Sexually Transmitted Infections in Adolescents and Young Adults: A Cross Section of Public Health.

INTRODUCTION: Sexually transmitted infections (STIs) can be caused by a number of microorganisms that vary greatly in size, life cycle, clinical manifestations, and sensitivity to available treatments. Transmission of STIs can occur during unprotected (or condomless) sexual contact and through the exchange of body fluids during any type of activity. The prevalence of sexually transmitted diseases remains high in the world, despite diagnostic and therapeutic improvements for these infectious diseases that rapidly eliminate the contagiousness of patients. Our study determines the prevalence of STI pathogens in adolescents and young adults in the population of the Province of Macerata (Italy). We will analyze data in correspondence to age and gender, and we will compare our results to international studies. MATERIALS AND METHOD: We analyzed STI test results from the entire database of a Provincial Health Authority for the period 2021-2022. The samples came from the following age groups: 0-12, 13-18, 19-25, and 26-35 from 2021 to 2022. The results came from vaginal and cervical swabs (for females); urethral, rectal, and pharyngeal swabs (for males and females); and seminal fluid (for males) for the following infections: HPV, Chlamydia trachomatis, Mycoplasma genitalium, Ureaplasmas, Gardnerella, Trichomonas vaginalis, Neisseria gonorrhoeae, and Treponema pallidum. The results also came from blood tests for HIV, hepatitis C, hepatitis B, and Treponema pallidum (TPHA, VDRL). In addition, we examined results from urine tests for chlamydia, Neisseria gonorrhoeae, trichomonas, and Treponema pallidum. CONCLUSIONS: The literature for other countries reports the need for comprehensive, culturally and developmentally sensitive care to address sexuality-related issues in adolescents and young adults, a need that also applies to Italy. These data will be of great importance in adopting evidence-based STI control programs in Marche Region. This study could, indeed, represent a landmark for public health officials and professionals, with the aim of promoting adolescents' access to sexual health services to receive useful information, strengthening preventive measures in younger age groups, and designing sexual education programs.

Efficient T-Cell Compartment in HIV-Positive Patients Receiving Orthotopic Liver Transplant and Immunosuppressive Therapy.

BACKGROUND: In patients undergoing orthotopic liver transplant (OLT), immunosuppressive treatment is mandatory and infections are leading causes of morbidity/mortality. Thus, it is essential to understand the functionality of cell-mediated immunity after OLT. The aim of the study was to identify changes in T-cell phenotype and polyfunctionality in human immunodeficiency virus-positive (HIV+) and -negative (HIV-) patients undergoing immunosuppressive treatment after OLT. METHODS: We studied peripheral blood mononuclear cells from 108 subjects divided into 4 groups of 27: HIV+ transplanted patients, HIV- transplanted patients, HIV+ nontransplanted patients, and healthy subjects. T-cell activation, differentiation, and cytokine production were analyzed by flow cytometry. RESULTS: Median age was 55 years (interquartile range, 52-59 years); the median CD4 count in HIV+ patients was 567 cells/mL, and all had undetectable viral load. CD4+ and CD8+ T-cell subpopulations showed different distributions between HIV+ and HIV- OLT patients. A cluster representing effector cells expressing PD1 was abundant in HIV- transplanted patients and they were characterized by higher levels of CD4+ T cells able to produce interferon-γ and tumor necrosis factor-α. CONCLUSIONS: HIV- transplanted patients have more exhausted or inflammatory T cells compared to HIV+ transplanted patients, suggesting that patients who have already experienced a form of immunosuppression due to HIV infection respond differently to anti-rejection therapy.

Effects of whole-body cryotherapy on the innate and adaptive immune response in cyclists and runners.

