Humanized mice for studying HIV latency and potentially its eradication.

PURPOSE OF THE REVIEW: The quest for an HIV cure faces a formidable challenge: the persistent presence of latent viral infections within the cells and tissues of infected individuals. This review provides a thorough examination of discussions surrounding HIV latency, the use of humanized mouse models, and strategies aimed at eliminating the latent HIV reservoir. It explores the hurdles and advancements in understanding HIV pathogenesis, mainly focusing on establishing latent reservoirs in CD4 + T cells and macrophages. Introducing the concepts of functional and sterile cures, the review underscores the indispensable role of humanized mouse models in HIV research, offering crucial insights into the efficacy of cART and the ongoing pursuit of an HIV cure. RECENT FINDINGS: Here, we highlight studies investigating molecular mechanisms and pathogenesis related to HIV latency in humanized mice and discuss novel strategies for eradicating latent HIV. Emphasizing the importance of analytical cART interruption in humanized mouse studies to gauge its impact on the latent reservoir accurately, the review underlines the ongoing progress and challenges in harnessing humanized mouse models for HIV research. SUMMARY: This review suggests that humanized mice models provide valuable insights into HIV latency and potential eradication strategies, contributing significantly to the quest for an HIV cure.

The Humanized Mouse Model: What Added Value Does It Offer for HIV Research?

In the early 2000s, novel humanized mouse models based on the transplantation of human hematopoietic stem and progenitor cells (HSPCs) into immunocompromised mice were introduced (hu mice). The human HSPCs gave rise to a lymphoid system of human origin. The HIV research community has greatly benefitted from these hu mice. Since human immunodeficiency virus (HIV) type 1 infection results in a high-titer disseminated HIV infection, hu mice have been of great value for all types of HIV research from pathogenesis to novel therapies. Since the first description of this new generation of hu mice, great efforts have been expended to improve humanization by creating other immunodeficient mouse models or supplementing mice with human transgenes to improve human engraftment. Many labs have their own customized hu mouse models, making comparisons quite difficult. Here, we discuss the different hu mouse models in the context of specific research questions in order to define which characteristics should be considered when determining which hu mouse model is appropriate for the question posed. We strongly believe that researchers must first define their research question and then determine whether a hu mouse model exists, allowing the research question to be studied.

RNA with chemotherapeutic base analogues as a dual-functional anti-cancer drug.

Nanoparticles of different sizes formulated with unmodified RNA and Protamine differentially engage Toll-like Receptors (TLRs) and activate innate immune responses in vitro. Here, we report that similar differential immunostimulation that depends on the nanoparticle sizes is induced in vivo in wild type as well as in humanized mice. In addition, we found that the schedule of injections strongly affects the magnitude of the immune response. Immunostimulating 130 nm nanoparticles composed of RNA and Protamine can promote lung metastasis clearance but provides no control of subcutaneous tumors in a CT26 tumor model. We further enhanced the therapeutic capacity of Protamine-RNA nanoparticles by incorporating chemotherapeutic base analogues in the RNA; we coined these immunochemotherapeutic RNAs (icRNAs). Protamine-icRNA nanoparticles were successful at controlling established subcutaneous CT26 and B16 tumors as well as orthotopic glioblastoma. These data indicate that icRNAs are promising cancer therapies, which warrants their further validation for use in the clinic.

cART Restores Transient Responsiveness to IFN Type 1 in HIV-Infected Humanized Mice.

