Analysis of the intestinal microbiota and profiles of blood amino acids and acylcarnitines in neonates with hyperbilirubinemia.

OBJECTIVE: This study aimed to discuss the distinctive features of the intestinal microbiota in neonates with hyperbilirubinemia and to comprehensively analyse the composition of the intestinal microbiota as well as the levels of free amino acids and acylcarnitines in the peripheral blood of neonates experiencing hyperbilirubinemia. RESULTS: At the phylum level, Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Chloroflexi were the five predominant microbial groups identified in both the hyperbilirubinemia and control groups. Alpha diversity analysis, encompassing seven indices, showed no statistically significant differences between the two groups. However, Beta diversity analysis revealed a significant difference in intestinal microbiota structure between the groups. Linear discriminant analysis effect size (LEfSe) indicated a significant reduction in the abundance of Gammaproteobacteria and Enterobacteriaceae within the hyperbilirubinemia group compared to that in the control group. The heatmap revealed that the control group exhibited increased abundances of Escherichia and Bifidobacterium, while the hyperbilirubinemia group exhibited increased levels of Enterococcus and Streptococcus. Regarding blood amino acids and acylcarnitines, there were greater concentrations of citrulline (Cit), arginine (Arg), ornithine (Orn), and valine (Val) in the hyperbilirubinemia group than in the control group. The hyperbilirubinemia group also exhibited significant increases in medium-chain fatty acids (C6, C8), long-chain fatty acids (C18), and free carnitine (C0). CONCLUSION: By comparing neonates with hyperbilirubinemia to those without, a significant disparity in the community structure of the intestinal microbiota was observed. The intestinal microbiota plays a crucial role in the bilirubin metabolism process. The intestinal microbiota of neonates with hyperbilirubinemia exhibited a certain degree of dysbiosis. The abundances of Bacteroides and Bifidobacterium were negatively correlated with the bilirubin concentration. Therefore, the fact that neonates with hyperbilirubinemia exhibit some variations in blood amino acid and acylcarnitine levels may provide, to a certain degree, a theoretical basis for clinical treatment and diagnosis.

Highly expressed miR-144-3p promotes the proliferation, migration and invasion of colon carcinoma cells by activating the Wnt/ß-catenin signaling pathway through targeting SFRP1.

Background: To investigate the influence of miR-144-3p on the proliferation, migration and invasion of colon carcinoma by targeting secreted frizzled-related protein 1 (SFRP1) as well as of the Wnt/ß-catenin signaling pathway. Methods: Based on the TCGA database, the association between the expression of miR-144-3p and the clinical information and prognosis of patients with colon carcinoma was examined, and SFRP1 was selected as the target gene for further studies based on bioinformatics prediction tools. CCK8 assay, wound healing assay and transwell invasion assay were employed to examine the impact of miR-144-3p on colon carcinoma cells. The regulation of SFRP1 by miR-144-3p was investigated using a dual-luciferase reporter system, and a rescue experiment was conducted to further elucidate whether miR-144-3p promotes the migration of colon carcinoma cells through targeting SFRP1 or not. The Wnt/ß-catenin signaling pathway-mediated effect of miR-144-3p in colon carcinoma was finally validated through the targeting of SFRP1. Results: The bioinformatics analysis showed that the miR-144 expression levels were substantially greater in colon carcinoma tissue than in para-carcinoma tissue and were closely with clinical stage and prognosis. The findings obtained from the trial indicated that miR-144-3p substantially expressed in colon carcinoma tissue sample and the colon carcinoma cells, and the overexpressed miR-144-3p boosted the colon carcinoma cells' proliferation, migration and invasion. The results of dual-luciferase reporter gene assay revealed that miR-144-3p targeted SFRP1, and rescue experiment was carried out and its results indicated that miR-144-3p increased colon carcinoma cells' migration through targeting SFRP1. In addition, the molecular axis of miR-144-3p/SFRP1 may over-activate the Wnt/ß-catenin signaling pathway. Conclusions: The present study has identified a novel malignant biological behavior, namely the ability of miR-144-3p to enhance the proliferation, migration and invasion of colon carcinoma cells by targeting SFRP1 and activating the Wnt/ß-catenin signaling pathway. Consequently, miR-144-3p emerges as a promising diagnostic and therapeutic target for colon carcinoma.

Recombinant Endoglin-Single-Chain Variable Fragment/ Induced Protein 10 Fusion Protein Potently Boosts the Anti-Tumor Efficacy of Adoptively Transferred TRP2-Specific CD8+ CD28+ Cytotoxic T Lymphocytes in Mice.

