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BIOLS 451 June 7, 2012

Bisma Bashir Mohammad ID # 20094704

Loss of CD4 T Lymphocytes in Patients Infected with HIV Type 1 is More Pronounced in the Duodenal Mucosa than in the Peripheral Blood
T Schneider, H-U Jahn, W Schmidt, E-O Riecken, M Zeitz, R Ullrich, and the Berlin Diarrhea/Wasting Syndrome Study Group.

Abstract:
HIV infects T cell subsets but how it changes the lymphoid cells is not known. This study was done to analyse these changes in the lymphoid cells comparing them to peripheral blood. Three colour cytometry was done on eight asymptomatic HIV infected patients, 26 AIDS patients, and 23 controls. No relation was found between the proportion of T cell subset between the between circulating and duodenal T cells but the CD4 T cells and its ratio between the duodenal and circulating CD4+ T cells, was reduced in duodenum more than that in peripheral blood of AIDS and asymptomatic HIV patients. From these findings, the loss of duodenal CD4 T cells can be correlated with early HIV infection. Other immunological abnormalities and immunodeficiency can occur due to loss of this CD4 T cells in lymphoid cells as the intestinal immune system is not enough even though circulating T cells are present.

Introduction:
Human immunodeficiency virus (HIV) infects the body through gastrointestinal tract. Due to HIV infection, T cells subset are most affected. This affect was studied in peripheral blood and other lymphoid compartments of HIV infected persons. However, HIV is more common in intestine and lymphoid organs as compared to peripheral blood. Activation and differentiation of T cells affect the replication and the pathological changes of the HIV. Three Colour Flow Cytometry was done to isolate lymphocytes from peripheral blood and duodenal mucosa and it was found that HIV infected patients lack CD4+ T lymphocyte more in the duodenal mucosa than peripheral blood.

Methods:
Patients: Thirty four HIV infected, white patients with gastrointestinal symptoms were studied. Of these 8 had asymptomatic HIV infection, the other patients had AIDS. These 34 patients had different symptoms such as, diarrhoea, dysphagia, weight loss, nausea, abdominal pain, and epigastric pain. Duodenal biopsy revealed different infectious agents in these patients. Thirty one of these patients were bisexual men and 3 were drugaddicts. Median age for all the patients was 38. Twenty eight patients were given zidovudine treatment at the time of study. Control group was set compromising of 23 patients with an age median of 39 years. They were not at the risk of HIV but were undergoing upper endoscopy due to epigastric pain or weight loss. Investigations: specimens for duodenal biopsy and venous blood samples were taken from the normal areas of distal duodenum from all patients and controls. Isolation of lymphocytes: Ficoll density gradient centrifugation was done to isolate blood lymphocytes whereas to isolate intestinal lymphocytes, a modified method used previously was adopted. Isolates samples were washed with PBS and excised. After 2nd washing, the excised samples were incubated overnight at 4C in RPMI 1640 medium. This medium is a mixture of many reagents, buffer and seras. Mucosal fragments were then incubated on a shaker for 3h at 37C. The cells were separated from one another by passing them through a spinal needle and then through nylon net. They were then washed and resuspended 30% isotonic density
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BIOLS 451 June 7, 2012

gradient solution and under-layered with 70% isotonic density gradient solution. This was centrifuged and intestinal lymphocytes consisting of lamina propria lymphocytes and intraepithelial lymphocytes was separated and then washed with RPMI. 0.9-29 x 106 of the mononuclear cells was obtained and trypan blue dye exclusion was done to visualize them. Immunofluorescence studies: T cells were isolated at the same time from peripheral blood and intestinal biopsy specimens and then stained with fluoresceinated antibodies to CD4, CD8 and CD3. Each sample was incubated with all the 3 antibodies at 4C for 30min. in a dark room with a buffer. Their proportion was determined by 3 color flow cytometry. They showed no significant difference for the expression of the surface antigens. Flow cytometric analysis: Flow cytometry and LYSIS II program were used to analyse 5000 cells. Spectral overlap was eliminated by electronic compensation and the lymphocyte populations and CD3+ cells were supplied by forward-sideward scatter light. 98% of cells stained with conjugated isotype matched control antibodies were excluded and results were given as percentages of positive cells per CD3+ T cells. Statistical analysis: Results were described as medians and range as they were not normally distributed. To determine the correlation between the two compartments studied, non-parametric correlation coefficient was calculated. To evaluate comparative statistical significance non-parametric test fro paired and unpaired data was done and the p values les then 0.05 were considered as significant.

