Jose M. Rivera

Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans.

Lymphocytes Layer It On Cells of the immune system begin to develop from hematopoietic stem cells (HSCs) during fetal life. In the adult, HSCs continue to produce immune cells to replenish dying cells or in response to an infection. In mice and birds, immune cell development occurs in a “layered” manner, whereby distinct populations of HSCs that arise at different times during development generate distinct immune cell lineages. In contrast, development of human immune cells, and T lymphocytes in particular, is thought to be linear. Mold et al. (p. 1695; see the Perspective by Betz) now show that T lymphocyte development in humans is also “layered,” and strategically so. T cells that arise from fetal HSCs are enriched in regulatory T cells, which promote immune tolerance, rather than classical T cells, which readily respond to foreign antigen. By favoring the development of regulatory T cell populations during fetal life, the immune system is perhaps better able to keep responses to maternal antigens in check. The development of large numbers of classical T cells is delayed until after birth when infectious agents represent a more imminent threat. Distinct fetal T cell lineages help explain the tolerogenic properties of the fetus and immune responsiveness at birth. Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or “layering”) of distinct hematopoietic stem cells (HSCs) that give rise to distinct lymphocyte lineages at different stages of development. Here we provide evidence of an analogous layered immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSCs that are present at different stages of development. We also provide evidence that the fetal T cell lineage is biased toward immune tolerance. These observations offer a mechanistic explanation for the tolerogenic properties of the developing fetus and for variable degrees of immune responsiveness at birth.

Superior human leukocyte reconstitution and susceptibility to vaginal HIV transmission in humanized NOD-scid IL-2Rγ(-/-) (NSG) BLT mice.

Humanized Bone marrow/Liver/Thymus (BLT) mice recapitulate the mucosal transmission of HIV, permitting study of early events in HIV pathogenesis and evaluation of preexposure prophylaxis methods to inhibit HIV transmission. Human hematopoiesis is reconstituted in NOD-scid mice by implantation of human fetal liver and thymus tissue to generate human T cells plus intravenous injection of autologous liver-derived CD34(+) hematopoietic stem cells to engraft the mouse bone marrow. In side-by-side comparisons, we show that NOD-scid mice homozygous for a deletion of the IL-2Rγ-chain (NOD-scid IL-2Rγ(-/-)) are far superior to NOD-scid mice in both their peripheral blood reconstitution with multiple classes of human leukocytes (e.g., a mean of 182 versus 14 CD4(+) T cells per μl 12 weeks after CD34(+) injection) and their susceptibility to intravaginal HIV exposure (84% versus 11% viremic mice at 4 weeks). These results should speed efforts to obtain preclinical animal efficacy data for new HIV drugs and microbicides.

Validation of the SCID-hu Thy/Liv mouse model with four classes of licensed antiretrovirals.

Background The SCID-hu Thy/Liv mouse model of HIV-1 infection is a useful platform for the preclinical evaluation of antiviral efficacy in vivo. We performed this study to validate the model with representatives of all four classes of licensed antiretrovirals. Methodology/Principal Findings Endpoint analyses for quantification of Thy/Liv implant viral load included ELISA for cell-associated p24, branched DNA assay for HIV-1 RNA, and detection of infected thymocytes by intracellular staining for Gag-p24. Antiviral protection from HIV-1-mediated thymocyte depletion was assessed by multicolor flow cytometric analysis of thymocyte subpopulations based on surface expression of CD3, CD4, and CD8. These mice can be productively infected with molecular clones of HIV-1 (e.g., the X4 clone NL4-3) as well as with primary R5 and R5X4 isolates. To determine whether results in this model are concordant with those found in humans, we performed direct comparisons of two drugs in the same class, each of which has known potency and dosing levels in humans. Here we show that second-generation antiretrovirals were, as expected, more potent than their first-generation predecessors: emtricitabine was more potent than lamivudine, efavirenz was more potent than nevirapine, and atazanavir was more potent than indinavir. After interspecies pharmacodynamic scaling, the dose ranges found to inhibit viral replication in the SCID-hu Thy/Liv mouse were similar to those used in humans. Moreover, HIV-1 replication in these mice was genetically stable; treatment of the mice with lamivudine did not result in the M184V substitution in reverse transcriptase, and the multidrug-resistant NY index case HIV-1 retained its drug-resistance substitutions. Conclusion Given the fidelity of such comparisons, we conclude that this highly reproducible mouse model is likely to predict clinical antiviral efficacy in humans.