In this research, we studied the therapeutic efficacy of a newly designed fusion protein containing Endoglin single-chain variable fragment and IP10 (Endoglin-scFv/IP10), together with our recently generated TRP2-specific CD8+ CD28+ CTLs (CD8+ CD28+ CTLs) in controlling melanoma growth in mice. The recombinant Endoglin-scFv/IP10 was expressed in E. coli, purified by affinity chromatography, and characterized in vitro for its chemotactic movement and immunoreactivity with endoglin-expressing cells. In vivo, melanoma xenografts were established in mice (C57BL/6) using B16F10 cells. After that, mice were treated with intravenous injections of vehicle (PBS), Endoglin-scFv/IP10 alone, CD8+ CD28+ CTLs alone, or Endoglin-scFv/IP10+ CD8+ CD28+ CTLs. The therapeutic efficacy was assessed by monitoring tumor growth, mouse survival and cellular biomarkers. Endoglin-scFv/IP10 fusion protein combined with CD8+ CD28+ CTLs observed a reduction in tumor growth, resulting in improved survival. On the cellular level, the combination treatment dramatically reduced the number of systemic and tumor associated myeloid-derived suppressor cells or regulatory T cells, increased tumor-responsive interferon-γ-producing lymphocytes and tumor-associated CD8+ CXCR3+ T cells, and inhibited proliferation and angiogenesis but stimulated apoptosis within melanoma tissue. This study demonstrates the therapeutic potential of Endoglin-scFv/IP10 fusion protein in combination with CD8+ CD28+ CTLs in melanoma treatment.

Chloroquine and Rapamycin Augment Interleukin-37 Expression via the LC3, ERK, and AP-1 Axis in the Presence of Lipopolysaccharides.

IL-37 is a cytokine that plays critical protective roles in many metabolic inflammatory diseases, and its therapeutic potential has been confirmed by exogenous IL-37 administration. However, its regulatory mechanisms remain unclear. U937 cells were treated with autophagy-modifying reagents (3-MA, chloroquine, and rapamycin) with or without LPS stimulation. Thereafter, IL-37 expression and autophagic markers (Beclin1, P62/SQSTM1, and LC3) were determined. For regulatory signal pathways, phosphorylated proteins of NF-κB (p65 and IκBα), AP-1 (c-Fos/c-Jun), and MAPK signal pathways (Erk1/2 and p38 MAPK) were quantified, and the agonists and antagonists of MAPK and NF-κB pathways were also used. Healthy human peripheral blood mononuclear cells were treated similarly to confirm our results. Four rhesus monkeys were also administered chloroquine to evaluate IL-37 induction in vivo and its bioactivity on CD4 proliferation and activation. IL-37 was upregulated by rapamycin and chloroquine in both U937 cells and human PBMCs in the presence of LPS. IL-37 was preferentially induced in autophagic cells associated with LC3 conversion. AP-1 and p65 binding motifs could be deduced in the sequence of the IL-37 promoter. Inductive IL-37 expression was accompanied with increased phosphorylated Erk1/2 and AP-1 and could be completely abolished by an Erk1/2 inhibitor or augmented by Erk1/2 agonists. In monkeys, chloroquine increased IL-37 expression, which was inversely correlated with CD4 proliferation and phosphorylated STAT3. IL-37 levels were induced by rapamycin and chloroquine through the LC3, Erk1/2, and NF-κB/AP-1 pathways. Functional IL-37 could also be induced in vivo.

Study on miRNAs in Pan-Cancer of the Digestive Tract Based on the Illumina HiSeq System Data Sequencing.

OBJECTIVE: miRNA has gained attention as a therapeutic target in various malignancies. The proposal of this study was to investigate the biological functions of key miRNAs and target genes in cancers of the digestive tract which include esophageal carcinoma (ESCA), gastric adenocarcinoma (GAC), colon adenocarcinoma (COAD), and rectal adenocarcinoma (READ). MATERIALS AND METHODS: After screening differentially expressed miRNAs (DEMIs) and differentially expressed mRNAs (DEMs) in four digestive cancers from The Cancer Genome Atlas (TCGA) database, the diagnostic value of above DEMIs was evaluated by receiver-operating characteristic (ROC) curve analysis. Then, corresponding DEMIs' target genes were predicted by miRWalk 2.0. Intersection of predicted target genes and DEMs was taken as the target genes of DEMIs, and miRNA-mRNA regulatory networks between DEMIs and target genes were constructed. Meanwhile, the univariate Cox risk regression model was used to screen miRNAs with distinct prognostic value, and Kaplan-Meier analysis was used to determine their significance of prognosis. Furthermore, we performed bioinformatics methods including protein-protein interaction (PPI) networks, gene ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and gene group RIDA analysis by Gene-Cloud of Biotechnology Information (GCBI) to explore the function and molecular mechanisms of DEMIs and predicted target genes in tumor development. RESULTS: Eventually, 3 DEMIs (miR-7-3, miR-328, and miR-323a) with significant prognostic value were obtained. In addition, 3 DEMIs (miR-490-3p, miR-133a-3p, and miR-552-3p) and 281 target genes were identified, and the 3 DEMIs showed high diagnostic value in READ and moderate diagnostic value in ESCA, GAC, and COAD. Also, the miRNA-mRNA regulatory network with 3 DEMIs and 281 overlapping genes was successfully established. Functional enrichment analysis showed that 281 overlapping genes were mainly related to regulation of cell proliferation, cell migration, and PI3K-Akt signaling pathway. CONCLUSION: The diagnostic value and prognostic value of significant DEMIs in cancers of the digestive tract were identified, which may provide a novel direction for treatment and prognosis improvement of cancers of the digestive tract.

