SERPING1 mRNA overexpression in monocytes from HIV+ patients.

OBJECTIVE: The HIV-1 virus activates the complement system, an essential element of the immune system. SERPING1 is a protease inhibitor that disables C1r/C1s in the C1 complex of the classical complement pathway. METHODS: In this paper, we performed an analysis of several microarrays deposited in GEO dataset to demonstrate that SERPING1 mRNA is modulated in CD14+ monocytes from HIV-1-infected individuals. In addition, data were validated on monocytes isolated from seronegative healthy volunteers, treated with IFNs. RESULTS: Our analysis shows that SERPING1 mRNA is overexpressed in monocytes from HIV-1+ patients and the expression levels correlate positively with viral load and negatively with the CD4+ T-cell count. Of note, anti-retroviral therapy is able to reduce the levels of SERPING1 mRNA, ex vivo. In addition, we found that 30% of the SERPING1 genes network is upregulated in monocytes from HIV-1+ patients. Noteworthy, the expression levels of IFITM1-an antiviral molecule belonging to the genes network-correlate positively with SERPING1 expression. Interestingly, the monocytes treatment with IFN-gamma, IFN-beta and IFN-alpha significantly upregulates the SERPING1 mRNA expression levels. CONCLUSIONS: From the outcome of our investigation, it is possible to conclude that SERPING1 and its network serve as important components of the innate immune system to restrict HIV-1 infection.

CD4+ T-cell gene expression of healthy donors, HIV-1 and elite controllers: Immunological chaos.

OBJECTIVES: T-cell repertoire dysfunction characterizes human immunodeficiency virus type 1 (HIV-1) infection, but the pathogenic mechanisms remain unclear. Disease progression is probably due to a profound dysregulation of Th1, Th2, Th17 and Treg patterns. The aim of this study was to analyze the features of CD4+ T cells in HIV-positive patients with different viroimmunological profile. METHODS: we used a gene expression dataset of CD4+ T cells from healthy donors, HIV+ naive patients and Elite Controllers (EC), obtained from the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/, accession number GSE18233). RESULTS: Principal Component Analysis (PCA) showed an almost complete overlap between the HIV-infected and EC patients, which cannot easily explain the different responses to HIV infection of these two group of patients. We have found that HIV patients and the EC showed an upregulation of the Th1 pro-inflammatory cytokines and chemokines, compared to the controls. Also, we have surprisingly identified IL28B, which resulted downregulated in HIV and EC compared to healthy controls. We focused attention also on genes involved in the constitution of the immunological synapse and we showed that HLA class I and II genes resulted significantly upregulated in HIV and in EC compared to the control. In addition to it, we have found the upregulation of others syncytial molecules, including LAG3, CTLA4, CD28 and CD3, assisting the formation of syncytia with APC cells. CONCLUSIONS: Understanding the mechanisms of HIV-associated immunological chaos is critical to strategically plan focused interventions.

LPS and HIV gp120 modulate monocyte/macrophage CYP27B1 and CYP24A1 expression leading to vitamin D consumption and hypovitaminosis D in HIV-infected individuals.

AIM: Vitamin D deficiency is very common among HIV-infected subjects. We cross-sectionally evaluated the prevalence and risk factors for hypovitaminosis D in 91 HIV-infected Italian patients. PATIENTS AND METHODS: We studied in a cohort of 91 HIV-infected Italian patients the metabolism of Vitamin D by evaluating the in vitro expression of CYP27B1, CYP24A1 and vitamin D receptor (VDR) by monocytes and macrophages stimulated with the viral envelope protein gp120 or lipopolysaccharide (LPS). RESULTS: The prevalence of vitamin D deficiency (25OHD < 10 ng/ml) and vitamin D insufficiency (25OHD 10-30 ng/ml) was 31% and 57%, respectively. In univariate analysis, female sex (p = 0.01), increasing age (p = 0.05), higher highly sensitive-C reactive protein (p = 0.025), higher parathyroid hormone (PTH) (p = 0.043) and lower BMI (p = 0.04) were associated with vitamin D deficiency. In multivariate analysis, the association was still significant only for PTH (p = 0.03) and female sex (p = 0.03). Monocyte stimulation with LPS (100 ng/ml) or gp120 (1 µg/ml) significantly upregulated CYP27B1 mRNA expression. Moreover, gp120 significantly increased VDR mRNA levels. On the contrary, neither LPS nor gp120 modified CYP24A1 levels. Macrophage stimulation with LPS (100 ng/ml) significantly upregulated CYP27B1 and CYP24A1 mRNA expression. When monocytes were cultured in the presence of 25OHD (40 ng/ml) and stimulated with LPS we detected significantly lower levels of 25OHD in the supernatant. CONCLUSIONS: Vitamin D deficiency was very common in our cohort of HIV-infected patients. Chronic inflammation, including residual viral replication, may contribute to hypovitaminosis D, by modulating vitamin D metabolism and catabolism. Systematic screening may help identifying subjects requiring supplementation.

