[Analysis of HIV transmission hotspots and characteristics of cross-regional transmission in Guangxi Zhuang Autonomous Region based on molecular network].

Objective: To analyze HIV transmission hotspots and characteristics of cross-regional transmission in Guangxi Zhuang autonomous region (Guangxi) based on the molecular network analysis, and provide evidence for optimization of precise AIDS prevention and control strategies. Methods: A total of 5 996 HIV pol sequences sampled from Guangxi between 1997 and 2020 were analyzed together with 165 534 published HIV pol sequences sampled from other regions. HIV-TRACE was used to construct molecular network in a pairwise genetic distance threshold of 0.5%. Results: The proportion of HIV sequences entering the molecular network of HIV transmission hotspots in Guangxi was 31.5% (1 886/5 996). In the molecular network of HIV cross-regional transmission, the links within Guangxi accounted for 51.6% (2 613/5 062), the links between Guangxi and other provinces in China accounted for 48.0% (2 430/5 062), and the links between Guangxi and other countries accounted for 0.4% (19/5 062). The main regions which had cross-regional linked with Guangxi were Guangdong (49.5%, 1 212/2 449), Beijing (17.5%, 430/2 449), Shanghai (6.9%, 168/2 449), Sichuan (5.7%, 140/2 449), Yunnan (4.2%, 102/2 449), Shaanxi (3.8%, 93/2 449), Zhejiang (2.8%, 69/2 449), Hainan (2.0%, 49/2 449), Anhui (1.5%, 37/2 449), Jiangsu (1.3%, 33/2 449), and other regions (each one <1.0%), respectively. The risk factors of entering the molecular network of HIV transmission hotspots in Guangxi included being aged ≥50 years (compared with being aged 25-49 years, aOR=1.68,95%CI:1.46-1.95), males (compared with females, aOR=1.21,95%CI:1.05-1.40), being single (compared with being married, aOR=1.18,95%CI:1.00-1.39), having education level of high school or above (compared with having education level of junior high school or below, aOR=1.21,95%CI:1.04-1.42), acquired HIV through homosexual intercourse (compared with acquired with HIV through heterosexual intercourse, aOR=1.77, 95%CI:1.48-2.12). The risk factors of cross-regional transmission included males (compared with females, aOR=1.74,95%CI:1.13-2.75), having education level of high school or above (compared with having education level of junior high school or below, aOR=1.96,95%CI:1.43-2.69), being freelancer/unemployed/retired (compared with being farmers, aOR=1.50,95%CI:1.07-2.11), acquired HIV through homosexual intercourse (compared with acquired with HIV through heterosexual intercourse, aOR=3.28,95%CI:2.30-4.72). Conclusion: There are HIV transmission hotspots in Guangxi. Guangxi and other provinces in China form a complex cross-regional transmission network. Future studies should carry out social network surveys in high-risk populations inferred from the molecular network analysis for the timely identification of hidden transmission chains and reduction of the second-generation transmission of HIV.

TSPY is a cancer testis antigen expressed in human hepatocellular carcinoma.

In search for genes associated with hepatocellular carcinoma (HCC) by cDNA microarray, we found that the transcription of TSPY, 'testis-specific protein Y-encoded', was upregulated in HCC. Investigation of a broad spectrum of normal and malignant tissues by RT-PCR revealed the TSPY transcript selectively expressed in normal testis, different histological types of human neoplastic tissues, and tumour cell lines. The expression of TSPY in cancer cells was further confirmed by in situ hybridisation. Indirect immunofluorescence microscopy analysis showed that TSPY was localised mainly in the cytoplasm of transiently transfected cells. Testis-specific protein Y-encoded was detected in 50% (16 of 32) of well- and moderately differentiated HCC patients, in 16% (four of 25) of poorly differentiated HCC patients, and in 5% (one of 19) of renal cell cancer patients. A serological survey revealed that 6.6% (seven of 106) HCC patients had anti-TSPY antibody response, demonstrating the immunogenicity of TSPY in humans. In conclusion, these data suggest that TSPY is a novel cancer/testis (CT) antigen and may be a potential candidate in vaccine strategy for immunotherapy in HCC patients.