Anti-Tumor Activity of Mannose-CpG-Oligodeoxynucleotides-Conjugated and Hepatoma Lysate-Loaded Nanoliposomes for Targeting Dendritic Cells In Vivo.

Dendritic cell (DC)-based tumor vaccines are a promising immunotherapeutic method of cancer treatment. However, their therapeutic applications are significantly limited by their weak immunogenicity, costly culturing steps, and easily degradable properties. Thus, the anti-tumor activity for the vaccines should be improved. In this study, a novel lipid nanoparticle (M/CpG-ODN-H22-Lipo) was developed, which was conjugated with synthetic CpG oligodeoxynucleotides (CpG-ODN) and mannose and then loaded with H22 hepatoma lysate. Our data corroborate that M/CpG-ODN-H22-Lipo selectively targeted DCs and significantly increased their induced-maturation. Besides, the vaccine halted tumor growth and extended survival of mice with hepatocellular carcinoma. Moreover, M/CpG-ODN-H22-Lipo treatment reduced the percentages of myeloid-derived suppressor cells (in the tumor and bone marrow) and regulatory T cells (Treg) in the spleen. In contrast, the number of IFN-gamma-positive cells in the spleen along with the serum IgG levels were up-regulated. Moreover, tumor angiogenesis and tumor-cell proliferation were halted by the treatment of M/CpG-ODN-H22-Lipo, whereas tumor cell apoptosis was up-regulated. Our data revealed that CD8 + T cells and NK cells were vital to mediate the anti-tumor immunity of M/CpG-ODN-H22-Lipo treatment. In sum, the results here proved M/CpG-ODN-H22-Lipo vaccine a safe, specific and effective DC-based anti-tumor immunotherapy with great potential for clinical applications.

Vincristine-Loaded and sgc8-Modified Liposome as a Potential Targeted Drug Delivery System for Treating Acute Lymphoblastic Leukemia.

Cytotoxic compounds vincristine sulphate (VCR) is widely used to against hemato-oncology, and especially the acute lymphoblastic leukemia (ALL). However, VCR's full therapeutic potential has been limited by its dose-limiting neurotoxicity, classically resulting in autonomic and peripheral sensory-motor neuropathy. Therefore, we developed a targeted liposomal drug delivery system (sgc8/VCR-Lipo) for improving the therapeutic effects of VCR against leukemia and reducing its systematic adverse effects. sgc8/VCR-Lipo could specifically bind to CCRF-CEM cells and significantly inhibit proliferation of cancer cells in vitro and tumor growth in vivo. The sgc8/VCR-Lipo nanoparticles may improve the anti-tumor efficacy of VCR and reduce side effects induced by non-specific drug release. These results suggest that our findings provide scientific evidence for developing novel aptamer-based targeted drug delivery systems for leukemia treatment.

Zidovudine-Based Treatments Inhibit the Glycosylation of ADAM17 and Reduce CD163 Shedding From Monocytes.

