Enhanced levels of functional HIV-1 co-receptors on human mucosal T cells demonstrated using intestinal biopsy tissue.

OBJECTIVE: To examine compartmental differences in co-receptor expression on CD4 lymphocytes between blood and gut using endoscopic biopsies. DESIGN: Mucosal and peripheral CD4 T cells from healthy controls were compared for co-receptor expression and vulnerability to infection by HIV-1. METHODS: Expression of CCR5 and CXCR4 was quantified by flow cytometry on isolated mucosal CD4 lymphocytes obtained from endoscopic biopsies and blood from healthy controls. Vulnerability to in vitro infection by both R5 and X4 strains was assessed by measuring p24. RESULTS: Biopsies yielded sufficient lymphocytes for flow cytometric characterization and infectivity studies. The percentage of mucosal CD4 T lymphocytes that expressed CCR5 and the per cell expression of CCR5 were both significantly increased compared with that in peripheral blood CD4 T lymphocytes. CXCR4 was expressed on the majority of CD4 lymphocytes in both compartments. In vitro infection of mucosal mononuclear cells supported greater viral replication of both R5 and X4 strains than peripheral blood mononuclear cells. CONCLUSIONS: Enhanced expression of CXCR4 and CCR5 on CD4 lymphocytes in normal intestinal mucosa predicts increased vulnerability to infection by both R5 and X4 HIV-1. Endoscopic biopsies provide a useful mucosal tissue sampling technique to identify compartmental immunologic differences that may be exploited by HIV-1 in establishing initial mucosal infection.

Decreased bone density in inflammatory bowel disease is related to corticosteroid use and not disease diagnosis.

Although corticosteroid therapy is associated with the development of osteopenia, it is unclear whether the cause of osteopenia in inflammatory bowel disease (Crohn's disease and ulcerative colitis) is related to corticosteroid therapy or other disease-related variables. Patients with Crohn's disease (a diffuse gastrointestinal disease) could have greater osteopenia than patients with ulcerative colitis because of small bowel disease and secondary malabsorption of calcium and vitamin D. A cross-sectional analysis of consecutive patients with Crohn's disease and ulcerative colitis was undertaken. Bone density was determined by measurements of the L2-L4 spine, the total hip, and Ward's triangle using dual energy X-ray absorptiometry (DXA). A number of clinical parameters were recorded prior to bone density evaluation and analyzed by univariate and subsequently multivariate analysis to determine possible predictors of osteopenia. Of the 26 patients with Crohn's disease, diminished bone density (a Z score of at least -1) was found at the hip in 64% and at the spine in 44%; and of the 23 patients with ulcerative colitis diminished bone density was found at the hip in 43% and at the spine in 48%. Among all the variables tested, only corticosteroid use was a statistically significant predictor of diminished bone density (p = 0.025 for the spine and hip and p = 0.005 for Ward's triangle). Disease diagnosis (Crohn's disease compared with ulcerative colitis) did not predict or correlate with diminished bone density. No obvious associations were seen between the measurements of any serum hormones or biochemistries and bone density, although the patients using corticosteroids had lower serum calcium levels than the nonusers.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of interleukin-1 by microglia in response to substance P: role for a non-classical NK-1 receptor.

Substance P (SP) is a central and peripheral neurotransmitter which has been found in multiple sclerosis plaques. SP stimulates peripheral immune cells and may play a role in some chronic inflammatory diseases. Human peripheral monocyte/macrophages have been shown to produce the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) in response to SP. Therefore, in this study we examined rat brain microglia for the presence of SP receptors and production of IL-1 and TNF alpha in response to SP. Microglia had 4900 +/- 950 (mean +/- SE) receptors per cell fitting a two-site model. Four percent of these were high-affinity receptors with a Kd of 8.2 x 10(-8) M +/- 3.6 x 10(-8) M (mean +/- SE), and 96% of them were low-affinity receptors with a Kd of 2.1 x 10(-6) M +/- 5.2 x 10(-7) M (mean +/- SE). Competitive studies with CP 96,345 and other SP analogs demonstrate these to be non-classical NK-1 receptors. SP alone did not stimulate IL-1 or TNF alpha production. However, SP in synergy with lipopolysaccharide (LPS) quadrupled IL-1 production compared to LPS alone, but did not affect TNF alpha production. These results have implications for certain inflammatory conditions in the central nervous system.