The lack of a human immunodeficiency virus (HIV) cure has heightened interest in immunotherapy. As such, type I interferons (IFNs), in particular, IFN alpha (IFN-α), have gained renewed attention. However, HIV pathogenesis is driven by sustained IFN-mediated immune activation, and the use of IFNs is rather controversial. The following questions therein remain: (i) which IFN-α subtype to use, (ii) at which regimen, and (iii) at what time point in HIV infection it might be beneficial. Here, we used IFN-α14 modified by PASylation for its long half-life in vivo to eventually treat HIV infection. We defined the IFN dosing regimen based on the maximum increase in interferon-stimulated gene (ISG) expression 6 h after its administration and a return to baseline of ubiquitin-specific protease 18 (USP18) prior to the next dose. Notably, USP18 is the major negative regulator of type I IFN signaling. HIV infection resulted in increased ISG expression levels in humanized mice. Intriguingly, high baseline ISG levels correlated with lower HIV load. No effect was observed on HIV replication when PASylated IFN-α14 was administered in the chronic phase. However, combined antiretroviral therapy (cART) restored responsiveness to IFN, and PASylated IFN-α14 administered during analytical cART interruption resulted in a transiently lower HIV burden than in the mock-treated mice. In conclusion, cART-mediated HIV suppression restored transient IFN responsiveness and provided a potential window for immunoenhancing therapies in the context of analytical cART interruption. IMPORTANCE cART is highly efficient in suppressing HIV replication in HIV-infected patients and has resulted in a dramatic reduction in morbidity and mortality in HIV-infected people, yet it does not cure HIV infection. In addition, cART has several disadvantages. Thus, the HIV research community is exploring novel ways to control HIV infection for longer periods without cART. Here, we explored novel, long-acting IFN-α14 for its efficacy to control HIV replication in HIV-infected humanized mice. We found that IFN-α14 had no effect on chronic HIV infection. However, when mice were treated first with cART, we observed a transiently restored responsiveness to INF and a transiently lower HIV burden after stopping cART. These data emphasize (i) the value of cART-mediated HIV suppression and immune reconstitution in creating a window of opportunity for exploring novel immunotherapies, (ii) the potential of IFNs for constraining HIV, and (iii) the value of humanized mice for exploring novel immunotherapies.

Fetal-Maternal Surgery for Spina Bifida in a HIV-Infected Mother.

INTRODUCTION: In select cases, in utero surgery for myelomeningocele (MMC) leads to better outcomes than postnatal repair. However, maternal HIV infection constitutes a formal exclusion criterion due to the potential of vertical HIV transmission. Encouraged by a previous case of a successful fetal spina bifida repair in a Hepatitis Bs antigen-positive woman, a plan was devised allowing for fetal surgery. CASE REPORT: In utero MMC repair was performed although the mother was HIV-infected. To minimize the risk of in utero HIV transmission, the mother was treated by highly active antiretroviral therapy throughout gestation as well as intravenous zi-dovudine administration during maternal-fetal surgery. The mother tolerated all procedures very well without any sequelae. The currently 20 month-old toddler is HIV negative and has significantly benefitted from fetal surgery. DISCUSSION/CONCLUSION: This case shows that maternal HIV is not a priori a diagnosis that excludes fetal surgery. Rather, it might be a surrogate for moving towards personalized medicine and away from applying too rigorous exclusion criteria in the selection of candidates for maternal-fetal surgery.

Immunophenotypic characterization of TCR γδ T cells and MAIT cells in HIV-infected individuals developing Hodgkin's lymphoma.

BACKGROUND: Despite successful combined antiretroviral therapy (cART), the risk of non-AIDS defining cancers (NADCs) remains higher for HIV-infected individuals than the general population. The reason for this increase is highly disputed. Here, we hypothesized that T-cell receptor (TCR) γδ cells and/or mucosal-associated invariant T (MAIT) cells might be associated with the increased risk of NADCs. γδ T cells and MAIT cells both serve as a link between the adaptive and the innate immune system, and also to exert direct anti-viral and anti-tumor activity. METHODS: We performed a longitudinal phenotypic characterization of TCR γδ cells and MAIT cells in HIV-infected individuals developing Hodgkin's lymphoma (HL), the most common type of NADCs. Cryopreserved PBMCs of HIV-infected individuals developing HL, matched HIV-infected controls without (w/o) HL and healthy controls were used for immunophenotyping by polychromatic flow cytometry, including markers for activation, exhaustion and chemokine receptors. RESULTS: We identified significant differences in the CD4+ T cell count between HIV-infected individuals developing HL and HIV-infected matched controls within 1 year before cancer diagnosis. We observed substantial differences in the cellular phenotype mainly between healthy controls and HIV infection irrespective of HL. A number of markers tended to be different in Vδ1 and MAIT cells in HIV+HL+ patients vs. HIV+ w/o HL patients; notably, we observed significant differences for the expression of CCR5, CCR6 and CD16 between these two groups of HIV+ patients. CONCLUSION: TCR Vδ1 and MAIT cells in HIV-infected individuals developing HL show subtle phenotypical differences as compared to the ones in HIV-infected controls, which may go along with functional impairment and thereby may be less efficient in detecting and eliminating malignant cells. Further, our results support the potential of longitudinal CD4+ T cell count analysis for the identification of patients at higher risk to develop HL.

