Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection.

BACKGROUND: SARS-CoV-2 is associated with an increased risk of venous and arterial thrombosis, but the underlying mechanism is still unclear. METHODS: We performed a cross-sectional analysis of platelet function in 25 SARS-CoV-2 and 10 healthy subjects by measuring Nox2 (NADPH oxidase 2)-derived oxidative stress and thromboxane B2, and investigated if administration of monoclonal antibodies against the S protein (Spike protein) of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the S protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation. RESULTS: Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and thromboxane B2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared with controls; both effects were lowered by Nox2 and TLR4 (Toll-like receptor 4) inhibitors. Two hours after administration of monoclonal antibodies, a significant inhibition of platelet activation was observed in patients with SARS-CoV-2 compared with untreated ones. In vitro study showed that S protein per se did not elicit platelet activation but amplified the platelet response to subthreshold concentrations of agonists and functionally interacted with platelet TLR4. A docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains; furthermore, immunoprecipitation and immunofluorescence showed S protein-TLR4 colocalization in platelets from SARS-CoV-2. Plasma from patients with SARS-CoV-2 enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF (tumor necrosis factor) α inhibitor; this effect was recapitulated by an in vitro study documenting that TNFα alone promoted platelet activation and amplified the platelet response to S protein via p47phox (phagocyte oxidase) upregulation. CONCLUSIONS: The study identifies 2 TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4. or p47phox as a tool to counteract thrombosis in SARS-CoV-2.

TTV and CMV viral load dynamics: Which emerges first during immunosuppression?

Novel biomarkers reflecting the degree of immunosuppression in transplant patients are required to ensure eventual personalized equilibrium between rejection and infection risks. With the above aim, Torque Teno Virus (TTV) viremia was precisely examined in a large cohort of transplanted immunocompromised patients (192 hematological and 60 solid organ transplant recipients) being monitored for Cytomegalovirus reactivation. TTV load was measured in 2612 plasma samples from 448 patients. The results revealed a significant increase in TTV viral load approximately 14 days following CMV reactivation/infection in solid organ transplant (SOT) patients. No recognizable difference in TTV load was noted among hematological patients during the entire timeframe analyzed. Furthermore, a temporal gap of approximately 30 days was noted between the viral load peaks reached by the two viruses, with Cytomegalovirus (CMV) preceding TTV. It was not possible to establish a correlation between CMV reactivation/infection and TTV viremia in hematological patients. On the other hand, the SOT patient cohort allowed us to analyze viral kinetics and draw intriguing conclusions. Taken together, the data suggest, to our knowledge for the first time, that CMV infection itself could potentially cause an increase in TTV load in the peripheral blood of patients undergoing immunosuppressive therapy.

High frequency of neutralizing antibodies to type I Interferon in HIV-1 patients hospitalized for COVID-19.

The presence of anti-IFN neutralizing antibodies (NAB) has been reported in critically ill COVID-19 patients. We found that 87.5% (7/8) of HIV-1 patients co-infected with SARS-CoV-2 had serum anti-IFN-I NAB against IFN-α subtypes, IFN-ß and/or IFN-ω. Anti-IFN-I NAB were also detected in oropharyngeal samples. Patients with NAB were males, and those with high serum anti-IFN-α/ω NAB titer had severe illness and exhibited reduction in the expression of IFN-stimulated genes. Thus, high titer of anti-IFN-α/ω NAB may contribute to the greater severity of COVID-19 in HIV-1 infected patients.

CRISPR/Cas9 Ablation of Integrated HIV-1 Accumulates Proviral DNA Circles with Reformed Long Terminal Repeats.

Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9. IMPORTANCE The excision of HIV-1 provirus from the host cell genome has proven feasible in vitro and, to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may reactivate. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of the HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation.

Convalescent plasma for haematological patients with SARS-CoV-2 pneumonia and severe depletion of B-cell lymphocytes following anti-CD20 therapy: a single-centre experience and review of the literature.