A randomized, placebo-controlled trial of calcium supplementation for decreased bone density in corticosteroid-using patients with inflammatory bowel disease: a pilot study.

BACKGROUND: Patients with inflammatory bowel disease (IBD) have a high prevalence of osteoporosis. A number of studies have found that corticosteroid use is associated with the development of osteoporosis in these patients. Calcium supplementation may be of benefit in corticosteroid-induced osteoporosis and calcium may be a nutrient that patients with IBD lack. AIM: To test the benefit of calcium supplementation on bone density in a pilot study over a 1-year period, in a group of corticosteroid-using patients with IBD, in a randomized, double-blind, placebo-controlled treatment study. METHODS: Corticosteroid-using patients with IBD including males over the age of 18 years and premenopausal females, were randomized to receive either calcium carbonate 1000 mg plus vitamin D 250 IU (Oscal) or an identically matched placebo. Dual energy X-ray absorptiometry measurements of bone density were obtained at entry and at 1 year. At entry, and every 3 months thereafter, serum was collected for the measurement of haemoglobin, biochemistry and bone hormones. Simultaneously a 24-h urine collection was analysed for calcium excretion and creatinine clearance, and a 4-day food record was collected to document dietary calcium and vitamin D ingestion. RESULTS: We found a high prevalence of moderately severe decreased bone density in corticosteroid-using patients with IBD. The dose of prednisone in the year prior to study entry was inversely correlated with bone density at the hip (R = -0.67, P = 0.004). At study entry serum osteocalcin was inversely correlated with corticosteroid dose in the year prior to the study (R = -0.64, P = 0.02) and at study end, directly correlated with the percentage change in spine bone density (R = 0.59, P = 0.01). The dietary calcium intake of these patients was close to the current RDA (recommended daily intake) for premenopausal, post-adolescent adults. Calcium supplementation with small extra doses of vitamin D conferred no obvious benefit to bone density at the end of 1 year. There was no correlation between oral calcium ingestion and bone mass measurements. Both the treatment and placebo groups' bone density remained relatively stable at 1 year, suggesting that bone loss in corticosteroid-using patients may peak early into the use of the corticosteroids. CONCLUSIONS: Calcium supplementation (1000 mg/day) conferred no significant benefit to bone density at 1 year in patients with corticosteroid-using IBD patients with osteoporosis. Future investigations should explore other therapeutic avenues that may have greater effects on increasing bone density in patients who already have considerable osteoporosis.

Neurotransmitters and cytokines in CNS pathology.

In summary, we have demonstrated an in vitro model for oligodendrocyte cell death that may be relevant to events in formation of lesions in MS. It involves cell contact to oligodendrocytes with activated, viable microglia (or inflammatory macrophages), surface TNF-alpha, surface adhesion molecules, and production of NO. Precise mechanisms of TNF-alpha and ICAM-1/LFA-1 participation and the nature of the susceptibility of the oligodendrocyte are currently being studied.

Neuroimmunomodulation in inflammatory bowel disease. How far from "bench" to "bedside"?

The chronic inflammatory bowel diseases (BID), Crohn's disease and ulcerative colitis, are characterized by recurrent periods of inflammation and tissue destruction. The clinical course is influenced by genetics, environmental factors, and the immune system. Recent insights (bench trials) benefiting from advances in genetic engineering and molecular biology have contributed to clinical care (bedside) in terms of actual or potential therapies. Does the neuroendocrine system significantly modify disease activity? Although conceptually appealing, evidence remains circumstantial. Compelling anecdotal reports exist that "stress" affects disease activity in terms of the frequency and severity of IBD flares (bedside), but the mechanisms underlying these observations are unknown. Evidence that neuroendocrine factors play a significant role in immunomodulation is progressing (bench). (i) Trinitrobenzene sulfonic acid (TNB)-induced colitis, although similar in unstressed Fisher and Lewis rats, shows marked worsening in stressed Lewis rats. (ii) Early studies of rectal pain perception suggest there are specific differences in neuroimaging studies (PET scans) in IBD patients compared to controls. (iii) Levels of substance P (SP) and its receptor are altered. (iv) Preliminary clinical studies with SP receptor antagonists show a trend toward improvement. (v) Importantly, the placebo response in clinical trials is as high as 45%. Evidence that neuroendocrine systems significantly modulate local inflammation is rapidly accumulating (bench), which will facilitate enhanced coordination of clinically relevant therapies (bedside).