Non-AIDS defining cancers: a comprehensive update on diagnosis and management.

The increasing incidence of chronic pathologies and especially non-AIDS defining cancers, such as lung cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, prostate cancer, and Hodgkin's lymphoma after the introduction of combined antiretroviral therapy requires the infectious diseases specialist to know how and when to suspect and diagnose cancer in people living with HIV. The aim of this review is to provide updated studies and information about non-AIDS defining cancers and their management in PLWH sheading a light on possible futures scenarios.

Calcineurin primes immature gonadotropin-releasing hormone-secreting neuroendocrine cells for migration.

During development, many neurons display calcium-dependent migration, but the role of this messenger in regulating gene expression leading to this event has not yet been elucidated. Among the decoders of calcium signals is calcineurin, a Ca(2+)/calmodulin serine/threonine phosphatase that has been involved in both short-term and long-term cellular changes. By using immortalized GnRH-secreting neurons, we now show that, in vitro, Ca(2+)-dependent gene expression, proceeding via calcineurin and the transcription factor nuclear factor of activated T cells, is a key player controlling the chemomigratory potential of developing GnRH-secreting neurons. Furthermore, our data highlight the switch nature of this phosphatase, whose activation or inactivation guides cells to proceed from one genetic program to the next.

Inhibitors of histone deacetylase (HDAC) restore the p53 pathway in neuroblastoma cells.

BACKGROUND AND PURPOSE: Inhibitors of histone deacetylase (HDAC) are emerging as a promising class of anti-cancer drugs, but a generic deregulation of transcription in neoplastic cells cannot fully explain their therapeutic effects. In this study we evaluated alternative molecular mechanisms by which HDAC inhibitors could affect neuroblastoma viability. EXPERIMENTAL APPROACH: Effects of HDAC inhibitors on survival of the I-type SK-N-BE and the N-type NB SH-SY5Y neuroblastoma cell lines were assessed by the MTT assay. Molecular pathways leading to this were examined by western blot, confocal microscopy and cytofluorometry. The mRNA levels of apoptotic mediators were assessed semi-quantitatively by RT-PCR. Tumour-suppressor p53 trans activity was assessed in EMSA experiments. HDAC inhibitors were also studied in cells subjected to plasmid-based p53 interference (p53i). KEY RESULTS: HDAC inhibitors induced cell death via the mitochondrial pathway of apoptosis with recruitment of Bcl-2 family members. Bcl-2 overexpression rendered neuroblastoma cells resistant to HDAC inhibitor treatment. Low concentrations of HDAC inhibitors (0.9 mM) caused a G(2) cell-cycle arrest and a marked upregulation of the p21/Waf1/Cip1 protein. HDAC inhibitors also activate the p53 protein via hyper-acetylation and nuclear re-localization, without affecting its protein expression. Accordingly, HDAC inhibitor-induced cell-killing and p21/Waf1/Cip1 upregulation is impaired in p53i-cells. CONCLUSIONS AND IMPLICATIONS: In neuroblastoma cells, HDAC inhibitors may overcome the resistance to classical chemotherapeutic drugs by restoring the p53 tumour-repressor function via its hyper-acetylation and nuclear migration, events usually impaired in such tumours. In neuroblastoma cells, HDAC inhibitors are not able to induce p21/Waf1/Cip1 in the absence of a functional p53.

