Showing papers by "Andrea Cossarizza published in 2013"

Association study of common genetic variants and HIV-1 acquisition in 6,300 infected cases and 7,200 controls

Abstract: Multiple genome-wide association studies (GWAS) have been performed in HIV-1 infected individuals, identifying common genetic influences on viral control and disease course. Similarly, common genetic correlates of acquisition of HIV-1 after exposure have been interrogated using GWAS, although in generally small samples. Under the auspices of the International Collaboration for the Genomics of HIV, we have combined the genome-wide single nucleotide polymorphism (SNP) data collected by 25 cohorts, studies, or institutions on HIV-1 infected individuals and compared them to carefully matched population-level data sets (a list of all collaborators appears in Note S1 in Text S1). After imputation using the 1,000 Genomes Project reference panel, we tested approximately 8 million common DNA variants (SNPs and indels) for association with HIV-1 acquisition in 6,334 infected patients and 7,247 population samples of European ancestry. Initial association testing identified the SNP rs4418214, the C allele of which is known to tag the HLA-B*57:01 and B*27:05 alleles, as genome-wide significant (p = 3.6×10−11). However, restricting analysis to individuals with a known date of seroconversion suggested that this association was due to the frailty bias in studies of lethal diseases. Further analyses including testing recessive genetic models, testing for bulk effects of non-genome-wide significant variants, stratifying by sexual or parenteral transmission risk and testing previously reported associations showed no evidence for genetic influence on HIV-1 acquisition (with the exception of CCR5Δ32 homozygosity). Thus, these data suggest that genetic influences on HIV acquisition are either rare or have smaller effects than can be detected by this sample size.

The monocytic population in chronic lymphocytic leukemia shows altered composition and deregulation of genes involved in phagocytosis and inflammation.

Abstract: Macrophages reside in tissues infiltrated by chronic lymphocytic leukemia B cells and the extent of infiltration is associated with adverse prognostic factors. We studied blood monocyte population by flow cytometry and whole-genome microarrays. A mixed lymphocyte reaction was performed to evaluate proliferation of T cells in contact with monocytes from patients and normal donors. Migration and gene modulation in normal monocytes cultured with CLL cells were also evaluated. The absolute number of monocytes increased in chronic lymphocytic leukemia patients compared to the number in normal controls (792±86 cells/μL versus 485±46 cells/μL, P=0.003). Higher numbers of non-classical CD14+CD16++ and Tie-2-expressing monocytes were also detected in patients. Furthermore, we performed a gene expression analysis of monocytes in chronic lymphocytic leukemia patients, showing up-regulation of RAP1GAP and down-regulation of tubulins and CDC42EP3, which would be expected to result in impairment of phagocytosis. We also detected gene alterations such as down-regulation of PTGR2, a reductase able to inactivate prostaglandin E2, indicating immunosuppressive activity. Accordingly, the proliferation of T cells in contact with monocytes from patients was inhibited compared to that of cells in contact with monocytes from normal controls. Finally, normal monocytes in vitro increased migration and up-regulated CD16, RAP1GAP, IL-10, IL-8, MMP9 and down-regulated PTGR2 in response to leukemic cells or conditioned media. In conclusion, altered composition and deregulation of genes involved in phagocytosis and inflammation were found in blood monocytes obtained from chronic lymphocytic leukemia patients, suggesting that leukemia-mediated “education” of immune elements may also include the establishment of a skewed phenotype in the monocyte/macrophage population.

N-acetyl-cysteine prevents toxic oxidative effects induced by IFN-α in human neurons.

