Evaluation of the cervical cytobrush sampling technique for the preparation of CD45+ mononuclear cells from the human cervix.

A cytobrush technique developed to prepare mononuclear cells from the intraepithelial layer of the endocervix has been evaluated. Specimens yielded approximately 4-6x10(6) cells, of which 10-15% were CD45+. Between 10% and 15% of these CD45+ cells were mononuclear leukocytes. The non-leukocyte cell fraction exhibited high levels of autofluorescence and for flow cytometry analysis, it was necessary to exclude these cells by gating. Macrophages constituted approximately 60% and T lymphocytes, 40% of the mononuclear cells in cytobrush samples. The CD4/CD8 T-cell ratio was similar to that observed in blood. In 9 of 13 specimens, B lymphocytes constituted less than 1% of the mononuclear cell fraction suggesting that the mononuclear cells were derived from the intraepithelial compartment rather then the deeper lamina propria. Lack of B lymphocytes also indicates minimal blood contamination in these samples, a conclusion supported by labelling for the red blood cell (RBC) glycoprotein glycophorin A. However, the need to monitor all samples for possible blood contamination was indicated by 4 of 13 samples in which B lymphocytes accounted for 2-8% of the mononuclear cells.

Higher levels of activation markers and chemokine receptors on T lymphocytes in the cervix than peripheral blood of normal healthy women.

Heterosexual transmission of human immunodeficiency virus (HIV-1) is the predominant mode of infection world-wide. To better understand sexual transmission of HIV-1 in women we have analysed virus co-receptor and cellular activation marker expression on T lymphocyte subsets from the cervical epithelium and have made comparisons with peripheral blood T cells. Intraepithelial cervical T lymphocytes were obtained with a cytobrush, immunolabelled and analysed by flow cytometry. Activation markers (CD69, CD25 and HLA-DR) were found to be more highly expressed on cervical than on blood T lymphocytes. These higher levels of activation on cervical T lymphocyte subsets could facilitate HIV-1 infection. CXCR4 was expressed at marginally higher levels than CCR5 on T cells from the cervical epithelium and peripheral blood. Thus, the preferential transmission of macrophage tropic strains of HIV-1 following sexual contact cannot be explained solely on the expression of chemokine co-receptors by T lymphocyte subsets.

Macrophages are increased in cervical epithelium of women with cervicitis.

BACKGROUND: Sexually transmitted diseases (STIs) are major causes of morbidity in women. The mechanisms involved in establishment of genital mucosal infection are poorly defined. OBJECTIVE: To investigate changes in cervical epithelial (CE) CD45+ cell subpopulations in women with microscopic evidence of cervicitis (n=9) and those without (n=12). METHODS: CE samples were obtained using cytobrush including matched venous blood. CE and peripheral blood (PB) mononuclear cells were analysed by flow cytometry for CD3+, CD4+, CD8+, CD14+,CD19+, and HLA-DR+ expression. RESULTS: Women with cervicitis had increased CE macrophages compared with those without (p<0.05). MHC class II+ cells were predominant in all cervical samples. Considerably fewer B lymphocytes were found in cervical samples in both groups of women. No changes were observed in cervical T lymphocyte subsets. However, a relative CD8+ lymphocytosis in PB was noted in women with cervicitis. CONCLUSION: The increased numbers of CE macrophages in women with cervicitis may have important implications for pathogenesis of STIs including human immunodeficiency virus infection.

HIV enteropathy: comparative morphometry of the jejunal mucosa of HIV infected patients resident in the United Kingdom and Uganda.

AIMS: To compare jejunal mucosal morphometry in HIV infected patients resident in London and Uganda. PATIENTS: Twenty HIV positive patients from London and 16 from Uganda were studied, and compared with HIV negative control subjects from both sites. METHODS: Stools and biopsy specimens were examined for enteropathogens. Surface area to volume (S:V) ratio was estimated morphometrically, mean crypt length of jejunal biopsy specimens was measured, and HIV infected cells detected immunohistochemically were quantified. RESULTS: Enteric pathogens were detected in none of the London patients, and in three Ugandan patients. S:V ratio was lower, and mean crypt length higher, in the specimens of London patients than in normal subjects, but there was no difference in S:V ratio or mean crypt length between Ugandan patients and controls. A negative correlation was present between S:V ratio and mean crypt length in all biopsy specimens analysed. HIV infected cells were detected only in lamina propria. CONCLUSION: Infection of cells in the lamina propria of the jejunum with HIV stimulates crypt cell proliferation, and a fall in villous surface area. The mucosal response to HIV is masked by other pathogens in the African environment.

