Release of HIV-1 particles from macrophages is promoted by an anchored cytoskeleton and driven by mechanical constraints.

A feature of HIV-1 replication in macrophages is that viral assembly occurs at the limiting membrane of a compartment often named the virus-containing compartment (VCC). Assembled virions accumulate in the lumen of the VCC, from where they can be released into the extracellular medium via mechanisms that remain poorly described. Here, we show that the actin cytoskeleton contributes to this process by performing experiments combining pharmacological and mechanical perturbations with imaging and biochemical analysis. We found that jasplakinolide inhibited HIV-1 release from macrophages and led to scattering of the compartment. Concomitantly, both the integrin CD18 (ß2-integrin) and the phosphorylated form of PYK2 (also known as PTK2B) were displaced away from the VCC. Inhibition of PYK2 activity promoted retention of viral particles in VCCs that lost their connections to the surface. Finally, in infected macrophages undergoing frustrated phagocytosis, VCCs rapidly trafficked to the basal membrane and released their viral content, in a manner dependent on their association with the actin cytoskeleton. These results highlight that the trafficking of VCCs and virus release are intimately linked to a reorganization of the macrophage actin cytoskeleton that can be modulated by external physical cues.

Antileishmanial Drugs Modulate IL-12 Expression and Inflammasome Activation in Primary Human Cells.

Leishmaniases are neglected tropical diseases. The treatment of leishmaniasis relies exclusively on chemotherapy including amphotericin B (AmB), miltefosine (hexadecylphosphocholine), and pentamidine. Besides the fact that these molecules are harmful for patients, little is known about the impact of such antileishmanial drugs on primary human cells in relation to immune function. The present study demonstrates that all antileishmanial drugs inhibit CD4 and CD8 T cell proliferation at the doses that are not related to increased cell death. Our results highlight that antileishmanial drugs have an impact on monocytes by altering the expression of IL-12 induced by LPS, whereas only AmB induced IL-10 secretion; both cytokines are essential in regulating Th1 cell-mediated immunity. Interestingly, IL-12 and anti-IL-10 Abs improved T cell proliferation inhibited by AmB. Furthermore, our results show that in contrast to hexadecylphosphocholine and pentamidine, AmB induced gene expression of the inflammasome pathway. Thus, AmB induced IL-1ß and IL-18 secretions, which are reduced by specific inhibitors of caspase activation (Q-VD) and NLRP3 activation (MCC950). Our results reveal previously underestimated effects of antileishmanial drugs on primary human cells.

Prognostic Factors in Penile Cancer: Should We Avoid Inguinal Lymph Node Staging?

INTRODUCTION: Penile cancer (PC) is a rare neoplasm, mostly in developed countries. Herewith, we evaluate the main prognostic factors of patients with PC undergoing surgery. METHODS: This is a retrospective analysis of prognostic factors of overall survival in 65 patients with PC treated at a tertiary referral center over the last 15 years (2004-2018). RESULTS: Almost half (48%) of the patients were diagnosed at an advanced local stage pT3/4. Thirty-eight (58%) patients underwent inguinal lymphadenectomy, and 25 (66%) were negative for lymph node (LN) invasion. Overall survival was 80% at a median follow-up of 31 months. In the multivariate analysis, the main factors of poor prognosis were nodal staging (pN) (p = 0.008) and perineural invasion (p = 0.023). The presence of LN metastasis and perineural invasion in the primary tumor increased the risk of death by 29 (hazard ratio 29.0, 95% confidence interval 2.4-354.2) and 13 (hazard ratio 12.7, 95% confidence interval 1.4-112.0) times, respectively. DISCUSSION/CONCLUSION: Late diagnosis of PC has a negative impact on overall survival, as nodal invasion correlates with survival. Despite the high number of negative inguinal lymphadenectomy, we continue to advocate aggressive surgical treatment of this disease due to the poor prognosis associated with LN metastasis.

Sensing of HIV-1 Entry Triggers a Type I Interferon Response in Human Primary Macrophages.

