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Objective To investigate the features of immune response of human immunodeficiency virus type-1 (HIV-1) antigen specific T lymphocytes in HIV-1 monoinfected or HIV 1/hepatitis C virus (HCV) coinfected individuals.Methods Twenty-six HIV-1 monoinfected and 23 HIV-1/HCV coinfected individuals were enrolled.Immunomagnetic microbeads were used to isolate T lymphocyte subpopulation CD4+ T cells and CD8+ T cells from human peripheral blood mononuclear cells (PBMC).Frequencies of interferon-γ (IFN-γ) secreting cells of CD4+,CD8+ T lymphocytes and PBMC stimulated by a peptide pool containing 12 overlapping peptides in HIV-1 P24 from 49 patients were assessed by enzyme-linked immunospot (ELISPOT) assay.HIV-1 RNA levels of these patients were also detected by real-time fluorescence quantitative polymerase chain reaction.The data were compared by one-way ANOVA and Mann-Whitney U test,and Spearman test was used for correlation analysis.Results Frequencies of HIV-1 antigen specific CD4+ T lymphocytes [median =25 spot-forming cells (SFC)/106 cells] were significantly lower than those of CD8+T lymphocytes (median=38SFC/106 cells,F=4.592,P=0.037) and PBMC (median=53 SFC/106 cells,F=5.436,P=0.025) in HIV-1 monoinfected group.Frequencies of HIV-1 antigen specific CD4+ T lymphocytes (median=5 SFC/106 cells,Z=-2.432,P=0.015),CD8+T lymphocytes (median=5 SFC/106 cells,Z=-1.996,P=0.046) and PBMC (median=10 SFC/106 cells,Z=-2.306,P=0.021) in HIV-1/HCV coinfected group were significantly lower than those in HIV-1 monoinfected group.Conclusions In HIV-1 infection,antigen specific immune response of CD4+ T cells can be activated,but weaker than that of CD8+ T cells.Co-infection with HCV might down-regulate the responses of HIV-1 antigen specific T lymphocytes in HIV-1 infected individuals.
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Objective To investigate impacts of chronic hepatitis B virus (HBV) co-infection on immune responses and clinical manifestations in acute infection of dengue virus type 1.Methods The serum levels of interferon (IFN)a,IFNβ,IFNγ,interleukin (IL)-10 and tumor necrosis factor (TNF)α of 310 dengue serotype 1 (DENV1)-infected patients were assessed by enzyme linked immunosorbent assay (ELISA),among which 8％ (25/310) were chronic HBV co-infection.Meanwhile,serum samples from 41 healthy adults and 47 chronic HBV infected subjects were recruited as controls.Comparisons among groups were done by one factor analysis of variance and correlation analysis of results was done by spearman rank correlation test.Results The serum level of IFN-α [(95.1 ± 279.3) pg/mL] was significantly higher than IFN-β[(2.8 ± 16.2) pg/mL] during acute dengue infection,while IFN-α level [(86.5±358.1) pg/mL] reduced in patients with HBV co-infection.The secretion kinetics of IFNα,IFNγ (pro-inflammatory cytokine) and IL-10 (anti-inflammatory cytokine)were analyzed.The medians of IFNα,IFNβ and IL-10 level were elevated to peak on day 2,day 3-4and day 6 after fever onset,respectively.Additionally,IFNα levels in patients with only dengue infection were negatively correlated with the platelet counts and serum alanine aminotransferase levels (r=-0.2327,0.2122,both P＜0.01).Conclusions Chronic HBV co-infection alters human immune responses elicited by acute dengue viral infection.Moreover,IFNα secretion may be associated with hemorrhagic tendency,while protective against inflammatory damage of liver.