The study aimed to identify the effects of whole-body cryotherapy (WBC) on immunological, hormonal, and metabolic responses of non-professional male athletes. Ten cyclists and ten middle-distance runners received 3 once-a-day sessions of WBC. Before initiating and after the final WBC session, a full set of hematologic parameters, serum chemistry profile, hormones, circulating mitochondrial (mt) DNA levels, cytokines, and chemokines concentration were evaluated. The phenotype of monocyte, T cells, and B cells was analyzed. mRNA expression of 6 genes involved in inflammasome activation (NAIP, AIM2, NLRP3, PYCARD, IL-1ß, and IL-18) was quantified. WBC reduced glucose and C and S protein and increased HDL, urea, insulin-like growth factor (IGF)-1, follicle-stimulating hormone, IL-18, IL-1RA, CCL2, and CXCL8. Intermediate and non-classical monocyte percentages decreased, and the CD14, CCR5, CCR2, and CXCR4 expressions changed in different subsets. Only IL-1ß mRNA increased in monocytes. Finally, a redistribution of B and T cell subsets was observed, suggesting the migration of mature cells to tissue. WBC seems to induce changes in both innate and adaptive branches of the immune system, hormones, and metabolic status in non-professional male athletes, suggesting a beneficial involvement of WBC in tissue repair.

Mitochondrial damage-associated molecular patterns stimulate reactive oxygen species production in human microglia.

Microglia are the resident innate immune cells of the central nervous system and exert functions of host defense and maintenance of normal tissue homeostasis, along with support of neuronal processes in the healthy brain. Chronic and dysregulated microglial cell activation has increasingly been linked to the status of neuroinflammation underlying many neurodegenerative diseases, including multiple sclerosis (MS). However, the stimulus (or stimuli) and mechanisms by which microglial activation is initiated and maintained MS are still debated. The purpose of our research was to investigate whether the endogenous mitochondrial (mt)-derived damage-associated molecular patterns (MTDs) mtDNA, N-formyl peptides and cardiolipin (CL) contribute to these phenomena. We characterized the effects of the abovementioned MTDs on microglia activation in vitro (i.e. using HMC3 cells) by evaluating the expression of gene coding for proteins involved in their binding and coupled to downstream signaling pathways, the up-regulation of markers of activation on the cell surface and the production of pro-inflammatory cytokines and reactive oxygen species. At the transcriptional level, significant variations in the mRNA relative expression of five of eleven selected genes were observed in response to stimulation. No changes in activation of antigenic profile or functional properties of HMC3 cells were observed; there was no up-regulation of HLA-DR expression or increased secretion of tumor necrosis factor-α and interleukin-6. However, after stimulation with mtDNA and CL, an increase in cellular oxidative stress, but not in the mt ROS O2-, compared to control cells, were observed. There were no effects on cell viability. Overall, our data suggest that MTDs could cause a failure in microglial activation toward a pro-inflammatory phenotype, possibly triggering an endogenous regulatory mechanism for the resolution of neuroinflammation. This could open a door for the development of drugs selectively targeting microglia and modulating its functionality to treat MS and/or other neurodegenerative conditions in which MTDs have a pathogenic relevance.

Impaired Mitochondrial Morphology and Functionality in Lonp1wt/- Mice.

LONP1 is a nuclear-encoded mitochondrial protease crucial for organelle homeostasis; mutations of LONP1 have been associated with Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome. To clarify the role of LONP1 in vivo, we generated a mouse model in which Lonp1 was ablated. The homozygous Lonp-/- mouse was not vital, while the heterozygous Lonp1wt/- showed similar growth rate, weight, length, life-span and histologic features as wild type. Conversely, ultrastructural analysis of heterozygous enterocytes evidenced profound morphological alterations of mitochondria, which appeared increased in number, swollen and larger, with a lower complexity. Embryonic fibroblasts (MEFs) from Lonp1wt/- mice showed a reduced expression of Lonp1 and Tfam, whose expression is regulated by LONP1. Mitochondrial DNA was also reduced, and mitochondria were swollen and larger, albeit at a lesser extent than enterocytes, with a perinuclear distribution. From the functional point of view, mitochondria from heterozygous MEF showed a lower oxygen consumption rate in basal conditions, either in the presence of glucose or galactose, and a reduced expression of mitochondrial complexes than wild type. In conclusion, the presence of one functional copy of the Lonp1 gene leads to impairment of mitochondrial ultrastructure and functions in vivo.

In vitro efficacy and safety of a system for sorbent-assisted peritoneal dialysis.