Transduction of dendritic cells with recombinant adenovirus encoding HCA661 activates autologous cytotoxic T lymphocytes to target hepatoma cells.

Transduction of recombinant adenovirus into dendritic cells (DCs) is a promising new tool for cancer vaccine development. Here, we report that an adenovirus vector carrying hepatocellular carcinoma (HCC) antigen HCA661 and infected into DCs generates T-cell immunity against hepatoma cells. HCA661 is a novel cancer/testis (CT) antigen screened by SEREX from sera of an HCC patient. We constructed a recombinant adenovirus expressing the full-length cDNA of HCA661 gene and then transduced immature DCs, which had been generated with GM-CSF and IL-4 from peripheral blood mononuclear cell of HLA-A2(+) healthy donors. The resulting adenovirus-transduced DCs differentiated in the presence of monocyte-conditioned medium and poly [I] : poly [C], expressing the surface markers of mature DCs, including CD83, CD80, CD86 and HLA-DR. After maturation, the transduced DCs transcribed HCA661 mRNA and were able to prime the naïve T cells to become cytotoxic T lymphocytes (CTLs). Intracellular flow cytometry and enzyme-linked immunospot assay showed that these CTLs were able to target a hepatoma cell line, HepG2, which is HLA-A2 and HCA661 positive. In summary, we found that this recombinant adenovirus can help to induce DC maturation and these mature DCs can activate T cells to target hepatoma cells. Therefore, this recombinant adenovirus may have potential for use in liver cancer immunotherapy.

Functional supertype of HLA-A2 in the presentation of Flu matrix p58-66 to induce CD8+ T-cell response in a Northern Chinese population.

The functional supertype of HLA-A2 was investigated in the presentation of the A*0201-restricted Flu matrix p58-66 peptide to activate recall CD8+ T-cell response. In healthy Northern Chinese, the HLA-A2 supertype was mainly composed of the six alleles, A*0201 (26.4%), A*0206 (12.7%), A*0203 (8.2%), A*0207 (7.3%), A*0210 (1.8%) and A*0205 (0.9%), as analyzed by PCR using sequence specific primer (PCR-SSP) and sequence based typing (SBT). The IFN-gamma release Elispot assay was employed to assess effector CD8+ T cells. In A*0201-bearing individuals, the CD8+ T-cell response was potent when stimulated with autologous CD8- PBMCs. The frequency of the effector CD8+ T cells was 96.6% with the magnitude of effector CD8+ T cells of 225 SFC/5 x 104 CD8+ T cells and the RI of 25.7. In non-A*0201 individuals, the effector CD8+ T cells were minimally detectable while the peptide was presented by the autologous CD8- PBMCs. However, the induction of the response of CD8+ T cells obtained from non-A*0201 individuals was remarkably improved when the peptide was presented by autologous dendritic cells instead of CD8- PBMCs. The HLA-A2 alleles possessing cross-reactivity in the peptide presentation were mainly of A*0206 and non-A*0201 heterozygotes of A*0206 and A*0210. Moreover, A*0206 as the HLA-A2 functional supertype was further confirmed by tetramer assay. In two A*0206+ donors with CD8+ T-cell response to the peptide, the CD8+ T-cell frequency assessed by specific binding of peptide HLA-A*0201 tetramer was 4.62% and 1.66%, respectively. Thus, our results have substantiated the immunological relevance of the HLA-A2 supertype, which may benefit the design of peptide vaccines with the potential to be applicable in broader populations.

Identification of two novel CT antigens and their capacity to elicit antibody response in hepatocellular carcinoma patients.