Abstract: Currently IFN-α is widely used for effective treatment of viral infections and several malignancies. However, IFN-α can cause neuropsychiatric disturbances and mental impairments, including fatigue, insomnia, depression, irritability and cognitive deficits. Molecular and cellular mechanisms leading to such side-effects are still poorly understood. Neurons seem to be an important target in mediating cellular effects induced by exposure to this cytokine, but so far little is known about IFN-α-induced effects on these cells. We have investigated the ability of IFN-α (2–100ng/ml) to induce damage and toxicity to the human neuroblastoma SH-SY5Y cell line, commonly used for studying such phenomena, and the mechanisms underlying these effects. After 24 h treatment, IFN-α increased mitochondrial activity, whereas cell density was reduced in a dose- and time-dependent manner. This effect did not depend on reduced cell proliferation, but rather the activation of apoptosis, as revealed by an increased Bax:Bcl-2 mRNA ratio after 72-h IFN-α exposure. At this time-point, IFN-α also reduced the expression of the brain-derived neurotrophic factor gene, and induced an increase in reactive oxygen species (ROS). A co-treatment with N-acetyl-cysteine (NAC; 5 mM), a potent antioxidant and mitochondrial modulator, was able to counteract all of these IFN-α-induced effects. These findings demonstrated that IFN-α induces neurotoxicity and apoptosis that is, in part, very likely due to mitochondrial damages and production of ROS. We suggest that NAC, already tested for the treatment of psychiatric disorders, may be useful to prevent IFN-α-induced central side-effects in a safe and effective way.

Daunomycin, an antitumor DNA intercalator, influences histone-DNA interactions.

Abstract: Although daunomycin and adriamycin are considered effective antitumor drugs and have been used in the clinic for over 40 years, their mechanism of action is still a matter of debate. We investigated the influence of daunomycin on interaction between linker or core histones and DNA in live HeLa cells in vitro, using image and flow cytometry. Exposure to daunomycin at clinically relevant concentrations (25–250 nM) caused dissociation of wild-type H1.1 as well as 4 H1 point mutants from DNA, followed by their accumulation in nucleoli and aggregation of chromatin. A detectable dissociation of H2B core histones occurred only at much higher concentrations of the drug (500 nM). Replication of DNA and synthesis of RNA were not halted by daunomycin (up to 2500 nM); however the characteristic subnuclear distribution of sites of transcription and replication was lost. Dissociation of the H1.1 linker histones and subsequent loss of higher order chromatin structures may constitute an important component of the mechanism of cytotoxicity of daunomycin.

Life expectancy in the immune-recovery era: the evolving scenario of HIV epidemic in Northern Italy

Abstract: Introduction:National cohort and intercohort studies have been set to describe the differences of life expectancy (LE) of HIV-infected individuals. Objective:The aim of this study was to assess the impact of immune recovery (IR) on LE of patients with HIV undergoing combination antiretroviral therapy. Methods:In this retrospective observational study, outcome measure was LE of patients with HIV compared with LE of northern Italian population. Group categorizations were as follows: patients with no immune recovery (nIR), patients with IR, patients who are immune maintained, and pre-highly active antiretroviral therapy (HAART) and post-HAART. Abridged life tables were constructed from age-specific mortality rates (per 1000 person years) to estimate LE from the age of 20–55 years. Results:A total of 9671 patients, 71% men, were included. After 2005, we assisted to a rapid increase in the overall rate of patients attaining IR in the community coupled with a progressive decrease of AIDS death, but not of non-AIDS deaths. In a 40-year-old patient, LE was 38.10 years [standard error (SE) = 2.60], 30.08 years (SE = 0.98), and 22.9 (SE = 0.69) in the IR, post-HAART group and nIR, respectively, compared with 41.38 years of the general Italian population. An approximately 5-year gap in LE was observed in IR patients. Discussion:We describe IR at a “community” level, related to calendar year and apparent 10 years after HAART introduction. HAART community IR is significantly influencing LE and is associated with the changing clinical picture of HIV disease. An increasing gradient of LE exists between nIR, post-HAART, and IR groups, with the latter, above the age of 40 years only, reaching LE of general population.

Randomized trial to evaluate cardiometabolic and endothelial function in patients with plasma HIV-1 RNA suppression switching to darunavir/ritonavir with or without nucleoside analogues.

Abstract: Background: We performed a study to evaluate change in cardiometabolic and endothelial function in HIV-infected patients switching to darunavir/ritonavir (DRV/r) monotherapy versus triple therapy. Methods: The MONARCH trial recruited 30 patients who were taking triple combination therapy and with HIV RNA .05), with no statistically significant difference between arms (10.7% ...