The ADAM17-directed Inhibitory Antibody MEDI3622 Antagonizes Radiotherapy-induced VEGF Release and Sensitizes Non-Small Cell Lung Cancer for Radiotherapy.

The cellular response to ionizing radiation (IR) depends on tumor cell and microenvironmental factors. Here, we investigated the role of IR-induced ADAM17 matrix metalloproteinase activity for the intercellular communication between tumor cells and the tumor vasculature in non-small cell lung cancer (NSCLC) tumor models. Factors shed by ADAM17 from NSCLC tumor cells (A549, H358) and relevant for endothelial cell migration were investigated using transwell migration assays, ELISA, and flow cytometry. Tumor angiogenesis-related endpoints were analyzed with the chorio-allantoic membrane assay and in murine NSCLC tumor models. Efficacy-oriented experiments were performed in a murine orthotopic NSCLC tumor model using irradiation with an image-guided small-animal radiotherapy platform alone and in combination with the novel ADAM17-directed antibody MEDI3622. In vitro, VEGF was identified as the major factor responsible for IR-induced and ADAM17-dependent endothelial cell migration toward attracting tumor cells. IR strongly enhanced tumor cell-associated ADAM17 activity, released VEGF in an ADAM17-dependent manner, and thereby coordinated the communication between tumor and endothelial cells. In vivo, tumor growth and microvessel size and density were strongly decreased in response to the combined treatment modality of IR and MEDI3622 but not by either treatment modality alone and thus suggest that the supra-additive effect of the combined treatment modality is in part due to abrogation of the ADAM17-mediated IR-induced protective effect on the tumor vasculature. Furthermore, we demonstrate that the novel ADAM17-inhibitory antibody MEDI3622 potently improves the radiotherapy response of NSCLC. Significance: The tumor response to radiotherapy is influenced by several factors of the tumor microenvironment. We demonstrate that inhibition of the sheddase ADAM17 by the novel antibody MEDI3622 reduces IR-induced VEGF release from tumor cells relevant for endothelial cell migration and vasculature protection, thereby enhancing radiotherapy treatment outcome of NSCLC.

EBV renders B cells susceptible to HIV-1 in humanized mice.

HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combination, these viruses are linked to AIDS-associated lymphomas. We found that EBV, which transforms B cells, renders them susceptible to HIV-1 infection in a CXCR4 and CD4-dependent manner in vitro and that CXCR4-tropic HIV-1 integrates into the genome of these B cells with the same molecular profile as in autologous CD4+ T cells. In addition, we established a humanized mouse model to investigate the in vivo interactions of EBV and HIV-1 upon coinfection. The respective mice that reconstitute human immune system components upon transplantation with CD34+ human hematopoietic progenitor cells could recapitulate aspects of EBV and HIV immunobiology observed in dual-infected patients. Upon coinfection of humanized mice, EBV/HIV dual-infected B cells could be detected, but were susceptible to CD8+ T-cell-mediated immune control.

Polymorphisms of SOCS-1 Are Associated With a Rapid HIV Progression Rate.

