BACKGROUNDD– individuals with previous D‐incompatible pregnancies and/or blood transfusions, as well as those who are actively immunized with small‐volume D+ red blood cells (RBCs), are stimulated to produce RhIG. Many factors could influence the stimulation of immunoglobulin production in response to foreign antigen (such as antigen immunogenicity and genetic factors), and it is unknown whether genetic markers could potentially identify responder anti‐D donors.STUDY DESIGN AND METHODSAnti‐D donors were assigned a responder profile based on their serum RhIG levels (n = 431). A subset of donors (n = 272) had DNA extracted for polymerase chain reaction genotyping assays for target genes in antigen presentation and pathogen recognition receptors (TLR2, TLR4, CD14, FcγRIIA, and the MHC Class II locus HLA‐DRB1). Statistical tests for associations between anti‐D donor responder profiles and genetic factors were performed.RESULTSA large proportion of our donors (38.7%) were classified as nonresponder donors, despite receiving multiple D+ RBC immunizations, whereas female sex was significantly associated with an all‐responder profile (p < 0.001). The presence of the DRB1*15 allele and absence of the DRB1*04 allele were more likely to be associated with a responder anti‐D donor, although not significantly after Bonferroni correction. A combination of the DRB1*15 allele and female sex was significantly associated with an anti‐D donor responder profile.CONCLUSIONThis study has identified female sex and the HLA‐DRB1*15 allele as potentially useful markers that could be used to screen donors before entry into D immunization programs.

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) can lead to significant changes to the HIV reservoir and HIV immune responses, indicating that further characterization of HIV-infected patients undergoing HSCT is warranted.Methods: We studied 3 patients who underwent HSCT after either reduced intensity conditioning or myeloablative conditioning regimen. We measured HIV antigens and antibodies (Ag/Ab), HIV-specific CD4(+) T-cell responses, HIV RNA, and DNA in plasma, peripheral blood mononuclear cells, isolated CD4(+) T cells from peripheral blood, and lymph node cells. The patients remained on antiretroviral therapy throughout the follow-up period.Results: All patients have been in continued remission for 4-6 years post-HSCT. Analyses of HIV RNA and DNA levels showed substantial reductions in HIV reservoir-related measurements in all 3 patients, changes in immune response varied with pronounced reductions in 2 patients and a less dramatic reduction in 1 patient. One patient experienced unexpected viral rebound 4 years after HSCT.Conclusions: These 3 cases highlight the substantial changes to the HIV reservoir and the HIV immune response in patients undergoing allogeneic HSCT. The viral rebound observed in 1 patient indicates that replication competent HIV can re-emerge several years after HSCT despite these marked changes.

Background Disruption of gastrointestinal tract epithelial and immune barriers contribute to microbial translocation, systemic inflammation, and progression of HIV-1 infection. Antiretroviral therapy (ART) may lead to reconstitution of CD4+ T cells in gut-associated lymphoid tissue (GALT), but its impact on humoral immunity within GALT is unclear. Therefore, we studied CD4+ subsets, including T follicular helper cells (Tfh), as well as resident B cells that have switched to IgA production, in gut biopsies, from HIV+ subjects on suppressive ART compared to HIV-negative controls (HNC). Methods Twenty-three HIV+ subjects on ART and 22 HNC undergoing colonoscopy were recruited to the study. Single-cell suspensions were prepared from biopsies from left colon (LC), right colon (RC), and terminal ileum (TI). T and B lymphocyte subsets, as well as EpCAM+ epithelial cells, were accurately enumerated by flow cytometry, using counting beads. Results No significant differences in the number of recovered epithelial cells were observed between the two subject groups. However, the median TI CD4+ T cell count/106 epithelial cells was 2.4-fold lower in HIV+ subjects versus HNC (19,679 versus 47,504 cells; p = 0.02). Similarly, median LC CD4+ T cell counts were reduced in HIV+ subjects (8,358 versus 18,577; p = 0.03) but were not reduced in RC. Importantly, we found no significant differences in Tfh or IgA+ B cell counts at either site between HIV+ subjects and HNC. Further analysis showed no difference in CD4+, Tfh, or IgA+ B cell counts between subjects who commenced ART in primary compared to chronic HIV-1 infection. Despite the decrease in total CD4 T cells, we could not identify a selective decrease of other key subsets of CD4+ T cells, including CCR5+ cells, CD127+ long-term memory cells, CD103+ tissue-resident cells, or CD161+ cells (surrogate marker for Th17), but there was a slight increase in the proportion of T regulatory cells. Conclusion While there were lower absolute CD4+ counts in the TI and LC in HIV+ subjects on ART, they were not associated with significantly reduced Tfh cell counts or IgA+ B cells, suggesting that this important vanguard of adaptive immune defense against luminal microbial products is normalized following ART.