Cytometry, immunology, and HIV infection: Three decades of strong interactions

Abstract: Flow cytometry (FCM) has been extensively used to investigate immunological changes that occur from infection with the human immunodeficiency virus (HIV) This review describes some of the most relevant cellular and molecular changes in the immune system that can be detected by FCM during HIV infection Finally, it will be discussed how this technology has facilitated the understanding not only of the biology of the virus but also of the mechanisms that the immune system activates to fight HIV and is allowing to monitor the efficacy of antiretroviral therapy

Recurrent varicella following steroids and fingolimod in a Multiple Sclerosis patient.

Abstract: Dear Sir, We found the recent article on fingolimod significantly increasing the frequencies of T regulatory cells (Treg) in Multiple Sclerosis (MS) patients (Serpero et al. 2013) to be of particular interest for the assessment of the drug’s mechanism of action. However, there is also increasing interest on fingolimod-induced mechanisms of reduced antiviral immunologic competence following the occurrence of encephalitides and deaths due to herpes viruses during treatment with fingolimod (Cohen et al. 2010; Ratchford et al. 2012) and the growing concern on the effects of fingolimod on immunosurveillance. We would like to share our observation of a case of recurrent varicella during treatment with fingolimod following treatment with steroids and briefly explore possible mechanisms which may have facilitated a viral reactivation, including the combination of fingolimod-induced lymphopenia and steroids. In addition, we present cytofluorimetric data, which are in accordance with those by Serpero et al. (2013), and hypothesize that the relative increase of Treg, which inhibit immune responses, may be involved in the reduced antiviral immunity in fingolimod-treated patients. A 52-year-old woman who had presented with two Multiple Sclerosis (MS) relapses during treatment with betainterferon in the preceding year, started treatment with natalizumab on January 23rd, 2013. On February 27th, shortly after initiating the second natalizumab infusion, she had an anaphylactic reaction which lead to hospitalization. She was treated with high-dose iv methylprednisolone for 2 days followed by oral prednisone (50 mg, which was tapered off in 12 days and discontinued on March 12th 2013). Her medical history was otherwise uneventful and she was not taking any concomitant medication. She had a moderate disability (Expanded Disability Status Scale: 3; range 0–10) due to left hemiparesis and mild gait ataxia. She reported a history of varicella disease. Following preliminary exams, inclusive of lymphocyte typing, which was normal, and of anti-Varicella Zoster Virus (VZV) IgG and IgM serum titers which revealed positive IgG (0,86) and negative IgM (0,04) titers (range of positivity: 0.21–6.00), confirming acquired varicella immunity, she started treatment with fingolimod on April 22nd. On April 27th a few pruriginous papular lesions gradually started appearing on her abdomen and right arm. Repeat VZV serology (May 7th) showed unchanged IgG (1.03) and IgM (0.03) serum titers and blood chemistry and full blood count only revealed a drug-related leucopenia with marked lymphopenia (0.56× 10 cells/mm). In the following days the number of papules gradually increased, spreading to other regions (trunk, lower limbs), and vesicles appeared. Fingolimod was discontinued D. Ferraro (*) : F. Vitetta :A. M. Simone : L. Federzoni : P. F. Nichelli : P. Sola Neurology Unit, Department of Neurosciences, University of Modena and Reggio Emilia, Nuovo Ospedale Civile Sant’Agostino Estense, Via Pietro Giardini, 1355, 41126 Modena, Italy e-mail: perdiana@tin.it

Cytometry for immunology: a stable and happy marriage.