OBJECTIVES: Immune activation, among others driven by interferon (IFN)-α and IFN-γ activation, is a main feature of progressive HIV infection. Suppressor of cytokine signaling (SOCS) 1 and 3 are negative feedback regulators of the IFN-α and IFN-γ axis. Here, we analyzed the role of 9 single-nucleotide polymorphisms (SNPs) within SOCS-1 and SOCS-3 genes for their association with an HIV progression rate in a cohort of 318 rapid vs 376 slow progressors from the Swiss HIV Cohort Study. DESIGN AND METHODS: We analyzed 9 SNPs, which we have identified in Swiss blood donors, in a cohort of HIV-infected patients (n = 1144), which have been categorized according to the decline in CD4 T-cell counts. In all the conducted analyses, we focused on the comparison between rapid and slow progressors with regard to SNPs in SOCS-1 and SOCS-3 and with regard to haplotypes using multivariate logistic regression models. RESULTS: Three SOCS-1 SNPs (rs193779, rs33989964, and rs4780355) are associated with a risk reduction for rapid progression. Two of these SNPs, rs33989964 and rs4780355, are in strong linkage disequilibrium, forming a frequent haplotype. Homozygous carriers of this haplotype are also associated with a risk reduction for rapid progression. By contrast, the minor TT genotype of rs33977706 is associated with twice the risk for rapid progression. No associations have been observed for the 4 SOCS-3 SNPs or the major SOCS-3 haplotypes. CONCLUSIONS: Our data suggest that SNPs in SOCS-1 are associated with HIV disease progression and speak in favor that immune activation is causal for the progressive immunodeficiency.

In utero Hepatitis B Immunization during Fetal Surgery for Spina Bifida.

BACKGROUND: Fetal surgery for spina bifida aperta may lead to significantly better outcomes than postnatal repair, particularly regarding shunt-dependent hydrocephalus, independent ambulation, and voiding functions. The "Management of Myelomeningocele Study" (MOMS) represents the current benchmark, also in terms of eligibility criteria. CASE REPORT: A positive maternal hepatitis B virus (HBV) status is a MOMS exclusion criterion. Here, we report on the first successful active and passive in utero HBV vaccination of a spina bifida fetus carried by a HBV-positive mother undergoing maternal-fetal surgery. The now 2-year-old infant is healthy, HBV negative, and drew maximal benefit from prenatal surgery. DISCUSSION AND CONCLUSION: Taken together, this patient benefitted maximally from fetal surgery for spina bifida, despite meeting an exclusion criterion. Thus, generally speaking, eligibility criteria for fetal surgery can be challenged under certain circumstances for the benefit of the patient.

Impact of Suboptimal APOBEC3G Neutralization on the Emergence of HIV Drug Resistance in Humanized Mice.

HIV diversification facilitates immune escape and complicates antiretroviral therapy. In this study, we take advantage of a humanized-mouse model to probe the contribution of APOBEC3 mutagenesis to viral evolution. Humanized mice were infected with isogenic HIV molecular clones (HIV-WT, HIV-45G, and HIV-ΔSLQ) that differ in their abilities to counteract APOBEC3G (A3G). Infected mice remained naive or were treated with the reverse transcriptase (RT) inhibitor lamivudine (3TC). Viremia, emergence of drug-resistant variants, and quasispecies diversification in the plasma compartment were determined throughout infection. While both HIV-WT and HIV-45G achieved robust infection, over time, HIV-45G replication was significantly reduced compared to that of HIV-WT in the absence of 3TC treatment. In contrast, treatment responses differed significantly between HIV-45G- and HIV-WT-infected mice. Antiretroviral treatment failed in 91% of HIV-45G-infected mice, while only 36% of HIV-WT-infected mice displayed a similar negative outcome. Emergence of 3TC-resistant variants and nucleotide diversity were determined by analyzing 155,462 single HIV reverse transcriptase gene (RT) and 6,985 vif sequences from 33 mice. Prior to treatment, variants with genotypic 3TC resistance (RT-M184I/V) were detected at low levels in over a third of all the animals. Upon treatment, the composition of the plasma quasispecies rapidly changed, leading to a majority of circulating viral variants encoding RT-184I. Interestingly, increased viral diversity prior to treatment initiation correlated with higher plasma viremia in HIV-45G-infected animals, but not in HIV-WT-infected animals. Taken together, HIV variants with suboptimal anti-A3G activity were attenuated in the absence of selection but displayed a fitness advantage in the presence of antiretroviral treatment.IMPORTANCE Both viral (e.g., RT) and host (e.g., A3G) factors can contribute to HIV sequence diversity. This study shows that suboptimal anti-A3G activity shapes viral fitness and drives viral evolution in the plasma compartment in humanized mice.

Efficient Human Cytomegalovirus Replication in Primary Endothelial Cells Is SOCS3 Dependent.

