Search

Traditionally, molecular mechanisms of pathogenesis for infectious agents were studied in cell culture or animal models but have limitations on the extent to which the resulting data reflect natural infection in humans. The COVID-19 pandemic has highlighted the urgent need to rapidly develop laboratory models that enable the study of host-pathogen interactions, particularly the relative efficacy of preventive measures. Recently, human and animal ex vivo tissue challenge models have emerged as a promising avenue to study immune responses, screen potential therapies and triage vaccine candidates. This approach offers the opportunity to closely approximate human disease from the perspective of pathology and immune response. It has advantages compared to animal models which are expensive, lengthy and often require containment facilities. Herein, we summarize some recent advances in the development of ex vivo tissue challenge models for COVID-19, HIV-1 and other pathogens. We focus on the contribution of these models to enhancing knowledge of host-pathogen interactions, immune modulation, and their value in testing therapeutic agents. We further highlight the advantages and limitations of using ex vivo challenge models and briefly summarize how the use of organoids provides a useful advancement over current approaches. Collectively, these developments have enormous potential for the study of infectious diseases.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) ˂14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

The penile epithelial microbiome remains underexplored. We sequenced human RNA and a segment of the bacterial 16S rRNA gene from the foreskin tissue of 144 adolescents from South Africa and Uganda collected during penile circumcision after receipt of 1-2 doses of placebo, emtricitabine + tenofovir disoproxil fumarate, or emtricitabine + tenofovir alafenamide to investigate the microbiome of foreskin tissue and its potential changes with antiretroviral use. We identified a large number of anaerobic species, including Corynebacterium acnes, which was detected more frequently in participants from South Africa than Uganda. Bacterial populations did not differ by treatment received, and no differentially abundant taxa were identified between placebo versus active drug recipients. The relative abundance of specific bacterial taxa was negatively correlated with expression of genes downstream of the innate immune response to bacteria and regulation of inflammation. Our results show no difference in the tissue microbiome of the foreskin with short-course antiretroviral use but that bacterial taxa were largely inversely correlated with inflammatory gene expression, consistent with commensal colonization., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

With the onset of COVID-19, the development of ex vivo laboratory models became an urgent priority to study host-pathogen interactions in response to the pandemic. In this study, we aimed to establish an ex vivo mucosal tissue explant challenge model for studying SARS-CoV-2 infection and replication. Nasal or oral tissue samples were collected from eligible participants and explants generated from the tissue were infected with various SARS-CoV-2 strains, including IC19 (lineage B.1.13), Beta (lineage B.1.351) and Delta (lineage B.1.617.2). A qRT-PCR assay used to measure viral replication in the tissue explants over a 15-day period, demonstrated no replication for any viral strains tested. Based on this, the ex vivo challenge protocol was modified by reducing the viral infection time and duration of sampling. Despite these changes, viral infectivity of the nasal and oral mucosa was not improved. Since 67% of the enrolled participants were already vaccinated against SARS-CoV-2, it is possible that neutralizing antibodies in explant tissue may have prevented the establishment of infection. However, we were unable to optimize plaque assays aimed at titrating the virus in supernatants from both infected and uninfected tissue, due to limited volume of culture supernatant available at the various collection time points. Currently, the reasons for the inability of these mucosal tissue samples to support replication of SARS-CoV-2 ex vivo remains unclear and requires further investigation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gordhan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: As topical pre-exposure prophylaxis (PrEP) has been shown to cause immune modulation in rectal or cervical tissue, our aim was to examine the impact of oral PrEP on lymphoid and myeloid changes in the foreskin in response to dosing and timing of drug administration., Design: HIV-negative male individuals ( n  = 144) were recruited in South Africa and Uganda into an open-label randomized controlled trial in a 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 ratio to control arm (with no PrEP) or one of eight arms receiving emtricitabine-tenofovir disoproxil fumarate (F/TDF) or emtricitabine-tenofovir alafenamide (F/TAF) at one of two different doses, 5 or 21 h before undergoing voluntary medical male circumcision (VMMC)., Methods: After dorsal-slit circumcision, foreskin tissue sections were embedded into Optimal Cutting Temperature media and analysed, blinded to trial allocation, to determine numbers of CD4 + CCR5 + , CD1a + cells and claudin-1 expression. Cell densities were correlated with tissue-bound drug metabolites and p24 production after ex-vivo foreskin challenge with HIV-1 bal ., Results: There was no significant difference in CD4 + CCR5 + or CD1a + cell numbers in foreskins between treatment arms compared with the control arm. Claudin-1 expression was 34% higher ( P  = 0.003) in foreskin tissue from participants receiving PrEP relative to controls, but was no longer statistically significant after controlling for multiple comparisons. There was neither correlation of CD4 + CCR5 + , CD1a + cell numbers, or claudin-1 expression with tissue-bound drug metabolites, nor with p24 production after ex-vivo viral challenge., Conclusion: Oral doses and timing of on-demand PrEP and in-situ drug metabolite levels in tissue have no effect on numbers or anatomical location of lymphoid or myeloid HIV target cells in foreskin tissue., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The efficacy of on-demand HIV pre-exposure prophylaxis (PrEP) for men in sub-Saharan Africa has not been evaluated, and the on-demand PrEP dosing requirement for insertive sex remains unknown., Methods: HIV-negative males 13-24 years, requesting voluntary medical male circumcision (VMMC), were enrolled into an open-label randomised controlled trial (NCT03986970), and randomised 1:1:1:1:1:1:1:1:1 to control arm or one of eight arms receiving emtricitabine-tenofovir disoproxil fumarate (F/TDF) or emtricitabine-tenofovir alafenamide (F/TAF) over one or two days, and circumcised 5 or 21 h thereafter. The primary outcome was foreskin p24 concentrations following ex vivo HIV-1 BaL challenge. Secondary outcomes included peripheral blood mononuclear cell (PBMC) p24 concentration, and drug concentrations in foreskin tissue, PBMCs, plasma and foreskin CD4+/CD4-cells. In the control arm, post-exposure prophylaxis (PEP) activity of non-formulated tenofovir-emtricitabine (TFV-FTC) or TAF-FTC was assessed with ex vivo dosing 1, 24, 48 or 72 h post-HIV-1 challenge., Findings: 144 participants were analysed. PrEP with F/TDF or F/TAF prevented ex vivo infection of foreskins and PBMCs both 5 and 21 h after PrEP dosing. There was no difference between F/TDF and F/TAF (p24 day15 geometric mean ratio 1.06, 95% confidence interval: 0.65-1.74). Additional ex vivo dosing did not further increase inhibition. In the control arm, PEP ex vivo dosing was effective up to 48 post-exposure diminishing thereafter, with TAF-FTC showing prolonged protection compared to TFV-FTC. Participants receiving F/TAF had higher TFV-DP concentrations in foreskin tissue and PBMCs compared with F/TDF, irrespective of dose and sampling interval; but F/TAF did not confer preferential TFV-DP distribution into foreskin HIV target cells. FTC-TP concentrations with both drug regimens were equivalent and ∼1 log higher than TFV-DP in foreskin., Interpretation: A double dose of either F/TDF or F/TAF given once either 5 or 21 h before ex vivo HIV-challenge provided protection across foreskin tissue. Further clinical evaluation of pre-coital PrEP for insertive sex is warranted., Funding: EDCTP2, Gilead Sciences, Vetenskapsrådet., Competing Interests: Declaration of interests CH has received research grants from EDCTP, Vetenskapsrådet and Gilead Sciences. LE has received research grants from EDCTP, and Gilead Sciences. LL has received research grants from EDCTP, Gilead Sciences, Roche Diagnostic, DO has received research grants from EDCTP, AS has received research grants from EDCTP, AP has received research grants from EDCTP, PN has received research grants from EDCTP, PS has received research grants from EDCTP, DS has received research grants from EDCTP, RM has received research grants from EDCTP, BA has received research grants from EDCTP, SP has received research grants from EDCTP, CC is an employee of Gilead Sciences, JS has received research grants from EDCTP, HW has received research grants from EDCTP, SK has received research funding, speaker honoraria and consulting fees from EDCTP, Gilead Sciences, ViiV, Merck, GSK, and Ridgeback. FC has received research grants from EDCTP and Vetenskapsrådet. ELW has received grants from EDCTP, MRC, and NIH. CG has received research grants from EDCTP. PK has received research grants from EDCTP. EW has received research grants from EDCTP, NIH and MRC. NM has received research grants from EDCTP, and Gilead Sciences and provided unpaid advice and leadership in the DSMB and Setshaba boards. CC is a full-time employee of Gilead Sciences. All other authors declare no competing interests aside from the research grant received for this study by EDCTP., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