FATE and TPTE genes were originally reported to be specifically expressed in the adult testis. We searched for the databases of Unigene and serial analysis of gene expression (SAGE) implying that these two gene transcripts might also be expressed in tumours. Herein, we demonstrated that FATE and TPTE mRNA transcripts were expressed in different histological types of tumours and normal testis. Both are cancer-testis (CT) antigens and renamed as FATE/BJ-HCC-2 and TPTE/BJ-HCC-5, respectively. Comparison at nucleotide sequence, the FATE/BJ-HCC-2 cDNA, was identical to that of FATE, whereas the TPTE/BJ-HCC-5 was found to have two isoforms in both cancers and testis: one was identical in cDNA sequence to TPTE, encoding a protein of 551 amino acids, and the other variant lacked an exon of 54 bp, encoding a protein of 533 amino acids. The mRNA expression was analysed by RT-PCR and real-time PCR. FATE/BJ-HCC-2 mRNA was detected in 66% (41 out of 62) in hepatocellular carcinoma (HCC) samples and 21% (three out of 14) in colon cancer samples, whereas the TPTE/BJ-HCC-5 mRNA was detected in 39% (24 out of 62) and 36% (five out of 14) in HCC and non-small lung cancer samples, respectively. The recombinant proteins were prepared and the reactivity of allogenic sera to these two antigens was screened. The frequency of antibody response against FATE/BJ-HCC-2 and TPTE/BJ-HCC-5 proteins was 7.3% (three out of 41) and 25.0% (six out of 24), respectively, in HCC patients bearing respective gene transcripts. Therefore, FATE/BJ-HCC-2 and TPTE/BJ-HCC-5 are the novel CT antigens capable of eliciting antibody response in cancer patients.

Evaluation of MAGE-1 and MAGE-3 as tumour-specific markers to detect blood dissemination of hepatocellular carcinoma cells.

The members of MAGE gene family are highly expressed in human hepatocellular carcinoma (HCC). In the present study, we tested the tumour-specific MAGE-1 and MAGE-3 transcripts in the peripheral blood of HCC patients by nested RT-PCR to detect the circulating tumour cells and evaluate their potential clinical implication. Of 30 HCC patients, the positive rate of MAGE-1 and MAGE-3 transcripts was 43.3% (13 out of 30) and 33.3% (10 out of 30) in PBMC samples, whilst the positive rate was 70% (21 out of 30) and 53.3% (16 out of 30) in the resected HCC tissue samples, respectively. The positivity for at least one MAGE gene transcript was 63.3% (19 out of 30) in PBMC samples of HCC patients and 83.3% (25 out of 30) in the resected HCC tissue samples. MAGE-1 and/or MAGE-3 mRNA were not detected in the PBMC of those patients from whom the resected HCC tissues were MAGE-1 or MAGE-3 mRNA negative, nor in the 25 PBMC samples from healthy donors. The detection of MAGE transcripts in PBMC was correlated with the advanced stages and tumour size of the HCC, being 82.4% (14 out of 17) in tumour stages III and IVa, 56.6% (five out of nine) in stage II, and null (nought out of four) in stage I. The serum alpha-FP in 33.3% (10 out of 30) of HCC patients was normal or slightly elevated (< 40 ng ml(-1)). However, six of these 10 patients (alpha-FP < 40 ng ml(-1)) were MAGE-1 and /or MAGE-3 mRNA positive in their PBMC. The follow-up survey of MAGE mRNA in PBMC was performed in 12 patients. Seven patients with persistent MAGE-1 and/or MAGE-3 mRNA positive or from negative turned to positive died because of metastasis and/or recurrence. In striking contrast, all four patients with MAGE-1 and/or MAGE-3 mRNA from positive turned to negative and one patient with persistent MAGE-3 transcript negative are alive after last test. Collectively, detection of MAGE transcripts with follow-up survey in PBMC is a feasible and reliable assay for the early prediction of the relapse and prognosis of the HCC patients.