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Objective To investigate the characteristics of V3 loop amino acid sequences of human immunodeficiency virus type 1 (HIV-1) quasi-species in long-term non-progressors (LTNP)infected with HIV. Methods End-point limiting dilution polymerase chain reaction (EPLD PCR) was used to amplify the env gene c2-v3-c3 region of single HIV-1 provirus from five LTNPs at sequential time points. The PCR products were then sequenced and the amino acid sequences of V3 loop were analyzed by sequence confirm analysis technology. Results The results showed that there were one to ten kinds of polymorphisms in the V3 region of HIV-1 quasi-species which were found from the serial samples of the five LTNP. However, the sequences of the predominant strains were either completely consistent or at most changed at one or two residues in the serial samples of individual patient. The tetramer compositions of the tip of V3 loop were consistent in each patient. It was GPGR in four patients and GPGK in one patient. It was speculated the co-receptor of HIV-1 was CC chemokine receptor (CCR)-5 based on the amino acids at the residue 11 and residue 25 of V3 loop and the net charge of V3 loop. Conclusions There are various polymorphisms at the HIV V3 loop in LTNP. However, the tetramer composition of the tip part of V3 loop is stable. The LTNP are very likely infected with non-syncytium inducing (NSI) strain.
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Objective To investigate the effect of highly active antiretroviral therapy (HAART) on human immunodeficiency virus type-1 ( HIV-1 ) antigen specific cytotoxic T lymphocyte (CTL) immune responses. Methods Peripheral blood mononuclear cells (PBMCs) were collected from 38 HIV-1 infected individuals receiving HAART ( HAART group) and 31 HIV-1 infected individuals not receiving HAART (non-HAART group), and stimulated with a peptide pool containing 12 overlapping peptides in HIV-1 P24;then the frequency of interferon γ ( IFNγ ) secreting cells were assessed by enzyme-linked immunospot (ELISPOT) method. Difference in HIV-1 antigen specific CTL immune response between non-HAART group and HAART group was analyzed by χ2 and Mann-Whitney U tests. Results Positive response rate of HIV-1 antigen specific CTL immune responses in HAART group ( 65.8%, 24/38 ) was higher than that of non-HAART group (32.3%, 10/31, χ2 = 6. 522, P ＜ 0.05 ). For HIV-1 infected individuals with blood CD4 +T cells ＞ 350/μL, the frequency of HIV-1 antigen specific CTL responses in HAART group was higher than that in non-HAART group (Z = -2. 819, P ＜0.05 ). In the HAART group, those receiving HAART more than 12 months were of higher frequency of HIV-1 antigen specific CTL responses ( Z =-2. 195, P ＜ 0. 05 ). Conclusion HAART especially long-term treatment may enhance HIV-1 specific CTL responses in HIV-1 infected individuals.
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detection of HIV-1 quasispecies in HIV-1 infected populations with low level viral load.
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Objective To identify the pathogens that cause hand, foot and mouth disease (HFMD) in adults and analyze the nucleotide sequences characteristics of enterovirus 71 (EV71). Methods The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the enterovirus from the samples of four adult HFMD patients. The 227 bp amplified segments of EVT1 were then sequenced and compared with the sequences of previously isolated EVT1 strains available from GenBank by homogeneity and phylogenetic tree analyses. Results All the results of RT-PCR with enterovirus universal primers and EVT1 specific primers were positive. The EV71 sequences analysis showed that the four new sequences (named as GZ19610, GZ99310, GZ99355 and GZ46477) shared 96.0% to 99.1% nucleotide identify themselves and shared 96.9% to 100.0% homology with the strain Fuyang/17.08/3 isolated in 2008 from Fuyang, Anhui Province. Phylogenetic tree analysis showed that the genotype of the four new sequences was all subtype C4, they were the same sub-genotype as those strains isolated from Chinese mainland and Chinese Taiwan in 2004, and the genetic distance between them was most closely. Conclusions EV71 can cause adult HFMD. Compared with the nucleotide sequences of EV71 strains that isolated now and formerly in China, there is no large variation of the EV71 sequences isolated from four adult HFMD patients in Guangzhou this time. The adult HFMD patients should be isolated for treatment to avoid them transmitting the virus and causing disease spreading.