Oral Administration of the Nucleoside EFdA (4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine) Provides Rapid Suppression of HIV Viremia in Humanized Mice and Favorable Pharmacokinetic Properties in Mice and the Rhesus Macaque

ABSTRACT Like normal cellular nucleosides, the nucleoside reverse transcriptase (RT) inhibitor (NRTI) 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) has a 3′-hydroxyl moiety, and yet EFdA is a highly potent inhibitor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication with activity against a broad range of clinically important drug-resistant HIV isolates. We evaluated the anti-HIV activity of EFdA in primary human cells and in HIV-infected humanized mice. EFdA exhibited excellent potency against HIVJR-CSF in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), with a 50% inhibitory concentration of 0.25 nM and a selectivity index of 184,000; similar antiviral potency was found against 12 different HIV clinical isolates from multiple clades (A, B, C, D, and CRF01_AE). EFdA was readily absorbed after oral dosing (5 mg/kg of body weight) in both mice and the rhesus macaque, with micromolar levels of the maximum concentration of drug in serum (Cmax) attained at 30 min and 90 min, respectively. Trough levels were at or above 90% inhibitory concentration (IC90) levels in the macaque at 24 h, suggesting once-daily dosing. EFdA showed reasonable penetration of the blood-brain barrier in the rhesus macaque, with cerebrospinal fluid levels at approximately 25% of plasma levels 8 h after single oral dosing. Rhesus PBMCs isolated 24 h following a single oral dose of 5 mg/kg EFdA were refractory to SIV infection due to sufficiently high intracellular EFdA-triphosphate levels. The intracellular half-life of EFdA-triphosphate in PBMCs was determined to be >72 h following a single exposure to EFdA. Daily oral administration of EFdA at low dosage levels (1 to 10 mg/kg/day) was highly effective in protecting humanized mice from HIV infection, and 10 mg/kg/day oral EFdA completely suppressed HIV RNA to undetectable levels within 2 weeks of treatment.

Cytomegalovirus Impairs Cytotrophoblast-Induced Lymphangiogenesis and Vascular Remodeling in an in Vivo Human Placentation Model

We investigated human cytomegalovirus pathogenesis by comparing infection with the low-passage, endotheliotropic strain VR1814 and the attenuated laboratory strain AD169 in human placental villi as explants in vitro and xenografts transplanted into kidney capsules of SCID mice (ie, mice with severe combined immunodeficiency). In this in vivo human placentation model, human cytotrophoblasts invade the renal parenchyma, remodel resident arteries, and induce a robust lymphangiogenic response. VR1814 replicated in villous and cell column cytotrophoblasts and reduced formation of anchoring villi in vitro. In xenografts, infected cytotrophoblasts had a severely diminished capacity to invade and remodel resident arteries. Infiltrating lymphatic endothelial cells proliferated, aggregated, and failed to form lymphatic vessels. In contrast, AD169 grew poorly in cytotrophoblasts in explants, and anchoring villi formed normally in vitro. Likewise, viral replication was impaired in xenografts, and cytotrophoblasts retained invasive capacity, but some partially remodeled blood vessels incorporated lymphatic endothelial cells and were permeable to blood. The expression of both vascular endothelial growth factor (VEGF)-C and basic fibroblast growth factor increased in VR1814-infected explants, whereas VEGF-A and soluble VEGF receptor-3 increased in those infected with AD169. Our results suggest that viral replication and paracrine factors could undermine vascular remodeling and cytotrophoblast-induced lymphangiogenesis, contributing to bleeding, hypoxia, and edema in pregnancies complicated by congenital human cytomegalovirus infection.

Identification of Essential Sequences for Cellular Localization in BRMS1 Metastasis Suppressor

Background Breast cancer metastasis suppressor 1 (BRMS1) reduces the number and the size of secondary tumours in a mouse model without affecting the growth of the primary foci upon its re-expression. Knockdown of BRMS1 expression associates with metastasis. The molecular details on BRMS1 mechanism of action include its ability to function as a transcriptional co-repressor and consistently BRMS1 has been described as a predominantly nuclear protein. Since cellular distribution could represent a potential mechanism of regulation, we wanted to characterize BRMS1 sequence motifs that might regulate its cellular distribution. According to its amino acids sequence, BRMS1 contain two putative nuclear localization signals, however none of them has been proved to work so far. Methodology/Principal Findings By using well known in vivo assays to detect both nuclear import and export signal, we have characterized, in the present study, one functional nuclear localisation signal as necessary and sufficient to promote nuclear transport. Additionally, the outcome of a directed yeast two-hybrid assay identify importin α6 as a specific partner of BRMS1 thus speculating that BRMS1 nuclear import could be specifically mediated by the reported nuclear transporter. Besides, the combination of a computational searching approach along the utilization of a nuclear export assay, identified a functional motif within the BRMS1 sequence responsible for its nuclear export, that resulted not affected by the highly specific CRM1 inhibitor Leptomycin-B. Interspecies heterokaryon assay demonstrate the capability of BRMS1 to shuttle between the nuclear and cytosolic compartments Conclusions/Significance Our results show for the first time that BRMS1 contains both nuclear import and export signals enabling its nucleo-cytoplasmic shuttling. These findings contributes new data for the understanding of the BRMS1 functions and allow us to speculate that this phenomenon could represent a novel mechanism for regulating the activity of BRMS1 or its associated cytosolic partners

Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction

Although invasive cytomegalovirus (CMV) disease is uncommon in the era of antiretroviral therapy (ART), asymptomatic CMV coinfection is nearly ubiquitous in HIV infected individuals. While microbial translocation and gut epithelial barrier dysfunction may promote persistent immune activation in treated HIV infection, potentially contributing to morbidity and mortality, it has been unclear whether CMV replication in individuals with no symptoms of CMV disease might play a role in this process. We hypothesized that persistent CMV replication in the intestinal epithelium of HIV/CMV-coinfected individuals impairs gut epithelial barrier function. Using a combination of state-of-the-art in situ hybridization technology (RNAscope) and immunohistochemistry, we detected CMV DNA and proteins and evidence of intestinal damage in rectosigmoid samples from CMV-positive individuals with both untreated and ART-suppressed HIV infection. Two different model systems, primary human intestinal cells differentiated in vitro to form polarized monolayers and a humanized mouse model of human gut, together demonstrated that intestinal epithelial cells are fully permissive to CMV replication. Independent of HIV, CMV disrupted tight junctions of polarized intestinal cells, significantly reducing transepithelial electrical resistance, a measure of monolayer integrity, and enhancing transepithelial permeability. The effect of CMV infection on the intestinal epithelium is mediated, at least in part, by the CMV-induced proinflammatory cytokine IL-6. Furthermore, letermovir, a novel anti-CMV drug, dampened the effects of CMV on the epithelium. Together, our data strongly suggest that CMV can disrupt epithelial junctions, leading to bacterial translocation and chronic inflammation in the gut and that CMV could serve as a target for therapeutic intervention to prevent or treat gut epithelial barrier dysfunction during HIV infection.

Efficacy of Broadly Neutralizing Monoclonal Antibody PG16 in HIV-infected Humanized Mice

Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clades A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70-80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV inoculation. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier.

BRMS151-98 and BRMS151-84 are crystal oligomeric coiled coils with different oligomerization states, which behave as disordered protein fragments in solution.

The breast cancer metastasis suppressor 1 (BRMS1) gene suppresses metastasis without affecting the primary tumor growth. Cellular localization of BRMS1 appears to be important for exerting its effects on metastasis inhibition. We recently described a nucleo-cytoplasmic shuttling for BRMS1 and identified a nuclear export signal within the N-terminal coiled coil. The structure of these regions shows an antiparallel coiled coil capable of oligomerizing, which compromises the accessibility to the nuclear export signal consensus residues. We have studied the structural and biophysical features of this region to further understand the contribution of the N-terminal coiled coil to the biological function of BRMS1. We have observed that residues 85 to 98 might be important in defining the oligomerization state of the BRMS1 N-terminal coiled coil. The fragments are mainly disordered in solution, with evidence of residual structure. In addition, we report the presence of a conformational dynamic equilibrium (oligomeric folded species ↔ oligomeric unfolded) in solution in the BRMS1 N-terminal coiled coil that might facilitate the nuclear export of BRMS1 to the cytoplasm.

Comprehensive comparison of neonate and adult human platelet transcriptomes

Understanding the underlying mechanisms of the well-substantiated platelet hyporeactivity in neonates is of interest given their implications for the clinical management of newborns, a population at higher bleeding risk than adults (especially sick and preterm infants), as well as for gaining insight into the regulatory mechanisms of platelet biology. Transcriptome analysis is useful in identifying mRNA signatures affecting platelet function. However, human fetal/neonatal platelet transcriptome analysis has never before been reported. We have used mRNA expression array for the first time to compare platelet transcriptome changes during development. Microarray analysis was performed in pure platelet RNA obtained from adult and cord blood, using the same platform in two independent laboratories. A high correlation was obtained between array results for both adult and neonate platelet samples. There was also good agreement between results in our adult samples and outcomes previously reported in three different studies. Gene enrichment analysis showed that immunity- and platelet function-related genes are highly expressed at both developmental stages. Remarkably, 201 genes were found to be differentially expressed throughout development. In particular, neonatal platelets contain higher levels of mRNA that are associated with protein synthesis and processing, while carrying significantly lower levels of genes involved in calcium transport/metabolism and cell signaling (including GNAZ). Overall, our results point to variations in platelet transcriptome as possibly underlining the hypo-functional phenotype of neonatal platelets and provide further support for the role of platelets in cellular immune response. Better characterization of the platelet transcriptome throughout development can contribute to elucidate how transcriptome changes impact different pathological conditions.