Improvement of sperm morphology after surgical varicocele repair.

BACKGROUND: A causative relationship between varicocele and impairment of semen quality has been largely investigated in the context of male infertility, although its clinical benefit remains controversial. OBJECTIVE: To investigate the effect of varicocele correction on detailed morphologic microscopic semen parameters in a large homogeneous cohort of patients and to evaluate which factors could predict semen improvement after the surgical treatment. MATERIALS AND METHODS: An observational, retrospective cohort study was carried out including all patients undergoing surgical treatment for varicocele from September 2011 to March 2020 in the same clinical centre. Enrolled males performed at least one semen analysis before and one after surgical varicocele correction. Primary outcome was the detailed morphologic microscopic sperm evaluation. Secondary outcomes were conventional semen analyses. RESULTS: A total of 121 males (mean age 24.6 ± 6.1 years) were enrolled. Using detailed morphologic microscopic sperm evaluation, a significant morphological improvement was recorded, with a reduction in head and tail abnormalities. Moreover, a significant increase in sperm concentration (p = 0.015) and percentage of progressive and total motility (p = 0.022 and p = 0.039) were observed after surgery. The multivariate logistic analysis identified the ultrasonography varicocele degree before surgery as a main predictor of the sperm concentration improvement (p = 0.016), with the highest improvement for varicocele of I and II degree. DISCUSSION: For the first time, the detailed morphologic microscopic sperm evaluation highlights a relevant reduction in sperm abnormalities after varicocele surgery, showing its potential application in clinical practice.

Daratumumab induces mechanisms of immune activation through CD38+ NK cell targeting.

Daratumumab (Dara), a multiple myeloma (MM) therapy, is an antibody against the surface receptor CD38, which is expressed not only on plasma cells but also on NK cells and monocytes. Correlative data have highlighted the immune-modulatory role of Dara, despite the paradoxical observation that Dara regimens decrease the frequency of total NK cells. Here we show that, despite this reduction, NK cells play a pivotal role in Dara anti-MM activity. CD38 on NK cells is essential for Dara-induced immune modulation, and its expression is restricted to NK cells with effector function. We also show that Dara induces rapid CD38 protein degradation associated with NK cell activation, leaving an activated CD38-negative NK cell population. CD38+ NK cell targeting by Dara also promotes monocyte activation, inducing an increase in T-cell costimulatory molecules (CD86/80) and enhancing anti-MM phagocytosis activity ex vivo and in vivo. In support of Dara's immunomodulating role, we show that MM patients that discontinued Dara therapy because of progression maintain targetable unmutated surface CD38 expression on their MM cells, but retain effector cells with impaired cellular immune function. In summary, we report that CD38+ NK cells may be an unexplored therapeutic target for priming the immune system of MM patients.

MiR-16 regulates crosstalk in NF-κB tolerogenic inflammatory signaling between myeloma cells and bone marrow macrophages.

High levels of circulating miR-16 in the serum of multiple myeloma (MM) patients are independently associated with longer survival. Although the tumor suppressor function of intracellular miR-16 in MM plasma cells (PCs) has been elucidated, its extracellular role in maintaining a nonsupportive cancer microenvironment has not been fully explored. Here, we show that miR-16 is abundantly released by MM cells through extracellular vesicles (EVs) and that differences in its intracellular expression as associated with chromosome 13 deletion (Del13) are correlated to extracellular miR-16 levels. We also demonstrate that EVs isolated from MM patients and from the conditioned media of MM-PCs carrying Del13 more strongly differentiate circulating monocytes to M2-tumor supportive macrophages (TAMs), compared with MM-PCs without this chromosomal aberration. Mechanistically, our data show that miR-16 directly targets the IKKα/ß complex of the NF-κB canonical pathway, which is critical not only in supporting MM cell growth, but also in polarizing macrophages toward an M2 phenotype. By using a miR-15a-16-1-KO mouse model, we found that loss of the miR-16 cluster supports polarization to M2 macrophages. Finally, we demonstrate the therapeutic benefit of miR-16 overexpression in potentiating the anti-MM activity by a proteasome inhibitor in the presence of MM-resident bone marrow TAM.

The Bcl-2 inhibitor venetoclax inhibits Nrf2 antioxidant pathway activation induced by hypomethylating agents in AML.