Convalescent plasma (CP) therapy might be effective in patients with haematological malignanciesand B-cell depletion. We report a single-centre experience of COVID-19 patients with non-Hodgkinlymphoma and absence of B-cells as a consequence of anti-CD20 therapy successfully treated withCP from October 2020 to May 2021. CP was given in the presence of pneumonia with respiratoryfailure despite standard treatment and consisted of three infusions on an alternate-day basis. A reviewof the current literature on this topic was also performed. Six patients were identified (medianage 59.5 years (range 50-73)). The last anti-CD20 drug administration occurred 60 days before infection(range 0-360). CP was administered after a median of 51 days (range 9-120) from SARS-CoV-2diagnosis, with an early improvement in all but one subject. We suggest a possible clinical benefitof convalescent CP treatment in COVID-19 patients with haematological malignancies and B-celldepletion having persistent/recurrent pneumonia.

Cytokine storm and histopathological findings in 60 cases of COVID-19-related death: from viral load research to immunohistochemical quantification of major players IL-1ß, IL-6, IL-15 and TNF-α.

This study involves the histological analysis of samples taken during autopsies in cases of COVID-19 related death to evaluate the inflammatory cytokine response and the tissue localization of the virus in various organs. In all the selected cases, SARS-CoV-2 RT-PCR on swabs collected from the upper (nasopharynx and oropharynx) and/or the lower respiratory (trachea and primary bronchi) tracts were positive. Tissue localization of SARS-CoV-2 was detected using antibodies against the nucleoprotein and the spike protein. Overall, we tested the hypothesis that the overexpression of proinflammatory cytokines plays an important role in the development of COVID-19-associated pneumonia by estimating the expression of multiple cytokines (IL-1ß, IL-6, IL-10, IL-15, TNF-α, and MCP-1), inflammatory cells (CD4, CD8, CD20, and CD45), and fibrinogen. Immunohistochemical staining showed that endothelial cells expressed IL-1ß in lung samples obtained from the COVID-19 group (p < 0.001). Similarly, alveolar capillary endothelial cells showed strong and diffuse immunoreactivity for IL-6 and IL-15 in the COVID-19 group (p < 0.001). TNF-α showed a higher immunoreactivity in the COVID-19 group than in the control group (p < 0.001). CD8 + T cells where more numerous in the lung samples obtained from the COVID-19 group (p < 0.001). Current evidence suggests that a cytokine storm is the major cause of acute respiratory distress syndrome (ARDS) and multiple organ failure and is consistently linked with fatal outcomes.

The role of teicoplanin in the treatment of SARS-CoV-2 infection: A retrospective study in critically ill COVID-19 patients (Tei-COVID study).

Teicoplanin has a potential antiviral activity expressed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was suggested as a complementary option to treat coronavirus disease 2019 (COVID-19) patients. In this multicentric, retrospective, observational research the aim was to evaluate the impact of teicoplanin on the course of COVID-19 in critically ill patients. Fifty-five patients with severe COVID-19, hospitalized in the intensive care units (ICUs) and treated with best available therapy were retrospectively analysed. Among them 34 patients were also treated with teicoplanin (Tei-COVID group), while 21 without teicoplanin (control group). Crude in-hospital Day-30 mortality was lower in Tei-COVID group (35.2%) than in control group (42.8%), however not reaching statistical significance (p = .654). No statistically significant differences in length of stay in the ICU were observed between Tei-COVID group and control group (p = .248). On Day 14 from the ICU hospitalization, viral clearance was achieved in 64.7% patients of Tei-COVID group and 57.1% of control group, without statistical difference. Serum C-reactive protein level was significantly reduced in Tei-COVID group compared to control group, but not other biochemical parameters. Finally, Gram-positive were the causative pathogens for 25% of BSIs in Tei-COVID group and for 70.6% in controls. No side effects related to teicoplanin use were observed. Despite several limitations require further research, in this study the use of teicoplanin is not associated with a significant improvement in outcomes analysed. The antiviral activity of teicoplanin against SARS-CoV-2, previously documented, is probably more effective at early clinical stages.

SARS-CoV-2 diagnostics in the virology laboratory of a University Hospital in Rome during the lockdown period.