BACKGROUND: In immunocompromised patients, human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality. Suppressor of cytokine signaling (SOCS) proteins are very potent negative regulators of the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. We hypothesized that HCMV exploits SOCS1 and/or SOCS3 to its advantage. METHODS: All experiments were carried out with primary human lung-derived microvascular endothelial cells (HMVEC). SOCS1 and SOCS3 were silenced by transfecting the cells with siRNA. HCMV was propagated and titered on human lung-derived fibroblasts MRC5. Real-time PCR and Western blot were used to detect mRNA and protein levels, respectively. RESULTS: The data presented show that an efficient replication of HCMV in HMVEC is dependent on SOCS3 protein. Time course analysis revealed an increase in SOCS3 protein levels in infected cells. Silencing of SOCS3 (siSOCS3) resulted in inhibition of viral immediate early, early, and late antigen production. Consistently, HCMV titers produced by siSOCS3 cultures were significantly decreased when compared to control transfected cultures (siCNTRs). STAT1 and STAT2 phosphorylation was increased in siSOCS3-infected cells when compared to siCNTR-treated cells. CONCLUSION: These findings indicate the implication of SOCS3 in the mechanism of HCMV-mediated control of cellular immune responses.

Dose-Dependent Differences in HIV Inhibition by Different Interferon Alpha Subtypes While Having Overall Similar Biologic Effects.

Type I interferons (IFNs) are key players in the antiviral immune response. Interferon alpha (IFN-α) belongs to this class of IFNs and comprises 12 subtypes that differ from each other in their binding affinities for a common receptor and, thus, in their signaling potencies. Recent data suggest that IFN-α6 and -α14 are the most potent IFN-α subtypes in restricting HIV replication when applied exogenously. However, in the context of antiviral therapy, IFNs are administered at high doses, which may compensate for differences in potency seen between IFN-α subtypes. In this study, we reexamined whether IFN-α subtypes induce different biological activities, with a focus on how IFN-α treatment dose affects cellular responses to HIV in primary CD4+ T cells, peripheral blood mononuclear cells (PBMCs), and macrophages. We found that the subtypes' antiviral activities were dose dependent, with >90% inhibition of HIV replication at a high dose of all IFN-αs except the weak IFN-α/ß receptor (IFNAR) binder, IFN-α1. The quality of the responses engendered by IFN-α1, -α2, -α6, and -α14 was highly comparable, with essentially the same set of genes induced by all four subtypes. Hierarchal cluster analysis revealed that the individual donors were stronger determinants for the IFN-stimulated-gene (ISG) responses than the specific IFN-α subtype used for stimulation. Notably, IFN-α2-derived mutants with substantially reduced IFNAR2 binding still inhibited HIV replication efficiently, whereas mutants with increased IFNAR1 binding potentiated antiviral activity. Overall, our results support the idea that IFN-α subtypes do not induce different biological responses, given that each subtype is exogenously applied at bioequivalent doses.IMPORTANCE Elucidating the functional role of the IFN-α subtypes is of particular importance for the development of efficacious therapies using exogenous IFN-α. Specifically, this will help define whether IFN therapy should be based on the use of pathogen-dependent IFN subtypes or, rather, IFN mutants with optimized IFNAR binding properties.

Optimizing Synthetic miRNA Minigene Architecture for Efficient miRNA Hairpin Concatenation and Multi-target Gene Knockdown.

Synthetic microRNA (miRNA) minigenes (SMIGs) have a major potential for molecular therapy; however, their optimal architecture still needs to be determined. We have previously optimized the stem structure of miRNA hairpins for efficient gene knockdown. Here, we investigate the overall architecture of SMIGs driven by polymerase II-dependent promoters. When miRNA hairpins were placed directly behind the promoter, gene knockdown was inefficient as compared with constructs containing an intercalated sequence ("spacer"). Spacer sequence was relevant for knockdown efficiency and concatenation potential: GFP-based sequences (even when truncated or including stop codons) were particularly efficient. In contrast, a spacer of similar length based on a CD4 intronic sequence was entirely inefficient. Spacer sequences influenced miRNA steady-state levels without affecting transcript stability. We demonstrate that with an optimized spacer, up to five concatenated hairpins targeting two different genes are efficiently expressed and able to knock down their respective targets. Transplantation of hematopoietic stem cells containing a CCR5 knockdown SMIG demonstrated a sustained in vivo efficacy of our approach. In summary, we have defined features that optimize SMIG efficiency. Based on these results, optimized knockdown of genes of interest, such as the HIV co-receptor CCR5 and the NADPH oxidase subunit p22phox, was achieved.