[The capability of IL-1 in the induction of chemokine production by murine thymic epithelial cell (MTEC1) clones].

To analyze the capability of IL-1 and IL-6 in the induction of chemokine (CF) production by mouse thymus epithelial cell (MTEC1) clones, MTEC1 cells were cloned through one cell culture and individual cell clones were established in long term culture referred to as MTEC1-DW clones. The constitutive production of IL-1, IL-6 and CF by MTEC1-DW clones was evaluated and the patterns of the cytokine production determined. Addition of exogenous IL-1 or IL-6 or both to the cultures of those MTEC1-DW clones that are unable to produce CF, and incubated for 2 days, then, to assess the chemotactic activity in the cell culture supernatants (SNs). In the opposite, addition of anti-IL-1 mAb(s) to the cultures of those MTEC1-DW clones that can produce IL-1 and CF to neutralize secreted IL-1 then, to test chemotactic activity in the SNs after 2-day incubation. The results showed that in the MTEC1-DW clones which were unable to constitutively produce IL-1 or CF, addition of IL-1 could induce these cloned cells to produce CF with high chemotactic activity. By constrast, addition of anti-IL-1 mAb(s) to those MTEC1-DW clones that constitutively produce IL-1 and CF could significantly inhibit them to produce CF. IL-6 only exhibited weak activity in the induction of CF production by those cloned cells. Therefore, in the cytokine network regulation, CF production is mainly induced by endogenously produced IL-1 in MTEC1 cells.

[Characterization of expression of the apoptosis associated PF18-3 molecule in thymocyte apoptosis following ConA activation].

The expression pattern of McAb PF18-3 recognized molecule in activation induced thymocyte apoptosis was analysed. Results indicated that following activation by ConA, thymocytes underwent activation induced apoptosis identified by delayed production of DNA ladder and TUNEL positive staining of thymocytes in late stage of activation. PF18-3 molecule was found to express specifically in subset of apoptotic thymocytes with diploid. Kinetic comparison of expression between PF18-3 molecule and Fas or translocated membrane phospholipid suggested that PF18-3 molecule is different from them and likely to be a novel molecule related to thymocyte apoptosis.

[Multiple types of cytokines secreted by a mouse fetal liver stromal cell line].

A murine fetal liver stromal cell line, called MFLC, has been established and maintained in DME supplemented with 10%NCS for 2 years. Without exogenous stimulation, the MFLC cells spontaneously secreted several types of cytokines, including IL-6, GM-CSF and chemotactic factor. Of which, IL-6 and chemotactic factor were abundant, GM-CSF at low level. Biological activities of IL-7 and IL-3 were not detected in the MFLC cell's supernatants. MFLC-SN induced colony formation of murine bone marrow cells in a dose dependent fashion in which mixed granulocyte/macrophage/megakaryocyte colonies(CFU-GMM) and granulocyte/macrophage colonies(CFU-GM) were dominant. MFLC-SN also sustained colony formation of bone marrow cells taken from 5-Fu injected mice, suggesting that there was a biological activity similar to SCF in MFLC-SN. The cytokines secreted by MFLC might play an important role in T cell early development in fetal liver. These results will be useful for us to analyse the mechanism of T cell development in fetal liver and to study the cytokine network regulation.

Isolation of a lymphocyte chemotactic factor produced by the murine thymic epithelial cell line MTEC1: identification as a 30 kDa glycosylated form of MCP-1.