Italy was one of the most affected nations by coronavirus disease 2019 outside China. The infections, initially limited to Northern Italy, spread to all other Italian regions. This study aims to provide a snapshot of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) epidemiology based on a single-center laboratory experience in Rome. The study retrospectively included 6565 subjects tested for SARS-CoV-2 at the Laboratory of Virology of Sapienza University Hospital in Rome from 6 March to 4 May. A total of 9995 clinical specimens were analyzed, including nasopharyngeal swabs, bronchoalveolar lavage fluids, gargle lavages, stools, pleural fluids, and cerebrospinal fluids. Positivity to SARS-CoV-2 was detected in 8% (527/6565) of individuals, increased with age, and was higher in male patients (P < .001). The number of new confirmed cases reached a peak on 18 March and then decreased. The virus was detected in respiratory samples, in stool and in pleural fluids, while none of gargle lavage or cerebrospinal fluid samples gave a positive result. This analysis allowed to gather comprehensive information on SARS-CoV-2 epidemiology in our area, highlighting positivity variations over time and in different sex and age group and the need for a continuous surveillance of the infection, mostly because the pandemic evolution remains unknown.

Chest computed tomography score, cycle threshold values and secondary infection in predicting COVID-19 mortality.

This retrospective and observational cohort study investigated chest computed tomography (CT) findings, cycle threshold (Ct) values in RT-PCR of SARS-CoV-2 and secondary infection occurrence to predict prognosis in COVID-19 patients. At hospital admission, CT findings and Ct values were collected. Microbiology tests performed after 48 hours from hospitalization were reviewed. According to in-hospital mortality, patients were grouped into non-survivors and survivors. Among 283 patients evaluated, in-hospital mortality rate was 13.8% (39/283). Secondary infection occurrence was 15.2% (43/283). Cut-off values for CT score >13.5 (AUC=0.682 p=0.0009) and for Ct <23.4 (AUC=0.749, p<0.0001) were predictive of death. Super-additive and synergic effects between high CT score plus secondary infection occurrence as well as between high CT score plus low Ct values affecting patient's outcome were observed. Chest CT score and Ct values in RT-PCR of SARS-CoV-2 could have a combination role for severity stratification of COVID-19 patients.

Activation of Latent HIV-1 T Cell Reservoirs with a Combination of Innate Immune and Epigenetic Regulators.

The presence of T cell reservoirs in which human immunodeficiency virus (HIV) establishes latency by integrating into the host genome represents a major obstacle to an HIV cure and has prompted the development of strategies aimed at the eradication of HIV from latently infected cells. The "shock-and-kill" strategy is one of the most pursued approaches to the elimination of viral reservoirs. Although several latency-reversing agents (LRAs) have shown promising reactivation activity, they have failed to eliminate the cellular reservoir. In this study, we evaluated a novel immune system-mediated approach to clearing the HIV reservoir, based on a combination of innate immune stimulation and epigenetic reprogramming. The combination of the STING agonist cGAMP (cyclic GMP-AMP) and the FDA-approved histone deacetylase inhibitor resminostat resulted in a significant increase in HIV proviral reactivation and specific apoptosis in HIV-infected cells in vitro Reductions in the proportion of HIV-harboring cells and the total amount of HIV DNA were also observed in CD4+ central memory T (TCM) cells, a primary cell model of latency, where resminostat alone or together with cGAMP induced high levels of selective cell death. Finally, high levels of cell-associated HIV RNA were detected ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from individuals on suppressive antiretroviral therapy (ART). Although synergism was not detected in PBMCs with the combination, viral RNA expression was significantly increased in CD4+ T cells. Collectively, these results represent a promising step toward HIV eradication by demonstrating the potential of innate immune activation and epigenetic modulation for reducing the viral reservoir and inducing specific death of HIV-infected cells.IMPORTANCE One of the challenges associated with HIV-1 infection is that despite antiretroviral therapies that reduce HIV-1 loads to undetectable levels, proviral DNA remains dormant in a subpopulation of T lymphocytes. Numerous strategies to clear residual virus by reactivating latent virus and eliminating the reservoir of HIV-1 (so-called "shock-and-kill" strategies) have been proposed. In the present study, we use a combination of small molecules that activate the cGAS-STING antiviral innate immune response (the di-cyclic nucleotide cGAMP) and epigenetic modulators (histone deacetylase inhibitors) that induce reactivation and HIV-infected T cell killing in cell lines, primary T lymphocytes, and patient samples. These studies represent a novel strategy for HIV eradication by reducing the viral reservoir and inducing specific death of HIV-infected cells.