A system for sorbent-assisted peritoneal dialysis (SAPD) was designed to continuously recirculate dialysate via a tidal mode using a single lumen peritoneal catheter with regeneration of spent dialysate by means of sorbent technology. We hypothesize that SAPD treatment will maintain a high plasma-to-dialysate concentration gradient and increase the mass transfer area coefficient of solutes. Thereby, the SAPD system may enhance clearance while reducing the number of exchanges. Application is envisaged at night as a bedside device (12 kg, nighttime system). A wearable system (2.0 kg, daytime system) may further enhance clearance during the day. Urea, creatinine, and phosphate removal were studied with the daytime and nighttime system (n = 3 per system) by recirculating 2 liters of spent peritoneal dialysate via a tidal mode (mean flow rate: 50 and 100 mL/min, respectively) for 8 h in vitro. Time-averaged plasma clearance over 24 h was modeled assuming one 2 liter exchange/day, an increase in mass transfer area coefficient, and 0.9 liters ultrafiltration/day. Urea, creatinine, and phosphate removal was 33.2 ± 4.1, 5.3 ± 0.5, and 6.2 ± 1.8 mmol, respectively, with the daytime system and 204 ± 28, 10.3 ± 2.4, and 11.4 ± 2.1 mmol, respectively, with the nighttime system. Time-averaged plasma clearances of urea, creatinine and phosphate were 9.6 ± 1.1, 9.6 ± 1.7, and 7.0 ± 0.9 mL/min, respectively, with the nighttime system and 10.8 ± 1.1, 13.4 ± 1.8, and 9.7 ± 1.6 mL/min, respectively, with the daytime and nighttime system. SAPD treatment may improve removal of uremic toxins compared with conventional peritoneal dialysis, provided that peritoneal mass transport will increase.

Increased plasma levels of mitochondrial DNA and pro-inflammatory cytokines in patients with progressive multiple sclerosis.

The role of damage-associated molecular patterns in multiple sclerosis (MS) is under investigation. Here, we studied the contribution of circulating high mobility group box protein 1 (HMGB1) and mitochondrial DNA (mtDNA) to neuroinflammation in progressive MS. We measured plasmatic mtDNA, HMGB1 and pro-inflammatory cytokines in 38 secondary progressive (SP) patients, 35 primary progressive (PP) patients and 42 controls. Free mtDNA was higher in SP than PP. Pro-inflammatory cytokines were increased in progressive patients. In PP, tumor necrosis factor-α correlated with MS Severity Score. Thus, in progressive patients, plasmatic mtDNA and pro-inflammatory cytokines likely contribute to the systemic inflammatory status.

Mitochondrial functionality and metabolism in T cells from progressive multiple sclerosis patients.

Patients with primary progressive (PP) and secondary progressive (SP) forms of multiple sclerosis (MS) exhibit a sustained increase in the number of Th1, T cytotoxic type-1 and Th17 cells in peripheral blood, suggesting that the progressive phase is characterized by a permanent peripheral immune activation. As T cell functionality and activation are strictly connected to their metabolic profile, we investigated the mitochondrial functionality and metabolic changes of T cell subpopulations in a cohort of progressive MS patients. T cells from progressive patients were characterized by low proliferation and increase of terminally differentiated/exhausted cells. T cells from PP patients showed lower Oxygen Consumption Rate and Extracellular Acidification Rate, lower mitochondrial mass, membrane potential and respiration than those of SP patients, a downregulation of transcription factors supporting respiration and higher tendency to shift towards glycolysis upon stimulation. Furthermore, PP effector memory T cells were characterized by higher Glucose transporter -1 levels and a higher expression of glycolytic-supporting genes if compared to SP patients. Overall, our data suggest that profound differences exist in the phenotypic and metabolic features of T cells from PP and SP patients, even though the two clinical phenotypes are considered part of the same disease spectrum.

Altered Expression of PYCARD, Interleukin 1ß, Interleukin 18, and NAIP in Successfully Treated HIV-Positive Patients With a Low Ratio of CD4+ to CD8+ T Cells.

The expression and activity of main inflammasome components in monocytes from successfully treated human immunodeficiency virus (HIV)-positive patients are poorly studied. Thus, we enrolled 18 patients with a low and 17 with a normal ratio of CD4+ T cells to CD8+ T cells and 11 healthy donors. We found that patients with a low ratio had decreased CCR2 expression among classical and intermediate monocytes and increased CCR5 expression among classical monocytes, compared with patients with a normal ratio. Patients with a low ratio also had higher NAIP and PYCARD messenger RNA levels after lipopolysaccharide stimulation, suggesting an altered ability to control immune activation that could affect their immune reconstitution.