Biotinylation of a bombesin/gastrin-releasing peptide analogue for use as a receptor probe.

The development of a biotinylated bombesin/gastrin-releasing peptide (GRP) for use as a receptor probe is reported. The lysine13 of a GRP-27 was substituted by arginine and lysine was added to the amino terminus. Biotinylation of the N-terminal lysine was performed. The biotinylated peptide was purified by HPLC and characterized by mass spectral analysis. Binding studies with murine Swiss 3T3 fibroblasts, cells known to express bombesin/GRP receptors, yielded a dissociation curve for the biotinylated GRP-27 analogue (biotin-Lysyl[Asp12,Arg13]GRP-27) which was nearly identical to that of native GRP. Using studies of gastrin release from isolated canine G cells, equipotent functional activity of the biotinylated probe and unmodified GRP was demonstrated. Measurements of retained 125I-avidin confirmed that the biotin/avidin interaction could occur once the biotin-peptide complex was bound. Applicability of the probe was demonstrated with fluorescent microscopy using avidin-FITC on Swiss 3T3 fibroblasts. In conclusion, a novel biotinylated bombesin/GRP analogue has been developed which retains the functional characteristics of the native peptide and is a useful probe for receptor studies.

Sensitive and reproducible quantitation of mucosal HIV-1 RNA and DNA viral burden in patients with detectable and undetectable plasma viral HIV-1 RNA using endoscopic biopsies.

Mucosal tissue is the main portal of entry for HIV-1 infection and, in macaques, has been demonstrated to be a significant compartment for viral replication and CD4+ T lymphocyte depletion. Quantitating tissue viral burden in addition to plasma viral load provides insights into HIV-1 pathogenesis and an additional means to gauge antiretroviral response. The aim of this study was to develop reliable, reproducible, and sensitive assays to quantitate tissue viral burden of HIV-1 RNA and DNA using 1-3 endoscopically acquired, rectosigmoid biopsies. Total DNA and RNA were simultaneously extracted following homogenization from the same tissue samples. Quantitative polymerase chain reaction (PCR) assay in the HIV-1 LTR region was used to detect viral DNA and RT-PCR for viral RNA. It was determined that HIV-1 RNA and DNA can be reproducibly quantified from a single rectosigmoid biopsy with minimal intra-assay or intra-patient variability. These results reflect high recovery of extracted nucleic acids with calculated results accurately reflecting in vivo levels. The techniques outlined differ from currently available approaches by incorporating control standards to identify loss or degradation of RNA and DNA from acquisition through the in vitro assay and permit extraction with high yields of RNA and DNA from the same tissue sample.

Gastrointestinal mucosal biopsy in HIV disease and AIDS.

The role of the gastroenterologist as consultant for patients with HIV infection is reviewed, with a particular focus on when endoscopy with biopsy may be helpful in the diagnostic evaluation. Suggestions on where to biopsy, how to collect samples, and what pathologies might be anticipated are included. In the clinical setting of new antiviral therapies, there has been a dramatic change in the etiologic factors for common presentations such as diarrhea. A review of suspect infections and malignancies is included.

Preferential depletion of gut CD4-expressing iNKT cells contributes to systemic immune activation in HIV-1 infection.

Chronic inappropriate immune activation is the central defect-driving loss of CD4(+) T helper cells and progression to AIDS in persons with HIV-1 infection, but the mechanisms remain controversial. We examined key regulatory invariant receptor natural killer T (iNKT) cells in the gut, the largest reservoir of lymphocytes and a key arena of HIV-1 pathogenesis. In healthy control persons, the anti-inflammatory CD4(+) iNKT-cell subset predominated over the pro-inflammatory CD4(-) iNKT-cell subset in the gut, but not in the blood, compartment. HIV-1 infection resulted in a preferential loss of this anti-inflammatory CD4(+) iNKT-cell subset within the gut. The degree of loss of the CD4(+) iNKT-cell subset in the gut, but not in the blood, correlated to the systemic immune activation and exhaustion that have been linked to disease progression. These results suggest a potentially important contribution of gut iNKT-cell imbalance in determining the systemic immune activation that is the hallmark of HIV-1 pathogenesis.

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in irritable bowel syndrome.