Abstract: THE XV International Congress of Immunology (ICI) held in Milan, Italy, August 22–27, 2013 is likely a perfect occasion to celebrate a marriage that was officiated in the mid-1970s, when monoclonal antibodies (mAbs) were created and became available for research. After a very brief period of engagement, the “official exchange of rings” was represented by the possibility to conjugate mAbs with adequate fluorochromes. This changed almost completely the main use of an instrument, that is, the flow cytometer, that had been created and commercialized few years before for the measurement of cellular DNA by fluorescent intercalating dyes such as ethidium bromide. The recognition of immune cells stained with fluorescent mAbs, typically performed by fluorescence microscopy, was then passed to complex (and at that time quite esoteric) instruments that could analyze thousands of cells in a few seconds, and could provide informative and highly repetitive results. In the early 1980s, the dramatic epidemics of human immunodeficiency virus (HIV)/AIDS pointed out the importance of CD41 T cells for most activities of the cognate immune system, and the importance of the loss of this cell type due to the action of the virus or excessive immune activation. The quantitation of CD41 T cells in a growing number of patients required the development of cytofluorimeters that were much easier to use than the original instruments they derived from. Such instruments are now more user-friendly than ever and can be used by almost all researchers after a relatively short period of training. As a result, in the last decades, immunologists have been using cytometric techniques to dissect almost all components of the immune response. The best demonstration is that the large majority, if not almost all, articles published in main immunological journals make use of flow cytometry. On the other side, in recent years, a large space in the journal Cytometry A has been dedicated to all the novel immunological techniques and approaches that are continuously developed to investigate, among others, cell phenotype, the quality or the amount of molecules produced or secreted by the cell populations of interest, different signaling processes, cell differentiation, proliferation or death, killing capacities, cell–cell interactions, response against tumour cells or to immunosuppressive drugs, transcription factors’ activity, quantification of soluble molecules, drug uptake (1–9). Clearly, flow cytometry is essential to sort the cells of interest for further investigations, such as cell cloning, analysis of gene expression, functional studies at the single cell level. It is also notable that the most recent advancements, and in particular mass cytometry (10), are allowing measurement of an unprecedented number of immune parameters at the single cell level, and to analyze in a relatively simple manner incredible amounts of data (11–14). A rapid check in PubMed shows that, as of July 2013, typing “flow cytometry immunology” gives more than 54,000 articles, the first of which, curiously, is not related to lymphocytes (15). However, during the 5th International Symposium of Flow Cytometry, Clinical and Biological Applications, held in Rome, Italy (December 2–5, 1980), a poster session was dedicated to Immunology (16).

Early alterations of B cells in patients with septic shock: another piece in the complex puzzle of the immune response in sepsis.

Abstract: Impairment of the inflammatory-immune response is currently accepted as a hallmark of severe sepsis even in the early stages of the disease. In this context, the alterations of the circulating B-lymphocytes have never been described in detail. The study by Monserrat and colleagues in the previous issue of Critical Care indicated that, in patients with septic shock, the B-cell compartment is early and deeply altered with different patterns in subset distribution and activation between survivors and non-survivors.

Absence of liver steatosis in HIV-HCV co-infected patients receiving regimens containing tenofovir or abacavir.

Abstract: Background In human immunodeficiency virus–hepatitis C virus (HIV–HCV) co-infected patients, steatosis has been independently associated with a number of antiretroviral drugs, including stavudine, especially in patients with non-3 HCV genotypes. We retrospectively investigated the presence of steatosis among HIV–HCV co-infected and HCV mono-infected patients, and the role of tenofovir disoproxil fumarate (TDF) or abacavir (ABC) in determining hepatic steatosis.

Frequent early multiple sclerosis relapses during treatment with fingolimod: a paradoxical effect?