In spite of prolonged and intensive treatment with combined antiretroviral therapy (cART), which efficiently suppresses plasma viremia, the integrated provirus of HIV-1 persists in resting memory CD4+ T cells as latent infection. Treatment with cART does not substantially reduce the burden of latent infection. Once cART is ceased, HIV-1 replication recrudesces from these reservoirs in the overwhelming majority of patients. There is increasing evidence supporting a role for noncoding RNAs (ncRNA), including microRNAs (miRNAs), antisense (as)RNAs, and short interfering (si)RNA in the regulation of HIV-1 transcription. This appears to be mediated by interaction with the HIV-1 promoter region. Viral miRNAs have the potential to act as positive or negative regulators of HIV transcription. Moreover, inhibition of virally encoded long-asRNA can induce positive transcriptional regulation, while antisense strands of siRNA targeting the NF-κB region suppress viral transcription. An in-depth understanding of the interaction between ncRNAs and the HIV-1 U3 promoter region may lead to new approaches for the control of HIV reservoirs. This review focuses on promoter associated ncRNAs, with particular emphasis on their role in determining whether HIV-1 establishes active or latent infection.

The current treatment strategy for HIV-1 involves prolonged and intensive combined antiretroviral therapy (cART), which successfully suppresses plasma viremia. It has transformed HIV-1 infection into a chronic disease. However, despite the success of cART, a latent form of HIV-1 infection persists as integrated provirus in resting memory CD4(+) T cells. Virus can reactivate from this reservoir upon cessation of treatment, and hence HIV requires lifelong therapy. The reservoir represents a major barrier to eradication. Understanding molecular mechanisms regulating HIV-1 transcription and latency are crucial to develop alternate treatment strategies, which impact upon the reservoir and provide a path toward a "functional cure" in which there is no detectable viremia in the absence of cART. Numerous reports have suggested ncRNAs are involved in regulating viral transcription and latency. This review will discuss the latest developments in ncRNAs, specifically short interfering (si) RNA and short hairpin (sh)RNA, targeting molecular mechanisms of HIV-1 transcription, which may represent potential future therapeutics. It will also briefly address animal models for testing potential therapeutics and current gene therapy clinical trials.

Transcriptional gene silencing (TGS) of mammalian genes can be induced by short interfering RNA (siRNA) targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA), which targets tandem NF-κB motifs within the viral 5'LTR. In this study, we screened a siRNA panel with the aim of identifying novel 5'LTR targets, to provide multiplexing potential with enhanced viral silencing and application toward developing alternate therapeutic strategies. Systematic examination identified a novel siRNA target, si143, confirmed to induce TGS as the silencing mechanism. TGS was prolonged with virus suppression >12 days, despite a limited ability to induce post- TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by chromatin immunoprecipitation analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac, and recruitment of argonaute-1, all characteristic marks of heterochromatin and TGS. Together, these epigenetic changes mimic those associated with HIV-1 latency. Further, robust resistance to reactivation was observed in the J-Lat 9.2 cell latency model, when transduced with shPromA and/or sh143. These data support si/shRNA-mediated TGS approaches to HIV-1 and provide alternate targets to pursue a functional cure, whereby the viral reservoir is locked in latency following antiretroviral therapy cessation.

