Probes for INS

Worldwide, nearly two million children are infected with HIV, with breastfeeding accounting for the majority of contemporary HIV transmissions. Antiretroviral therapy (ART) has reduced HIV-related morbidity and mortality but is not curative. The main barrier to a cure is persistence of latent HIV in long-lived reservoirs. However, our understanding of the cellular and anatomic sources of the HIV reservoir during infancy and childhood is limited. Here, we developed a pediatric model of ART suppression in orally SIV-infected rhesus macaque (RM) infants, with measurement of virus persistence in blood and tissues after 6-9 months of ART. Cross-sectional analyses were conducted to compare SIV RNA and DNA levels in adult and infant RMs naïve to treatment and on ART. We demonstrate efficient viral suppression following ART initiation in SIV-infected RM infants with sustained undetectable plasma viral loads in the setting of heterogeneous penetration of ART into lymphoid and gastrointestinal tissues and low drug levels in the brain. We further show reduction in SIV RNA and DNA on ART in lymphoid tissues of both infant and adult RMs, but stable (albeit low) levels of SIV RNA and DNA in the brains of viremic and ART-suppressed infants. Finally, we report a large contribution of naïve CD4+ T-cells to the total CD4 reservoir of SIV in blood and lymph nodes of ART-suppressed RM infants, that differs from what we show in adults. These results reveal important aspects of HIV/SIV persistence in infants and provide insight into strategic targets for cure interventions in a pediatric population.IMPORTANCE While antiretroviral therapy (ART) can reduce HIV replication, the virus cannot be eradicated from an infected individual and our incomplete understanding of HIV persistence in reservoirs greatly complicates the generation of a cure for HIV. Given the immaturity of the infant immune system, it is of critical importance to study HIV reservoirs specifically in this population. Here, we established a pediatric animal model to simulate breastfeeding transmission and study SIV reservoirs in rhesus macaques (RM) infants. Our study demonstrates that ART can be safely administered to infant RM for prolonged periods of time and efficiently controls viral replication in this model. SIV persistence was shown in blood and tissues with a similar anatomic distribution of SIV reservoirs in infant and adult RMs. However, in the peripheral blood and lymph nodes, a higher contribution of the naïve CD4+ T-cells to the SIV reservoir was observed in infants compared to adults.

African green monkeys (AGMs) are a natural host of SIV that do not develop simian AIDS. Adult AGMs naturally have low numbers of CD4+T cells and a large population of MHC class II-restricted CD8αα T cells that are generated through CD4 downregulation in CD4+ T cells. In this article, we study the functional profiles and SIV infection status in vivo of CD4+ T cells, CD8αα T cells, and CD8αβ T cells in lymph nodes, peripheral blood, and bronchoalveolar lavage fluid of AGMs and rhesus macaques (in which CD4 downregulation is not observed). We show that, although CD8αα T cells in AGMs maintain functions associated with CD4+ T cells (including Th follicular functionality in lymphoid tissues and Th2 responses in bronchoalveolar lavage fluid), they also accumulate functions normally attributed to canonical CD8+ T cells. These hyperfunctional CD8αα T cells are found to circulate peripherally, as well as reside within the lymphoid tissue. Due to their unique combination of CD4 and CD8 T cell effector functions, these CD4- CD8αα T cells are likely able to serve as an immunophenotype capable of Th1, follicular Th, and CTL functionalities, yet they are unable to be infected by SIV. These data demonstrate the ambiguity of CD4/CD8 expression in dictating the functional capacities of T cells and suggest that accumulation of hyperfunctional CD8αα T cells in AGMs may lead to tissue-specific antiviral immune responses in lymphoid follicles that limit SIV replication in this particular anatomical niche.

Abstract

Purpose

High-risk human papillomavirus (HR-HPV) infections was the causal factor in the development of cervical cancer, but the significance of HPV viral load in the prediction of the response to current therapeutic approaches had not reached consensus. The present study was performed to assess the high risk HPV viral load of cervical cancer patients who underwent radiotherapy alone or in combination with chemotherapy or hyperthermotherapy or both in correlation to long-term survival.

Methods

116 cervical cancer patients were recruited and assigned into four groups of different therapeutic modalities. The prevalent high risk types of HPV 16, 18, 58 were detected by type specific in situ hybridization (ISH), and HPV mRNA was detected by RNA scope assay using RNA scope 2.0 FFPE Reagent Kit. Semi-quantification of the HR-HPV viral load was measured based on the intensity of ISH signal captured from the tumor nests in the grey scale.

Results

The HR-HPV viral load had a significant negative correlation with survival (r_s = −0.368,P = 0.001). The 15-year survival rate of low viral load group was 68.18%, moderate viral load group was 52.17%, and high viral load group was 34.69% (P = 0.001). HPV mRNA expression was strongly consistent with HPV viral load. The 15-year survival rates of different therapeutic groups were 39.29%, 58.62%, 50.00%, 55.17%, respectively (P = 0.545). Combinatorial treatment modalities improved the actual survival, which demonstrated no significant difference among 5,10 and 15 years comparison. Cox regression analysis showed that the relative risk of death was obviously higher in the HPV 18 single positive group and high HPV viral load group.