Monitoring HIV DNA and cellular activation markers in HIV-infected humanized mice under cART.

BACKGROUND: The major obstacle to cure of HIV type-1 infection is the presence of the HIV reservoir, hidden from the immune system and insensitive to combined antiretroviral therapy (cART). Eradication approaches have been hindered by the difficulty for accurately monitoring its size in vivo, especially in the lymphoid organs. Humanized mouse models are a valuable tool for systematically assess the efficacy of therapeutic interventions in reducing the HIV reservoir. Nonetheless, persistence of the HIV reservoir over time, in the presence of cART, has yet to be analyzed in this in vivo model. FINDINGS: We found that the proviral DNA as well as the total DNA were very stable in the spleen and mesenteric lymph node irrespective of the length of cART. Notably, the amount of proviral DNA was very similar in the spleen and lymph node. Furthermore, we observed a correlation between the percentage of splenic human CD4+ T-cells with total HIV DNA, between the number of human CD38 + CD8+ T-cells in the spleen with the amount of integrated HIV DNA, and eventually between the hCD4/hCD8 ratio in the spleen with integrated as well as total HIV DNA implying that the CD8+ T cells influence the size of the HIV reservoir. CONCLUSIONS: Here, we demonstrated the stability of this reservoir in humanized mice irrespective of the length of cART, confirming the relevancy of this model for HIV latency eradication investigations. Notably, we also found correlates between the frequency of CD4+ T-cells, their activation status and viral parameters, which were analogous to the ones in HIV-infected patients. Thus, hu-mice represent a very valuable HIV latency model.

Antiviral Activity of HIV gp120-Targeting Bispecific T Cell Engager Antibody Constructs.

Today's gold standard in HIV therapy is combined antiretroviral therapy (cART). It requires strict adherence by patients and lifelong medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency but cannot cure patients. The bispecific T cell-engaging (BiTE) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. Here, we generated BiTE antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1 + 2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3ε scFv. These engineered human BiTE antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in peripheral blood mononuclear cells (PBMCs) as well as in macrophages cocultured with autologous CD8+ T cells, the most potent being the human CD4(1 + 2) BiTE [termed CD(1 + 2) h BiTE] antibody construct and the CD4(1 + 2)L17b BiTE antibody construct. The CD4(1 + 2) h BiTE antibody construct promoted HIV infection of human CD4-/CD8+ T cells. In contrast, the neutralizing B12 and the VRC01 BiTE antibody constructs, as well as the CD4(1 + 2)L17b BiTE antibody construct, did not. Thus, BiTE antibody constructs targeting HIV gp120 are very promising for constraining HIV and warrant further development as novel antiviral therapy with curative potential.IMPORTANCE HIV is a chronic infection well controlled with the current cART. However, we lack a cure for HIV, and the HIV pandemic goes on. Here, we showed in vitro and ex vivo that a BiTE antibody construct targeting HIV gp120 resulted in substantially reduced HIV replication. In addition, these BiTE antibody constructs display efficient killing of gp120-expressing cells and inhibited replication in ex vivo HIV-infected PBMCs or macrophages. We believe that BiTE antibody constructs recognizing HIV gp120 could be a very valuable strategy for a cure of HIV in combination with cART and compounds which reverse latency.

Long-term leukocyte reconstitution in NSG mice transplanted with human cord blood hematopoietic stem and progenitor cells.