Despite prolonged and intensive application, combined antiretroviral therapy cannot eradicate human immunodeficiency virus (HIV)-1 because it is harbored as a latent infection, surviving for long periods of time. Alternative approaches are required to overcome the limitations of current therapy. We have been developing a short interfering RNA (siRNA) gene silencing approach. Certain siRNAs targeting promoter regions of genes induce transcriptional gene silencing. We previously reported substantial transcriptional gene silencing of HIV-1 replication by an siRNA targeting the HIV-1 promoter in vitro. In this study, we show that this siRNA, expressed as a short hairpin RNA (shRNA) (shPromA-JRFL) delivered by lentiviral transduction of human peripheral blood mononuclear cells (PBMCs), which are then used to reconstitute NOJ mice, is able to inhibit HIV-1 replication in vivo, whereas a three-base mismatched variant (shPromA-M2) does not. In shPromA-JRFL-treated mice, HIV-1 RNA in serum is significantly reduced, and the ratio of CD4(+)/CD8(+) T cells is significantly elevated. Expression levels of the antisense RNA strand inversely correlates with HIV-1 RNA in serum. The silenced HIV-1 can be reactivated by T-cell activation in ex vivo cultures. HIV-1 suppression is not due to offtarget effects of shPromA-JRFL. These data provide "proof-of principle" that an shRNA targeting the HIV-1 promoter is able to suppress HIV-1 replication in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e137; doi:10.1038/mtna.2013.64; published online 3 December 2013.

Streptococcus pneumoniae is the most common cause of community‐acquired pneumonia and a major cause of morbidity and mortality throughout the world. It has been a major research priority to identify gene polymorphisms responsible for/associated with susceptibility and severity of S. pneumoniae infection to gain a better understanding of host genetic variants and their influence and clinical relevance to pneumococcal infections. In the present study, polymorphisms in several candidate genes, including TLR2‐Arg/Gln753, TLR4‐Asp/Gly299, TLR4‐Thr/Ile399, CD14‐159C/T and FcγRIIA‐R/H131, were examined in 85 children with pneumococcal sepsis as an invasive pneumococcal disease and 409 healthy blood donors as controls. The prevalence of the TLR4‐299/399 polymorphisms was significantly lower in the patient population than in controls (4 vs 11%; P <0.05; odds ratio (OR) 0.3; 95% confidence interval (CI) 0.1–1), while the prevalence of the CD14‐159CC and FcγRIIA‐R/R131 genotypes was significantly higher (35 vs 25%; P <0.05; OR 1.7; 95% CI 1–2.8 and 39 vs 21%; P <0.001; OR 2.5; 95% CI 1.4–4, respectively). Further, only 35% of patients carried either low‐risk genotypes or protective genotypes in contrast to 61% of controls ( P <0.0001; OR 2.8; 95% CI 1.7–4.6). We conclude that genetic variability in the TLR4, CD14 and FcγRIIA genes is associated with an increased risk of developing invasive disease in patients who are infected with S. pneumoniae .

ABSTRACT

ABSTRACT To investigate whether genetic factors of innate immunity might influence susceptibility and/or progression in individuals infected with SARS, we evaluated the CD14 gene polymorphism in 198 Hong Kong blood donors and 152 Hong Kong severe acute respiratory syndrome (SARS) patients who were previously genotyped for FcγRIIA polymorphisms. The prevalence of the CD14-159CC polymorphism was significantly higher in the patients with severe SARS than in the those with mild SARS or controls (31% versus 15% [mild SARS] or 20% [controls]; mild SARS: P = 0.029; odds ratio, 2.74; 95% confidence interval, 1.15 to 6.57; controls, P = 0.04; odds ratio, 2.41; 95% confidence interval, 1.05 to 5.54), and both CD14-159CC and FcγRIIA-RR131 are risk genotypes for severe SARS-CoV infection.