Seroprevalence of group B Coxsackieviruses: Retrospective study in an Italian population.

Group B Coxsackieviruses (CVB) include six serotypes (B1-6) responsible for a wide range of clinical diseases. Since no recent seroepidemiologic data are available in Italy, the study aim was to investigate CVB seroprevalence in a wide Italian population. The study retrospectively included 2459 subjects referring to a large academic hospital in Rome (Italy) in the period 2004-2016. Seroprevalence rates and neutralizing antibodies (nAb) titers were evaluated in relation to years of observation and subjects' characteristics. Positivity for at least one serotype was detected in 69.1% of individuals. Overall, the prevalent serotype was B4, followed by B3 (33.3%), B5 (26.2%), B1 (12.7%), B2 (11.0%), and B6 (1.7%). For B2, a significant decrease in seroprevalence over years was observed. Positivity to at least one virus was 25.2% in children aged 0 to 2 years, but significantly increased in preschool (3-5 years) (50.3%) and school (6-10 years) children (70.4%). Higher nAb responses for B3 and B4 were observed in children aged 3 to 5 years. A high overall CVB prevalence was found. Type-specific variations in prevalence over time probably reflect the fluctuations in circulation typical of Enteroviruses. Children are at greater risk for CVB infection given the high number of seronegative subjects aged 0 to 10 years.

Chest CT score in COVID-19 patients: correlation with disease severity and short-term prognosis.

OBJECTIVES: To correlate a CT-based semi-quantitative score of pulmonary involvement in COVID-19 pneumonia with clinical staging of disease and laboratory findings. We also aimed to investigate whether CT findings may be predictive of patients' outcome. METHODS: From March 6 to March 22, 2020, 130 symptomatic SARS-CoV-2 patients were enrolled for this single-center analysis and chest CT examinations were retrospectively evaluated. A semi-quantitative CT score was calculated based on the extent of lobar involvement (0:0%; 1, < 5%; 2:5-25%; 3:26-50%; 4:51-75%; 5, > 75%; range 0-5; global score 0-25). Data were matched with clinical stages and laboratory findings. Survival curves and univariate and multivariate analyses were performed to evaluate the role of CT score as a predictor of patients' outcome. RESULTS: Ground glass opacities were predominant in early-phase (≤ 7 days since symptoms' onset), while crazy-paving pattern, consolidation, and fibrosis characterized late-phase disease (> 7 days). CT score was significantly higher in critical and severe than in mild stage (p < 0.0001), and among late-phase than early-phase patients (p < 0.0001). CT score was significantly correlated with CRP (p < 0.0001, r = 0.6204) and D-dimer (p < 0.0001, r = 0.6625) levels. A CT score of ≥ 18 was associated with an increased mortality risk and was found to be predictive of death both in univariate (HR, 8.33; 95% CI, 3.19-21.73; p < 0.0001) and multivariate analysis (HR, 3.74; 95% CI, 1.10-12.77; p = 0.0348). CONCLUSIONS: Our preliminary data suggest the potential role of CT score for predicting the outcome of SARS-CoV-2 patients. CT score is highly correlated with laboratory findings and disease severity and might be beneficial to speed-up diagnostic workflow in symptomatic cases. KEY POINTS: • CT score is positively correlated with age, inflammatory biomarkers, severity of clinical categories, and disease phases. • A CT score ≥ 18 has shown to be highly predictive of patient's mortality in short-term follow-up. • Our multivariate analysis demonstrated that CT parenchymal assessment may more accurately reflect short-term outcome, providing a direct visualization of anatomic injury compared with non-specific inflammatory biomarkers.