Induction of reactive oxygen species (ROS), an important process for the cytotoxicity of various acute myeloid leukemia (AML) therapies including hypomethylating agents (HMAs), concurrently activates the NF-E2-related factor 2 (Nrf2) antioxidant response pathway which in turn results in induction of antioxidant enzymes that neutralize ROS. In this study, we demonstrated that Nrf2 inhibition is an additional mechanism responsible for the marked antileukemic activity in AML seen with the combination of HMAs and venetoclax (ABT-199). HMA and venetoclax combined treatment augmented mitochondrial ROS induction and apoptosis compared with treatment HMA alone. Treatment of AML cell lines as well as primary AML cells with venetoclax disrupted HMA decitabine-increased nuclear translocation of Nrf2 and induction of downstream antioxidant enzymes including heme oxygenase-1 and NADP-quinone oxidoreductase-1. Venetoclax treatment also leads to dissociation of B-cell lymphoma 2 from the Nrf2/Keap-1 complex and targets Nrf2 to ubiquitination and proteasomal degradation. Thus, our results here demonstrated an undiscovered mechanism underlying synergistic effect of decitabine and venetoclax in AML cells, elucidating for impressive results in antileukemic activity against AML in preclinical and early clinical studies by combined treatment of these drugs.

Daratumumab induces CD38 internalization and impairs myeloma cell adhesion.

Daratumumab (Dara), a human immunoglobulin G1 kappa (IgG1κ) monoclonal anti-CD38 antibody, has been approved by the U.S. Food and Drug Administration for the treatment of relapsed multiple myeloma (MM) as a single agent as well as in combination with immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI). Although the scientific rationale behind the use of Dara in combination with IMiDs has been extensively explored, the molecular mechanisms underlying Dara-PI regimens have not yet been investigated. Here, we demonstrate that CD38 on the surface of MM cells is rapidly internalized after Dara treatment; we also show that Dara treatment impairs MM cell adhesion, an effect that can be rescued by using the endocytosis inhibitor Dynasore. Finally, we show that Dara potentiates bortezomib (BTZ) killing of MM cells in vitro and in vivo, independent of its function as an immune activator. In conclusion, our data show that Dara impairs MM cell adhesion, which results in an increased sensitivity of MM to proteasome inhibition.

IL-18 and Stem Cell Factor affect hematopoietic progenitor cells in HIV-infected patients treated during primary HIV infection.

The impact of early antiretroviral therapy (ART) during Primary HIV Infection (PHI) on the hematopoietic progenitor cells (HPCs) homeostasis is not available. This study aimed to characterize HPCs and their relationship with cytokines regulating progenitors function in ART-treated patients with PHI. We enrolled HIV infected patients treated with ART during PHI. Circulating HPCs, Lymphoid-HPCs (L-HPCs) frequency and plasmatic concentrations of IL-7, IL-18 and Stem Cell Factor (SCF) were analysed at baseline and after 6 months of therapy. ART introduction during PHI restored the decline of L-HPCs, induced a decrease in the level of pro-inflammatory IL-18 cytokine and a parallel increase of SCF. Moreover, L-HPCs frequency positively correlated with IL-18 at baseline, and with SCF after 6 months of therapy, suggesting that different signals impact L-HPCs expansion and maintenance before and after treatment. Finally, the SCF receptor expression on HPCs decreased after early ART initiation. These insights may open new perspectives for the evaluation of cytokine-driven L-HPCs expansion and their impact on the homeostasis of hematopoietic compartment during HIV infection.

Copper 64-labeled daratumumab as a PET/CT imaging tracer for multiple myeloma.

As a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose (18F-FDG) is a function of the metabolic activity of both malignant and nonmalignant cells, the results frequently lack sufficient specificity. Radiolabeled antibodies targeting MM tissue may detect disease irrespective of cell metabolism. Hence, we conjugated the clinically significant CD38-directed human antibody daratumumab (Darzalex [Dara]) to the DOTA chelator and labeled it with the positron-emitting radionuclide copper 64 (64Cu; 64Cu-DOTA-Dara). Here, we show that 64Cu-DOTA-Dara can efficiently bind CD38 on the surface of MM cells and was mainly detected in the bones associated with tumor in a MM murine model. We also show that PET/CT based on 64Cu-DOTA-Dara displays a higher resolution and specificity to detect MM cell dissemination than does 18F-FDG PET/CT and was even more sensitive than were bioluminescence signals. We therefore have supporting evidence for using 64Cu-DOTA-Dara as a novel imaging agent for MM.

Different features of Vδ2 T and NK cells in fatal and non-fatal human Ebola infections.