Intraoperative Spinal Navigation for the Removal of Intradural Tumors: Technical Notes.

BACKGROUND: In recent years, spinal surgery has incorporated the many advantages of navigation techniques to facilitate the placement of pedicle screws during osteosynthesis, mainly for degenerative diseases. However, spinal intradural tumors are not clearly visible by intraoperative fluoroscopy or computed tomography scans, thereby making navigation necessary. OBJECTIVE: To evaluate the use of spinal navigation for the removal of intradural and spinal cord tumors using spinal magnetic resonance imaging (MRI) merged with intraoperative 3-dimensional (3-D) fluoro images. METHODS: After fixing the patient reference frame on the spinous process, the 3-D fluoro images were obtained in the surgical room. Using this image as the reference, the preoperative volumetric MRI images and intraoperative 3-D fluoro images were merged using automated software or manually. RESULTS: From January to July 2016, we performed 10 navigated procedures for intradural spinal tumors by merging MRI and 3-D fluoro images. Nine patients had an intradural extramedullary tumor, 6 had neurinomas, and 3 had meningiomas; 1 patient had an intramedullary spinal cord metastasis. CONCLUSION: The surgically demonstrated benefits of spinal navigation for the removal of intradural tumors include the decreased risk of surgery at the wrong spinal level, a minimal length of skin incision and muscle strip, and a reduction in bone removal extension. Furthermore, this technique offers the advantage of opening the dura as much as is necessary and, in the case of intrinsic spinal cord tumors, it allows the tumor to be centered. Otherwise, this would not be visible, thus enabling the precise level and the posterior midline sulcus to be determined when performing a mielotomy.

Exploring viral reservoir: The combining approach of cell sorting and droplet digital PCR.

Combined antiretroviral therapy (cART) blocks different steps of HIV replication and maintains plasma viral RNA at undetectable levels. The virus can remain in long-living cells and create a reservoir where HIV can restart replicating after cART discontinuation. A persistent viral production triggers and maintains a persistent immune activation, which is a well-known feature of chronic HIV infection, and contributes either to precocious aging, or to the increased incidence of morbidity and mortality of HIV positive patients. The new frontier of the treatment of HIV infection is nowadays eradication of the virus from all host cells and tissues. For this reason, it is crucial to have a clear and precise idea of where the virus hides, and which are the cells that keep it silent. Important efforts have been made to improve the detection of viral reservoirs, and new techniques are now giving the opportunity to characterize viral reservoirs. Among these techniques, a strategic approach based upon cell sorting and droplet digital PCR (ddPCR) is opening new horizons and opportunities of research. This review provides an overview of the methods that combine cell sorting and ddPCR for the quantification of HIV DNA in different cell types, and for the detection of its maintenance.

High speed flow cytometry allows the detection of circulating endothelial cells in hemangioblastoma patients.

Circulating endothelial cells (CECs) detach from the intima monolayer after endothelial damages. Their circulating endothelial progenitors (CEPs) represent less than 0.01% of nucleated blood cells. Increased levels of CECs and CEPs have been detected in patients with several types of cancer, suggesting that they could be a useful blood-based marker for detecting a tumor, or for monitoring its clinical course. However, their routine monitoring is time consuming and technically challenging. Here, we present a flow cytometry method for quantifying such cells in a cohort of patients with hemangioblastoma (HB). HB is a rare benign tumor, responsible for 1-2.5% of primary intracranial tumors and up to 10% of spinal cord tumors, and for which no tools are available to predict the onset or recurrence in patients undergoing surgical removal of tumor mass. This method allowed us to accurately quantifying CEC and CEP before and after surgery. CEPs are present at high levels in HB patients than control before intervention, and decrease after tumor removal, suggesting that their percentage could represent a valid tool to monitor the disease onset and recurrence.

Label-free detection of interleukin-6 using electrolyte gated organic field effect transistors.