Enhanced stress responsiveness has been implicated as a potential mechanism contributing to the pathophysiology of irritable bowel syndrome (IBS), and should be reflected in altered function of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system. Both of these systems can modulate mucosal immune function. The aims of this study were: (i) to characterize the basal circadian rhythm of adrenocorticotropin hormone (ACTH) and cortisol in IBS vs healthy controls; (ii) to compare stimulated ACTH, cortisol and noradrenaline responses to a pelvic visceral stressor (sigmoidoscopy) in IBS and controls; and (iii) to correlate neuroendocrine responses with colonic mucosal cytokine expression and symptoms in IBS. Two separate studies were conducted in women. In Study 1, basal cortisol levels were analysed in 41 IBS and 25 controls using 24-h collections of plasma ACTH and cortisol (q10 min sampling). In Study 2, 10 IBS patients with diarrhoea (IBS-D) and 10 controls underwent sigmoidoscopy with measurements of stimulated neuroendocrine responses and cytokine mRNA expression in colonic tissue. Basal ACTH levels were significantly blunted (P < 0.05), while basal and stimulated plasma cortisol levels were higher in patients. Basal cortisol levels prior to an experimental visceral stressor positively correlated with anxiety symptoms (P < 0.004), but not IBS symptoms. Irritable bowel syndrome patients with diarrhoea had significantly decreased mRNA expression of mucosal cytokines [interleukin (IL)-2, IL-6] in the sigmoid colon vs controls (P < 0.05). Although dysregulations in stress-responsive systems such as the HPA axis and mucosal immune function are demonstrated in IBS, they do not appear to have a primary role in modulating IBS severity and abdominal pain.

Stress and mind-body impact on the course of inflammatory bowel diseases.

At present, the medical management of inflammatory bowel diseases (IBD) including Crohn's disease and ulcerative colitis, are focused on topical, locally active antiinflammatories and systemic immunosuppressives, which are thought to exert their targeted effects in the gastrointestinal mucosa. There is a paucity of controlled trials assessing the impact of mind, central nervous system (CNS), and neuromodulation on the overly active immune response in the intestinal mucosa. Patients and their physicians have long been aware of a strong association between attitude, stress, and flares of their IBD. Although reports to date remain mostly anecdotal, the degree to which mind-body influences and stress impact levels of local inflammation deserves closer attention with the aim of identifying contributing mechanisms, which may highlight new therapeutic interventions, as well as assist in identifying particular subsets of patients that may respond to novel forms of adjunctive treatments for IBD, including hypnosis, meditation, neuropeptide receptor modulation, and cortisol-releasing factor (CRF) modulation.

Adult polycystic renal disease presenting in infancy: a report emphasizing the bilateral involvement.

We describe a girl with adult polycystic renal disease that presented as a unilateral abdominal mass on an excretory urogram. The child had a 4-generation family history of this disorder. Clinical course had been unremarkable until she was 12 years old and died of a ruptured intracranial aneurysm. Despite the clinical impression of unilateral renal involvement, postmortem examination showed that the clinically normal kidney had developed early cyst formation in several areas, thus, underscoring the bilateral nature of adult polycystic disease.

Increased neutrophil receptors for and response to the proinflammatory bacterial peptide formyl-methionyl-leucyl-phenylalanine in Crohn's disease.

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.

Chylothorax due to Mycobacterium tuberculosis.

Chylothorax in an adult is a rare cause of pleural effusion. Mycobacterium tuberculosis may cause chylous effusion, but usually in association with extensive intrapulmonary involvement. A case of chylothorax is presented in which M tuberculosis was isolated from the pleural fluid and was the only intrathoracic manifestation of tuberculosis.

A preponderance of CCR5(+) CXCR4(+) mononuclear cells enhances gastrointestinal mucosal susceptibility to human immunodeficiency virus type 1 infection.

The gastrointestinal mucosa harbors the majority of the body's CD4(+) cells and appears to be uniquely susceptible to human immunodeficiency virus type 1 (HIV-1) infection. We undertook this study to examine the role of differences in chemokine receptor expression on infection of mucosal mononuclear cells (MMCs) and peripheral blood mononuclear cells (PBMCs) by R5- and X4-tropic HIV-1. We performed in vitro infections of MMCs and PBMCs with R5- and X4-tropic HIV-1, engineered to express murine CD24 on the infected cell's surface, allowing for quantification of HIV-infected cells and their phenotypic characterization. A greater percentage of MMCs than PBMCs are infected by both R5- and X4-tropic HIV-1. Significant differences exist in terms of chemokine receptor expression in the blood and gastrointestinal mucosa; mucosal cells are predominantly CCR5(+) CXCR4(+), while these cells make up less than 20% of the peripheral blood cells. It is this cell population that is most susceptible to infection with both R5- and X4-tropic HIV-1 in both compartments. Regardless of whether viral isolates were derived from the blood or mucosa of HIV-1-infected patients, HIV-1 p24 production was greater in MMCs than in PBMCs. Further, the chemokine receptor tropism of these patient-derived viral isolates did not differ between compartments. We conclude that, based on these findings, the gastrointestinal mucosa represents a favored target for HIV-1, in part due to its large population of CXCR4(+) CCR5(+) target cells and not to differences in the virus that it contains.