BACKGROUND: sCD163, a biomarker of monocyte-macrophage activation, has been identified as a predictor of all-cause mortality in treated HIV-infected individuals. Nevertheless, little is known about whether different antiretroviral drugs differentially regulate sCD163 levels and monocyte activation. METHODS: A total of 123 patients receiving zidovudine (ZDV)-based (n = 55) or tenofovir disoproxil fumarate (TDF)-based (n = 68) antiretroviral regimens were enrolled, and their viral loads, CD4 counts, as well as plasma sCD163 and sCD14 levels were quantified. Twenty-eight (14 in each group) patients donated additional blood samples for flow cytometry and gene expression analyses using purified monocytes. THP-1 cultures were also used to investigate the effect of ZDV on ADAM17, which is responsible for CD163 shedding. RESULTS: As compared to the TDF-treated group, the ZDV-treated group had lower plasma sCD163 levels and higher CD163 expression on CD14++CD16 monocytes. Five metabolic-inflammatory genes exhibited significantly different expression levels between purified monocytes of the ZDV and TDF groups (IL-6, 2.90-fold lower in ZDV group, P < 0.001; iNOS, 1.81-fold higher; CX3CR1, 1.72-fold lower; MIP-1ß, 1.10-fold lower; and PPARγ-1, 1.36-fold higher, P < 0.05). Moreover, we show that ZDV treatment increases the surface expression of CD163 in cultured THP-1 cells, accompanied by the inhibition of glycosylation and surface expression of ADAM17. CONCLUSIONS: Compared with TDF treatment, ZDV treatment causes lower plasma sCD163 levels, probably by inhibiting the glycosylation of ADAM17 and CD163 shedding. Our results show that ZDV functions as an ADAM17 inhibitor in vivo and extend our understanding of its immune-modulatory effects and adverse effects.

The enhanced antitumor-specific immune response with mannose- and CpG-ODN-coated liposomes delivering TRP2 peptide.

PURPOSE: Dendritic cell (DC)-based cancer vaccines is a newly emerging and potent form of immune therapy. As for any new technology, there are still considerable challenges that need to be addressed. Here, we investigate the antitumor potential of a novel liposomal vaccine, M/CpG-ODN-TRP2-Lipo. METHODS: We developed a vaccination strategy by assembling the DC-targeting mannose and immune adjuvant CpG-ODN on the surface of liposomes, which were loaded with melanoma-specific TRP2180-188 peptide as liposomal vaccine. M/CpG-ODN-TRP2-Lipo treatment was used to intendedly induce activation of DCs and antitumor- specific immune response in vivo. RESULTS: Our results demonstrated in vitro that the prepared liposomal particles were efficiently taken up by DCs. This uptake led to an enhanced activation of DCs, as measured by the upregulation of MHC II, CD80, and CD86. Furthermore, M/CpG-ODN-TRP2-Lipo effectively inhibited the growth of implanted B16 melanoma and prolonged the survival of mice. This therapy significantly reduced the number of myeloid-derived suppressor cells (MDSCs) and regulatory T cells, while simultaneously increasing the number of activated T cells, tumor antigen-specific CD8+ cytotoxic T cells, and interferon-γ-producing cells. At the same time, it was found to suppress tumor angiogenesis and tumor cell proliferation, as well as up-regulate their apoptosis. Interestingly, MyD88-knockout mice had significantly shorter median survival times compared to wild-type mice following the administration of M/CpG-ODN-TRP2-Lipo. CONCLUSIONS: The results suggested that the antitumor activities of the vaccine partially rely on the Myd88 signaling pathway. Interestingly, compared to whole tumor cell lysate-based vaccine, M/CpG-ODN-TRP2-Lipo, tumor specific antigen peptide-based vaccine, improved survival of tumor-bearing mice as well as enhanced their antitumor responses. All in all, we describe a novel vaccine formulation, M/CpG-ODN-TRP2-Lipo, with the aim of improving antitumor responses by alleviating the immunosuppressive environment in tumors.

Folate-Modified Chitosan Nanoparticles Coated Interferon-Inducible Protein-10 Gene Enhance Cytotoxic T Lymphocytes' Responses to Hepatocellular Carcinoma.

Adoptive therapy using tumor antigen-specific cytotoxic T lymphocytes (CTLs) is a promising approach for treatment of human cancers. Due to immune suppression in cancer patients, it is difficult for tumor antigen-specific CTLs to arrive at tumor tissues. Interferon-inducible protein-10 (IP-10) is a powerful chemokine that effectively attracts CTLs to tumor tissues and improves their anti-tumor activity. Increase over expression of IP-10 in tumor tissues can efficiently promote efficacy of adoptive therapy. Folate-modified chitosan nanoparticles coating the human IP-10 gene (FA-CS-hIP-10) were therefore developed in this study. The FA-CS-hIP-10 nanoparticles were specifically bound to folate receptors on hepatoma cells and promoted the expression of IP-10, to improve the activity of pMAGE-A1(278-286) specific CTLs. Combination of the FA-CS-hIP-10 and pMAGE-A1(278-286) specific CD8+ CTLs efficiently increased secretion of IFN-γ, inhibited tumor growth and extended survival of nude mice with subcutaneously transplanted human hepatocellular carcinoma. Our results demonstrated that the mechanism behind this novel therapeutic approach involved inhibition of angiogenesis and proliferation, and also promoted apoptosis of tumor cells. Our study provides a potentially novel approach for treatment of human hepatocellular carcinoma by improving the activity of tumor antigen-specific CTLs.