BACKGROUND: Humanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor cells into immunodeficient mice and have become important pre-clinical models for biomedical research. However, data about their hematopoiesis over time are scarce. We therefore characterized leukocyte reconstitution in NSG mice, which were sublethally irradiated and transplanted with human cord blood-derived CD34+ cells at newborn age, longitudinally in peripheral blood and, for more detailed analyses, cross-sectionally in peripheral blood, spleen and bone marrow at different time points. RESULTS: Human cell chimerism and absolute human cell count decreased between week 16 and 24 in the peripheral blood of hu mice, but were stable thereafter as assessed up to 32 weeks. Human cell chimerism in spleen and bone marrow was maintained over time. Notably, human cell chimerism in peripheral blood and spleen as well as bone marrow positively correlated with each other. Percentage of B cells decreased between week 16 and 24, whereas percentage of T cells increased; subsequently, they levelled off with T cells clearly predominating at week 32. Natural killer cells, monocytes and plasmacytoid dendritic cells (DCs) as well as CD1c + and CD141+ myeloid DCs were all present in hu mice. Proliferative responses of splenic T cells to stimulation were preserved over time. Importantly, the percentage of more primitive hematopoietic stem cells (HSCs) in bone marrow was maintained over time. CONCLUSIONS: Overall, leukocyte reconstitution was maintained up to 32 weeks post-transplantation in our hu NSG model, possibly explained by the maintenance of HSCs in the bone marrow. Notably, we observed great variation in multi-lineage hematopoietic reconstitution in hu mice that needs to be taken into account for the experimental design with hu mice.

Impairment of CCR6+ and CXCR3+ Th Cell Migration in HIV-1 Infection Is Rescued by Modulating Actin Polymerization.

CD4+ T cell repopulation of the gut is rarely achieved in HIV-1-infected individuals who are receiving clinically effective antiretroviral therapy. Alterations in the integrity of the mucosal barrier have been indicated as a cause for chronic immune activation and disease progression. In this study, we present evidence that persistent immune activation causes impairment of lymphocytes to respond to chemotactic stimuli, thus preventing their trafficking from the blood stream to peripheral organs. CCR6+ and CXCR3+ Th cells accumulate in the blood of aviremic HIV-1-infected patients on long-term antiretroviral therapy, and their frequency in the circulation positively correlates to levels of soluble CD14 in plasma, a marker of chronic immune activation. Th cells show an impaired response to chemotactic stimuli both in humans and in the pathogenic model of SIV infection, and this defect is due to hyperactivation of cofilin and inefficient actin polymerization. Taking advantage of a murine model of chronic immune activation, we demonstrate that cytoskeleton remodeling, induced by okadaic acid, restores lymphocyte migration in response to chemokines, both in vitro and in vivo. This study calls for novel pharmacological approaches in those pathological conditions characterized by persistent immune activation and loss of trafficking of T cell subsets to niches that sustain their maturation and activities.

Similar efficacy of broad-range ITS PCR and conventional fungal culture for diagnosing fungal infections in non-immunocompromised patients.

BACKGROUND: Broad-range fungal inter spacer region (ITS) polymerase chain reaction (PCR) has been evaluated for the detection and identification of fungi in clinical specimens from severely immunocompromised patients, but not in non-selected patients. Thus, the aim of this study was to compare the diagnostic performance of ITS PCR with that of fungal culture and to investigate its clinical impact on the diagnosis of fungal infections in non-immunocompromised patients. The corresponding patients' data were retrieved by detailed medical chart reviews. RESULTS: Results from 251 specimens showed a high concordance of 89.6 % for ITS PCR and fungal culture. The analytical sensitivity and specificity of ITS PCR considering culture as gold standard were 87.7 and 90.3 %, respectively, the positive and negative predictive value (PPV and NPV) were 76 and 95.5 %, respectively. Assessing the clinical probability of a fungal infection based on detailed chart reviews, PCR had a clinical sensitivity of 88.9 %, a specificity of 86.3 %, a PPV of 64.0 % and a NPV of 96.6 %. The overall performance of fungal broad-range PCR was similar to that of culture. CONCLUSIONS: Our data show that, in non-selected and non-immunocompromised patients, the performance of ITS PCR is similar to that of culture for detecting fungal infections, not the least because sensitivity of culture in patients under antifungal treatment is surprisingly high. Compared to culture, PCR has the advantage of a rapid time-to-result (approximately two working days), proper identification of rare pathogens, prompt initiation of a species-targeted antifungal treatment, and prospects for automation.