COVID-19 in Patients with Hematologic Disorders Undergoing Therapy: Perspective of a Large Referral Hematology Center in Rome.

INTRODUCTION: Patients with cancer may be more susceptible to and have higher morbidity and mortality rates from COVID-19 than the general population, while epidemiologic data specifically addressed to hematologic patients are limited. To investigate whether patients with hematologic diseases undergoing therapy are at increased risk for acquiring SARS CoV-2 infection compared to the general population, a retrospective study was carried out at a referral hematologic center in Rome, Italy, during the period of the greatest epidemic spread (March 8 to May 14, 2020). METHODS: All adult and pediatric patients with a diagnosis of a neoplastic or a nonneoplastic hematologic disease who underwent treatment (chemotherapy or immunosuppressive or supportive therapy) during the study period or in the previous 6 months were considered. The prevalence of COVID-19 in the overall outpatient and inpatient population undergoing hematologic treatment compared to that of the general population was analyzed. The measures taken to manage patients during the epidemic period are described. RESULTS: Overall, 2,513 patients with hematological diseases were considered. Out of 243 (9.7%) patients who were screened for SARS CoV-2, three of 119 (2.5%) outpatients with fever or respiratory symptoms and none of 124 asymptomatic patients were diagnosed with COVID-19. Three further patients were diagnosed with COVID-19 and managed in other hospitals in Rome. As of May 14, 2020, the prevalence of COVID-19 in our hematologic population accounted for 0.24% (95% CI 0.23-0.25; 6 of 2,513 patients: 1 case in every 419 patients) as compared to 0.12% (7,280 of 5,879,082 residents; 1 case in every 807 residents) in the general population (p = 0.14). Three of 6 patients diagnosed with COVID-19 required critical care and 2 died while still positive for SARS CoV-2. Out of 225 healthcare providers on duty at our Institution during the study period, 2 (0.9%) symptomatic cases were diagnosed with COVID-19. CONCLUSION: In our experience, the prevalence of COVID-19 in hematologic patients, mainly affected by malignancies, was not significantly higher compared to that of the general population. Definition of adapted strategies for healthcare services, while continuing to administer the standard hematologic treatments, represents the crucial challenge for the management of hematologic diseases in the COVID-19 era.

Increased expression of IL-32 correlates with IFN-γ, Th1 and Tc1 in virologically suppressed HIV-1-infected patients.

Following recent attention focused on IL-32 as an important component involved in the inflammatory cytokine network, we speculated that IL-32's action on IFN-γ and IFN-γ secreting T cell subsets may help sustain the immune activation and dysregulation found in patients with HIV-1 achieving viral suppression. To explore this hypothesis, transcript levels of IL-32 and IFN-γ were evaluated in PBMC from 139 virologically suppressed HIV-1-infected patients and from 63 healthy individuals by Real Time RT-PCR assays. IL-32 and IFN-γ mRNA levels were also analyzed in CD4+ T cells, CD14+ monocytes and lamina propria lymphocytes (LPL) of the gut district in a subgroup of HIV-1-infected subjects. IFN-γ secreting CD4+ (Th1) and CD8+ (Tc1) T cell subset frequencies were evaluated in LPL by multiparametric flow cytometry. Gene expression results revealed that IL-32 and IFN-γ levels in PBMC from HIV-1-positive patients were significantly elevated compared to those from healthy donors, correlated with each other and increased with patient age. Both IL-32 and IFN-γ genes were also more strongly expressed in CD4+ T cells than in CD14+ monocytes. By contrast, IL-32 levels in LPL were comparable to those measured in PBMC, while IFN-γ levels were higher in PBMC than those in LPL. Negative correlations were found between IL-32 levels and the frequencies of Th1 and Tc1 subsets in gut mucosa. Collectively, our results provide the first evidence that IL-32 levels remain elevated in treated HIV-1-infected patients and correlate with IFN-γ, Th1 and Tc1 subsets.

Characterisation of HIV-1 molecular transmission clusters among newly diagnosed individuals infected with non-B subtypes in Italy.

