis of cirrhosis has not been clearly defined. Among 126 cases recorded in the literature, in only eight (6 per cent), including the two reported here, was bacterascites associated with anaerobic or microaerophilic bacteria. Clinical features in these cases were indistinguishable from those associated with aerobic bacteria. However, polymicrobial bacterascites occurred in four of eight cases associated with anaerobes, as contrasted with only 10 of 118 cases (8 per cent) associated with aerobes alone. On the other hand, concurrent bacteremia occurred in only one of eight cases associated with anaerobes as contrasted with 52 of 118 cases (44 per cent) of aerobic spontaneous peritonitis. Experimental evidence is cited in an attempt to explain this relatively low incidence of spontaneous peritonitis associated with anaerobic or microaerophilic bacteria, despite the high density of these organisms in the normal bowel flora.

The previous sections illustrate that we are still defining (a) which sets of lymphoid cells are present in the intestine and which are not, (b) which sets are peculiar to the intestine, and (c) how the sets that are there function in the intestinal microenvironment. An understanding of the latter point is going to require knowledge of how these sets communicate with and regulate one another via cell surface molecules such as MHC class I and class II molecules, and via soluble mediators or lymphokines. The recent advances in various technologies make this a particularly exciting time in this field because the tools are now available to address and answer some of these basic and important questions in mucosal immunology. At the same time these advances hold great promise for our eventual understanding of chronic inflammatory diseases of the intestine. As was mentioned at the outset, the immune system has considerable power for both protection and destruction. It remains a puzzle how this latter potential is contained and controlled in the intestine of most individuals, such that they do not have inflammatory disease even in the setting of intense stimulation by substances, such as endotoxin, that are phlogistic elsewhere in the body. An answer to the question of why everyone does not have intestinal inflammation could provide new insights into the mechanisms involved in chronic intestinal inflammatory diseases. The recent advances just detailed, as well as others sure to come, suggest that it is only a matter of time before such questions are answered.

Human intestinal lamina propria mononuclear cells have been shown to mediate mitogen-induced cellular cytotoxicity, antibody-dependent cellular cytotoxicity, and lymphokine activated killer cell function. However, although natural killer cells have been demonstrated in the gut mucosa of rodents, recent reports found little or no spontaneous cytotoxic activity in the lamina propria of the human gut. Using the natural killer cell-related monoclonal antibody NKH-1, which has not previously been applied to studies of mucosal killer cell function, we have shown by immunofluorescence that 2%-3% of enzymatically dispersed lamina propria lymphocytes are NKH-1+. A "panning" technique was then used to enrich for the NKH-1+ cells. Panned cells were consistently greater than or equal to 80% NKH-1+ by indirect immunofluorescence. Unlike their counterparts in the peripheral blood, the mucosal NKH-1+ cells were Leu-11-. Although unseparated lamina propria lymphocytes failed to exhibit natural killer activity against K562 targets in 4-h chromium release assays at effector to target ratios of up to 100:1, the NKH-1+ cells were cytolytically active at ratios of less than 5:1. Mucosal lymphocytes depleted of natural killer cells (NKH-1-) exhibited cytotoxic activity when cultured for 72 h with interleukin-2. The precursors of the lymphokine culture activated phenomenon were NKH-1-, Leu-11-, T4-, T3-, T11+, and T8+. Although lamina propria T3+ cells did not exhibit spontaneous or culture activated cytotoxicity, they were shown to exhibit nonspecific anti-CD3 (anti-T3)-induced T-cell cytotoxicity. In conclusion, functional natural killer and lymphokine activated killer cells are both present in the human gut mucosa and represent distinct populations of cytotoxic cells. In addition, anti-CD3-induced cytotoxicity is a feature of mucosal T cells. These mucosal killer cell subsets differ phenotypically from those previously described in the peripheral blood.

Neutrophils have an important role in mediating tissue injury in inflammatory bowel disease. The proinflammatory peptide formyl-methionyl-leucyl-phenylalanine (FMLP), which is produced by intestinal bacteria, is a potent chemotactic factor for human neutrophils and has been used experimentally to induce acute colitis in animal models. Its action involves specific receptors on the surface of the neutrophil. Increased mucosal permeability, which has been described in Crohn's disease, might contribute to the pathogenesis of the intestinal lesions in this disease by means of increased absorption of FMLP and other gut-derived bacterial products. To evaluate the interaction between FMLP and neutrophils in patients with inflammatory bowel disease, we have studied the binding characteristics and responsiveness of circulating neutrophils to this peptide. Neutrophils from patients with Crohn's disease but not those with ulcerative colitis were found to have significantly increased numbers of receptors for FMLP with similar affinity constants. This was associated with a significantly increased functional response to FMLP in chemiluminescence assays. Differences in neutrophils between Crohn's disease and ulcerative colitis are probably not intrinsic to the neutrophil but reflect their conditioning by the different inflammatory milieu of these diseases.