Cytokines are small proteins that play fundamental roles in inflammatory processes in the human body. In particular, interleukin (IL)-6 is a multifunctional cytokine, whose increased levels are associated with infection, cancer, and inflammation. The quantification of IL-6 is therefore of primary importance in early stages of inflammation and in chronic diseases, but standard techniques are expensive, time-consuming, and usually rely on fluorescent or radioactive labels. Organic electronic devices and, in particular, organic field-effect transistors (OFETs) have been proposed in the recent years as novel platforms for label-free protein detection, exploiting as sensing unit surface-immobilized antibodies or aptamers. Here, the authors report two electrolyte-gated OFETs biosensors for IL-6 detection, featuring monoclonal antibodies and peptide aptamers adsorbed at the gate. Both strategies yield biosensors that can work on a wide range of IL-6 concentrations and exhibit a remarkable limit of detection of 1 pM. Eventually, electrolyte gated OFETs responses have been used to extract and compare the binding thermodynamics between the sensing moiety, immobilized at the gate electrode, and IL-6.

HIV-DNA content in different CD4+ T-cell subsets correlates with CD4+ cell :  CD8+ cell ratio or length of efficient treatment.

OBJECTIVES: HIV establishes a latent infection at different degrees within naïve (TN) or central (TCM) and effector memory (TEM) CD4 T cell. Studying patients in whom HIV production was suppressed by combined antiretroviral therapy, our main aim was to find which factors are related or can influence intracellular viral reservoir in different CD4 T-cell subsets. METHODS: We enrolled 32 HIV patients successfully treated for more than 2 years, with a CD4 T-cell count more than 500 cells/µl and plasma viremia undetectable from at least 1 year. Proviral HIV-DNA, the amount of cells expressing signal-joint T-cell receptor rearrangement excision circles and telomere length were quantified by droplet digital PCR in highly purified, sorted CD4 T-cell subsets; plasma IL-7 and IL-15 were measured by ELISA. RESULTS: HIV-DNA was significantly lower in TN cells compared with TCM or to TEM. Conversely, TN cells contained more signal-joint T-cell receptor rearrangement excision circles compared with TCM or to TEM; no appreciable changes were observed in telomere length. HIV-DNA content was significantly higher in TN and TCM cells, but not in TEM, from patients with shorter time of treatment, or in those with lower CD4 : CD8 ratio. CONCLUSION: Length of treatment or recovery of CD4 : CD8 ratio significantly influences viral reservoir in both TN and TCM. Measuring HIV-DNA in purified lymphocyte populations allows a better monitoring of HIV reservoir and could be useful for designing future eradication strategies.

Invariant natural killer T cells and mucosal-associated invariant T cells in multiple sclerosis.

Multiple sclerosis (MS) is a chronic progressive inflammatory demyelinating disorder of the central nervous system, and in several countries is a leading cause of permanent neurological disability in young adults, particularly women. MS is considered an autoimmune disease, caused by an aberrant immune response to environmental triggers in genetically susceptible subjects. However, the contribution of the innate or of the adaptive immune system to the development and progression of the disease has not yet been fully elucidated. Innate-like T lymphocytes are unconventional T cells that bridge the innate and adaptive arms of the immune system, because they use a T cell receptor to sense external ligands, but behave like innate cells when they rapidly respond to stimuli. These cells could play an important role in the pathogenesis of MS. Here, we focus on invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells, and we review the current knowledge on their biology and possible involvement in MS. Although several studies have evaluated the frequency and functions of iNKT and MAIT cells both in MS patients and in experimental mouse models, contradictory observations have been reported, and it is not clear whether they exert a protective or a pro-inflammatory and harmful role. A better understanding of how immune cells are involved in MS, and of their interactions could be of great interest for the development of new therapeutic strategies.

Anti-TNF-α Drugs Differently Affect the TNFα-sTNFR System and Monocyte Subsets in Patients with Psoriasis.