Suppression of HIV Replication by CD8(+) Regulatory T-Cells in Elite Controllers.

We previously demonstrated in the Chinese macaque model that an oral vaccine made of inactivated SIV and Lactobacillus plantarum induced CD8(+) regulatory T-cells, which suppressed the activation of SIV(+)CD4(+) T-cells, prevented SIV replication, and protected macaques from SIV challenges. Here, we sought whether a similar population of CD8(+) T-regs would induce the suppression of HIV replication in elite controllers (ECs), a small population (3‰) of HIV-infected patients with undetectable HIV replication. For that purpose, we investigated the in vitro antiviral activity of fresh CD8(+) T-cells on HIV-infected CD4(+) T-cells taken from 10 ECs. The 10 ECs had a classical genomic profile: all of them carried the KIR3DL1 gene and 9 carried at least 1 allele of HLA-B:Bw4-80Ile (i.e., with an isoleucine residue at position 80). In the nine HLA-B:Bw4-80Ile-positive patients, we demonstrated a strong viral suppression by KIR3DL1-expressing CD8(+) T-cells that required cell-to-cell contact to switch off the activation signals in infected CD4(+) T-cells. KIR3DL1-expressing CD8(+) T-cells withdrawal and KIR3DL1 neutralization by a specific anti-killer cell immunoglobulin-like receptor (KIR) antibody inhibited the suppression of viral replication. Our findings provide the first evidence for an instrumental role of KIR-expressing CD8(+) regulatory T-cells in the natural control of HIV-1 infection.

Folate-modified Chitosan Nanoparticles Containing the IP-10 Gene Enhance Melanoma-specific Cytotoxic CD8(+)CD28(+) T Lymphocyte Responses.

BACKGROUND: Adoptive immunotherapy with cytotoxic T lymphocytes (CTLs) has great potential for the treatment of some malignant cancers. Therefore, augmenting the responses of tumor-specific CTLs is significant for the adoptive immunotherapy of melanoma. This study aimed to investigate the anti-tumor response of a combination therapy employing folate-modified chitosan nanoparticles containing IP-10 (interferon-γ-inducible protein-10) plus melanoma TRP2-specific CD8(+)CD28(+) T cells. METHODS: We prepared folate-modified chitosan nanoparticles containing the mouse IP-10 gene (FA-CS-mIP-10), and induced melanoma TRP2-specific CD8(+)CD28(+) T cells by co-culturing them with artificial antigen-presenting cells. B16-bearing mice were treated with FA-CS-mIP-10, melanoma TRP2-specific CD8(+)CD28(+) T cells, a combination of both, and the saline control. Tumor volumes and the survival time of mice were recorded. The proportion of myeloid-derived suppressor cells (MDSCs) infiltrating the tumor microenvironment and regulatory T cells (Tregs) in the spleen was analyzed by flow cytometry. We also detected the proliferation and angiogenesis of tumors by immunohistochemistry and apoptosis by TUNEL. RESULTS: The combination therapy inhibited the progression of melanoma in vivo. Compared with other treatments, it more efficiently inhibited tumor growth and increased the survival time of mice. After treatment with combination therapy, the proportion of MDSCs and Tregs decreased, while the percentage of CXCR3(+)CD8(+) T cells increased. Furthermore, combination therapy inhibited proliferation and promoted apoptosis of tumor cells and significantly inhibited tumor angiogenesis in vivo. CONCLUSION: We describe a novel strategy for improving the anti-tumor response of CD8(+)CD28(+) CTLs by combining them with FA-CS-mIP-10 nanoparticles.

Evolving molecular epidemiological profile of human immunodeficiency virus 1 in the southwest border of China.