A medullary-type murine thymic epithelial cell line (MTEC1) established in our laboratory constitutively produces multiple species of chemotactic factors, which attract lymphocytes, neutrophils, and monocytes. Chemotactic proteins were isolated from MTEC1 supernatant and purified to homogeneity by adsorption to controlled pore glass, heparin-Sepharose chromatography, cation-exchange FPLC and RP-HPLC. A chemotactic factor for both lymphocytes and monocytes was identified as a 30 kDa protein by SDS-PAGE analysis under reducing conditions. After cleavage of the NH2-terminally blocked protein with formic acid, the amino acid sequence of the internal fragment was analysed and found to be identical to the amino acid sequence of mouse MCP-1/JE. The protein is hence identified as a glycosylated natural form of MCP-1/JE secreted by thymic epithelial cells. The 30 kDa glycosylated form of MCP-1 shows lower specific chemotactic activity (CA) for both lymphocytes and monocytes than the 6-7 kDa unglycosylated form of MCP-1.

Increased production of interleukin 4 in children with simple idiopathic nephrotic syndrome.

Sera and peripheral blood mononuclear cells (PBMC) were collected from 42 children with simple idiopathic nephrotic syndrome (INS) aged from 1.5 to 14 years and 28 age and sex-matched healthy children. The levels of IgE in serum and interleukin 4 (IL-4) in supernatant of PHA-activated PBMC were tested by ELISA. A significantly high incidence of allergic diseases was noted in INS children and their first-degree relatives, and there was a close association between atopy and INS. Besides, the levels of IgE and IL-4 were increased in nephrotic state (P < 0.001 both). IgE normal and IL-4 slightly lower in remission state (P < 0.2, P < 0.01 respectively). A positive correlation was noticed between IL-4 and IgE, and between IL-4 and 24-hour proteinuria (r = 0.472, r = 0.562; P < 005, P < 0.01 respectively). These showed a close association between atopy and INS. Both IL-4 and IgE, the important mediators in atopy, showed abnormal change in patients. The increased IL-4 production of T cells might account for the elevated serum IgE level. IL-4 may have a pathogenetic role in INS, but it still remains speculative.

[Cytokine production by MTSC 4 cells, an established mouse thymic dendritic cell line].

The cytokine production by MTSC 4 cells, a murine thymic dendritic cell line established in our laboratory, has been investigated. Without exogenous stimulation, the MTSC 4 cells constitutively produce multiple types of cytokines, including IL-1, IL-6, IL-7, CSF, IFN and Chemotactic factor (CF). Of which, IL-6 and IFN were abundant: IL-1 and Chemokine, moderate: IL-7 and CSF at low level. MTSC4 cells could not produce detectable either IL-3 of TNF alpha. The establishment of MTSC 4 may be useful in the analysis of the mechanism of thymus selection, and in the study of the network regulation of autocrined cytokines.

The capacity of lymphokine production by peripheral blood lymphocytes from aged humans.

The functional capability to produce interleukin 2 (IL2) and interferon gamma (IFN gamma) by peripheral blood lymphocytes (PBL) from elderly humans was evaluated. Thirty-nine samples of PBL derived from donors aged from 56 to 79 yr were cultured with phytohaemagglutinin (PHA) as stimulus at 37 degrees C for 48 hours. The IL2 activity in the supernatants was measured by its ability to sustain the IL2-dependent CTLL growth. Cultures of elder human T cells produced low level of IL2 activity, that is on the average of 9.7 +/- 9.8 mu/ml, equivalent to 28.1% the IL2 activity in parallel cultures of PBL derived from young donors (34.2 +/- 8.3 mu/ml, assessed on the basis of 145 samples). In the elder humans, the IL2 production by PBL decreased progressively as the increase of the age of the donors. In contrast to the impairment of IL2 production, the amount of IFN gamma secretion by elder human T cells was almost at the same level as that by young human T cells. The average IFN gamma activity of 39 samples of elder PBL cultures and 128 samples of young PBL cultures was 2,961 +/- 736 mu/ml and 3133 +/- 950 mu/ml, respectively. There was no significantly positive correlation between the level of IL2 and IFN gamma when comparison was made based on each individual samples. This implies that it may exist an alternative way of IFN gamma production which does not require the induction by IL2, and the relatively high level of IFN gamma may not impose adverse effect on the IL2 production.