BACKGROUND: Human Ebola infection is characterized by a paralysis of the immune system. A signature of αß T cells in fatal Ebola infection has been recently proposed, while the involvement of innate immune cells in the protection/pathogenesis of Ebola infection is unknown. Aim of this study was to analyze γδ T and NK cells in patients from the Ebola outbreak of 2014-2015 occurred in West Africa, and to assess their association with the clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: Nineteen Ebola-infected patients were enrolled at the time of admission to the Ebola Treatment Centre in Guinea. Patients were divided in two groups on the basis of the clinical outcome. The analysis was performed by using multiparametric flow cytometry established by the European Mobile Laboratory in the field. A low frequency of Vδ2 T-cells was observed during Ebola infection, independently from the clinical outcome. Moreover, Vδ2 T-cells from Ebola patients massively expressed CD95 apoptotic marker, suggesting the involvement of apoptotic mechanisms in Vδ2 T-cell loss. Interestingly, Vδ2 T-cells from survivors expressed an effector phenotype and presented a lower expression of the CTLA-4 exhaustion marker than fatalities, suggesting a role of effector Vδ2 T-cells in the protection. Furthermore, patients with fatal Ebola infection were characterized by a lower NK cell frequency than patients with non fatal infection. In particular, both CD56bright and CD56dim NK frequency were very low both in fatal and non fatal infections, while a higher frequency of CD56neg NK cells was associated to non-fatal infections. Finally, NK activation and expression of NKp46 and CD158a were independent from clinical outcome. CONCLUSIONS/SIGNIFICANCES: Altogether, the data suggest that both effector Vδ2 T-cells and NK cells may play a role in the complex network of protective response to EBOV infection. Further studies are required to characterize the protective effector functions of Vδ2 and NK cells.

HIV-Specific CD8 T Cells Producing CCL-4 Are Associated With Worse Immune Reconstitution During Chronic Infection.

BACKGROUND: Immunological nonresponse represents the Achilles heel in the combination antiretroviral therapy (cART) effectiveness, and increases risk of clinical events and death. CD8 T cells play a crucial role in controlling HIV replication, and polyfunctional HIV-specific CD8 T cells have been associated with nonprogressive HIV infection. However, the possible role of polyfunctional CD8 T cells in predicting posttreatment immune reconstitution has not yet been explored. The aim of this study was to identify functional markers predictive of immunological response to cART in chronic HIV-infected patients. METHODS: A cohort of chronic HIV-infected individuals naive to cART were enrolled in the ALPHA study. CD4/CD8 T-cell subsets, their differentiation/activation, as well as susceptibility to apoptosis were analyzed before and after 12 months of cART. Moreover, CD8 T cells polyfunctional response after HIV antigenic stimulation was also assessed. RESULTS: Results showed a significant correlation between worse CD4 T-cell restoration and low frequency of naive CD4 T cells, high frequency of effector memory CD4 T cells, and high susceptibility to apoptosis of CD4 T cells all before cART. Moreover, CD8 functional subsets expressing total C-C motif chemokine ligand 4 (CCL-4) or in combination with CD107a and interferon gamma (IFNγ) were negatively associated with immune reconstitution. CONCLUSIONS: In conclusion, our study shows that a more differentiated phenotype of CD4 T cells and CCL-4-producing CD8 T cells could represent valuable predictors of worse immune reconstitution. These parameters may be used as tools for identifying patients at risk of immunological failure during cART and eventually represent the basis for innovative therapeutic strategies.

Bone Marrow CD34+ Progenitor Cells from HIV-Infected Patients Show an Impaired T Cell Differentiation Potential Related to Proinflammatory Cytokines.

The impact of HIV infection on the frequency and differentiation capability of CD34+ bone marrow hematopoietic progenitor cells (BM-HPCs) is still debated, having a possible primary role in antiretroviral-induced immunoreconstitution. We investigated the influence of HIV replication or proinflammatory cytokines on lymphopoietic capability of BM-HPCs from seven viremic (VR) and five nonviremic (NVR) HIV-infected patients. We found that BM-HPCs from VR patients were unable to differentiate in vitro toward T cells, and produced proinflammatory cytokines in the absence of viral replication. In contrast, the lymphoid differentiation potential of BM-HPCs was partially restored in successfully antiretroviral therapy-treated patients. We also showed that TLR8 triggering induced BM-HPCs from healthy donors to release proinflammatory cytokines affecting T cell differentiation. These data suggest that in HIV-infected patients, the lymphopoiesis capability of BM-HPCs may be modulated by a virus-driven autocrine mechanism involving proinflammatory cytokines.