A new method is presented to measure strain over a large area of a single crystal. The 4D-ED data are collected by recording a 2D diffraction pattern at each position in the 2D area of the TEM lamella scanned by the electron beam of STEM. Data processing is completed with a new computer program (available free of charge) that runs under the Windows operating system. Previously published similar methods are either commercial or need special hardware (electron holography) or are based on HRTEM, which involves limitations with respect to the size of the field of view. All these limitations are overcome by our approach. The presence of defects results in small local changes in orientation that change the subset of experimentally available diffraction spots in the individual patterns. Our method is based on a new principle, namely fitting a lattice to (a subset of) measured diffraction spots to improve the precision of the measurement. Although a spot to be measured may be missing in some of the patterns even the missing spot can be precisely measured by the lattice determined from the available spots. Application is exemplified by heavily boron-doped silicon with intended usage as a low-temperature superconductor in qubits.

Introduction: Chronic myeloid leukemia (CML) is a hematopoietic myeloproliferative disorder that accounts for 20% of all leukemias of adults. The introduction of tyrosine kinase inhibitors (TKIs) (imatinib, bosutinib, dasatinib, nilotinib, ponatinib) has yielded significant benefits for patients with CML in terms of survival and quality of life. This real-world analysis evaluated the economic burden for managing patients with CML in 2nd or ≥ 3rd TKI lines in Italian settings of clinical practice., Methods: A retrospective observational analysis was performed exploiting the administrative databases of a sample of entities covering around 15 million inhabitants. From 2015 to 2018, the study included adult patients with at least one prescription for TKIs, (and for some TKI with at least one hospitalization discharge diagnosis for CML, or at least one prescription for BCR-ABL examination). The index date was the first TKI prescription. Healthcare resource consumption and costs for patients with CML in 2nd and ≥ 3rd line treatment with TKIs were analyzed for drug prescriptions, hospitalizations, specialist visits, and diagnostic services., Results: In total 635 patients were included, 491 in 2nd line and 144 in 3rd line with TKIs. Dasatinib was the most frequently prescribed drug in 2nd line (28.9%) and imatinib in later lines (26.4%). With progressing lines of treatment, healthcare consumption showed a trend towards increased non-TKI prescriptions per patient (8 for 2nd line and 9.7 for ≥ 3rd line). The management of patients with CML in later lines resulted in increased overall healthcare burden, with hospitalizations accounting for about half of total expenditure, whatever the treatment line and type of TKI., Conclusions: This analysis in Italian real-life clinical practice reported economic expenditure for patients with CML in 2nd or ≥ 3rd lines with TKIs, mostly burdened by hospitalizations. Such clinical complexity suggests that further efforts are needed to improve the therapeutic management of later lines of CML., (© 2022. The Author(s).)

The mucosal environment of the upper respiratory tract is the first barrier of protection against SARS-CoV-2 transmission. However, the mucosal factors involved in viral transmission and potentially modulating the capacity to prevent such transmission have not fully been identified. In this pilot proteomics study, we compared mucosal and systemic compartments in a South African cohort of vaccinated and unvaccinated individuals undergoing maxillofacial surgery with previous history of COVID-19 or not. Inflammatory profiles were analyzed in plasma, nasopharyngeal swabs, and nasal and oral tissue explant cultures, using Olink and Luminex technologies. SARS-CoV-2-specific antibody levels were measured in serum and tissue explants. An increased pro-inflammatory proteomic profile was measured in the nasal compartment compared to plasma. However, IP-10 and MIG levels were higher in secretions than in nasal tissue, and the opposite was observed for TGF-β. Nasal anti-SARS-CoV-2 spike IgG correlated with mucosal MIG expression for all participants. A further positive correlation was found with IP-10 in BioNTech/Pfizer-vaccinated individuals. Systemic levels of anti-SARS-CoV-2 spike IgG elicited by this vaccine correlated with plasma IL-10, IL-6 and HBD4. Proteomic profiles measured in mucosal tissues and secretions using combined technologies could reveal correlates of protection at the mucosal portals of viral entry.

Preventing new HIV infections remains a global challenge. Young women continue to bear a disproportionate burden of infection. Oral pre-exposure prophylaxis (PrEP), offers a novel women-initiated prevention technology and PrEP trials completed to date underscore the importance of their inclusion early in trials evaluating new HIV PrEP technologies. Data from completed topical and systemic PrEP trials highlight the role of gender specific physiological and social factors that impact PrEP uptake, adherence and efficacy. Here we review the past and current developments of HIV-1 prevention options for women with special focus on PrEP considering the diverse factors that can impact PrEP efficacy. Furthermore, we highlight the importance of inclusion of female scientists, clinicians, and community advocates in scientific efforts to further improve HIV prevention strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Karim, Archary, Barré-Sinoussi, Broliden, Cabrera, Chiodi, Fidler, Gengiah, Herrera, Kharsany, Liebenberg, Mahomed, Menu, Moog, Scarlatti, Seddiki, Sivro and Cavarelli.)

Whilst short-term oral pre-exposure prophylaxis (PrEP) with antiretroviral drugs in men who have sex with men has shown protection against HIV-1 infection, the impact of this regimen on the in vivo foreskin transcriptome is unknown. We collected foreskin tissue after voluntary medical male circumcision from 144 young men (72 from Uganda and 72 from South Africa) randomized to one to two doses of either oral tenofovir (TFV) disoproxil fumarate (FTC-TDF) or tenofovir alafenamide (FTC-TAF) or no drug (untreated controls). This novel approach allowed us to examine the impact of short-term oral PrEP on transcriptome of the male genital tract. A single dose of FTC-TDF did not affect the foreskin transcriptome in relation to control arm, however one dose of FTC-TAF induced upregulation of four genes AKAP8 , KIAA0141 , HSCB and METTL17 . Following two doses of either FTC-TDF or FTC-TAF, there was an increase in 34 differentially expressed genes for FTC-TDF and 15 for FTC-TAF, with nine DEGs in common: KIAA0141 , SAFB2 , CACTIN , FXR2 , AKAP8 , HSCB , CLNS1A , DDX27 and DCAF15 . Functional analysis of differentially expressed genes revealed modulation of biological processes related to mitochondrial stress (KIAA0141, HSCB and METTL17) , anti-viral and anti-inflammatory pathways ( CACTIN and AKAP8) . Our results show that short-course on-demand oral PrEP in men modulates genes in foreskin tissue which are likely unfavorable to HIV acquisition and replication. We also describe an upregulated expression of genes involved in diverse mitochondria biology which may potentially result in worsened mitochondria-related. These results warrant further studies to assess the role of short-course and prolonged oral PrEP on biological processes of the foreskin mucosa., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Petkov, Herrera, Else, Lebina, Opoka, Seiphetlo, Pillay, Mugaba, Namubiru, Odoch, Ssemata, Serwanga, Kaleebu, Webb, Khoo, Martinson, Gray, Fox and Chiodi.)