OAS Gene Family Expression Is Associated with HIV-Related Neurocognitive Disorders.

HIV-associated neurocognitive disorders are common in HIV-infected individuals, even in the combination antiretroviral therapy (c-ART) era. Several mechanisms are involved in neuronal damage, including chronic inflammation immune activation. Mammalian 2'-5'-oligoadenylate synthetase (OAS) genes are produced in response to interferon (IFN), mainly by monocytes, and exert their antiviral functions by activation of RNase L that degrades viral and cellular RNAs. In this study, we aimed at exploring OAS gene family RNA expression in simian immunodeficiency virus encephalitis (SIVE), in HIV-associated neurocognitive disorders (HAND), and in HIV-associate dementia (HAD). We analyzed three microarray datasets obtained from the NCBI in order to assess the expression levels of OAS gene family network in brain biopsies of macaques with SIVE vs uninfected animals, as well as post-mortem brain of individuals with HAND (on or off ART) vs uninfected controls and three brain regions of HIV-infected individuals with both neurocognitive impairment (HAD) and encephalitis (HIVE). All OAS genes were upregulated both in SIVE and in HAND. OAS expression was significantly higher in high-viremic individuals; increased expression levels persisted in cART subjects when compared to healthy controls. OAS gene network analysis showed that several genes belonging to the type I IFN pathway, especially CXCL10 and IFIT3, were similarly upregulated in SIVE/HAND. Furthermore, we identified a significant upregulation of OAS gene family RNA expression in basal ganglia, white matter, and frontal cortex of HIV-1, HAD, and HAD/HIVE patients compared to healthy subjects. OAS gene family expression is increased in brain sections from individuals with HAND, HAD, and HIVE as well as macaques with SIVE. OAS family expression is likely to be induced by IFN as a consequence of viral replication in the CNS. Its long-term upregulation may contribute to the chronic inflammatory status and neurocognitive impairment we still observe in virologically suppressed individuals on c-ART.

Caspase cleavage enhances the apoptosis-inducing effects of BAD.

The function of BAD, a proapoptotic member of the Bcl-2 family, is regulated primarily by rapid changes in phosphorylation that modulate its protein-protein interactions and subcellular localization. We show here that, during interleukin-3 (IL-3) deprivation-induced apoptosis of 32Dcl3 murine myeloid precursor cells, BAD is cleaved by a caspase(s) at its N terminus to generate a 15-kDa truncated protein. The 15-kDa truncated BAD is a more potent inducer of apoptosis than the wild-type protein, whereas a mutant BAD resistant to caspase 3 cleavage is a weak apoptosis inducer. Truncated BAD is detectable only in the mitochondrial fraction, interacts with BCL-X(L) at least as effectively as the wild-type protein, and is more potent than wild-type BAD in inducing cytochrome c release. Human BAD, which is 43 amino acids shorter than its mouse counterpart, is also cleaved by a caspase(s) upon exposure of Jurkat T cells to anti-FAS antibody, tumor necrosis factor alpha (TNF-alpha), or TRAIL. Moreover, a truncated form of human BAD lacking the N-terminal 28 amino acids is more potent than wild-type BAD in inducing apoptosis. The generation of truncated BAD was blocked by Bcl-2 in IL-3-deprived 32Dcl3 cells but not in Jurkat T cells exposed to anti-FAS antibody, TNF-alpha, or TRAIL. Together, these findings point to a novel and important role for BAD in maintaining the apoptotic phenotype in response to various apoptosis inducers.

Hepatitis C-related hepatocellular carcinoma: diagnostic and therapeutic management in HIV-patients.

The efficacy of the current HIV therapy has led to increased survival and prolongation of the average life expectancy of people living with HIV (PLWH), as well as the emergence of comorbidities and non-AIDS related cancer. Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Current evidence suggests that HCC is an important cause of morbidity and mortality in HIV infected patients. In fact, HCC prevalence rate is indeed higher with respect to the general population average. In this paper, we review the diagnostic and therapeutic management of Hepatitis C-related hepatocellular carcinoma in HCV-HIV co-infected patients. Several therapeutic options are available depending on several factors as HCC stage, liver functions, comorbidities and they have been divided into three groups: potentially curative, proven effective but not curative, and unproven or ineffective therapy. In HIV-infected patients, surgical options are preferred compared to non-surgical therapies. Further studies, especially multicenter ones, are needed in order to define the most appropriate, evidence-based therapeutic approach to PLWH suffering from HCC. It also appears necessary to develop appropriate care guidelines for PLWH.