Unique human immune signature of Ebola virus disease in Guinea.

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4(+) and CD8(+) T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

Endothelial and Metabolic Function Interactions in Overweight/Obese Children.

AIM: Although the underlined mechanisms are still unknown, metabolic/coagulation alterations related to childhood obesity can induce vascular impairments. The aim of this study was to investigate the relationship between metabolic/coagulation parameters and endothelial function/vascular morphology in overweight/obese children. METHODS: Thirty-five obese/overweight children (22 pre-pubertal, mean age: 9.52±3.35 years) were enrolled. Body mass index (BMI), homeostasis model assessment index (HOMAIR), metabolic and coagulation parameters, [adiponectin, fibrinogen, high molecular weight adiponectin (HMW), endothelin-1, and vonWillebrand factor antigen] ultrasound early markers of atherosclerosis [flow-mediated dilatation (FMD), common carotid intima-media thickness (C-IMT), and anteroposterior diameter of infra-renal abdominal aorta (APAO)] were assessed. RESULTS: APAO was related to anthropometric (age: r=0.520, p=0.001; height: r=0.679, p＜0.001; weight: r=0.548, p=0.001; BMI: r=0.607, p＜0.001; SBP: r=0.377, p=0.026) and metabolic (HOMAIR: r=0.357, p=0.035; HMW: r=－0.355, p=0.036) parameters. Age, height, and systolic blood pressure were positively related to increased C-IMT (r=0.352, p=0.038; r=0.356, p=0.036; r=0.346, p=0.042, respectively). FMD was not related to any clinical and biochemical characteristics of the pediatric population. Age, HOMAIR, fasting glucose levels, and HMW were independent predictors for APAO increase. Each unit decrease in HMW concentrations (1 µg/ml) induced a 0.065 mm increase in APAO. CONCLUSION: High molecular weight adiponectin is related to cardiovascular risk in overweight/obese children.

Vγ9Vδ2 T-Cell Polyfunctionality Is Differently Modulated in HAART-Treated HIV Patients according to CD4 T-Cell Count.

Alteration of γδ T-cell distribution and function in peripheral blood is among the earliest defects during HIV-infection. We asked whether the polyfunctional response could also be affected, and how this impairment could be associated to CD4 T-cell count. To this aim, we performed a cross-sectional study on HIV-infected individuals. In order to evaluate the polyfunctional-Vγ9Vδ2 T-cell response after phosphoantigen-stimulation, we assessed the cytokine/chemokine production and cytotoxicity by flow-cytometry in HAART-treated-HIV+ persons and healthy-donors. During HIV-infection Vγ9Vδ2-polyfunctional response quality is affected, since several Vγ9Vδ2 T-cell subsets resulted significantly lower in HIV+ patients in respect to healthy donors. Interestingly, we found a weak positive correlation between Vγ9Vδ2 T-cell-response and CD4 T-cell counts. By dividing the HIV+ patients according to CD4 T-cell count, we found that Low-CD4 patients expressed a lower number of two Vγ9Vδ2 T-cell subsets expressing MIP-1ß in different combinations with other molecules (CD107a/IFNγ) in respect to High-CD4 individuals. Our results show that the Vγ9Vδ2 T-cell-response quality in Low-CD4 patients is specifically affected, suggesting a direct link between innate Vγ9Vδ2 T-cells and CD4 T-cell count. These findings suggest that Vγ9Vδ2 T-cell quality may be indirectly influenced by HAART therapy and could be included in a new therapeutical strategy which would perform an important role in fighting HIV infection.

Insulin resistance and endothelial function in children and adolescents.