Along with CD4+ T lymphocytes, macrophages are a major cellular source of HIV-1 replication and a potential viral reservoir. Following entry and reverse transcription in macrophages, cloaking of the viral cDNA by the HIV-1 capsid limits its cytosolic detection, enabling efficient replication. However, whether incoming HIV-1 particles are sensed by macrophages prior to reverse transcription remains unclear. Here, we show that HIV-1 triggers a broad expression of interferon (IFN)-stimulated genes (ISG) in monocyte-derived macrophages within a few hours after infection. This response does not require viral reverse transcription or the presence of HIV-1 RNA within particles, but viral fusion is essential. This response is elicited by viruses carrying different envelope proteins and thus different receptors to proceed for viral entry. Expression of ISG in response to viral entry requires TBK1 activity and type I IFNs signaling. Remarkably, the ISG response is transient but affects subsequent viral spread. Together, our results shed light on an early step of HIV-1 sensing by macrophages at the level of entry, which confers an early protection through type I IFN signaling and has potential implications in controlling the infection.IMPORTANCE HIV infection is restricted to T lymphocytes and macrophages. HIV-1-infected macrophages are found in many tissues of infected patients, even under antiretroviral therapy, and are considered a viral reservoir. How HIV-1 is detected and what type of responses are elicited upon sensing remain in great part elusive. The kinetics and localization of the production of cytokines such as interferons in response to HIV is of critical importance to understanding how the infection and the immune response are established. Our study provides evidence that macrophages can detect HIV-1 as soon as it enters the cell. Interestingly, this sensing is independent of the presence of viral nucleic acids within the particles but requires their fusion with the macrophages. This triggers a low interferon response, which activates an antiviral program protecting cells against further viral challenge and thus potentially limiting the spread of the infection.

Correction: Early Loss of Splenic Tfh Cells in SIV-Infected Rhesus Macaques.

[This corrects the article DOI: 10.1371/journal.ppat.1005287.].

Early Loss of Splenic Tfh Cells in SIV-Infected Rhesus Macaques.

Follicular T helper cells (Tfh), a subset of CD4 T lymphocytes, provide crucial help to B cells in the production of antigen-specific antibodies. Although several studies have analyzed the dynamics of Tfh cells in peripheral blood and lymph nodes (LNs) during Aids, none has yet addressed the impact of SIV infection on the dynamics of Tfh cells in the spleen, the primary organ of B cell activation. We show here a significant decrease in splenic Tfh cells in SIVmac251-infected rhesus macaques (RMs) during the acute phase of infection, which persists thereafter. This profound loss is associated with lack of sustained expression of the Tfh-defining transcription factors, Bcl-6 and c-Maf but with higher expression of the repressors KLF2 and Foxo1. In this context of Tfh abortive differentiation and loss, we found decreased percentages of memory B cell subsets and lower titers of SIV-specific IgG. We further demonstrate a drastic remodeling of the lymphoid architecture of the spleen and LNs, which disrupts the crucial cell-cell interactions necessary to maintain memory B cells and Tfh cells. Finally, our data demonstrated the early infection of Tfh cells. Paradoxically, the frequencies of SIV DNA were higher in splenic Tfh cells of RMs progressing more slowly suggesting sanctuaries for SIV in the spleen. Our findings provide important information regarding the impact of HIV/SIV infection on Tfh cells, and provide new clues for future vaccine strategies.

Leishmania infantum modulates host macrophage mitochondrial metabolism by hijacking the SIRT1-AMPK axis.

Metabolic manipulation of host cells by intracellular pathogens is currently recognized to play an important role in the pathology of infection. Nevertheless, little information is available regarding mitochondrial energy metabolism in Leishmania infected macrophages. Here, we demonstrate that during L. infantum infection, macrophages switch from an early glycolytic metabolism to an oxidative phosphorylation, and this metabolic deviation requires SIRT1 and LKB1/AMPK. SIRT1 or LBK1 deficient macrophages infected with L. infantum failed to activate AMPK and up-regulate its targets such as Slc2a4 and Ppargc1a, which are essential for parasite growth. As a result, impairment of metabolic switch caused by SIRT1 or AMPK deficiency reduces parasite load in vitro and in vivo. Overall, our work demonstrates the importance of SIRT1 and AMPK energetic sensors for parasite intracellular survival and proliferation, highlighting the modulation of these proteins as potential therapeutic targets for the treatment of leishmaniasis.

Abortive T follicular helper development is associated with a defective humoral response in Leishmania infantum-infected macaques.

Leishmania infantum causes a chronic infectious disease named visceral leishmaniasis (VL). We employed a non-human primate model to monitor immune parameters over time and gain new insights into the disease. Rhesus macaques were infected with L. infantum and the T helper and B cell immunological profiles characterized during acute and chronic phases of infection. Parasite detection in visceral compartments during the acute phase was associated with differentiation of effector memory CD4 T cells and increased levels of Th1 transcripts. At the chronic phase, parasites colonized novel lymphoid niches concomitant with increased expression of IL10. Despite the occurrence of hypergammaglobulinemia, the production of parasite-specific IgG was poor, being confined to the acute phase and positively correlated with the frequency of an activated memory splenic B cell population. We noticed the expansion of a splenic CD4 T cell population expressing CXCR5 and Bcl-6 during acute infection that was associated with the differentiation of the activated memory B cell population. Moreover, the number of splenic germinal centers peaked at one month after infection, hence paralleling the production of specific IgG. However, at chronic infection these populations contracted impacting the production of parasite-specific IgG. Our study provides new insights into the immune events taking place in a physiologically relevant host and a mechanistic basis for the inefficient humoral response during VL.