HIV-1 pre-exposure prophylaxis (PrEP) relies on inhibition of HIV-1 replication steps. To understand how PrEP modulates the immunological environment, we derived the plasma proteomic profile of men receiving emtricitabine-tenofovir (FTC-TDF) or emtricitabine-tenofovir alafenamide (FTC-TAF) during the CHAPS trial in South Africa and Uganda (NCT03986970). The CHAPS trial randomized 144 participants to one control and 8 PrEP arms, differing by drug type, number of PrEP doses and timing from final PrEP dose to sampling. Blood was collected pre- and post-PrEP. The inflammatory profile of plasma samples was analyzed using Olink (N=92 proteins) and Luminex (N=33) and associated with plasma drug concentrations using mass spectrometry. The proteins whose levels changed most significantly from pre- to post-PrEP were CCL4, CCL3 and TNF-α; CCL4 was the key discriminator between pre- and post-PrEP samples. CCL4 and CCL3 levels were significantly increased in post-PrEP samples compared to control specimens. CCL4 was significantly correlated with FTC drug levels in plasma. Production of inflammatory chemokines CCL4 and CCL3 in response to short-term PrEP indicates the mobilization of ligands which potentially block virus attachment to CCR5 HIV-1 co-receptor. The significant correlation between CCL4 and FTC levels suggests that CCL4 increase is modulated as an inflammatory response to PrEP., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Petkov, Herrera, Else, Mugaba, Namubiru, Odoch, Opoka, Pillay, Seiphetlo, Serwanga, Ssemata, Kaleebu, Webb, Khoo, Lebina, Gray, Martinson, Fox and Chiodi.)

Real world data are becoming a crucial tool to understand how cancer is treated in routine daily practice. This real-world analysis aims to describe the characteristics of patients with CML in 2nd or ≥3rd tyrosine kinase inhibitors (TKI) lines of therapy, to evaluate their treatment sequence and utilization in settings of Italian clinical practice in Italy. A retrospective analysis was performed using an administrative databases covering around 15.3 million cases. All adult patients prescribed with TKI as 2nd or ≥3rd lines (L) of therapy for CML during January 2015-December 2018 were included. A total of 491 patients in 2nd and 144 in ≥3rd L was included. In both cohorts, hypertension was the most reported comorbidity, followed by metabolic and blood count alterations. In each calendar inclusion year, an increment of 97.6% was observed in the number of patients treated in ≥3rd L. In the 2nd L cohort, 18.7% had a switch to 3rd L, while 26.4% of ≥3rd L patients switched to a subsequent line. Around 40% in both lines discontinued their treatment after a median time of 5.5 (2nd L) and 4.3 (≥3rd L) years. The results provided insights into CML management clinical practice, indicating a heavy disease burden for patients in later lines that showed an increasing complex management, and suggest that a need for novel treatment strategies might exists.

Differentiation of CD4+ T naïve (T N ) into central memory (T CM ) cells involves extensive molecular processes. We compared the transcriptomes of CD4+ T N and T CM cells from HIV-1 infected patients receiving early anti-retroviral therapy (ART; EA; n = 13) and controls (n = 15). Comparison of protein coding genes between T CM and T N revealed 533 and 82 differentially expressed genes (DEGs) in controls and EA, respectively. A high degree of transcriptional complexity was detected during transition of CD4+ T N to T CM cells in controls involving 70 TFs, 20 master regulators of T cell differentiation (TBX21, GATA3, RARA, FOXP3, RORC); in EA only 7 TFs were modulated with expression of several master regulators remaining unchanged during differentiation. Analysis of interactions between modulated TFs and target genes revealed important regulatory interactions missing in EA group. We conclude that T cell differentiation in EA patients is impaired due to reduced modulation of genes involved in transition from CD4+ T N to T CM cells., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

HBV vaccination effectively prevents HBV transmission and the development of liver cancer. Disease progression and liver-related complications are more common in HIV-1/HBV co-infected than HBV mono-infected individuals. A considerable body of literature, which will be reviewed here, indicates that response to HBV vaccine is suboptimal in HIV-1-infected individuals and that the poor maintenance of protective immunity to HBV vaccines in these individuals is an important medical issue. Several factors affect HBV vaccine response during HIV-1 infection including CD4+ T cell counts, B cell response, vaccine formulation, schedules, and timing of antiretroviral therapy (ART). The initial response to HBV vaccination also plays a critical role in the sustainability of antibody responses in both HIV-1-infected and uninfected vaccinees. Thus, regular follow-up for antibody titer and a booster dose is warranted to prevent HBV transmission in HIV-1 infected people.

We analyzed the whole transcriptome characteristics of blood CD4+ T naïve (T N ) cells isolated from HIV-1 infected patients starting ART at acute (early ART = EA; n = 13) or chronic (late ART = LA; n = 11) phase of infection and controls (C; n = 15). RNA sequencing revealed 389 differentially expressed genes (DEGs) in EA and 810 in LA group in relation to controls. Comparison of the two groups of patients showed 183 DEGs. We focused on DEGs involved in apoptosis, inflammation and immune response. Clustering showed a poor separation of EA from C suggesting that these two groups present a similar transcriptomic profile of CD4+ T N cells. The comparison of EA and LA patients resulted in a high cluster purity revealing that different biological dysfunctions characterize EA and LA patients. The upregulated expression of several inflammatory chemokine genes distinguished the patient groups from C; CCL2 and CCL7, however, were downregulated in EA compared to LA patients. BCL2, an anti-apoptotic factor pivotal for naïve T cell homeostasis, distinguished both EA and LA from C. The expression of several DEGs involved in different inflammatory processes (TLR4, PTGS2, RAG1, IFNA16) was lower in EA compared LA. We conclude that although the transcriptome of CD4+ T N cells isolated from patients initiating ART at acute infection reveals a more quiescent phenotype, the survival profile of these cells still appears to be affected. Our results show that the detrimental process of inflammation is under more efficient control in EA patients., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Plasmodium falciparum parasites cause malaria and co-exist in humans together with B-cells for long periods of time. Immunity is only achieved after repeated exposure. There has been a lack of methods to mimic the in vivo co-occurrence, where cells and parasites can be grown together for many days, and it has been difficult with long time in vitro studies., Methods and Results: A new method for growing P. falciparum in 5% CO 2 with a specially formulated culture medium is described. This knowledge was used to establish the co-culture of live P. falciparum together with human B-cells in vitro for 10 days. The presence of B-cells clearly enhanced parasite growth, but less so when Transwell inserts were used (not allowing passage of cells or merozoites), showing that direct contact is advantageous. B-cells also proliferated more in presence of parasites. Symbiotic parasitic growth was verified using CESS cell-line and it showed similar results, indicating that B-cells are indeed the cells responsible for the effect. In malaria endemic areas, people often have increased levels of atypical memory B-cells in the blood, and in this assay it was demonstrated that when parasites were present there was an increase in the proportion of CD19 + CD20 + CD27 - FCRL4 + B-cells, and a contraction of classical memory B-cells. This effect was most clearly seen when direct contact between B-cells and parasites was allowed., Conclusions: These results demonstrate that P. falciparum and B-cells undoubtedly can affect each other when allowed to multiply together, which is valuable information for future vaccine studies.