Kaposi's sarcoma in HIV-infected patients in the era of new antiretrovirals.

Kaposi's Sarcoma (KS) is a multicentric angioproliferative cancer of endothelial cells (ECs) caused by Human Herpesvirus 8 (HHV8) characterized by clinical heterogeneity depending on the host immune conditions. Despite its incidence has dramatically decreased in developed countries after the introduction of Highly Active Antiretroviral Therapy (HAART), KS remains the most frequent tumor in HIV-infected patients worldwide. Clinical presentation varies from an indolent slowly progressive behavior, generally limited to the skin, to an aggressive and rapidly progressing disease. In more than 50% of cases, the skin lesions are often associated with a more or less important visceral involvement, particularly to the oral cavity and the gastrointestinal tract that are involved in 35% and 40% of cases respectively. A large number of treatments can be used both as local and as systemic therapy. Particularly, HAART represents the first treatment in patients with moderate lesions limited to skin, and it can be sufficient to reduce significantly the size of lesions and, often, the complete disappear in 35% of cases after 3-9 months of treatment. In case of a rapidly progressive disease with extensive cutaneous and/or visceral involvement systemic drugs are used such as the liposomal anthracyclines pegylated liposomal doxorubicin (PLD) and daunorubicin citrate liposome (DNX), the combined treatment adriamycin-bleomycin-vincristine (ABV) and bleomycin-vincristine (BV), Paclitaxel and Interferon-alfa. In patients with limited skin localization, the local treatment can play an important role. Local medical therapy is based on the use of alitretinoin, antineoplastic drugs vincristine, vinblastine and bleomycin and Sodium Tetradecyl Sulfate (STS). In addition to medical therapy, physical treatment, such as cryotherapy and radiotherapy, are also commonly used.

Mitogenic effect of nerve growth factor (NGF) in LNCaP prostate adenocarcinoma cells: role of the high- and low-affinity NGF receptors.

We have investigated the effect of nerve growth factor (NGF) in the androgen-dependent, prostate adenocarcinoma LNCaP cell line. Exposure of LNCaP cells to NGF resulted in a significant increase of cell proliferation. The effect was concentration dependent and equally present in serum- or charcoal-stripped serum-supplemented and serum-deprived conditions. The mitogenic action of NGF was accompanied by an enhanced expression of prostate-specific antigen (PSA) and resulted additive to the proliferative effect of dihydrotestosterone. The proliferative effect of NGF appeared to be mediated by the high-affinity NGF receptor, p140trka. Only p140trka, but not the low-affinity NGF receptor, p75LNGFR, was expressed in LNCaP cells; both the proliferative response and the phosphorylation of p140trka upon NGF treatment were prevented by the tyrosine kinase inhibitor K252a. LNCaP cells transiently transfected with the cDNA encoding for p75LNGFR appeared more sensitive to NGF, as demonstrated by the increased number of p75LNGFR-transfected LNCaP cells exposed for 72 h to NGF compared with wild LNCaP cultures. However, p75LNGFR-transfected LNCaP cells rapidly underwent apoptotic death when deprived of NGF. Our study demonstrates the physiological relevance of NGF in the regulation of prostate cell proliferation and the relative contribution of the high- and low-affinity NGF receptors in this control.

Tumor necrosis factor-alpha induces apoptosis in immortalized hypothalamic neurons: involvement of ceramide-generating pathways.