DRAM triggers lysosomal membrane permeabilization and cell death in CD4(+) T cells infected with HIV.

Productive HIV infection of CD4(+) T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells.

Complications after surgical correction of penile fractures-Is there a clinical impact?

BACKGROUND: Surgical exploration and closure of the tunica albuginea is the recommended treatment for penile fractures. The recovery of sexual function is the main result that surgeons and patients pursue. OBJECTIVE: We sought to evaluate the sexual health effects of a surgically corrected penile fracture. Secondarily, we sought to identify risk factors that may influence long-term sexual function and their effects on genital body image satisfaction. METHODS: A retrospective analysis of patients who underwent surgical correction of penile fractures between 2007 and 2022 in a tertiary center was performed. Lesion characteristics, weeks until the resumption of sexual activity, and post-operative sexual function were recorded. The presence of glans hypoesthesia, penile deformation, penile nodule palpation, and self-satisfaction with body image were assessed. RESULTS: Sixty-nine patients with a mean age of 42.30 ± 12.98 years and a median follow-up of 70 (20-134) months were identified. Sexual intercourse was recorded as a percentage. Penile deformation was the most common complication, appearing in 14.5% of patients, erectile dysfunction in 5.8%, penile nodules in 4.3%, and glans hypoesthesia in 2.9%. The median post-operative International Index of Erectile Function-5 was 24 (21.5-24). Self-satisfaction with body image had a median of 9 and was negatively associated with bilateral lesions and penile deformation. DISCUSSION AND CONCLUSION: Distal fractures could be linked to erectile dysfunction and glans hypoesthesia. Surgical correction of penile fractures shows positive functional and self-reported outcomes, and the potential andrological complications rarely necessitate specific treatment.

Eighteen years of experience in laparoscopic implantation of artificial urinary sphincter in women with intrinsic sphincter deficiency.

INTRODUCTION AND OBJECTIVES: Artificial urinary sphincter (AUS) is a treatment option for women with stress urinary incontinence (SUI) after failure of previous surgery or as a primary procedure in severe intrinsic sphincter deficiency (ISD). The aim of the study was to assess the long-term efficacy and risk factors for surgical revision and definitive explantation of AUS laparoscopic implantation in female patients. METHODS: A retrospective review of all women submitted to AUS implantation between April 2005 and March 2023 was conducted. The AUS was implanted via transperitoneal laparoscopic approach, by two experienced surgeons. The primary endpoint was postoperative continence. Continence was defined as no leakage and no pad usage or leakage and/or pad usage with no impact on social life and failure as leakage and/or pad usage impacting social life. As secondary outcomes, clinical predictive factors for AUS revision and definitive explantation were evaluated. RESULTS: In the last 18 years, females with a mean age of 68±12 years-old were submitted to laparoscopic implantation of AUS. Early overall complication rate was 16%, but only one case was Clavien-Dindo ≥3. After a median follow-up of 67 months, 22.2% of the patients needed a device revision, the majority due to mechanical device dysfunction. AUS definitive explantation was performed in 16%, mainly due to urethral/vaginal erosion (9.9%) and infection (6.2%). Patients with age ≥70 years and follow-up ≥10 years significantly predisposed for device revision. At the time of the last follow-up, 72% of the patients were keeping the urinary continency. CONCLUSIONS: Laparoscopic AUS implantation in females is an effective treatment for SUI due to ISD. Meanwhile, adequate patient selection, multidisciplinary evaluation and careful expectation management are essential to achieving good results, concerning their significant complication rate.

Prostatic Fossa Pseudoaneurysm After Robot-Assisted Radical Prostatectomy (RARP): A Case Report.