CXCL13 signals through the G protein-coupled chemokine receptor CXCR5 to drive development of secondary lymphoid tissue as well as B cell and Tfh cell trafficking to germinal centers (GC), which leads to the differentiation of B cells to plasma cells and memory B cells. CXCL13 has been proposed as a general plasma biomarker for GC activities. In HIV-1 infected individuals, plasma CXCL13 levels have been associated with the rate of disease progression to AIDS. Moreover, CXCL13 production has been reported to be increased in HIV-1-infected lymph nodes, which may drive increased downregulation of CXCR5. In this review, we address the role of CXCL13 in HIV-1 infected individuals with regard to GC formation, generation of broadly neutralizing antibodies after infection and vaccination, and AIDS-related B cell lymphoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bekele Feyissa, Chiodi, Sui and Berzofsky.)

People living with human immunodeficiency virus (HIV-1) are at increased risk of developing cancer, such as Kaposi sarcoma (KS), non-Hodgkin lymphoma (NHL), cervical cancer, and other cancers associated with chronic viral infections. Traditionally, this is linked to HIV-1-induced immune suppression with depletion of CD4+ T-helper cells, exhaustion of lymphopoiesis and lymphocyte dysfunction. However, the long-term successful implementation of antiretroviral therapy (ART) with an early start did not preclude the oncological complications, implying that HIV-1 and its antigens are directly involved in carcinogenesis and may exert their effects on the background of restored immune system even when present at extremely low levels. Experimental data indicate that HIV-1 virions and single viral antigens can enter a wide variety of cells, including epithelial. This review is focused on the effects of five viral proteins: envelope protein gp120, accessory protein negative factor Nef, matrix protein p17, transactivator of transcription Tat and reverse transcriptase RT. Gp120, Nef, p17, Tat, and RT cause oxidative stress, can be released from HIV-1-infected cells and are oncogenic. All five are in a position to affect "innocent" bystander cells, specifically, to cause the propagation of (pre)existing malignant and malignant transformation of normal epithelial cells, giving grounds to the direct carcinogenic effects of HIV-1.

Using mass cytometry, we investigated the expression of 28 markers on CD8+ and CD4+ T cells from HIV-1 infected patients with a variable size of HIV-1 reservoir defined as high (HR) and low (LR) reservoir; we aimed at identifying phenotypic associations of T cells with size of HIV-1 reservoir. We showed that the frequency of CD45+ CD8+ and CD4+ T cells was directly proportional to the size of HIV-1 reservoir; HR patients had a significantly larger frequency of blood CD45 high T cells and higher CD45 expression on both CD8+ and CD4+ T cells. CD45 is a receptor-type protein tyrosine phosphatase essential in TCR signaling. Functional and phenotypical analysis of CD45 high cells revealed that they express activation and proliferation markers (CD38 + HLA-DR + and Ki-67) and produce cytokines upon in vitro activation. CD45 high T cells also expressed high levels of immune check-point PD-1. Our results link CD45 expression on T cells to HIV-1 reservoir; PD-1 expression on CD45 high T cells may contribute to their exhaustion.

Background: HIV remains a major public health issue, especially in Eastern and Southern Africa. Pre-exposure prophylaxis is highly effective when adhered to, but its effectiveness is limited by cost, user acceptability and uptake. The cost of a non-inferiority phase III trial is likely to be prohibitive, and thus, it is essential to select the best possible drug, dose and schedule in advance. The aim of this study, the Combined HIV Adolescent PrEP and Prevention Study (CHAPS), is to investigate the drug, dose and schedule of pre-exposure prophylaxis (PrEP) required for the protection against HIV and the acceptability of PrEP amongst young people in sub-Saharan Africa, and hence to inform the choice of intervention for future phase III PrEP studies and to improve strategies for PrEP implementation., Methods: We propose a mixed-methods study amongst young people aged 13-24 years. The first component consists of qualitative research to identify the barriers and motivators towards the uptake of PrEP amongst young people in South Africa, Uganda and Zimbabwe. The second component is a randomised clinical trial (ClinicalTrials.gov NCT03986970, June 2019) using a novel ex vivo HIV challenge method to investigate the optimal PrEP treatment (FTC-TDF vs FTC-TAF), dose and schedule. We will recruit 144 amongst HIV-negative uncircumcised men aged 13-24 years from voluntary male medical circumcision clinics in two sites (South Africa and Uganda) and randomise them into one of nine arms. One group will receive no PrEP prior to surgery; the other arms will receive either FTC-TDF or FTC-TAF, over 1 or 2 days, and with the final dose given either 6 or 20 h prior to surgery. We will conduct an ex vivo HIV challenge on their resected foreskin tissue., Discussion: This study will provide both qualitative and quantitative results to help decide the optimum drug, dose and schedule for a future phase III trial of PrEP. The study will also provide crucial information on successful strategies for providing PrEP to young people in sub-Saharan Africa., Trial Registration: ClinicalTrials.gov NCT03986970 . Registered on 14 June 2019.

Treatment of HIV-1-infected patients results in improved clinical and immunological conditions, but severe non-AIDS-related conditions still persist. Novel proteomic platforms have identified inflammatory proteins where abundance is dysregulated in adult treated patients, whereas limited data are available in treated HIV-1 infection of children. Using a proteomic plasma profiling approach comprising 92 inflammation-related molecules, we analyzed specimens from 43 vertically HIV-1-infected children receiving antiretroviral treatment (ART) and matched controls in Ethiopia. The infected children were analyzed as a group and separately, according to age of treatment initiation. Proteins displaying a significantly different abundance between groups were hierarchically clustered and presented in heat maps. Random forest analysis was performed to pin-point proteins discriminating between groups; five proteins (STAMBP, CD5, TFG-α, TRANCE, AXIN1) were the strongest prediction factors for treated HIV-1 infection. TRANCE was previously linked to reduced bone mass levels in HIV-1-infected children. CCL4 chemokine, ligand to HIV-1 co-receptor CCR5, was the most critical protein for successful classification between children who initiated ART at different time points. Our data provide evidence that a dysregulated expression of proteins linked to immunological abnormalities and bone metabolism can be found in HIV-1-infected children with prolonged exposure to ART.

Streptococcus pneumoniae ( S. pneumoniae ) vaccines have substantially reduced the burden of invasive pneumococcal diseases (IPDs) worldwide. Despite high coverage with S. pneumoniae vaccination, upper-respiratory-tract colonization by S. pneumoniae is still common. We assessed maintenance of serological responses to S. pneumoniae serotypes included in PCV-10 by ELISA in HIV-1-infected children (n = 50) and age-matched controls (n = 50) in Ethiopia. We isolated S. pneumoniae in nasopharyngeal swabs and determined S. pneumoniae serotype by whole genome sequencing (WGS). Comparable levels of S. pneumoniae serotype-specific IgG concentrations were detected in plasma of HIV-1-infected children and matched controls, with geometric mean concentrations (GMCs) consistently higher than the protective threshold for PCV-10 serotypes of 0.35 μg/mL. We isolated S. pneumoniae from 38 (out of 97) nasopharyngeal swabs, 25 from HIV-1-infected children and 13 from controls. WGS based serotyping revealed 22 known S. pneumoniae serotypes and 2 nontypeable (NT) isolates. Non-PCV-10 serotypes represented >90% of isolates. We showed that HIV-1-infected children and matched controls in Ethiopia carry a level of maintained serological memory to PCV-10 considered protective for IPDs. We identified a higher proportion of nasopharyngeal carriage with highly pathogenic S. pneumoniae non-PCV strains among HIV-1-infected children compared to controls.