BACKGROUND: We have previously reported in Xishuangbanna (Banna) Dai Autonomous Prefecture, a well-developed tourist destination in the southwest border of China, that HIV-1 transmitted dominantly through heterosexual contact with less divergent genotypes and few drug resistant mutations. Due to the rapid increase of newly diagnosed HIV-1 cases per year in Banna in recent years, it's important to evaluate the evolution of HIV-1 molecular epidemiology for the better understanding of ongoing HIV-1 outbreak in this region. METHODOLOGY/PRINCIPAL FINDINGS: By sequencing of HIV-1 pol genes and phylogenetic analysis, we conducted a molecular epidemiologic study in 352 HIV-1-seropositive highly active antiretroviral treatment (HAART)-naïve individuals newly diagnosed at the Banna Center for Disease Control and Prevention between 2009 and 2011. Of 283 samples (84.1% taken from heterosexually acquired adults, 10.6% from needle-sharing drug users, 2.8% from men who have sex with men, 0.4% from children born from HIV-1-infected mothers, and 2.1% remained unknown) with successful sequencing for pol gene, we identified 108 (38.2%) HIV-1 subtype CRF08_BC, 101 (35.7%) CRF01_AE, 49 (17.3%) CRF07_BC, 5 (1.8%) C/CRF57_BC, 3 (1.1%) B', 1 (0.4%) B/CRF51_01B, and 16 (5.7%) unique recombinants forms. Among these infected individuals, 104 (36.7%) cases showed drug resistant or resistance-relevant mutations, and 4 of them conferring high-level resistance to 3TC/FTC, EFV/NVP or NFV. Phylogenetic analysis revealed 21 clusters (2-7 sequences) with only 21.2% (60/283) sequences involved. CONCLUSION/SIGNIFICANCE: In contrast to our previous findings, CRF08_BC, replaced CRF01_AE, became the dominant genotype of HIV-1 in Banna prefecture. The viral strains with drug resistance mutations were detected frequently in newly diagnosed HIV-1-infected individuals in this region.

Mucosal SIV Vaccines Comprising Inactivated Virus Particles and Bacterial Adjuvants Induce CD8(+) T-Regulatory Cells that Suppress SIV-Positive CD4(+) T-Cell Activation and Prevent SIV Infection in the Macaque Model.

A new paradigm of mucosal vaccination against human immunodeficiency virus (HIV) infection has been investigated in the macaque model. A vaccine consisting of inactivated simian immunodeficiency virus (SIV)mac239 particles together with a living bacterial adjuvant (either the Calmette and Guerin bacillus, Lactobacillus plantarum or Lactobacillus rhamnosus) was administered to macaques via the vaginal or oral/intragastric route. In contrast to all established human and veterinary vaccines, these three vaccine regimens did not elicit SIV-specific antibodies nor cytotoxic T-lymphocytes but induced a previously unrecognized population of non-cytolytic MHCIb/E-restricted CD8(+) T-regulatory cells that suppressed the activation of SIV-positive CD4(+) T-lymphocytes. SIV reverse transcription was thereby blocked in inactivated CD4(+) T-cells; the initial burst of virus replication was prevented and the vaccinated macaques were protected from a challenge infection. For 3-14 months after intragastric immunization, 24 macaques were challenged intrarectally with a high dose of SIVmac239 or with the heterologous strain SIV B670 (both strains grown on macaques PBMC). Twenty-three of these animals were found to be protected for up to 48 months while all 24 control macaques became infected. This protective effect against SIV challenge together with the concomitant identification of a robust ex vivo correlate of protection suggests a new approach for developing an HIV vaccine in humans. The induction of this new class of CD8(+) T-regulatory cells could also possibly be used therapeutically for suppressing HIV replication in infected patients and this novel tolerogenic vaccine paradigm may have potential applications for treating a wide range of immune disorders and is likely to may have profound implications across immunology generally.

Anti-tumor immune response of folate-conjugated chitosan nanoparticles containing the IP-10 gene in mice with hepatocellular carcinoma.

Immunotherapy is one of the most promising new therapies for hepatocellular carcinoma (HCC) in recent years. In this study, folate-conjugated chitosan nanoparticles (FA-CS-NPs) were loaded with mouse interferon-γ-inducible protein-10 (IP-10) plasmid, which were used for immunotherapy in HCC. H22 tumor-bearing mice were treated with FA-CS-NPs entrapped IP-10 plasmid and targeting efficiency was observed by optical imaging in vivo. Flow cytometry was used to measure the number of myeloid-derived suppressor cells (MDSCs) in the tumor and CD4+CD25+FoxP3+ T-regulatory cells (CD4+CD25+FoxP3+ Tregs) in the spleen. The enzyme-linked immunospot (ELISPOT) assay was used to quantify the number of interferon-γ (IFN-γ)-positive cells. IP-10 expression, tumor vessel density, cell proliferation and apoptosis were evaluated by immunohistochemistry. It was shown that FA-CS-NPs entrapped IP-10 plasmid displayed anti-tumor activity with inhibition of tumor growth and prolonging the survival time in H22 tumor-bearing mice. Treatment of H22 tumor-bearing mice with FA-CS-NPs entrapped IP-10 plasmid inhibited angiogenesis and promoted IP-10 expression and induced apoptosis in the tumor. FA-CS-NPs entrapped IP-10 plasmid-treated mice also had a lower proportion of Tregs in the spleen, a higher proportion of MDSCs in the tumor and higher number of IFN-γ-positive cells in the spleen compared with the mice from the other experimental groups. These data suggested that the gene delivery system of folate-conjugated chitosan nanoparticle loaded with IP-10 plasmid may be a promising strategy for immunotherapy of HCC.