AIMS: Insulin resistance (IR) impairs cellular response to insulin due to a dysfunction in glucose metabolism, associated with an increased cardiovascular risk. The aim of our study was to investigate the relationship among homeostasis model assessment index (HOMA index), endothelial function and vascular morphology in order to better stratify cardiovascular risk in children and adolescents. METHODS: A total of 150 children and adolescents (55 pre-pubertal, mean age 10.4 ± 3.1 years) were enrolled. Anthropometric [body mass index (BMI), waist circumference (WC)], laboratory [blood lipids, inflammatory markers, insulinemia, glycemia], HOMA index and ultrasound parameters [flow-mediated dilatation (FMD), common carotid intima-media thickness (cIMT) and antero-posterior diameter of infra-renal abdominal aorta (APAO)] were assessed. RESULTS: cIMT was positively related to age (r=0.274, p<0.01), BMI (r=0.318, p<0.01), WC (r=0.315, p<0.01) and triglycerides (r=0.230, p<0.01). APAO measurements showed a linear positive correlation with age (r=0.435, p<0.01), BMI (r=0.505, p<0.01), WC (r=0.487, p<0.01), triglycerides (r=0.280, p<0.01), C-reactive protein (r=0.209, p<0.05), fasting insulin (r=0.378, p<0.01) and HOMA index (r=0.345, p<0.01). FMD was inversely related to age (r=-0.251, p<0.01), rough BMI (r=-0.318, p<0.01), WC (r=-0.340, p<0.01), fasting insulin (r=-0.281, p<0.01) and HOMA index (r=-0.282, p<0.01). Multiple regression analysis found no influence of HOMA index on APAO and cIMT. HOMA index was an independent predictor for brachial artery FMD worsening after the statistical adjustment. CONCLUSION: HOMA index increase induced a worsening in endothelial function since childhood.

Chronic HIV-infected patients show an impaired dendritic cells differentiation of bone marrow CD34⁺ cells.

HIV infection affects dendritic cells (DCs) number, maturation, and function although the cause remains largely unknown. Purified CD34⁺ hematopoietic progenitor cells (HPCs) obtained from bone marrow of chronic HIV-infected patients were investigated for the differentiative capability toward mature DCs. HIV, although not in active replication, was found able to impair CD34⁺ HPC differentiation into mature DCs. These results suggest that DCs impairment found in HIV-infected patients may be related to a failure by bone marrow CD34 HPCs to produce an adequate number of DCs.

Sixteen years of experience with stone management in horseshoe kidneys.

INTRODUCTION: Horseshoe kidney is the commonest congenital renal fusion anomaly, and is often complicated by urolithiasis. We focus on our 16 years of experience with stone management in horseshoe kidneys. MATERIALS AND METHODS: We reviewed the progress of 44 patients treated between 1987 and 2002. Shock wave lithotripsy (SWL) was used in 25 patients; the average stone surface area was 91 (range 10-1,600) mm2 and average follow-up was 36.5 (range 1-91) months. 19 patients underwent percutaneous nephrolithotomy (PCNL); the average stone surface area was 197 (range 6-2,400) mm2. Follow-up data are available for 8 patients and the average follow-up was 42.3 (range 3-144) months. RESULTS: In the SWL group the 3-month stone-free rate (SFR) was only 31%. In the PCNL group the SFR was 75% on the postoperative day-1 KUB. Complications occurred in 9 patients. CONCLUSIONS: Stone management in horseshoe kidneys is challenging: PCNL produces a higher SFR with minimal major complications and failed access. PCNL thus appears to be the preferred management option in patients with urolithiasis in horseshoe kidneys.

Male bulbourethral sling after radical prostatectomy: intermediate outcomes at 2 to 4-year followup.

PURPOSE: We reviewed our experience with 49 consecutive patients undergoing a male sling procedure. MATERIALS AND METHODS: The 7-item International Prostate Symptom Score and 22-item incontinence quality of life questionnaire (although not validated in Italian) were used to assess the clinical impact of the bulbourethral sling. Surgical outcome was also assessed by videourodynamics at 1 year. Urodynamic success was defined as no leakage during videourodynamic evaluation. RESULTS: Mean followup was 32 months (range 26 to 48). Preoperatively 3 patients used 2 pads daily (mild incontinence), 34 used 3 to 5 (moderate incontinence) and the remaining 12 used more than 5 (severe incontinence). Clinical success was defined as a decrease in pad use to completely dry (no pad) or to social continence (1 pad daily) at followup. Of the 49 patients 38 (77%), 33 (67%) and 63% (31) were considered socially continent at the 3-month, 1-year and 3-year followup, respectively. Only 15 of the 49 patients (30%) were considered completely dry at the 3-year followup. Significant perineal pain was reported in the early postoperative period but it resolved in all patients. Infection occurred in 3 patients, while no erosion was found. CONCLUSIONS: The male perineal sling is a safe surgical option for post-prostatectomy urinary incontinence, especially when strict patient selection is done. Patient satisfaction is superior to urodynamic results and the procedure allows physiological voiding. Patients should be informed of the possibility of progressive failure with time and significant perineal pain in the early postoperative period.