Phospholipase A2 (PA-2) is known to be involved in many calcium-dependent cellular processes and inhibitors of PA-2 have been shown to inhibit natural killer cell-mediated cytotoxicity (NK CMC). Since the trigger stage is calcium dependent, it was postulated that this effector cell-associated enzyme may play a role in early calcium-dependent processes. To define how PA-2 might be involved in NK lysis, the effect of both PA-2 inhibitors and exogenous PA-2 on the stages of NK lysis was examined. PA-2 inhibitors, quinacrine and p-bromophenacyl bromide, inhibited NK CMC at the effector cell level, but affected neither initial target-effector cell binding nor dissociated conjugates during the length of the NK assay, suggesting that they block post-binding lytic events. A calcium pulse assay showed that PA-2 inhibitors inhibit only moderately when added after calcium and only within the first 15 min, demonstrating that these inhibitors blocked very early post-binding lytic events. Because this very early post-binding inhibitory effect was consistent with effects upon the NK trigger mechanism, the effect of exogenous PA-2 on NK lysis was tested. Pretreatment of K562 target cells but not pretreatment of peripheral blood lymphocytes (PBL) with 20 units/ml PA-2 enhanced lysis by two to eight-fold (based upon lytic units), showing its enhancing effect to be at the target cell level. Single cell assays using effector cells purified by indirect panning with monoclonal antibody NKH-1 showed that only the number of killer cells was increased. Calcium pulse assays showed that enhancement of lysis was maximum 15 min after addition of calcium and decreased rapidly thereafter, demonstrating its effect at an early post binding stage. Additionally, PA-2 was shown to overcome inhibition by the monoclonal antibody 13.3, which has been shown to affect the trigger stage of NK lysis (post-binding but prior to calcium dependent events). Thus, it appears that an NK cell-associated PA-2 could function by modulating the target cell surface, revealing a structure which acts as a "secondary" trigger, subsequent to the 13.3 "trigger", requisite for activation of the NK lytic process.

An unusual case of community-acquired necrotizing aspiration pneumonia with pulmonary abscess and empyema associated with Campylobacter fetus (formerly Vibrio fetus) in mixed culture is reported. This microaerophilic gram-negative curved bacillus has been increasingly associated with human disease. Unlike most pathogens associated with community-acquired aspiration pneumonia, this organism is usually resistant to penicillin. Infection associated with this organism is characteristically relapsing in nature and requires protracted antimicrobial therapy.

Purpose: The Hermansky-Pudlak syndrome is an autosomal recessive disorder consisting of the triad of oculocutaneous albinism, a storage pool platelet defect, and multisystem tissue deposition of ceroid pigment. Although the underlying metabolic defect has not been identified, secondary or associated effects on the immune system are suggested by reports of an association with disorders such as pulmonary fibrosis, granulomatous colitis, lupus, and frequent bacterial infections. We evaluated a large group of patients with the Hermansky-Pudlak syndrome to assess immune competence in this condition., Patients and Methods: Fifteen patients with this syndrome were studied. Control subjects included healthy volunteers in the same age range as the patients. Peripheral blood lymphocytes and neutrophils were isolated according to previously described methods. Immunofluorescent staining, flow cytometry, and cytotoxicity assays were performed. Determination of lymphocyte transformation, mixed lymphocyte response, and neutrophil function was made., Results: Immunoglobulin levels, complement, lymphocyte subsets, natural killer and lymphokine-activated cytotoxicity, mixed lymphocyte responses, and lectin-induced transformation were normal in all patients. In addition, there was no evidence for a lymphocyte proliferative response to a preparation of urinary ceroid pigment isolated from these patients. Neutrophil function, including luminol-dependent chemiluminescence, chemotaxis, and aggregation was not significantly different from control values., Conclusion: The results suggest that there is no generalized defect of peripheral blood lymphocyte or neutrophil function in the Hermansky-Pudlak syndrome. We propose that future studies should examine the possibility that associated disorders arise from a defect within the monocyte-macrophage system, perhaps secondary to ceroid accumulation.