Induction of CD8+ regulatory T cells protects macaques against SIV challenge.

Efforts to develop a vaccine against HIV have so far met with limited success. Given that CD4(+) T cell activation drives the initial burst of viral replication, we explored in macaques whether an oral vaccine comprised of Lactobacillus plantarum, a commensal bacterium that favors immune tolerance, and inactivated simian immunodeficiency virus mac239 (SIVmac239) would induce CD4(+) T cell unresponsiveness/tolerance toward SIV antigens and thereby prevent the establishment of SIV infection. The tolerogenic vaccine induced MHC-Ib/E-restricted CD8(+) regulatory T cells (Tregs) that suppressed SIV-harboring CD4(+) T cell activation and ex vivo SIV replication in 15 of 16 animals without inducing SIV-specific antibodies or cytotoxic T lymphocytes. Of 16 macaques that were intrarectally challenged with SIVmac239 or heterologous strain SIVB670, 15 were sterilely protected. In four macaques that were rechallenged intravenously, plasma SIV levels peaked slightly and then dropped to undetectable levels, although the animals subsequently harbored intracellular SIV DNA. Infusion of CD8 antibodies confirmed the role of CD8(+) Tregs in preventing/suppressing SIV in vivo. These findings suggest a new avenue of research toward developing an HIV-1 vaccine.

Molecular epidemiology of human immunodeficiency virus type 1 in Guangdong province of southern China.

BACKGROUND: Although the outbreak of human immunodeficiency virus type 1 (HIV-1) in Guangdong has been documented for more than a decade, the molecular characteristics of such a regional HIV-1 epidemic remained unknown. METHODOLOGY/PRINCIPAL FINDINGS: By sequencing of HIV-1 pol/env genes and phylogenetic analysis, we performed a molecular epidemiologic study in a representative subset (n  = 200) of the 508 HIV-1-seropositive individuals followed up at the center for HIV/AIDS care and treatment of Guangzhou Hospital of Infectious Diseases. Of 157 samples (54.1% heterosexual acquired adults, 20.4% needle-sharing drug users, 5.7% receivers of blood transfusion, 1.3% men who have sex with men, and 18.5% remained unknown) with successful sequencing for both pol and env genes, 105 (66.9%) HIV-1 subtype CRF01_AE and 24 (15.3%) CRF07_BC, 9 (5.7%) B', 5 (3.2%) CRF08_BC, 5 (3.2%) B, 1 (0.6%) C, 3 (1.9%) CRF02_AG, and 5 (3.2%) inter-region recombinants were identified within pol/env sequences. Thirteen (8.3%) samples (3 naïves, 6 and 5 received with antiretroviral treatment [ART] 1-21 weeks and ≥24 weeks respectively) showed mutations conferring resistance to nucleoside/nonnucleoside reverse transcriptase inhibitors or protease inhibitors. Among 63 ART-naïve patients, 3 (4.8%) showed single or multiple drug resistant mutations. Phylogenetic analysis showed 8 small clusters (2-3 sequences/cluster) with only 17 (10.8%) sequences involved. CONCLUSION/SIGNIFICANCE: This study confirms that sexual transmission with dominant CRF01_AE strain is a major risk for current HIV-1 outbreak in the Guangdong's general population. The transmission with drug-resistant variants is starting to emerge in this region.

Variability of bio-clinical parameters in Chinese-origin Rhesus macaques infected with simian immunodeficiency virus: a nonhuman primate AIDS model.