Impaired absorption of zidovudine in patients with AIDS-related small intestinal disease.

OBJECTIVES: To investigate the effect of small intestinal disease (SID) on the absorption of zidovudine (ZDV) in patients with AIDS. METHODS: Fourteen fasted homosexual men with AIDS received a single oral dose of ZDV (5 mg/kg). Nine subjects had clinical evidence of intestinal disease (chronic diarrhoea with wasting) confirmed by reduced fat absorption measured indirectly using the 14C-triolein test. Five subjects had AIDS-related symptoms other than those affecting the gastrointestinal tract with normal fat absorption. Sequential measurements of plasma ZDV including its glucuronide metabolite (GZDV) were obtained using radio-immunoassay and ZDV/GZDV concentrations-time profiles of both groups of subjects were compared. Comparisons were also made for each of the following computed variables: the maximum plasma concentration (Cmax), time to reach Cmax (Tmax), area under the plasma concentration-time curve (AUC0-6 h), the elimination half-life (t 1/2), and apparent oral clearance (CL0). RESULTS: In patients with SID, Cmax ZDV was reduced (6.39 +/- 3.39 versus 11.51 +/- 5.01 mumol/l; P < 0.05) and Tmax ZDV prolonged (0.81 +/- 0.51 versus 0.40 +/- 0.14 h; P < 0.05) but AUC0-6 h ZDV was no different from the non-SID group (8.03 +/- 2.73 versus 14.56 +/- 9.0 mumol/l-1xh; P = 0.06). There were no differences in t 1/2 ZDV (1.22 +/- 0.20 versus 1.13 +/- 0.30 h) or CL0 ZDV (3017 +/- 1158 versus 1700 +/- 889 ml/min; P > 0.05) between SID and non-SID groups, respectively, and GZDV values were comparable between the two groups. CONCLUSIONS: These data suggest delayed absorption rather than altered metabolism of ZDV in AIDS-related SID and raise the possibility of drug malabsorption. The clinical efficacy of orally administered low-dose ZDV regimens may require further evaluation in patients with chronic diarrhoea and AIDS.

Comparative assessment of small intestinal and colonic permeability in HIV-infected homosexual men.

OBJECTIVES: To investigate both small and large intestinal permeability in HIV-positive subjects, and correlate variation in intestinal mucosal abnormality with immunological and nutritional markers of HIV disease. METHODS: Small and large intestinal permeability studies were performed in 14 HIV-seropositive patients and eight healthy men. Eight out of the 14 patients had diarrhoea and all subjects were negative for enteropathogens. Small intestinal permeability was determined using the lactulose-mannitol test and large intestinal permeability using the colonic absorption of 51Cr-EDTA. In addition, CD4 cell count, beta 2-microglobulin, C-reactive protein estimation and anthropometry were carried out in all subjects. RESULTS: HIV-seropositive subjects had higher lactulose-mannitol ratios (LMR; 0.084 +/- 0.007 versus 0.013 +/- 0.0008) and lower 51Cr activity (1.986 +/- 0.066 versus 3.115 +/- 0.560) than controls (P < 0.0004 and P < 0.05, respectively). Colonic uptake of 51Cr-EDTA was no different between subjects with and those without diarrhoea (2.04 +/- 0.124 versus 1.92 +/- 0.143, P > 0.05). A negative correlation was found between LMR and 51Cr-EDTA, but only for patients with diarrhoea (r = -0.81; P = 0.015). CONCLUSION: Regional variation affecting intestinal absorptive function occurs in patients with HIV-related diarrhoea and is characterized by increased LMR and reduced colonic uptake of 51Cr-EDTA. The pathogenesis and clinical significance of such changes are unknown.