BACKGROUND RARP is an established procedure in treatment of localized prostate cancer. Hemorrhagic complications in the postoperative period are rare, but sometimes life-threatening. Adequate monitoring and prompt intervention in these unusual scenarios rely on clinical judgement and blood and imaging studies. Prostatic fossa pseudoaneurysm formation after RARP is very rare and its etiology is not well known; it may be related to small vessel trauma. It becomes apparent with the development of hematuria 1-6 weeks after surgery. CASE REPORT A 58-year-old man underwent RARP with extended lymph node dissection for intermediate-risk prostate cancer, with bilateral preservation of neurovascular bundles and puboprostatic ligaments. He was discharged on day 2 without complications. In the following 4 weeks he came to the Emergency Department 3 times with hematuria and acute urinary retention. Four weeks after surgery, a pelvic CT angiogram showed a 20-mm pseudoaneurysm in the prostatic fossa, which was embolized by percutaneous angiography, with resolution of symptoms. He was discharged soon thereafter. CONCLUSIONS This case study describes a patient with prostatic fossa pseudoaneurysm after RARP. It was diagnosed 1 month after surgery and effectively managed by percutaneous embolization. Despite being a very rare condition, it must be kept in mind, especially when postoperative hematuria develops 1-6 weeks after surgery. Use of a management algorithm including serial blood tests, CT angiogram, and percutaneous angiography can lead to early detection and avoid life-threatening hemorrhage and overall postoperative morbidity.

Development and Study of a New Silane Based Polyurethane Hybrid Flexible Adhesive-Part 1: Mechanical Characterization.

The need for more sustainable adhesive formulations has led to the use of silane-based adhesives in different industrial sectors, such as the automotive industry. In this work, the mechanical properties of a dual cure two-component prototype adhesive which combined silylated polyurethane resin (SPUR) with standard epoxy resin was characterized under quasi-static conditions. The characterization process consisted of tensile bulk testing, to determine the Young's modulus, the tensile strength and the tensile strain to failure. The shear stiffness and shear strength were measured by performing a thick adherend shear test. The in-plane strain field was obtained using a digital image correlation method. Double-cantilever beam and mixed-mode tests were performed to assess the fracture toughness under pure modes. The prototype adhesive showed promising but lower properties compared to commercial solutions. Furthermore, the adhesive was modified via the addition of three different resin modifier additives and characterized via measuring the shear and tensile properties, but no enhancements were found. Finally, the adhesive was formulated with three different SPUR viscosities. The critical energy release rate analysis showed an optimum value for the medium viscosity SPUR adhesive.

The Development and Study of a New Silylated Polyurethane-Based Flexible Adhesive-Part 2: Joint Testing and Numerical Modelling.

The need for more sustainable adhesive formulations has presented the possibility of using silane-based adhesives in the automotive industry. In this work, a dual-cure two-component silylated polyurethane resin (SPUR) adhesive was tested in single-lap joints, to assess in-joint behaviour at room temperature under quasi-static conditions for aluminium substrates. The effect of two different overlap lengths, 25 and 50 mm, was also considered. A numerical model was built using cohesive zone modelling in finite element software, to reproduce the mechanical behaviour of the joint. The model was fed with data experimentally withdrawn from the first part of this paper. A triangular-shaped cohesive zone model (CZM) law was chosen as the adhesive behaviour was highly elastic and lacked yielding phenomena. The experimental results served as the base for the numerical validation, allowing accurate CZM parameters to be successfully determined.

Undergraduate teaching of urology: Quo vadis?

BACKGROUND: The undergraduate teaching of urology is not uniform in the various European medical schools and even absent in some of them, despite the widespread adoption of the Bologna process, which advocates a standardization and harmonization of medical education. Our aim was to evaluate the perception of junior doctors about the undergraduate teaching of Urology and the exposure to the specialty of Urology in undergraduate education in Portuguese medical schools. METHODS: A questionnaire was emailed to all physicians who first enrolled in the Board of Portuguese Doctors in 2017 and 2018. The questionnaire consisted of several questions about specialty exposure, pathology, and basic urological procedures. A database for statistical analysis was created. RESULTS: One hundred and eighty-six answers were considered valid. Although almost all participant physicians attribute considerable importance to Urology specialty, most find their exposure to urological pathology and basic urological procedures to be inappropriate in medical school. Urinary lithiasis and lower urinary tract symptoms are the subjects on which doctors feel most prepared after graduating. Interestingly, 63.4% of doctors consider that the education they had in college was preponderant in choosing their specialty. CONCLUSIONS: The teaching of Urology in Portuguese Medical Schools is considered by junior doctors as inadequate, not reflecting the importance of this specialty in the clinical practice. These results are like those found in other countries. A reflection and consequent change of the teaching paradigm is necessary, namely at the practical teaching level.

Non-human primates and Leishmania immunity.