BACKGROUND: Although Chinese-origin Rhesus macaques (Ch RhMs) infected with simian immunodeficiency virus (SIV) have been used for many years to evaluate the efficacy of AIDS vaccines and therapeutics, the bio-clinical variability of such a nonhuman primate AIDS model was so far not established. METHODOLOGY/PRINCIPAL FINDINGS: By randomizing 150 (78 male and 72 female) Ch RhMs with diverse MHC class I alleles into 3 groups (50 animals per group) challenged with intrarectal (i.r.) SIVmac239, intravenous (i.v.) SIVmac239, or i.v. SIVmac251, we evaluated variability in bio-clinical endpoints for 118 weeks. All SIV-challenged Ch RhMs became seropositive for SIV during 1-2 weeks. Plasma viral load (VL) peaked at weeks 1-2 and then declined to set-point levels as from week 5. The set-point VL was 30 fold higher in SIVmac239 (i.r. or i.v.)-infected than in SIVmac251 (i.v.)-infected animals. This difference in plasma VL increased overtime (>100 fold as from week 68). The rates of progression to AIDS or death were more rapid in SIVmac239 (i.r. or i.v.)-infected than in SIVmac251 (i.v.)-infected animals. No significant difference in bio-clinical endpoints was observed in animals challenged with i.r. or i.v. SIVmac239. The variability (standard deviation) in peak/set-point VL was nearly one-half lower in animals infected with SIVmac239 (i.r. or i.v.) than in those infected with SIVmac251 (i.v.), allowing that the same treatment-related difference can be detected with one-half fewer animals using SIVmac239 than using SIVmac251. CONCLUSION/SIGNIFICANCE: These results provide solid estimates of variability in bio-clinical endpoints needed when designing studies using the Ch RhM SIV model and contribute to the improving quality and standardization of preclinical studies.

[Effectiveness of Ganoderma lucidum preparation in treating simian acquired immune deficiency syndrome].

OBJECTIVE: To explore the effectiveness of the traditional Chinese herbal medicine Lingzhi (Ganoderma lucidum) preparation in treating simian acquired immune deficiency syndrome (SAIDS). METHODS: Five female adult Chinese rhesus monkeys were inoculated rectally with SIVmac239, and were all diagnosed as SAIDS by laboratory and clinical examinations 17 months later. Of these 5 monkeys, 3 (#393, #374, and #381; treatment group) were orally administered with Ganoderma lucidum (2 spores powder capsules plus 2 spores oil capsules on a daily basis), and the remaining other two monkeys (#348 and #361) served as control and did not receive treatment. RESULTS: Animal #393 (treatment group), #361 (control group) and #348 (control group) died of SAIDS (opportunity infection) 3.5 months, 6 months, and 11 months later, respectively. Two animals (#374 and #381) survived. The necropsy revealed depletion and/or exhaustion of their lymphoid tissue. In the monkey #374, the peripheral CD4(+) T lymphocyte increased by 30% in the 6(th) month compared with the baseline level and then fluctuated. The plasma viral load gradually fell and reached about 1 log(10) in the treatment group, but remained stable in the control group. As shown by pathological examinations, the lymph node and spleen of monkeys #374 (treatment group) and #381 (treatment group) showed rehabilitation and reconstruction in the lymphatic tissue, thymus, nerve tissue of gyrus hippocampi, pituitary gland, pineal body, thyroid gland, adrenal gland, and ovary. In the control group, however, animals experienced depletion of lymph nodes, atrophy of spleen, disappearance of thymus, and other disorders in endocrine organs. CONCLUSION: Ganoderma lucidum preparation may have certain protective effect on the immune system, nervous system, and endocrine system of monkeys with SAIDS.

[Comparison of intravenous and intrarectal SIVmac239 infections in rhesus monkeys of Chinese origin].

OBJECTIVE: To investigate the biological and clinical features of Chinese rhesus monkeys after intravenous (IV) and intrarectal (IR) challenge with SIVmac239 in rhesus monkeys of Chinese origin, and compare the differences between the routes of infection. METHODS: Rhesus monkeys of Chinese origin were inoculated with SIVmac239 either by IV (n = 19) or IR (n = 6) routes. Simian immunodeficiency virus (SIV)-specific antibody titer, CD4 + T cell counting, plasma SIV load, lymph node pathology, and clinical manifestations were compared between these two groups 232 or 168 days after challenging. RESULTS: All SIVmac239-inoculated animals became seropositive for SIV-specific antibodies. SIV-specific IgM was detected in IV groups as from day 10 but was not detected in IR for all the time points. Although SIV-specific IgG was detected as from day 30 in both groups, the IgG titers were ten-fold higher in IV group than in IR group after day 168. CD4 + T-cell counting decreased progressively in IV group but remained stable in IR group over time. Plasma SIV RNA loads peaked in all animals between day 10 and day 14 (10(7) copies/ml), then declined to "setpoint" (10(3) - 10(6) copies/ml) about 2 months later. Most inoculated animals manifested lymphadenopathy. Two animals in IV group and one in IR group died of simian AIDS between day 150 and day 210, as evidenced by the autopsies showing the depletion of lymph tissues, Pneumocystis carinii pneumonia and other opportunity infections. Conclusion IV or IR inoculation of SIVmac239 in Chinese rhesus monkeys will result in chronic SIV infection with a similar clinical feature of natural HIV infection, which provides an excellent experimental animal model for AIDS.