TNF-α has a central role in the development and maintenance of psoriatic plaques, and its serum levels correlate with disease activity. Anti-TNF-α drugs are, however, ineffective in a relevant percentage of patients for reasons that are currently unknown. To understand whether the response to anti-TNF-α drugs is influenced by the production of anti-drug antibodies or by the modulation of the TNFα-TNFα receptor system, and to identify changes in monocyte phenotype and activity, we analysed 119 psoriatic patients who either responded or did not respond to different anti-TNF-α therapies (adalimumab, etanercept or infliximab), and measured plasma levels of TNF-α, TNF-α soluble receptors, drug and anti-drug antibodies. Moreover, we analyzed the production of TNF-α and TNF-α soluble receptors by peripheral blood mononuclear cells (PBMCs), and characterized different monocyte populations. We found that: i) the drug levels varied between responders and non-responders; ii) anti-infliximab antibodies were present in 15% of infliximab-treated patients, while anti-etanercept or anti-adalimumab antibodies were never detected; iii) plasma TNF-α levels were higher in patients treated with etanercept compared to patients treated with adalimumab or infliximab; iv) PBMCs from patients responding to adalimumab and etanercept produced more TNF-α and sTNFRII in vitro than patients responding to infliximab; v) PBMCs from patients not responding to infliximab produce higher levels of TNF-α and sTNFRII than patients responding to infliximab; vi) anti- TNF-α drugs significantly altered monocyte subsets. A complex remodelling of the TNFα-TNFα receptor system thus takes place in patients treated with anti-TNF-α drugs, that involves either the production of anti-drug antibodies or the modulation of monocyte phenotype or inflammatory activity.

iNKT Cells in Secondary Progressive Multiple Sclerosis Patients Display Pro-inflammatory Profiles.

BACKGROUND: Multiple sclerosis (MS), an autoimmune disease with neurodegeneration and inflammation is characterized by several alterations of different T cell subsets. However, few data exist on the role of iNKT lymphocytes. OBJECTIVE: To identify possible changes in the phenotype of iNKT cells in patients with different clinical forms of MS and find alterations in their polyfunctionality [i.e., ability to produce simultaneously up to four cytokines such as IL-17, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and IL-4]. METHODS: We studied a total of 165 patients, 91 with a relapsing-remitting form [RR; 31 were treated with interferon (IFN)1a-ß, 25 with natalizumab (NAT), 29 with glatiramer acetate; 17 were newly diagnosed RR without treatment, 19 not-active RR without treatment]. Forty-four patients had a progressive MS: 20 primary progressive (PP) and 24 secondary progressive (SP). A total of 55 age- and sex-matched subjects represented healthy controls (CTR). Among fresh peripheral blood mononuclear cells, iNKT cells were identified by flow cytometry. Moreover, the capability of iNKT cells to produce different cytokines (IL-17, TNF-α, IFN-γ, and IL-4) after in vitro stimulation were evaluated in 18 RR (11 treated with NAT and 7 with IFN), 4 PP, 6 SP, and 16 CTR. RESULTS: No main differences were found in iNKT cell phenotype among MS patients with different MS forms or during different treatments. However, the polyfunctional response of iNKT cells showed Th1 and Th17 profiles. This was well evident in patients with SP form, who are characterized by high levels of inflammation and neurodegeneration, and exhibited a sustained increase in the production of Th17 cytokines. Patients treated with NAT displayed lower levels of iNKT cells producing IL-17, TNF-α, and IFN-γ. CONCLUSION: Our data suggest that the progressive phase of the disease is characterized by permanent iNKT activation and a skewing towards an inflammatory phenotype. Compared to other treatments, NAT was able to modulate iNKT cell function.

Th1 and Th17 proinflammatory profile characterizes invariant natural killer T cells in virologically suppressed HIV+ patients with low CD4+/CD8+ ratio.

INTRODUCTION: Scanty data exist on the phenotype and functionality of invariant natural killer T (iNKT) cells in HIV-infected (HIV+) patients. METHODS: By flow cytometry, we studied iNKT cells from 54 HIV+ patients who started combined antiretroviral therapy and had undetectable viral load for more than 1 year. Twenty-five maintained a CD4/CD8 ratio less than 0.4, whereas 29 reached a ratio more than 1.1; 32 age-matched and sex-matched patients were healthy controls (CTR). RESULTS: Patients with low ratio had lower percentage of CD4 iNKT cells compared with patients with high ratio and higher CD8 iNKT cell percentage; double-negative iNKT cells were lower in HIV+ patients compared with CTR. Patients with low ratio had higher percentage of CD4 and double-negative iNKT cells expressing CD38 and HLA-DR compared with patients with high ratio. CD4 iNKT cells expressing PD-1 were higher in patients with CD4/CD8 ratio less than 0.4, whereas double-negative iNKT cells expressing PD-1 were lower compared with patients with ratio more than 1.1. Patients with low ratio had higher CD4 iNKT cells producing IL-17, CD8 iNKT cells producing IFN-γ, TNF-α or IFN-γ and TNF-α, and double-negative iNKT cells producing IL-17 or IL-17 and IFN-γ compared with CTR. Activated CD4 (or CD8) T cells correlated with activated CD4 (or CD8) iNKT cells, as well as the percentages of CD4 (or CD8) T cells expressing PD-1 was correlated to that of CD4 (or CD8) iNKT cells expressing PD-1. CONCLUSION: Low CD4/CD8 ratio despite effective combined antiretroviral therapy is associated with altered iNKT cell subsets, enhanced activation, and prominent Th1/Th17 proinflammatory profile.