In the context of infectious diseases, non-human primates (NHP) provide the best animal models of human diseases due to the close phylogenetic relationship and the similar physiology and anatomical systems. Herein, we summarized the contribution of NHP models for understanding the immunity to leishmaniases, which are a group of diseases caused by infection with protozoan parasites of the genus Leishmania and classified as one of the neglected tropical diseases.

HIV-2-Infected Macrophages Produce and Accumulate Poorly Infectious Viral Particles.

A significant proportion of HIV-2-infected patients exhibit natural virological control that is generally absent from HIV-1-infected patients. Along with CD4+ T cells, HIV-1 targets macrophages which may contribute to viral spreading and the latent reservoir. We have studied the relationship between macrophages and HIV-2, focusing on post-entry steps. HIV-2-infected monocyte-derived macrophages (MDMs) produced substantial amounts of viral particles that were largely harbored intracellularly. New viruses assembled at the limiting membrane of internal compartments similar to virus-containing compartments (VCCs) described for HIV-1. VCCs from MDMs infected with either virus shared protein composition and morphology. Strikingly, HIV-2 Gag was mostly absent from the cytosol and almost exclusively localized to the VCCs, whereas HIV-1 Gag was distributed in both locations. Ultrastructural analyses of HIV-2-infected MDMs revealed the presence of numerous VCCs containing both immature and mature particles in the lumen. HIV-2 particles produced de novo by MDMs were poorly infectious in reporter cells and in transmission to activated T cells through a process that appeared independent of BST2 restriction. Rather than being involved in viral spreading, HIV-2-infected macrophages may represent a cell-associated source of viral antigens that can participate in the immune control of HIV-2 infection.

Transcriptional Analysis of Human Skin Lesions Identifies Tryptophan-2,3-Deoxygenase as a Restriction Factor for Cutaneous Leishmania.

Disease manifestation after infection with cutaneous Leishmania species is the result of a complex interplay of diverse factors, including the immune status of the host, the infecting parasite species, or the parasite load at the lesion site. Understanding how these factors impact on the pathology of cutaneous leishmaniasis (CL) may provide new targets to manage the infection and improve clinical outcome. We quantified the relative expression of 170 genes involved in a diverse range of biological processes, in the skin biopsies from patients afflicted with CL caused by infection with either L. major or L. tropica. As compared to healthy skin, CL lesions bear elevated levels of transcripts involved in the immune response, and conversely, present a significant downregulation in the expression of genes involved in epidermal integrity and arginine or fatty acid metabolism. The expression of transcripts encoding for cytotoxic mediators and chemokines in lesions was inversely correlated with the expression of genes involved in epidermal integrity, suggesting that cytotoxicity is a major mediator of CL pathology. When comparing the transcriptional profiles of lesions caused by either L. major or L. tropica, we found them to be very similar, the later presenting an aggravated inflammatory/cytotoxic profile. Finally, we identified genes positively correlated with the parasite load in lesions. Among others, these included Th2 or regulatory cytokines, such as IL4 or IL10. Remarkably, a single gene among our dataset, encoding for tryptophan-2,3-deoxygenase (TDO), presented a negative correlation with the parasite load, suggesting that its expression may restrict parasite numbers in lesions. In agreement, treatment of macrophages infected with L. major in vitro with a TDO inhibitor led to an increase in parasite transcripts. Our work provides new insights into the factors that impact CL pathology and identifies TDO as a restriction factor for cutaneous Leishmania.

Mucosal T follicular helper cells in SIV-infected rhesus macaques: contributing role of IL-27.

Mesenteric lymph nodes (MLNs), that drain the large and small intestine, are critical sites for the induction of oral tolerance. Although depletion of CD4 T cells in the intestinal lamina propria is a hallmark of HIV infection, CD4 T cell dynamics in MLNs is less known due to the lack of accessibility to these LNs. We demonstrate the early loss of memory CD4 T cells, including T follicular helper cells (Tfh) and a remodeling of MLN architecture in SIV-infected rhesus macaques (RMs). Along with the loss of Tfh cells, we observe the loss of memory B cells and of germinal center B cells. Tfh cells display a Th1 profile with increased levels of the transcription factors that negatively impact on Tfh differentiation and of Stat5 phosphorylation. MLNs of SIV-infected RMs display lower mRNA transcripts encoding for IL-12, IL-23, and IL-35, whereas those coding for IL-27 are not impaired in MLNs. In vitro, IL-27 negatively impacts on Tfh cells and recapitulates the profile observed in SIV-infected RMs. Therefore, early defects of memory CD4 T cells, as well of Tfh cells in MLNs, which play a central role in regulating the mucosal immune response, may have major implications for Aids.