Localization of human intestinal defensin 5 in Paneth cell granules.

Antibiotic peptides of higher animals include the defensins, first discovered in phagocytic cells but recently also found to be produced by epithelial cells. We biosynthesized recombinant human intestinal defensin 5 (rHD-5) using the baculovirus-insect cell expression system. Since insect cells process defensin incompletely and secrete the precursor proHD-5, we substituted a methionine for an alanine at a likely processing site to allow selective chemical cleavage with cyanogen bromide, and rHD-5 was used to elicit polyclonal antibodies. By the immunoperoxidase-staining technique, the antibodies selectively stained Paneth cells of the normal adult small intestine. Immunogold electron microscopy further localized HD-5 to the Paneth cell secretory granules. Since some defensins exert activity cytotoxic to mammalian cells, we assayed the effect of rHD-5 on the human intestinal cell lines Caco2 and Int407. proHD-5 did not exert cytotoxic activity, and rHD-5 showed only minimal activity against Int407 and was inert against Caco2. Since Paneth cells release their granules adjacent to the mitotic cells of the intestinal crypts, HD could protect this cell population against invasion and parasitization by microbes.

Penicillin susceptibility testing of gonococci by disc diffusion.

This study investigated the use of various beta-lactam discs in the detection of penicillin sensitive, intermediate resistant and resistant strains (minimal inhibitory concentrations of penicillin G of less than 0.06, 0.06 to 0.25, and greater than 0.25 mg/l respectively). Discs containing 0.15 and 0.6 microgram of penicillin G, and 5 microgram of methicillin were the most useful in separating the three groups. Cut-off zones between sensitive, intermediate resistant and resistant strains are 12 and 24 mm for 0.15 microgram penicillin discs; 24 and 32 mm for 0.6 microgram penicillin discs; and 20 and 30 mm for 5 microgram methicillin discs. Oxacillin discs (1 microgram) were useful in separating resistant from both sensitive and intermediate resistant strains at a cut-off zone of 8 mm. Use of a combination of discs provides the best index of susceptibility group, and at least two should be used

Comparative efficacies of ceftriaxone, moxalactam, and ampicillin in experimental Salmonella typhimurium infection.

The activities of ceftriaxone, moxalactam, and ampicillin against Salmonella typhimurium LT-2 were compared in culture media at pH 5, 6, 7 and 8 and in mice inoculated intraperitoneally. The minimal inhibitory concentrations for strain LT-2 in Mueller-Hinton broth were 0.03 microgram of ceftriaxone per ml, 0.08 microgram of moxalactam per ml, and 0.4 microgram of ampicillin per ml. A comparison of minimal inhibitory concentrations in buffered broth at pH 5 with those in media at higher pH values showed that ceftriaxone was more acid stable than the other antibiotics. Groups of CF-1 female mice inoculated intraperitoneally with 3 X 10(4) colony-forming units received saline or each drug in fourfold decremental doses by the subcutaneous route every 8 h for 3 days, beginning at 24 h after challenge. The mean log 10 colony-forming units of S. typhimurium per spleen at the end of treatment and the mortality rates at 21 days after inoculation were measured for each treatment group. The mean log 10 colony-forming units per spleen was significantly reduced from that of the saline control by dosages of greater than or equal to 0.06 mg of ceftriaxone per kg, 64 mg of moxalactam per kg, or greater than or equal to 16 mg of ampicillin per kg (P less than 0.05). Mortality rates of infected mice were significantly reduced by dosages of greater than or equal to 1 mg of ceftriaxone per kg or greater than or equal to 64 mg of ampicillin per kg (P less than 0.05), whereas moxalactam in dosages as high as 16 mg/kg did not significantly reduce mortality rate. These results demonstrate the superiority of ceftriaxone to the other tested antibiotics on a weight basis in this model of experimental Salmonella infection.