Results:

According to table 1, flow cytometry studies on isolated duodenal and circulating T cells at the same time revealed decrease in the proportion of CD4 T cells whereas an increase in CD8 T cells when controls were compared to asymptomatic HIV and AIDS patients. The ration of CD4:CD8 was reversed from 2:1 to 1:2. In the duodenum the proportion of CD4T cells decreased whereas the proportion of CD8 T cells increased when controls were compared to asymptomatic HIV and AIDS patients. The ration of CD4:CD8 again decreased but the proportion of CD4-CD8- T cells in the duodenal mucosa was not different between the controls and HIV infected patients but increased in AIDS patients. The percentage of T cells was also not different in the duodenum of the three groups (Table I).
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BIOLS 451 June 7, 2012

The median proportion of CD4+ T cells in the blood and duodenum was reduced to 46% and 8% respectively, in asymptomatic HIV and AIDS patients. Thus the decrease in CD4 T cells in HIV infection is much more common in the duodenum than in the peripheral blood, especially at early stages of the disease. There was no significant relationship between the percentage of CD4+ T cells in the peripheral blood and in the duodenum of controls and HIV infected patients. Percentage of CD4 T cells in the peripheral blood and duodenum in patients with diarrhoea or secondary intestinal infection is shown in table II. Their intestinal T cell subsets were not different.

Discussion:
Results suggest that the proportion of CD4+ T cells in HIV infection is lower in the duodenal mucosa than in the peripheral blood. Intraepithelial and lamina propria cells are usually found with the duodenal lymphocytes with normally ratio CD4:CD8 of lamina propria similar to circulating T cells but in intraepithelial T cells CD8+ or CD4-CD8- is dominant. So it is expected to have reduced % of CD4+ T cells in the duodenum but in HIV infected patients this decrease is exceeded but the proportion of intraepithelial T cells is similar to the proportion of T cells of HIV infected patients and controls. So the decreased proportion of duodenal CD4+ T cells in HIV infected patients cant be explained by increase in intraepithelial T cells or loss of CD4+ T cells in cell isolation procedure. Decrease in CD4+ T cells in lamina propria of HIV infected patients with a decrease in CD4:CD8 from controls was shown in immunohistological studies. Studies also showed that CD4+ macrophages were the main component in CD4+ lamina propria cells in the duodenum of HIV infected patients. Also staining of fixed cells detected intracellular Ag and the CD4+ expression of HIV infected T cells were lost on the cell surface but was maintained intracellularly. Due to this loss of cell surface CD4+ T cells can lead to the increase of duodenal CD4- CD8- T cells in AIDS patients, as only the surface expression of CD4+ on CD3+ T cells was analysed by flow cytometry. The precise mechanism by which HIV infection leads to the CD4+ T cell depletion in vivo is unclear, but it is generally thought that the amount of virus present correlates with the extent of CD4+ depletion. HIV infected cells were more in lymph nodes and adrenal gland than in blood. Memory T cells were more affected with HIV which are common in intestine than in blood and these activated T cells promote the replication of HIV which causes excessive loss of CD4+ T cells in duodenum than in peripheral blood. In our study, the peripheral blood and the duodenal mucosa T cell subsets changes had no relationship in of controls or the HIV infected patients. To start antiviral treatment or primary prophylaxis of opportunistic infections for HIV infected patients, this correlation cannot be used as it is inappropriate. As in early infection of HIV, duodenal CD4+ T cells are affected, so intestinal immunity is not enough to prevent HIV. The depletion of T cells can cause other immunological abnormalities.

Summary:
No correlation exist between the T cells in peripheral blood and the duodenal mucosa but during HIV infection, duodenal CD4+ T cells are more affected than in blood. So peripheral blood CD4+ T cells count cannot be correlated for the presence of HIV.