Quantification of mitochondrial reactive oxygen species in living cells by using multi-laser polychromatic flow cytometry.

Reactive oxygen species (ROS) are constantly produced in cells, mainly by mitochondria, as a consequence of aerobic respiration. Most ROS derive from superoxide, which is rapidly converted to hydrogen peroxide. ROS are involved in the regulation of several physiological and pathological processes, and the possibility to measure them simultaneously is needed, when the redox status of the cells is modified by experimental/biological conditions. Flow cytometry is the main technology that generates multiple information at the single cell level in a high-throughput manner, and gives rapid and quantitative measurements of different ROS with high sensitivity and reproducibility. Here, we describe a novel approach to detect simultaneously mitochondrial hydrogen peroxide and mitochondrial superoxide in living cells. The staining has been performed by using the fluorescent dyes MitoSOX Red Mitochondrial Superoxide Indicator, Mitochondria Peroxy Yellow 1, Annexin-V Pacific Blue conjugate, TO-PRO-3 iodide, anti-CD4-APC-Cy7 and -CD8-Pacific Orange mAbs. We used this approach to quantify mitochondrial ROS in CD4+ and CD8+ T cells form patients affected by Down syndrome and age- and sex-matched healthy donors. © 2016 International Society for Advancement of Cytometry.

Levels of circulating endothelial cells are low in idiopathic pulmonary fibrosis and are further reduced by anti-fibrotic treatments.

BACKGROUND: It has been suggested that circulating fibrocytes and endothelial cells actively participate in the intense remodelling of the pulmonary vasculature in patients with idiopathic pulmonary fibrosis (IPF). Indeed, fibrotic areas exist that have fewer blood vessels, whereas adjacent non-fibrotic tissue is highly vascularized. The number of circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) might reflect the balance between vascular injury and repair. Thus, fibrocytes as well as endothelial cells could potentially be used as biomarkers of disease progression and treatment outcome. METHODS: Peripheral blood samples were collected from 67 patients with a multidisciplinary diagnosis of IPF and from 45 age-matched and sex-matched healthy volunteers. Buffy coat was isolated according to standard procedures and at least 20 million cells were stained with different monoclonal antibodies for the detection of CEC, EPC and circulating fibrocytes. For the detection of CEC and EPC, cells were stained with anti-CD45, anti-CD34, anti-CD133, anti-CD14, anti-CD309 and with the viability probe Far-Red LIVE/DEAD. For the detection of circulating fibrocytes, cells were first stained with LIVE/DEAD and the following monoclonal antibodies: anti-CD3, anti-CD19, anti-CD45, anti-CD34 and anti-CD14, then cells were fixed, permeabilized and stained with fluorochrome-conjugated anti-collagen I monoclonal antibodies. RESULTS: Patients with IPF displayed almost undetectable levels of circulating fibrocytes, low levels of CEC, and normal levels of EPC. Patients treated with nintedanib displayed higher levels of CEC, but lower levels of endothelial cells expressing CD309 (the type II receptor for vascular endothelial growth factor). Treatment with both nintedanib and pirfenidone reduced the percentage of CEC and circulating fibrocytes. CONCLUSIONS: Levels of CEC were reduced in patients with IPF as compared to healthy individuals. The anti-fibrotic treatments nintedanib and pirfenidone further reduced CEC levels. These findings might help explain the mechanism of action of these drugs and should be explored as predictive biomarkers in IPF.