OBJECTIVE: We evaluated the characteristics of HIV-1 molecular transmission clusters (MTCs) in 1890 newly diagnosed individuals infected with non-B subtypes between 2005 and 2017 in Italy. METHODS: Phylogenetic analyses were performed on pol sequences to characterise subtypes/circulating recombinant forms and identify MTCs. MTCs were divided into small (SMTCs, 2-3 sequences), medium (MMTCs, 4-9 sequences) and large (LMTCs, ≥10 sequences). Factors associated with MTCs were evaluated using logistic regression analysis. RESULTS: 145 MTCs were identified and involved 666 individuals (35.2%); 319 of them (16.9%) were included in 13 LMTCs, 111 (5.9%) in 20 MMTCs and 236 (12.5%) in 112 SMTCs. Compared with individuals out of MTCs, individuals involved in MTCs were prevalently Italian (72.7% vs 30.9%, p<0.001), male (82.9% vs 62.3%, p<0.001) and men who have sex with men (MSM) (43.5% vs 14.5%, p<0.001). Individuals in MTCs were also younger (median (IQR) years: 41 (35-49) vs 43 (36-51), p<0.001) and had higher CD4 cell count in comparison with individuals out of MTCs (median (IQR): 109/L: 0.4 (0.265-0.587) vs 0.246 (0.082-0.417), p<0.001). The viral load remained stable between the two groups (median (IQR) log10 copies/mL: 4.8 (4.2-5.5) vs 5.0 (4.3-5.5), p=0.87). Logistic regression confirmed that certain factors such as being MSM, of Italian origin, younger age and higher CD4 cell count were significantly associated with MTCs. CONCLUSIONS: Our findings show that HIV-1 newly diagnosed individuals infected with non-B subtypes are involved in several MTCs in Italy. These MTCs include mainly Italians and MSM and highlight the complex phenomenon characterising the HIV-1 spread. This is important especially in view of monitoring the HIV epidemic and guiding the public health response.

Lymphomagenic properties of a HIV p17 variant derived from a splenic marginal zone lymphoma occurred in a HIV-infected patient.

Despite antiretroviral therapy, HIV+ individuals still have increased risk to develop lymphomas, including marginal zone lymphomas, suggesting that factors other than HIV-related immunosuppression are probably acting as lymphomagenic factors in the HIV setting. The possible pathogenic involvement of HIV p17 protein variants was investigated in a particularly informative case of HIV-related splenic marginal zone lymphoma, which was negative for oncogenic virus infections, thus allowing us to assess the possible direct contribution of these HIV-encoded proteins to lymphomagenesis. The presence of p17 protein was analyzed by immunohistochemistry in lymphoma tissue. Recombinant p17 protein derived from the dominant sequence detected in plasma and lymphoma biopsy was characterized for B-cell proliferation, clonogenicity in soft agar, in vitro tube formation and wound healing. Intracellular signaling was investigated by immunoblotting. HIV p17 protein was detected in reactive lymphoid follicles but not within lymphoma cells. An identical dominant variant p17 sequence, p17-Lyrm, carrying a 117 to 118 Ala-Ala insertion was detected in both plasma and lymphoma tissue. Recombinant p17-Lyrm enhanced B-cell proliferation and clonogenicity promoted the formation of capillary-like structures and enhanced endothelial cell migration. Unlike reference p17, the p17-Lyrm variant enhanced the activation of Akt and ERK, critical kinases in lymphomagenesis. p17-Lyrm clonogenic activity was dependent on the activation of Akt but not of ERK1/2. These results indicated that HIV p17 variants with distinct molecular signatures and functional properties may accumulate in lymphoid tissues of HIV-infected individuals where they may act as a local stimulus promoting the development of lymphomas.

Increased SAMHD1 transcript expression correlates with interferon-related genes in HIV-1-infected patients.