Abstract: We read with interest the recent articles on early relapses and severe disease manifestations in multiple sclerosis (MS) patients during treatment with fingolimod (FTY).1–3 Min et al.1 describe the development of extensive brain lesions in a patient with neuromyelitis optica spectrum disorder two weeks after starting FTY, while different authors have reported early relapses during FTY therapy following a switch from natalizumab after a three-month wash-out period. In particular, Daelman et al.2 describe how FTY was discontinued in a patient after a severe relapse which occurred within 11 days of starting treatment, and that a subsequent re-introduction of the drug was followed by another relapse 10 days later. Rinaldi et al.3 observed a disease reactivation in 7/22 (32%) patients within one month of FTY therapy (four patients with clinical relapses and three patients with new magnetic resonance imaging (MRI) lesions). It has been argued that this may be due to FTY not acting quickly enough to stop disease reactivation after natalizumab discontinuation, but the timing of the events could also suggest a causative role of FTY therapy. We would therefore like to share our observations on frequent early relapses in patients commencing FTY at our Center (Center for Demyelinating Diseases of Modena, Italy). Eleven patients (10 female, 1 male, mean age: 41 years, mean disease duration: 11.5 years) started treatment with FTY between January 2012 and October 2012. All patients were switched from beta-interferon or glatiramer acetate to FTY because of clinical or MRI disease progression after a wash-out period of maximum one month. By the end of January 2013, five patients (45%) had experienced a relapse: of these relapses, two occurred within the first two weeks, two within approximately two months and one after five months. All patients recovered completely after a course of steroids. After six months of treatment, MRI disease activity (new T2 lesions and/or gadolinium-enhanced lesions) was present in 4/5 (80%) patients who had experienced a relapse. Although the timing of the relapses does not constitute evidence of causation and there are currently no data to support this notion, a hypothesis is that early relapses during FTY treatment could be explained by a transient reduction of T regulatory cells (Treg), at least in terms of absolute values, or of their immunosuppressive activity, although data is contradictory. On the one hand, there is evidence that FTY potently inhibits Treg proliferation in vitro and in vivo by inhibiting interleukin (IL)-2 mediated mitogenic signals: furthermore, the immunosuppressive effect of transferred Treg was abrogated in two mouse models of inflammation.4 On the other hand, FTY was shown to enhance the functional activity of CD4+/CD25+ Treg cells in vitro and in vivo.5 Future studies aimed at clarifying whether a drug-induced reduced egress or function of inhibitory immune cells may facilitate disease exacerbations, especially in highly 'active' MS patients, are warranted. The duration of these effects and whether they may be predictive of a non-response to FTY also need to be established.

A Janus role for MerTK in the outcome of septic shock.

Abstract: Severe sepsis remains a clinical challenge today, even indeveloped countries, because of the increased age of thepatients and the high burden of antibiotic resistance [1]. Inaddition, in recent years, we have become increasinglyaware that, even in the early stages of sepsis, a high pro-portion of patients experience a severe dysfunction ofnative and adaptive immunity, which is closely related topatients’ outcome [2]. In this context, understanding thefunctionality of receptors able to modulate the immuneresponse, and in particular to downregulate inflammation,has great importance for designing new therapeutic strat-egies. Among these molecules, a main role is occupied bythe TAM family of receptor tyrosine kinases, which isformed by three members, i.e., Tyro3, AxI, and Mer, thathave two vitamin K-dependent ligands, i.e., the growtharrest-specific protein (Gas)6 and Protein S [3]. In theSeptember 2013 issue of Intensive Care Medicine, Gui-gnant et al. [4] explored by an elegant experimental studythe expression of TAM receptors in circulating immunecells in patients with septic shock and after trauma, and inhealthy volunteers. A significant early alteration of MerTKexpression on monocytes and neutrophils was observed inpatients with septic shock, and a persistent MerTK over-expression after septic shock was associated with adverseoutcome. The ex vivo analysis showed also a differentMerTK surface expression of healthy monocytes after48 h incubation with plasma from septic or traumapatients, supporting the difference in TAM expressionbetween infection-induced inflammation and sterileinflammation (i.e., trauma). The study provides newimportant data on the mechanisms of immune response insepsis, but the true reasons for the observed alterations inMerKT expression and their role in the complex patho-physiological context of sepsis remain to be clarified.TAM receptors are under the control of Toll-likereceptors (TLR), and are widely expressed by cells of theinnateimmunesystem[5].ActivationofTAMreceptorsbytheir ligands triggers a cascade of intracellular events thatculminate in the inhibition of nuclear factor-kappaB (NF-jB), a crucial proinflammatory molecule, and results indownregulation of inflammation [6]. When expressed bymonocytes and granulocytes, these receptors (and in par-ticular MerTK) can also mediate the phagocytosis ofapoptoticcells[7],whichisfundamentalinordertoremovedead cells without triggering inflammation. Several datanow exist that underline the importance of MerTK activa-tion in the elimination of potential autoantigens derived byapoptotic cells, in the inhibition of the production of pro-inflammatory cytokines, and in the prevention of theexpansion of autoreactive lymphocytes [8–10]. The func-tional impairment of MerTK promotes not onlyautoimmunity but also atherosclerosis, while its overex-pression is linked to poor prognosis in cancer [11].The strong relationships existing between TAMreceptors and a proinflammatory molecule such as tumornecrosis factor-alpha (TNF-a) are reinforced by theobservation that they can interact with a common protein,the matrix metalloproteinase TNF-a converting enzyme,also known as ‘‘a disintegrin and a metalloproteinase 17’’(ADAM17). In order to provide the mature, soluble,secretable form of TNF-a, ADAM17 cleaves the mem-brane form of this important proinflammatory cytokine[12]. Interestingly, ADAM17 is also able to cleave the