Analysis of multiple sugar probes in urine and plasma by high-performance anion-exchange chromatography with pulsed electrochemical detection. Application in the assessment of intestinal permeability in human immunodeficiency virus infection.

Mannitol, 3-O-methylglucose and lactulose administered orally are used to investigate small intestinal absorption pathways and mucosal integrity. Current methods of analysis include thin-layer chromatography, gas chromatography (GC) and enzymatic analysis, which require separate estimation of mono- and disaccharides and for GC, prior derivatization. We describe a high-pressure anion-exchange chromatographic method coupled with pulsed electrochemical detection allowing simultaneous measurement of all three sugars and its clinical application in monitoring intestinal damage in human immunodeficiency virus (HIV) infection. Sample preparation is simple and fast. All sugars are resolved within 10 min. Mean recovery is 93.3% for all sugars and the overall relative standard deviation is 4.2%. Intestinal permeability (lactulose/mannitol ratio) rises with disease progression to AIDS, indicating mucosal damage. The greatest increase in permeability is associated with chronic diarrhoea. The method is an ideal non-invasive test to assess gut mucosal damage in HIV infection.

Direct in vitro infection of human intestine with HIV-1.

OBJECTIVE: To directly infect human fetal intestine with HIV in vitro. DESIGN: Human fetal intestinal explant cultures were exposed to HIV-1 and monitored for evidence of infection by biochemical assay, immunohistochemistry and in situ hybridization. METHODS: Human fetal intestinal explants (14-21 weeks) were established in culture and exposed to HIV-1. Tissue culture fluid was assayed for p24 antigen and reverse transcriptase activity over a 14-day period. Explants were removed from culture on days 4, 7, 10 and 14 postinoculation and subjected (1) to immunohistochemistry to detect p24 and gp160/41 antigens, and (2) to in situ hybridization to detect HIV-1 RNA. Explant tissue culture fluid was cocultured with Jurkat T-cells to detect infectious viral particles. RESULTS: Reverse transcriptase activity and p24 antigen levels in fetal explant culture fluid rose between 7 and 14 days after viral inoculation. Jurkat T-cell cocultures confirmed the presence of infectious virus. Cells in the lamina propria resembling lymphocytes and macrophages of both small intestine and colon stained positively for the viral proteins p24 and gp41. The same type of cells also stained positively for HIV-1 RNA using in situ hybridization. Dual-label immunohistochemistry, combined immunohistochemistry and in situ hybridization confirmed the presence of viral protein and RNA in cells bearing the CD3, CD4 (lymphocyte) or CD68 (macrophage) surface markers. There was no evidence at any time of HIV-1 infection of epithelial cells. CONCLUSIONS: Cells of the lamina propria of the small intestine and colon, bearing lymphocyte or macrophage markers, can be directly infected by and support the replication of HIV-1. Such infection may be implicated in the pathogenesis of HIV enteropathy.

Altered small-intestinal permeability associated with diarrhoea in human-immunodeficiency-virus-infected Caucasian and African subjects.

1. Small-intestine integrity in Caucasian and African patients infected with human immunodeficiency virus was determined by measuring the permeation across the mucosa of two sugars, lactulose and mannitol. 2. The sugars were assayed by h.p.l.c. and pulsed amperometric detection in 6 h urine samples. Stool microscopy for enteropathogens was performed in all patients. 3. The ratio of lactulose to mannitol recovered in urine was increased in Caucasian and African patients with advanced human immunodeficiency virus infection. Asymptomatic human-immunodeficiency-virus-infected subjects had a normal lactulose/mannitol ratio. African patients with diarrhoea showed a twofold reduction in mannitol excretion. Such a change in mannitol absorption was not detected in Caucasian patients and occurred regardless of the presence of enteropathogens. 4. Altered small-intestinal permeability is associated with symptomatic diarrhoea in human immunodeficiency virus infection in both Caucasian and African patients.

Ileal and jejunal absorptive function in patients with AIDS and enterococcidial infection.