To investigate possible effects that may contribute, together with a direct action on neurohormone secretion, to the impairment of gonadal axis function during inflammation, we evaluated the effect of TNF alpha on the growth and viability of GT1-7 hypothalamic neurons and the intracellular transduction pathways involved in these effects. TNF alpha caused a reduction of cell number and an induction of apoptotic death. These effects were mimicked by cell-permeable analogs of ceramide and by neutral or acidic sphingomyelinase. Exposure to acidic sphingomyelinase induced a persistent (up to 48 h) reduction of cell growth and apoptosis, whereas the effect of neutral sphingomyelinase was time limited. The involvement of acidic sphingomyelinase in TNF alpha action was demonstrated by the partial prevention of ceramide generation, apoptosis, and reduced cell growth by the inhibitor of the acidic sphingomyelinase-generating pathway, D609, whereas the involvement of ceramide was proved by complete prevention of TNF alpha-induced effects by treatment with okadaic acid at concentrations inhibiting ceramide-dependent protein phosphatase. The present data indicate that TNF alpha, through activation of ceramide-generating pathways, is able to affect GT1-7 cell viability, suggesting an additional effect that may contribute to the global action of this cytokine on neuroendocrine activities.

Distinct effects of ceramide-generating pathways in prostate adenocarcinoma cells.

Ceramide, generated by the hydrolysis of sphingomyelin, mediates the actions of several cytokines such as tumour necrosis factor-alpha (TNF-alpha) interferon-gamma and interleukin-1beta (IL-1beta), including their inhibitory effect on tumour proliferation. We have evaluated the role of ceramide in the proliferation of prostate cancer by using the human prostate adenocarcinoma LNCaP cell line. Treatment of LNCaP cells with neutral or acidic sphingomyelinase or addition of C8- or C2-ceramide, two cell permeable analogues of endogenous ceramide, induced a profound inhibition of cell proliferation. This effect appeared after 24 h, was still present after 72 h of exposure to the drugs and exhibited concentration-dependency (10-200 and 5-200 mU ml(-1) for neutral and acidic sphingomyelinase, respectively, and 1-25 microM for C8-ceramide). The inhibitory effect on cell growth caused by neutral sphingomyelinase and ceramides was rapidly reversible as LNCaP cells rapidly regained their previous proliferation rate following withdrawal of the treatment. IL-1beta produced profound inhibition of LNCaP cell proliferation and caused enhanced ceramide formation. No clear features of apoptotic cell death were detectable by either oligonucleosome formation, cytofluorimetric analysis or nuclear staining following exposure of LNCaP cells to neutral sphingomyelinase, ceramide or IL-1beta. However, clear changes in LNCaP cell cycle distribution were detectable following these treatments. In contrast, treatment with acidic sphingomyelinase or TNF-alpha induced apoptotic death detectable by flow cytometric analysis and bisbenzimide staining. In conclusion, our data demonstrate that preferential activation of distinct enzymatic pathways by cytokines may lead to different outcomes in the viability of LNCaP cells.

Detection of immunoglobulin G to measles virus, rubella virus, and mumps virus in serum samples and in microquantities of whole blood dried on filter paper.

Immunity to measles virus, rubella virus, and mumps virus was determined by EIA in serum samples and in dried whole blood specimens spotted on Whatman filter paper (5 mm in diameter). Both specimens were obtained from each patient by venepuncture and finger prick. Ten microliters of whole blood is enough to detect antibodies to these three different viruses. The comparison of the results obtained by EIA from 227 serum and whole blood samples have demonstrated close agreement: 98.6% for measles virus, 99.1% for rubella virus, and 96.0% for mumps virus. Moreover, 96 whole blood samples can be tested in a microtiter plate and can be stored at room temperature for 15 days or at +4 degrees C for several months. Therefore, whole blood dried on filter paper is a convenient alternative method for collecting and transporting specimens, it is easier and safer than venepuncture, and could be used for large-scale epidemiological studies, especially in newborns. This method could solve the problem of sampling, especially in young children, and could simplify studies of vaccine efficacy.

Improvement in establishing the period of rubella virus primary infection using a mild protein denaturant.