We compared outer and inner foreskin tissue from adolescent males undergoing medical male circumcision to better understand signals that increase HIV target cell availability in the foreskin. We measured chemokine gene expression and the impact of sexually transmitted infections (STIs) on the density and location of T and Langerhans cells. Chemokine C-C ligand 27 (CCL27) was expressed 6.94-fold higher in the inner foreskin when compared with the outer foreskin. We show that the density of CD4 + CCR5 + cells/mm 2 was higher in the epithelium of the inner foreskin, regardless of STI status, in parallel with higher CCL27 gene expression. In the presence of STIs, there were higher numbers of CD4 + CCR5 + cells/mm 2 cells in the sub-stratum of the outer and inner foreskin with concurrently higher number of CD207 + Langerhans cells (LC) in both tissues, with the latter cells being closer to the keratin surface of the outer FS in the presence of an STI. When we tested the ability of exogenous CCL27 to induce T-cell migration in foreskin tissue, CD4 + T cells were able to relocate to the inner foreskin epithelium in response. We provide novel insight into the impact CCL27 and STIs on immune and HIV-1 target cell changes in the foreskin.

Background: Successful vaccinations rely on antibody responses. Chemokine receptors play an important role in B cell homing to differentiation niches. We assessed CXCR4, CXCR5 and CCR6 expression on B cells during HIV-1 infection and relate it to antibody responses against a HBV vaccine., Methods: Blood was obtained from 54 healthy controls and 38 ART-treated HIV-1 infected children, aviremic (n = 25) or viremic (n = 13). Frequency of naïve and memory B cell subsets was studied by immunostaining. Homing capacity of blood B cells to lymphoid and inflamed tissues was evaluated through CXCR4, CXCR5 and CCR6 expression. Plasma CXCL12 and CXCL13 levels and antibody titers to HBV antigen were determined by ELISA., Results: The frequency of naïve and resting memory (RM) B cells in ART treated children was comparable to control subjects. Profound defects in the homing phenotypes of naïve and memory B cells were identified, with lower CXCR4 and CXCR5 expression. Increased CXCL13 levels were observed in infected children, inversely correlating to CXCR5 expressing B cell subpopulations. Antibody titers to HBV vaccine correlated with frequency of resting and switched memory B cells in HIV-1 infected children., Conclusions: Homing defects of B cells to germinal center may underlie impaired vaccine responses during HIV-1 infection., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The genital tract of African women has been shown to differ from what is currently accepted as 'normal', defined by a pH≤4.5 and lactobacilli-dominated microbiota. Adolescent girls and young women (AGYW) from sub-Saharan Africa are at high risk for HIV, and we hypothesized that specific biological factors are likely to be influential. This study aimed to compare characteristics of vaginal health in HIV-negative AGYW (16-22-years-old), from two South African communities, to international norms. We measured plasma hormones, vaginal pH, presence of BV (Nugent scoring), sexually transmitted infections (multiplex PCR for Chlamydia trachomatis, Neisseria gonorrhoea, Trichomonas vaginalis, Mycoplasma genitalium) and candidiasis (Gram stain) in AGYW (n = 298) from Cape Town and Soweto. Cervicovaginal microbiota was determined by 16S pyrosequencing; 44 genital cytokines were measured by Luminex; and cervical T-cell activation/proliferation (CCR5, HLA-DR, CD38, Ki67) was measured by multiparametric flow cytometry. 90/298 (30.2%) AGYW were negative for BV, candidiasis and bacterial STIs. L. crispatus and L. iners were the dominant bacteria in cervicovaginal swabs, and the median vaginal pH was 4.7. AGYW with L. crispatus-dominant microbiota (42.4%) generally had the lowest cytokine concentrations compared to women with more diverse microbiota (34/44 significantly upregulated cytokines). Frequencies of CCR5+CD4+ T-cells co-expressing CD38 and HLA-DR correlated positively with interleukin (IL)-6, TNF-α, GRO-α, macrophage inflammatory protein (MIP)-1α, and IL-9. While endogenous oestrogen had an immune-dampening effect on IL-6, TNF-related apoptosis-inducing ligand (TRAIL) and IL-16, injectable hormone contraceptives (DMPA and Net-EN) were associated with significantly lower endogenous hormone concentrations (p<0.0001 for oestrogen and progesterone) and upregulation of 34/44 cytokines. Since genital inflammation and the presence of activated CD4+ T cells in the genital tract have been implicated in increased HIV risk in South African women, the observed high levels of genital cellular activation and cytokines from AGYW may point towards biological factors increasing HIV risk in this region., Competing Interests: The authors have declared that no competing interests exist.

Recent guidelines recommend antiretroviral therapy (ART) to be administered as early as possible during HIV-1 infection. Few studies addressed the immunological benefit of commencing ART during the acute phase of infection. We used mass cytometry to characterize blood CD4+ T cells from HIV-1-infected patients who initiated ART during acute or chronic phase of infection. Using this method, we analyzed a large number of markers on millions of individual immune cells. The results revealed that CD4+ T cell clusters with high expression of CD27, CD28, CD127, and CD44, whose function involves T cell migration to inflamed tissues and survival, are more abundant in healthy controls and patients initiating ART during the acute phase; on the contrary, CD4+ T cell clusters in patients initiating ART during the chronic phase had reduced expression of these markers. The results are suggestive of a better preserved immune function in HIV-1-infected patients initiating ART during acute infection.

Optimization of DNA vaccine delivery improves the potency of the immune response and is crucial to clinical success. Here, we inquired how such optimization impacts the magnitude of the response, its specificity and type. BALB/c mice were DNA-immunized with two model immunogens, HIV-1 protease and reverse transcriptase by intramuscular or intradermal injections with electroporation. DNA immunogens were co-delivered with DNA encoding luciferase. Delivery and expression were monitored by in vivo bioluminescence imaging (BLI). The endpoint immune responses were assessed by IFN-γ/IL-2 FluoroSpot, multiparametric flow cytometry and antibody ELISA. Expression and immunogenicity were compared in relation to the delivery route. Regardless of the route, protease generated mainly IFN-γ, and reverse transcriptase, IL-2 and antibody response. BLI of mice immunized with protease- or reverse transcriptase/reporter plasmid mixtures, demonstrated significant loss of luminescence over time. The rate of decline of luminescence strongly correlated with the magnitude of immunogen-specific response, and depended on the immunogenicity profile and the immunization route. In vitro and in vivo BLI-based assays demonstrated that intradermal delivery strongly improved the immunogenicity of protease, and to a lesser extent, of reverse transcriptase. Immune response polarization and epitope hierarchy were not affected. Thus, by changing delivery/immunogen expression sites, it is possible to modulate the magnitude, but not the type or fine specificity of the induced immune response., Competing Interests: The authors have declared that no competing interests exist.