Conclusions

The semi-quantitive viral load assessment in situ is a feasible approach in clinical practice. The more the HPV viral load was, the worse the survival of patients would be. The combinational treatments were in favor of the disease-stabilization.

Human papillomavirus (HPV) infection modulates several host cytokines contributing to cancer development. Oncostatin M (OSM), an IL-6 family cytokine, acts to promote cell senescence and inhibit growth. Its dysregulation promotes cell survival, cell proliferation and metastasis in various malignancies. The effect of HPV on OSM dysregulation has not been investigated. To elucidate this, immunohistochemistry was used on formalin-fixed, paraffin-embedded oral squamous cell carcinoma (OSCC) tissues: HPV-positive (50) and HPV-negative (50) cases. Immortalized human cervical keratinocytes expressing HPV16E6 (HCK1T, Tet-On system) were used to demonstrate the role of HPV16E6 in OSM expression. In addition, a vector containing HPV16E6/E7 was transiently transfected into oral cancer cell lines. Cell viability, cell-cycle progression and cell migration were evaluated using flow cytometry and a wound healing assay, respectively. The results showed various intensities of OSM expression in OSCC. Interestingly, the median percentages of strongly stained cells were significantly higher in HPV-positive OSCCs than in HPV-negative OSCCs. To explore the role of HPV oncoproteins on OSM expression, the expression of HPV16E6 in the HCK1T Tet-On condition was induced by doxycycline and HPV16E6 was found to significantly upregulate levels of OSM mRNA and protein, with concomitant upregulation of c-Myc. In addition, the levels of OSM mRNA and protein in E6/E7 transiently transfected oral cancer cells also gradually increased in a time-dependent manner and these transfected cells showed greater viability and higher migration rates and cell-cycle progression than controls. This result demonstrates that HPV16 oncoproteins upregulate OSM and play an important role to promote OSCC development.

Persistent high-risk human papillomavirus (HPV) infection is strongly associated with development of high-grade cervical intraepithelial neoplasia or cancer (CIN3+). In single type infections serial type-specific viral-load measurements predict the natural history of the infection. In infections with multiple HPV-types, the individual type-specific viral-load profile could distinguish progressing HPV-infections from regressing infections. A case-cohort natural history study was established using samples from untreated women with multiple HPV-infections who developed CIN3+ (n=57) or cleared infections (n=88). Enriched cell pellet from liquid based cytology samples were subjected to a clinically validated real-time qPCR-assay (18 HPV-types). Using serial type-specific viral-load measurements (≥3) we calculated HPV-specific slopes and coefficient of determination (R2 ) by linear regression. For each woman slopes and R2 were used to calculate which HPV-induced processes were ongoing (progression, regression, serial transient, transient). In transient infections with multiple HPV-types, each single HPV-type generated similar increasing (0.27copies/cell/day) and decreasing (-0.27copies/cell/day) viral-load slopes. In CIN3+ at least one of the HPV-types had a clonal progressive course (R2 ≥0.85;0.0025copies/cell/day). In selected CIN3+ cases (n=6) immunostaining detecting type-specific HPV 16,31,33,58 and 67 RNA showed an even staining in clonal populations (CIN3+), whereas in transient virion-producing infections the RNA-staining was less in the basal layer compared to the upper layer where cells were ready to desquamate and release newly-formed virions. RNA-hybridization patterns matched the calculated ongoing processes measured by R2 and slope in serial type-specific viral-load measurements preceding the biopsy. In women with multiple HPV-types, serial type-specific viral-load measurements predict the natural history of the different HPV-types, and elucidates HPV-genotype attribution. 

CD4+ T cells are the primary HIV-1 target cell, with the vast majority of these cells residing within lymphoid tissue compartments throughout the body. Predictably, HIV-1 infection, replication, localization, reservoir establishment and persistence, as well as associated host immune and inflammatory responses and disease pathology principally take place within the tissues of the immune system. By virture of the fact that the virus-host struggle is played out within lymphoid and additional tissues compartments in HIV-1 infected individuals it is critical to understand HIV-1 infection and disease within these relevant tissue sites; however, there are obvious limitations to studying these dynamic processes in humans. Nonhuman primate (NHP) research has provided a vital bridge between basic and preclinical research and clinical studies, with experimental SIV infection of NHP models offering unique opportunities to understand key processes of HIV-1 infection and disease that are either not practically feasible or ethical in HIV-1 infected humans. In this review we will discuss current approaches to studying the tissue based immunopathogenesis of AIDS virus infection in NHPs, including both analyses of tissues obtained at biopsy or necropsy and complementary non-invasive imaging approaches that may have practical utility in monitoring HIV-1 disease in the clinical setting.

Abstract