In vivo booster immunization with tetanus toxoid normally results in the temporal development of circulating populations of B cells that secrete antibody after in vitro culture. In assessing the humoral immunoregulatory status in patients with inflammatory bowel disease, we found that the antibody production by the B cells that spontaneously secrete tetanus-specific immunoglobulin G in vitro, and which occur in the circulation 7 days postimmunization, was highly variable and below normal in the majority of Crohn's disease and ulcerative colitis patients. The decreased in vitro antibody responses correlated with the lack of an increase in the serum antitetanus immunoglobulin G titers, but did not correlate with disease activity, location, or steroid therapy. The majority of patients who did not produce an immunoglobulin G-antitetanus toxoid antibody response similarly failed to produce an immunoglobulin G-antidiphtheria antibody response after immunization. Lymphocytes from the patients who failed to produce normal levels of antitetanus toxoid antibody did, however, proliferate normally when stimulated by tetanus toxoid in vitro. These results suggest there are in vivo humoral immune defects in inflammatory bowel disease.

Two new preparations are under clinical trial for use in the treatment of ulcerative proctitis and left-sided colitis. One is 5-aminosalicylate, a component of sulfasalazine. This substance has virtually no immunomodulating capabilities. Evidence for efficacy equal to that of sulfasalazine in acute colitis has been obtained but its role in preventing recurrences is unknown. The other substance is tixocortol, a rapidly metabolized steroid, which has local antiinflammatory activity. Specific procedures of preparation and dosage remain to be established. Both preparations are administered as enemas.

Sulfasalazine and sulfapyridine but not 5-aminosalicylate inhibit spontaneous cytotoxicity mediated by human natural killer (NK) cells. The aim of this study was to determine which stage(s) of the NK cytotoxic reaction is inhibited by these compounds. Effector/target cell binding studies performed in parallel with cytotoxicity assays using purified large granular lymphocytes indicated that inhibition is a post-binding event. The kinetic profile of inhibition in a calcium pulse assay showed that inhibition continues long after the effector cell triggering stage and that although sulfasalazine may have some inhibitory effect on the calcium-dependent events of the programming phase, sulfapyridine continues to inhibit during the calcium-independent or lethal hit phase of the cytotoxic sequence. The NK soluble cytotoxic factor (NKCF) assay was used as a measure of the lethal hit since the time course of this assay permits study of the various substages of this terminal event in the lytic sequence. Sulfasalazine and sulfapyridine but not 5-aminosalicylate inhibited NKCF-mediated target cell lysis. Different substages of the NKCF-induced lytic reaction were affected by these agents. Sulfasalazine appears to inhibit binding of NKCF to the target cell whereas sulfapyridine predominantly inhibits early post-binding events.

Inhibitors of energy metabolism, 2-deoxyglucose and cyanide were shown to inhibit NKCF-mediated lysis of L929 target cells at the same molar concentrations that effectively inhibited cellular ATP levels and the toxic effects of pseudomonas toxin A. In addition, inhibitors of receptor-mediated endocytosis, cytochalasin B, a microtubule disrupter, and trifluoperazine, an inhibitor of clathrin-coat formation, inhibited NKCF-mediated lysis and expression of pseudomonas toxin activity, but had little effect upon cellular ATP. Lysomotropic agents chloroquine, ammonium chloride, and dansylcadavarine also inhibited both NKCF-mediated lysis and pseudomonas toxin activity. These results are similar to those involving diphtheria toxin and the plant toxins abrin, modeccin, and ricin, whose mode of action involves inhibition of protein synthesis following receptor-mediated endocytosis. However, it was determined that NKCF did not cause a decrease in the rate of protein synthesis up to the time of cell death. These results suggest that active target cell processes (possibly involving receptor-mediated endocytosis of NKCF) must occur for target cell lysis to be completed.

We present some new structural and functional data on the family of high mol. wt glycoproteins of human hematopoietic cells which strongly suggests they play a role proximate to certain Ca2+-dependent events. Structural data on this complex are presented which describe elements of its tertiary structure and associations between the different glycoproteins in the plasma membrane. The mol. wt profile of the T-200 structures on highly enriched natural killer (NK) cells is used to support an argument for a T-cell vs a myeloid nature for NK cells. Furthermore, certain murine monoclonal antibodies directed to a particular epitope of T-200 on NK cells are shown to block cytolysis. The specificity of this blockade strongly suggests that T-200 may be an NK receptor. A mapping of this effect has allowed us to define a new stage of the lytic cycle, termed "triggering". This very rapid event occurs subsequent to conjugation between the NK and the target cell, and prior to the Ca2+-dependent second-phase programming for lysis. A close association between "triggering", as defined by antibodies to T-200, and Ca2+-dependent stimulus-secretion events support the concept of T-200 as a receptor structure.