DNA vaccines require a considerable enhancement of immunogenicity. Here, we optimized a prototype DNA vaccine against drug-resistant HIV-1 based on a weak Th2-immunogen, HIV-1 reverse transcriptase (RT). We designed expression-optimized genes encoding inactivated wild-type and drug-resistant RTs (RT-DNAs) and introduced them into mice by intradermal injections followed by electroporation. RT-DNAs were administered as single or double primes with or without cyclic-di-GMP, or as a prime followed by boost with RT-DNA mixed with a luciferase-encoding plasmid ("surrogate challenge"). Repeated primes improved cellular responses and broadened epitope specificity. Addition of cyclic-di-GMP induced a transient increase in IFN-γ production. The strongest anti-RT immune response was achieved in a prime-boost protocol with electroporation by short 100V pulses done using penetrating electrodes. The RT-specific response, dominated by CD4+ T-cells, targeted epitopes at aa 199-220 and aa 528-543. Drug-resistance mutations disrupted the epitope at aa 205-220, while the CTL epitope at aa 202-210 was not affected. Overall, multiparametric optimization of RT strengthened its Th2- performance. A rapid loss of RT/luciferase-expressing cells in the surrogate challenge experiment revealed a lytic potential of anti-RT response. Such lytic CD4+ response would be beneficial for an HIV vaccine due to its comparative insensitivity to immune escape.

Adolescents in Africa are at high risk for HIV infection, other sexually transmitted infections (STIs) and bacterial vaginosis (BV). Since behavior and burden of STIs/BV may influence HIV risk, behavioral risk factors and prevalence of STIs/BV were compared in HIV-seronegative adolescent females (n = 298; 16-22 years) from two South African communities (Soweto and Cape Town). STIs ( Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, herpes simplex virus (HSV)-1, HSV-2, Treponema pallidum, and Haemophilus ducreyi) were detected by multiplex polymerase chain reaction, human papillomavirus (HPV) by Roche Linear Array, and BV by Nugent scoring. Rates of BV (Nugent ≥7; 46.6%) and HPV (66.8%) were high in both communities. Prevalence of C. trachomatis and N. gonorrhoeae were >2-fold higher in Cape Town than Soweto (Chlamydia: 42% [62/149] versus 18% [26/148], p < 0.0001; gonorrhoea 11% [17/149] versus 5% [7/148], p = 0.05). Only 24% of adolescents with vaginal discharge-causing STIs or BV were symptomatic. In South African adolescents, clinical symptoms compatible with vaginal discharge syndrome had a sensitivity of 23% and specificity of 85% for the diagnosis of discharge-causing STI or BV. In a region with high HIV prevalence and incidence, >70% of young women with treatable conditions that could enhance HIV risk would have been missed because they lacked symptoms associated with syndromic management.

Persistence of minimal residual disease (MRD) after treatment for myeloma predicts inferior outcomes, but within MRD-positive patients there is great heterogeneity with both early and very late relapses. Among different MRD techniques, flow cytometry provides additional information about antigen expression on tumor cells, which could potentially contribute to stratify MRD-positive patients. We investigated the prognostic value of those antigens required to monitor MRD in 1265 newly diagnosed patients enrolled in the GEM2000, GEM2005MENOS65, GEM2005MAS65 and GEM2010MAS65 protocols. Overall, CD19 pos , CD27 neg , CD38 lo , CD45 pos , CD81 pos , CD117 neg and CD138 lo expression predicted inferior outcomes. Through principal component analysis, we found that simultaneous CD38 low CD81 pos CD117 neg expression emerged as the most powerful combination with independent prognostic value for progression-free survival (HR:1.69; P=0.002). This unique phenotypic profile retained prognostic value among MRD-positive patients. We then used next-generation flow to determine antigen stability throughout the course of the disease, and found that the expression of antigens required to monitor MRD is mostly stable from diagnosis to MRD stages, except for CD81 whose expression progressively increased from baseline to chemoresistant tumor cells (14 vs 28%). Altogether, we showed that the phenotypic profile of tumor cells provides additional prognostic information, and could be used to further predict risk of relapse among MRD-positive patients.

Abnormal immune activation and expansion of CD8+ T cells, especially of memory and effector phenotypes, take place during HIV-1 infection, and these abnormal features persist during administration of antiretroviral therapy (ART) to infected patients. The molecular mechanisms for CD8+ T-cell expansion remain poorly characterized. In this article, we review the literature addressing features of CD8+ T-cell immune pathology and present an integrated view on the mechanisms leading to abnormal CD8+ T-cell expansion during HIV-1 infection. The expression of molecules important for directing the homing of CD8+ T cells between the circulation and lymphoid tissues, in particular CCR5 and CXCR3, is increased in CD8+ T cells in circulation and in inflamed tissues during HIV-1 infection; these disturbances in the homing capacity of CD8+ T cells have been linked to increased CD8+ T-cell proliferation. The production of IL-15, a cytokine responsible for physiological proliferation of CD8+ T cells, is increased in lymphoid tissues during HIV-1 infection as result of microbial translocation and severe inflammation. IL-15, and additional inflammatory cytokines, may lead to deregulated proliferation of CD8+ T cells and explain the accumulation of CD8+ T cells in circulation. The decreased capacity of CD8+ T cells to localize to gut-associated lymphoid tissue also contributes to the accumulation of these cells in blood. Control of inflammation, through ART administration during primary HIV-1 infection or therapies aimed at controlling inflammation during HIV-1 infection, is pivotal to prevent abnormal expansion of CD8+ T cells during HIV-1 infection., (© 2018 The Association for the Publication of the Journal of Internal Medicine.)

In weakly spin-orbit coupled materials, the spin-selective nature of recombination can give rise to large magnetic-field effects, e.g. on the electro-luminescence of molecular semiconductors. Although silicon has weak spin-orbit coupling, observing spin-dependent recombination through magneto-electroluminescence is challenging: silicon's indirect band-gap causes an inefficient emission and it is difficult to separate spin-dependent phenomena from classical magneto-resistance effects. Here we overcome these challenges and measure magneto-electroluminescence in silicon light-emitting diodes fabricated via gas immersion laser doping. These devices allow us to achieve efficient emission while retaining a well-defined geometry, thus suppressing classical magnetoresistance effects to a few percent. We find that electroluminescence can be enhanced by up to 300% near room temperature in a seven Tesla magnetic field, showing that the control of the spin degree of freedom can have a strong impact on the efficiency of silicon LEDs.

During anti-retroviral therapy (ART) HIV-1 persists in cellular reservoirs, mostly represented by CD4+ memory T cells. Several approaches are currently being undertaken to develop a cure for HIV-1 infection through elimination (or reduction) of these reservoirs. Few studies have so far been conducted to assess the possibility of reducing the size of HIV-1 reservoirs through vaccination in virologically controlled HIV-1-infected children. We recently conducted a vaccination study with a combined hepatitis A virus (HAV) and hepatitis B virus (HBV) vaccine in 22 HIV-1-infected children. We assessed the size of the virus reservoir, measured as total HIV-1 DNA copies in blood cells, pre- and postvaccination. In addition, we investigated by immunostaining whether the frequencies of CD4+ and CD8+ T cells and parameters of immune activation and proliferation on these cells were modulated by vaccination. At 1 month from the last vaccination dose, we found that 20 out of 22 children mounted a serological response to HBV; a majority of children had antibodies against HAV at baseline. The number of HIV-1 DNA copies in blood at 1 month postvaccination was reduced in comparison to baseline although this reduction was not statistically significant. A significant reduction of HIV-1 DNA copies in blood following vaccination was found in 12 children. The frequencies of CD4+ (naïve, effector memory) and CD8+ (central memory) T-cell subpopulations changed following vaccinations and a reduction in the activation and proliferation pattern of these cells was also noticed. Multivariate linear regression analysis revealed that the frequency of CD8+ effector memory T cells prior to vaccination was strongly predictive of the reduction of HIV-1 DNA copies in blood following vaccination of the 22 HIV-1-infected children. The results of this study suggest a beneficial effect of vaccination to reduce the size of virus reservoir in HIV-1-infected children receiving ART. A reduced frequency of activated CD4+ cells and an increase in central memory CD8+ T cells were associated with this finding. Further studies should assess whether vaccination is a possible tool to reduce HIV-1 reservoirs.