Immune Response and HIV Infection: Old Problems for New Challenges

Abstract:  Abstract— The infection with HIV, the virus that causes AIDS, triggers a number of immune responses that have the aim to control the production and propagation of the virus, mainly by blocking its binding to CD4+ T cells by neutralizing antibodies, or killing infected cells by cytotoxic T lymphocytes. However, in most cases the immune system is not able to control this retrovirus, that uses sophisticated evolutionary mechanisms to survive and propagate its progeny. In this review some immune mechanisms that try to contrast HIV are discussed, putting an accent on the variety of problems that immunologists have had, and actually have in designing and producing an effective vaccine.

A Janus role for MerTK in the outcome of septic

Abstract: Severe sepsis remains a clinical challenge today, even indeveloped countries, because of the increased age of thepatients and the high burden of antibiotic resistance [1]. Inaddition, in recent years, we have become increasinglyaware that, even in the early stages of sepsis, a high pro-portion of patients experience a severe dysfunction ofnative and adaptive immunity, which is closely related topatients’ outcome [2]. In this context, understanding thefunctionality of receptors able to modulate the immuneresponse, and in particular to downregulate inflammation,has great importance for designing new therapeutic strat-egies. Among these molecules, a main role is occupied bythe TAM family of receptor tyrosine kinases, which isformed by three members, i.e., Tyro3, AxI, and Mer, thathave two vitamin K-dependent ligands, i.e., the growtharrest-specific protein (Gas)6 and Protein S [3]. In theSeptember 2013 issue of Intensive Care Medicine, Gui-gnant et al. [4] explored by an elegant experimental studythe expression of TAM receptors in circulating immunecells in patients with septic shock and after trauma, and inhealthy volunteers. A significant early alteration of MerTKexpression on monocytes and neutrophils was observed inpatients with septic shock, and a persistent MerTK over-expression after septic shock was associated with adverseoutcome. The ex vivo analysis showed also a differentMerTK surface expression of healthy monocytes after48 h incubation with plasma from septic or traumapatients, supporting the difference in TAM expressionbetween infection-induced inflammation and sterileinflammation (i.e., trauma). The study provides newimportant data on the mechanisms of immune response insepsis, but the true reasons for the observed alterations inMerKT expression and their role in the complex patho-physiological context of sepsis remain to be clarified.TAM receptors are under the control of Toll-likereceptors (TLR), and are widely expressed by cells of theinnateimmunesystem[5].ActivationofTAMreceptorsbytheir ligands triggers a cascade of intracellular events thatculminate in the inhibition of nuclear factor-kappaB (NF-jB), a crucial proinflammatory molecule, and results indownregulation of inflammation [6]. When expressed bymonocytes and granulocytes, these receptors (and in par-ticular MerTK) can also mediate the phagocytosis ofapoptoticcells[7],whichisfundamentalinordertoremovedead cells without triggering inflammation. Several datanow exist that underline the importance of MerTK activa-tion in the elimination of potential autoantigens derived byapoptotic cells, in the inhibition of the production of pro-inflammatory cytokines, and in the prevention of theexpansion of autoreactive lymphocytes [8–10]. The func-tional impairment of MerTK promotes not onlyautoimmunity but also atherosclerosis, while its overex-pression is linked to poor prognosis in cancer [11].The strong relationships existing between TAMreceptors and a proinflammatory molecule such as tumornecrosis factor-alpha (TNF-a) are reinforced by theobservation that they can interact with a common protein,the matrix metalloproteinase TNF-a converting enzyme,also known as ‘‘a disintegrin and a metalloproteinase 17’’(ADAM17). In order to provide the mature, soluble,secretable form of TNF-a, ADAM17 cleaves the mem-brane form of this important proinflammatory cytokine[12]. Interestingly, ADAM17 is also able to cleave the