PURPOSE: To investigate the contribution of SAMHD1 to HIV-1 infection in vivo and its relationship with IFN response, the expression of SAMHD1 and IFN-related pathways was evaluated in HIV-1-infected patients. METHODS: Peripheral blood mononuclear cells (PBMC) from 388 HIV-1-infected patients, both therapy naïve (n = 92) and long-term HAART treated (n = 296), and from 100 gender and age-matched healthy individuals were examined. CD4+ T cells, CD14+ monocytes and gut biopsies were also analyzed in HIV-1-infected subjects on suppressive antiretroviral therapy. Gene expression levels of SAMDH1, ISGs (MxA, MxB, HERC5, IRF7) and IRF3 were evaluated by real-time RT-PCR assays. RESULTS: SAMHD1 levels in HIV-1-positive patients were significantly increased compared to those in healthy donors. SAMHD1 expression was enhanced in treated patients compared to naïve patients (p < 0.0001) and healthy donors (p = 0.0038). Virologically suppressed treated patients exhibited higher SAMHD1 levels than healthy donors (p = 0.0008), viraemic patients (p = 0.0001) and naïve patients (p < 0.0001). SAMHD1 levels were also increased in CD4+ T cells compared to those in CD14+ monocytes and in PBMC compared to those of GALT. Moreover, SAMHD1 was expressed more strongly than ISGs in HIV-1-infected patients and positive correlations were found between SAMHD1, ISGs and IRF3 levels. CONCLUSIONS: SAMHD1 is more strongly expressed than the classical IFN-related genes, increased during antiretroviral therapy and correlated with ISGs and IRF3 in HIV-1-infected patients.

Evaluation of HIV-DNA and inflammatory markers in HIV-infected individuals with different viral load patterns.

BACKGROUND: Persistent residual viremia (RV) and low grade inflammation and immune activation have been associated with non-AIDS defining events. The impact of persistent RV and HIV-DNA load on immune activation/inflammation remains unclear. The purpose of this study was to gain new insights into the relation between viremia, markers of inflammation and HIV-DNA levels. METHODS: Three hundred and twenty-one HIV-infected patients were studied. A retrospective analysis of viremia values, prospectively collected for 48 months, was performed. Patients were separated into three groups: 113 TND (Target Not Detected, patients with sustained undetectable viremia); 113 RV (Residual Viremia, patients who had at least three detectable viral load (VL) values <37 copies/ml); 95 LLV (Low Level Viremia, patients with at least two VL values >37 but <200 copies/ml). HIV-DNA, TNF-α, IL-6 and sCD14 were analyzed. RESULTS: HIV-DNA, sCD14 and TNF-α were significantly lower in the TND group than in the RV and LLV groups. In addition, RV patients showed lower levels of HIV-DNA and sCD14 than LLV individuals. HIV-DNA load was not related to markers of inflammation. The ordinal logistic analysis showed that two independent variables were significantly associated with VL pattern: sCD14, HIV-DNA. In addition NRTIs plus NNRTIs and NRTIs plus PIs were negatively associated to VL pattern compared to INI-containing regimen. CONCLUSIONS: Persistent undetectable viremia was associated with lower levels of inflammatory markers and HIV-DNA. However, the lack of normalization of these biomarkers in the TND group and the fact that HIV-DNA load was not associated with inflammation strongly suggest that other mechanisms play a major role in maintaining inflammation over time.

Lack of association of Chlamydia pneumoniae with cardiovascular diseases in virologically suppressed HIV patients.

Cardiovascular disease (CVD) is a major public health problem in developed countries with over 17 million deaths per year. In the last decade, several infectious agents rather than any single pathogen, including Chlamydia pneumoniae and human immunodeficiency virus (HIV), have been shown to contribute to the development of atherosclerosis and subsequent cardiovascular events by inducing systemic inflammation and/or acting directly on the vascular wall. For the first time, we evaluated C. pneumonia DNA in peripheral blood mononuclear cells from HIV patients by real-time polymerase chain reaction in order to shed light on C. pneumonia as a co-factor with HIV in the development of CVDs. C. pneumonia DNA was not detected in our virologically suppressed HIV patients (<37 copies/mL). This finding may be related to high CD4+T cell count (>500 cells/µl) found in HIV patients suggesting functional cell-mediated immunity as a fundamental mechanism for the clearance of chlamydial infection in this population. Larger studies are needed to confirm this hypothesis.