HBV vaccine has 95% efficacy in children to prevent HBV infection and related cancer. We conducted a prospective study in HIV-1 infected children receiving ART (n = 49) and controls (n = 63) to assess humoral and cellular responses to HBV vaccine provided with three doses under an accelerated schedule of 4 weeks apart. At 1 month post-vaccination all children, except 4 HIV-1 infected, displayed protective antibody (ab) titers to HBV vaccine; ab titers were lower in infected children (P < 0.0001). Ab titers decreased (P < 0.0001) in both HIV-1 infected and control children at 6 months. The frequency of circulating Tfh (cTFh) cells was 20.3% for controls and 20.8% for infected children prior to vaccination and remained comparable post-vaccination. Cytokine expression by cTfh cells upon activation with HBV antigen was comparable in the two groups at baseline and 1 month post-vaccination. Higher plasma levels (P < 0.0001) of CXCL13 were found in infected children which correlated with cTfh cell frequency at baseline. In conclusion, a lower ab response to HBV vaccine was measured in HIV-1 infected children. The frequency and activation profile of cTfh cells was comparable in infected children and controls suggesting that cells other than Tfh cells are responsible for impaired ab response to HBV vaccine.

IL-7 was previously shown to upregulate the expression of molecules important for interaction of CD4+ T cells with B cells. It is poorly studied whether IL-7 has a role in the biology of T follicular helper (Tfh) cells and whether IL-7 dysregulates the expression of B-cell costimulatory molecules on Tfh cells. We review the literature and provide arguments in favor of IL-7 being involved in the biology of human Tfh cells. The CD127 IL-7 receptor is expressed on circulating Tfh and non-Tfh cells, and we show that IL-7, but not IL-6 or IL-21, upregulates the expression of CD70 and PD-1 on these cells. We conclude that IL-7, a cytokine whose level is elevated during HIV-1 infection, may have a role in increased expression of B cell costimulatory molecules on Tfh cells and lead to abnormal B cell differentiation.

Dendritic cells (DCs) are potent antigen-presenting cells that might play contradictory roles during HIV-1 infection, contributing not only to antiviral immunity but also to viral dissemination and immune evasion. Although DCs are characterized by enormous functional diversity, it has not been analyzed how differentially programmed DCs interact with HIV-1. We have previously described the reprogramming of DC development by endogenously produced lactic acid that accumulated in a cell culture density-dependent manner and provided a long-lasting anti-inflammatory signal to the cells. By exploiting this mechanism, we generated immunostimulatory DCs characterized by the production of TH1 polarizing and inflammatory mediators or, alternatively, suppressed DCs that produce IL-10 upon activation, and we tested the interaction of these DC types with different HIV-1 strains. Cytokine patterns were monitored in HIV-1-exposed DC cultures. Our results showed that DCs receiving suppressive developmental program strongly upregulated their capacity to produce the TH1 polarizing cytokine IL-12 and the inflammatory chemokines CCL2 and CCL7 upon interaction with HIV-1 strains IIIB and SF162. On the contrary, HIV-1 abolished cytokine production in the more inflammatory DC types. Preincubation of the cells with the HIV-1 proteins gp120 and Nef could inhibit IL-12 production irrespectively of the tested DC types, whereas MyD88- and TRIF-dependent signals stimulated IL-12 production in the suppressed DC type only. Rewiring of DC cytokines did not require DC infections or ligation of the HIV-1 receptor CD209. A third HIV-1 strain, BaL, could not modulate DC cytokines in a similar manner indicating that individual HIV-1 strains can differ in their capacity to influence DCs. Our results demonstrated that HIV-1 could not induce definite and invariable modulatory programs in DCs. Instead, interaction with the virus triggered different responses in different DC types. Thus, the outcome of DC-HIV-1 interactions might be highly variable, shaped by endogenous features of the cells and diversity of the virus.

Objective: The frequency of immature transitional B cells is increased in blood of HIV-1-infected individuals. We investigated whether HIV-1 infection affects expression and function of chemokine receptors important for egress of immature transitional B cells from bone marrow and migration to lymphoid organs., Design: This is a cross-sectional study analysing the migratory phenotype and function of immature transitional B cells in HIV-1-infected individuals, in relation to antiretroviral treatment and age., Methods: Frequency of blood immature transitional B cells and their phenotypic characteristics, including chemokine receptors and a maturation marker, were determined by immunostainings. Migratory capacities were studied in a migration assay., Results: The increased frequency of immature transitional B cells in untreated HIV-1 infection was normalized in patients receiving antiretroviral treatment; in our cohorts, age did not have an impact on the frequency of circulating immature transitional B cells. Immature transitional B cells from nontreated patients expressed low levels of CD21 molecule. We found an elevated frequency of CXCR3 and CXCR4 expressing immature transitional B cells in treated and nontreated patients. CXCR4 receptor was unresponsive to CXCL12 ligand in in-vitro migration and internalization assays. In addition, CXCR5 expression was downregulated on immature transitional B cells from infected patients, and these cells migrated poorly in response to CXCR5 ligand., Conclusion: Circulating immature transitional B cells from HIV-1-infected patients are not fully mature, probably due to premature egress from bone marrow; these cells showed a phenotype which could impair entry into secondary lymphoid organs. Changes in migratory capacity of immature transitional B cells may affect B-cell maturation during HIV-1 infection.

Background: Follicular dendritic cells (FDCs) are important components in the organization of germinal centers in lymphoid tissue where, following antigen presentation, B cells differentiate into memory B cells. The possibility of establishing primary cell lines from FDCs isolated from lymphoid tissue paved the way for characterization of FDC biological properties. We exposed primary FDC cell lines to HIV-1 strains in vitro and studied changes in the chemo-attractive properties of FDCs and release of inflammatory cytokines., Results: FDC lines expressed several known and putative HIV-1 receptors; viral genome was amplified in HIV-1 exposed FDCs which released low levels of p24 HIV-1 protein in culture supernatants, but were not definitely proven to be productively infected. Exposure of FDCs to HIV-1 strains did not change the expression of markers used to characterize these cells. HIV-1 exposed FDCs, however, changed the expression of chemo-attractants involved in cell recruitment at inflammatory sites and increased the production of several inflammatory cytokines. The inflammatory milieu created upon HIV-1 exposure of FDCs led to impaired B cell survival in vitro and reduced Ig production., Conclusions: FDC lines exposed to different HIV-1 strains, although not able to support productive HIV-1 replication, show an increased production of inflammatory cytokines. Our in vitro model of interactions between HIV-1 exposed FDC lines and B cells suggest that exposure of FDCs to HIV-1 in vivo can contribute to inflammation within germinal centers and that this pathological event may impair B cell survival and contribute to impaired B cell responses during HIV-1 infection.