Small intestinal absorptive function was investigated in six patients with the acquired immunodeficiency syndrome (AIDS) who had diarrhoea and weight loss. Proximal function was assessed by [14C]Triolein test of fat absorption. Distal function was determined by a test of bile acid absorption in which the loss of radio-labelled synthetic bile acid, 75seleno-23-homocholic acid-taurine ([75Se]HCAT), from the enterohepatic circulation was quantified by abdominal gamma-scanning and by a vitamin B12-intrinsic factor absorption test. Concurrently indirect tests of small intestinal bacterial overgrowth ([14C]glycocholate and breath hydrogen) were carried out. In addition, jejunal histological examination and stool microscopy and culture for enteropathogens were performed. Fat absorption was reduced in all six patients, four of whom had jejunal villous atrophy. Bile acid and vitamin B12 absorption were normal in four subjects. Enteropathogens were not detected in any of the four subjects with normal terminal ileal absorptive function. In contrast, reduced bile acid and vitamin B12 absorption were detected in two of six subjects. Both patients had an enteropathogen (Cryptosporidium spp. and Isospora belli) present on stool and jejunal histological examination. Neither subject had evidence of small-intestinal bacterial overgrowth. AIDS patients therefore may have normal ileal absorptive function in the presence of jejunal disease. Infection with Cryptosporidium spp. or I. belli may however, be associated with severe ileal dysfunction.

Rapid and simultaneous determination of lactulose and mannitol in urine, by HPLC with pulsed amperometric detection, for use in studies of intestinal permeability.

The lactulose/mannitol dual sugar absorption test is a non-invasive test of intestinal permeability. Its widespread use has been limited by the difficulties of analysis for carbohydrates in urine at low concentrations. We describe a "high-pressure" liquid-chromatographic method for determining lactulose and mannitol in urine, in which anion-exchange chromatography and pulsed amperometric detection are used. Sample preparation is simple and fast, and lactulose and mannitol and the internal standards arabinose and cellobiose are well resolved within 15 min. Analytical response of the method is linear with concentrations to 3 g/L, and one can detect as little as 0.3 mg of lactulose per liter of urine. Analytical recovery was between 90% and 107% for all sugars analyzed, and there was good agreement with results by a gas-chromatographic method (r = 0.993 lactulose, 0.984 mannitol). The method may potentially be applied to the study of other carbohydrates present in biological fluids at low concentrations.

Fat absorption and exocrine pancreatic function in human immunodeficiency virus infection.

Exocrine pancreatic function and fat absorption were determined using a 'tubeless' test in 25 human immunodeficiency virus (HIV) antibody positive subjects (23 males, two females), CDC criteria groups II (four), III (one), and IV (20). In 12 fat absorption was poor but in only three of these were the results indicative of pancreatic insufficiency and in all three this was mild. In nine of the cases the results were compatible with small intestinal malabsorption. Mild, but not severe, exocrine pancreatic insufficiency may occur in acquired immune deficiency syndrome; however fat malabsorption is more commonly associated with a small intestinal cause.

Combined single day 14C-triolein breath test and PABA test in the diagnosis of malabsorption.

The 14C-triolein breath test, a recognised index of fat absorption, and the p-aminobenzoic acid (PABA) test, a 'tubeless' test of exocrine pancreatic function, have both been widely used in the diagnosis of malabsorption and exocrine pancreatic insufficiency. This study evaluates the potential of a combination of both tests in the investigation of fat absorption and exocrine pancreatic function. Combination of the tests has become technically feasible because of the introduction of high pressure liquid chromatography as the preferred method of analysis for PABA, and use of p-aminosalicylic acid (PAS) as the marker for PABA absorption and metabolism. We studied 25 healthy subjects, 11 patients with exocrine pancreatic disease and 12 patients with gastrointestinal disease. The combined test identified subjects with reduced fat absorption and distinguished subjects with exocrine pancreatic insufficiency from those with an intestinal cause of fat malabsorption. The test could be completed in 7 h and had high patient acceptability. These findings suggest that the combined 14C-triolein breath test and PABA test can be used as a non-invasive, 1-day investigation of fat absorption and exocrine pancreatic function.