| The innate immune system is the first line of defense against invading pathogens and from it the more sophisticated adaptive immune system was derived. Despite nearly two decades of research directed at inducing adaptive immunity or vaccine, there was no significant achievment has been made. On the basis of recent observations, it is suggested that the study on innate immune responses is very important in HIV infection. Natural killer cells ( NK) are the main cells in innate immune responses. The classical NK cells express CD56, lacking expression of TCRs, its phenotye is CD3- CD56 + ; Natural Killer T cells ( NKT) is a second set of NK cells expressing TCR molecules and NK cell surface markers CD56, its phenotype is CD3 + CD56 ". The responses and functions of NK cells and NKT cells in HIV infection are still not clear. In this work we have analysed the NK and NKT cells and their associated cell surface markers in HIV infection and investige the correlation between NK cells and HIV infection.Material and Methods1. Study populationDiagnosis of HIV - 1 infection was made on the basis of a positive anti -HIV ELISA ( Vironostika, Organon Tednika, The Netherlands ) and confirmed by a positive Western immunoblot ( Genelab Diagnostics, Singapore ). Treatment - naive subjects were asked to participate in the study. HIV - seronegative controls were randomly selected from healthy population with normal blood routine test, liver and renal function.2. Determination of lymphocyte counts and corresponding ratioPipette 20ul of TriTEST CD4FITC/CD8PE/CD3PerCP or CD3FITC/ CD56CD16PE/CD45PerCP reagent and add 50ul anticoagulated whole blood into the bottom of the TruCOUNT Tube using reverse pipetting and incubate for 15 minutes in dark at room temperature (20 -25℃). Add 450ul 1X FACS Lysing Solution to the tube, incubate for 15minutes in the dark at room temperature and analyze the samples on the flow cytometer using FACS MULTISET software to acquire T lymphocyte counts, NK cell counts and corresponding ratios. Gating on CD45+ cells, acquire CD3 VCD56CD16VCD45 + NKT counts and corresponding ratios using CELLQUEST software.3. Cell surface stainingAccording to the manual of cell surface staining kits of BD Company, 100, 000 cells were acquired and expression of cell surface markers on NK cells were analyzed using CELLQUEST software by FACSCalibur flow cytometer.4. Isolation of nucleic acidViral RNA or human leucocyte DNA were isolated automatic nucleic acid extraction system (MagNApure LC) and nucleic acid extraction kits ( ROC).5. Determination of viral load ( VL)Viral load was performed by Roch viral load kits and preformed according to the kit instruction.6. Amplification of KIR3DLland KIR3DS1 genePrimers for KIR3DL1 gene: KIR3DL1 ( Forward) CGCTGTGGTGCCTCGA and KIR3DL1 (Reverse) GGTGTGAACCCCGACATG.Primers for KIR3DSlgene: KIR3DS1 (Forward) AGCCTGCAGGGAA-CAGAAG and KIR3DS1 (Reverse) GCCTGACTGTGGTGTCG.DNA extracted from peripheral blood mononuclear cells was used as templates. PCR cycling was carried out in a GeneAmp PCR 9600thermal cycler(PE Applied Biosystems) as follows; 1 cycle of 94℃ for 3min; 4 cycles of 94℃ 15 sec,65℃15 sec,72℃30 sec; 21 cycles of 94℃15sec,60℃ 15 sec,72℃30 sec; 5 cycles of 94℃. 15 sec,55℃1 min, 72℃ 2 min, 72℃7 min. 5ul amplified product was applied to 1.5% agarose gel electrophoresis and the results was determined comparing to DNA maker.7. HAART regimen:. Patients with no obvious liver and renal function abnormality were chosen according to the " standard therapy management" issued by the Ministry of Health and treated with antiviral drugs: Indinavir, a kind of proteinase inhibitor, taken orally 1000mg every 8 hours; Efavirenz, a kind of non - nucleoside reverse tran-scriptase inhibitor, taken orally 600mg daily.Results1. Reference range of NK cell and T lymphocyte counts of healthy adults in the northeast ChinaThe reference range of NK cells is 121 -916 cell/ul,NK% is 6 -35% , CD4 is 373 - 1350 cell/ul,CD8 is 240 - 1320 ce...
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