A more precise determination of the date of primary rubella infection in pregnancy is obtained by application of the urea denaturation method. Two denaturation buffers containing 6 and 8 M urea were compared for the detection of rubella IgG avidity by enzyme immunoassay. The results demonstrate that IgG avidity detected by 6M urea increases more rapidly, from 16% on the third day to 51% on the 46th day after the rash, than that obtained by 8 M urea, from 13 to 36%. The specific rubella IgG avidity detected by 6 M urea denaturation buffer increased significantly in paired sera obtained at an interval of 15 days. The same samples did not show any significant increase of IgG avidity when an 8 M urea denaturation buffer was used. The use of a mild denaturant such as 6 M urea indicates the presumptive date of primary rubella infection in pregnancy within 2 months of sampling.

Porphyromonas gingivalis prevalence related to other micro-organisms in adult refractory periodontitis.

Forty-six adult periodontal patients, selected on the basis of clinical examination, and 46 adult healthy subjects were examined. The subgingival plaque samples from one inflammatory and one non-inflammatory site of each periodontal patient were studied to determine Porphyromonas gingivalis prevalence related to other periodontal micro-organisms and to periodontal tissue destruction. The results showed Porphyromonas gingivalis as the main pathogenic micro-organism isolated in the inflammatory sites together with Bacteroides forsythus. Peptostreptococcus sp., Actinomyces sp. and Prevotella sp. were found as a normal oral flora in the healthy subjects. Fusobacterium nucleatum, Prevotella intermedia, Campylobacter rectus and Eikenella corrodens were detected both in inflammatory and in non-inflammatory sites of periodontal patients as well as in the healthy subjects.

Anti-HCMV serologic response in patients with acute viral hepatitis.

The authors point out a high serological incidence of HCMV infection in the course of HCV hepatitis compared to a low incidence detected in HBV and HAV hepatitis. Three immunoglobulin classes (IgG, IgM, and IgA) specific to HCMV were investigated. Antibody titers of each immunoglobulin class are reported for all the cases of HCMV infection.

Detection of immunoglobulin G to a Sindbis-related virus by a membrane antigen enzyme immunoassay.

We determined the seroprevalence of a Sindbis-related virus isolated for the first time in 1975 from ticks in south-east Sicily and typed by Gresikova et al. in 1978. An indirect enzyme immunoassay based on viral membrane antigen for coating microtiter strips was used for the detection of immunoglobulin G to the Sindbis-related virus. The method appeared more sensitive than a similar enzyme immunoassay based on crude lysate antigen. Comparison of the results obtained from sera tested both by membrane antigen enzyme immunoassay and microneutralization test showed 92% agreement, while the agreement between microneutralization test and crude antigen enzyme immunoassay was 76%. An overall elevated seroprevalence (63.66%) was found in a population group living in and around the area of first isolation and seroprevalence in different age groups was also studied.

Coxsackie virus and urogenital pathology.

In 43 women (average age 49.6 years), and 70 men (average age 55.2 years) with pathology of the genito-urinary apparatus, seroantibodies to Coxsachie virus B were measured using the passive hemoagglutination method and the virus was isolated in the uterine. Viral isolation test was negative in all urine samples tested. Seropositivity for Coxsackie virus was reported in 26 women (60.46%) and in 51 men (72.85%). Positivity to B1 was 37.16% (42 cases), B2 in 38.05% (43 cases), and to B4 in 35.39% (40 cases). 17.69% (20 cases) of patients were seropositive to only one serotype, 17.69% (20 cases) to 5 serotypes, 14.15% (16 cases) to 3 serotypes, 9.73% (11 cases) to 2 serotypes, and 8.84% (10 cases) to 4 serotypes. B1, 2, 3, 4, 5 (25.97%; 20 cases) and B2, 3, 4, 5 (7.79%; 6 cases) were the most frequent associations. Seropositivity to Coxsackie virus was reported in 100% of patients (4 cases) with urethral caruncola, in 84.21% (16 cases out of 19) with cancer of the bladder, in 81.81% with cystitis (9 cases out of 11) and in 80% with prostatitis (8 out of 10 cases). In relation to sex, seropositivity was higher in males in cases of calculosis (75%; 9 cases out of 12 against 28.57%; 2 cases out of 7) and in cystitis (100%; 6 cases against 60%; 3 cases out of 5). Further studies are necessary to determine the clinical significance of serum Coxsackie virus antibodies in patients with urological pathology in the absence of urinary elimination of Coxsackie virus.