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Objective To identify the sequence of hepatitis B virus S gene"a"determinant in patients with positive HBsAg and anti-HBs.Methods Nested-PCR Was used to amplify the HBV S gene in 4 patients with positive HBsAg and anti-HBs,and the PCR products were sequenced directly or after cloning.The sequences of"a" determinants were then analyzed by sequence alignment.Results Direct sequencing of PCR products showed that there was one amino acid (aa)residue in"a"determinant less conserved region emerging polymorphism in all 4 patients.Clone sequencing showed that aa residue at 126 of "a"determinant in patient 1 miSht be Thr,Ile and Set,at 134 might be Phe and Set;the aa at 126 in patient 2 misht be Ala and Thr.and in patient 3 might be Ile and Asn;aa polymorphism was not found in patient 4.Conclusion The polymorphism of"a"determinant in HBV S gene might be associated with positivity of both HBsAg and anti-HBs in hepatitis B patients.
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Objective To study the occurring regulation of antibody of Urbani sever acute respiratory syndrome (SARS)-associated coronavirus after onset of illness in patients with SARS and investigate the co-infection status of Chlamydiae pneumoniae (Chlamydiae P.) , Mycoplasma pneumoniae (Mycoplasma P.), Adenovirus, Respiratory Syncytia virus (RSV). Methods Serum Antibody IgM and IgG of Urbani SARS-associated coronavirus of 43 patients with SARS and 10 patients with other diseases except SARS at the two different phases of illnesses were detected with immune fluorescent technique. Antibody IgM and IgG of Chlamydiae P., Mycoplasma P., Adenovirus and RSV in the above samples were detected with enzyme-linked immunosorbent assay (ELISA). Results 40 cases' infection of were Urbani SARS-associated coronavirus were determined (93.02%) and 3 cases were negative (6.98%). 10 patients with other diseases except SARS have negative serum Antibody IgM and IgG of Urbani SARS-associated coronavirus. Recent infection rates of Chlamydiae P., Mycoplasma P., Adenovirus and RSV were 25.58%, 16.28%, 6.98% and 4.65% , respectively, and former infection rates of these pathogens were 39.53%, 34.88%, 27.91% and 0, respectively. Antibody IgM of Urbani SARS-associated coronavirus occurred at the same time of onset of fever. Positive rates of IgM were respectively 69.57% and 62.96% in 8～14 days and 15～33 days after onset of fever, and there were no remarkable difference between them, but they were re-markably higher than that in 1～7 days after onset of fever (16.67%). Antibody IgG of Urbani SARS-as-sociated coronavirus occurred at the 6th day after onset of fever. Positive rates of IgG were respectively 19.44%, 65.22% and 92.59% in 1～7 days, 8～14 days and 15～33 days after onset of fever, and there were remarkable difference among them. Conclusions Antibody IgM and IgG of Urbani SARS-associated coronavirus may occur at the early stage of illness in patientswith SARS, which positive cases may increase remarkably 2 weeks later after onset of fever. There may be recent infection and/or former infection of Chlamydiae P., Mycoplasma P., Adenovirus and RSV in some patients with infectious atypical pneumonia. Detection of Antibody IgM and IgG of Urbani SARS-associated coronavirus in sera with immune fluorescent technique can be used on the early diagnosis of SARS.
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Fifty four male Wistar rats weighing 60-90g were divided into three groups of 18 each; rice-soybean basal diet with 0.2%DL-methionine as control, 2.0%D1-methionine supplemented group, 2.0%DL-methionine and 3.0%glycine supplemented group. All animals were housed in individual wire cages and both experimental diet and water were given ad libitum. After feeding 21 days, 0.037MBq/g bw of 3H-leucine was injected intraperitoneally. At 1,2, 4, 6, 8, 10 hours after injection, three rats per group were killed and the radioactivity of 3H-leucine per mg hepatic and brain tissue (dpm/mg tissue) were determined by liquid scintillation counter. The results showed that 3H-leuciue incorporarted during the entire 10 hours period of observation markedly declined and its peak was delayed in the hepatic tissue of excessive methionine supplemented group. However, 3H-leucine incorporated was improved significantly in liver in the rats fed excessive DL-methionine with glycine diet. In contrast, no significant difference of incorporation in the rat brain among the three groups were found. The results demonstrated that the addition of excess methionine retarded evidently protein synthesis of hepatocytes in the rats, but no similar toxic effect occurred in the rat brain tissue.