Early initiation of antiretroviral therapy (ART) is becoming a common clinical practice according to current guidelines recommending treatment to all HIV-1-infected patients. However, it is not known whether ART initiated during the early phase of infection prevents the establishment of abnormal phenotypic features previously reported in CD4+ and CD8+T cells during chronic HIV-1 infection. In this cross-sectional study, blood specimens were obtained from 17 HIV-1-infected patients who began ART treatment shortly after infection (early ART [EA]), 17 age-matched HIV-1-infected patients who started ART during chronic phase of infection (late ART [LA]), and 25 age-matched non-HIV-1-infected controls. At collection of specimens, patients in EA and LA groups had received ART for comparable periods of time. Total HIV-1 DNA was measured in white blood cells by quantitative PCR. The concentration of 9 inflammatory parameters and 1 marker of fibrosis, including sCD14 and β-2 microglobulin, was measured in plasma. Furthermore, expression of markers of abnormal immune activation (human leukocyte antigen - antigen D related [HLA-DR] and CD38), exhaustion (programmed death 1, CD28, CD57) and terminal differentiation (CD127) was measured on CD4+ and CD8+T cells. T-cell proliferation was measured through Ki67 expression. The copies of total HIV-1 DNA in blood were significantly lower (P = 0.009) in EA compared with that in LA group. Only the expression of HLA-DR on naïve CD4+ T cells distinguished EA from LA, whereas expression of 3 surface markers distinguished T-cell populations of HIV-1-infected patients from controls. These included HLA-DR distinguishing CD4+ T cells from EA compared with controls, and also CD38 and CD127 on CD4+ and CD8+ T cells, respectively, distinguishing both groups of patients from controls. The sCD14 levels were significantly higher in EA patients, and β-2 microglobulin levels were higher in LA group compared with that in controls. Our results demonstrate an equivalent abnormal expression of activation (HLA-DR and CD38 on CD4+ T cells) and terminal differentiation (CD127 on CD8+ T cells) markers in T cells from both EA and LA patients. The size of total HIV-1 DNA copies in blood of EA was lower compared with LA patients. These findings suggest that some abnormalities taking place in the T-cell compartment during primary HIV-1 infection may not be corrected by early ART.

Objective: CD70 molecules expressed by activated T cells provide potent B cell stimulatory signals. We hypothesized that an altered CD70 expression might contribute to B cell abnormalities during HIV-1 infection., Design: CD70 expression and the functional and migratory properties of the CD4CD70 T lymphocytes were analyzed in HIV-1-infected patients and in humanized mice. Correlations were tested between CD70 expression and features of B-cell activation, apoptosis sensitivity and functional exhaustion., Methods: CD4CD70 T cells were analyzed in cohorts of CD4 T-cell lymphopenic, viremic or nonlymphopenic, nonviremic HIV-1-infected patients and in noninfected individuals. CD70 upregulation was also followed in HIV-1-infected humanized mice. CD38, CD95, LAIR1 and PD-1 expressions were monitored on B-cell subpopulations, Ki67 was assessed to estimate B-cell proliferation and antibody levels were measured in plasma., Results: Blood CD4CD70 T-cell frequencies increased in response to CD4 T-cell depletion or high viremia levels as a possible consequence of increased activation and proliferation in this subset. CD4CD70 T cells produced T-helper 1-type cytokines and expressed chemokine receptors mobilizing toward sites of inflammation but not to lymphoid follicles. High CD70 expression was observed in HIV-1-infected humanized mice at extrafollicular sites (peritoneum, bone-marrow). CD4CD70 T-cell frequencies correlated with the expression of the activation marker CD38 and the death receptor CD95 on various memory B-cell subsets, with B-cell proliferation and with plasma IgG levels., Conclusions: CD4CD70 T cells may contribute to B cell hyperactivation and accelerated memory B-cell turnover during HIV-1 infection.

T follicular helper (Tfh) cells are important components in development of specific humoral immune responses; whether the number and biology of Tfh cells is impaired in HIV-1-infected children is not yet studied.The frequency, phenotype, and function of Tfh cells and B cells were determined in blood of HIV-1-infected children receiving antiretroviral therapy (ART) and age-matched controls. Flow cytometry was used to characterize the frequency of Tfh cells and B cell subsets. Cytokine expression was measured after in vitro activation of Tfh cells.A reduced frequency of memory Tfh cells (P < 0.001) was identified in HIV-1-infected children and, on these cells, a reduced expression of programmed death-1 (PD-1) and inducible T cell costimulator (ICOS) (P < 0.001 and P < 0.01). Upon activation, the capacity of Tfh cells to express IL-4, an important cytokine for B cell function, was impaired in HIV-1-infected children.B cell subpopulations in HIV-1-infected children displayed significant differences from the control group: the frequency of resting memory (RM) B cells was reduced (P < 0.01) whereas the frequency of exhausted memory B cells increased (P < 0.001). Interestingly, the decline of RM cells correlated with the reduction of memory Tfh cells (P = 0.02).Our study shows that function and phenotype of Tfh cells, pivotal cells for establishment of adaptive B cell responses, are impaired during HIV-1 infection in children. A consistent reduction of memory Tfh cells is associated with declined frequencies of RM B cells, creating a novel link between dysfunctional features of these cell types, major players in establishment of humoral immunity.

Ocular Adnexal Lymphomas are the first cause of primary ocular malignancies, and among them the most common are MALT Ocular Adnexal Lymphomas. Recently systemic immunotherapy with anti-CD20 monoclonal antibody has been investigated as first-line treatment; however, the optimal management for MALT Ocular Adnexal Lymphomas is still unknown. The present study evaluated retrospectively the outcome of seven consecutive patients with primary MALT Ocular Adnexal Lymphomas, of whom six were treated with single agent Rituximab. All patients received 6 cycles of Rituximab 375 mg/mq every 3 weeks intravenously. The overall response rate was 100%; four patients (67%) achieved a Complete Remission, and two (33%) achieved a partial response. In four patients an additional Rituximab maintenance every 2-3 months was given for two years. After a median follow-up of 29 months (range 8-34), no recurrences were observed, without of therapy- or disease-related severe adverse events. None of the patients needed additional radiotherapy or other treatments. Rituximab as a single agent is highly effective and tolerable in first-line treatment of primary MALT Ocular adnexal Lymphomas. Furthermore, durable responses are achievable with the same-agent maintenance. Rituximab can be considered the agent of choice in the management of an indolent disease in whom the "quality of life" matter is of primary importance.

Background: Aged individuals respond poorly to vaccination and have a higher risk of contracting infections in comparison to younger individuals; whether age impacts on the composition and function of B cell subpopulations relevant for immune responses is still controversial. It is also not known whether increased age during HIV-1 infection further synergizes with the virus to alter B cell subpopulations. In view of the increased number of HIV-1 infected patients living to high age as a result of anti-retroviral treatment this is an important issue to clarify., Results: In this work we evaluated the distribution of B cell subpopulations in young and aged healthy individuals and HIV-1 infected patients, treated and naïve to treatment. B cell populations were characterized for the expression of inhibitory molecules (PD-1 and FcRL4) and activation markers (CD25 and CD69); the capacity of B cells to respond to activation signals through up-regulation of IL-6 expression was also evaluated. Increased frequencies of activated and tissue-like memory B cells occurring during HIV-1 infection are corrected by prolonged ART therapy. Our findings also reveal that, in spite of prolonged treatment, resting memory B cells in both young and aged HIV-1 infected patients are reduced in number, and all memory B cell subsets show a low level of expression of the activation marker CD25., Conclusions: The results of our study show that resting memory B cells in ART-treated young and aged HIV-1 infected patients are reduced in number and memory B cell subsets exhibit low expression of the activation marker CD25. Aging per se in the HIV-1 infected population does not worsen impairments initiated by HIV-1 in the memory B cell populations of young individuals.