Izabela Bialuk

Requirement of the human T-cell leukemia virus p12 and p30 products for infectivity of human dendritic cells and macaques but not rabbits

The identification of the genes necessary for human T-cell leukemia virus (HTLV-1) persistence in humans may provide targets for therapeutic approaches. We demonstrate that ablation of the HTLV-1 genes encoding p12, p30, or the HBZ protein, does not affect viral infectivity in rabbits and in this species, only the absence of HBZ is associated with a consistent reduction in virus levels. We observed reversion of the HTLV-1 mutants to the HTLV-1 wild-type genotype in none of the inoculated rabbits. In contrast, in macaques, the absence of HBZ was associated with reversion of the mutant virus to the wild-type genotype in 3 of the 4 animals within weeks from infection. Similarly, reversion to the wild type was observed in 2 of the 4 macaque inoculated with the p30 mutant. The 4 macaques exposed to the p12 knock remained seronegative, and only 2 animals were positive at a single time point for viral DNA in tissues. Interestingly, we found that the p12 and the p30 mutants were also severely impaired in their ability to replicate in human dendritic cells. These data suggest that infection of dendritic cells may be required for the establishment and maintenance of HTLV-1 infection in primate species.

Hijacking the T-cell Communication Network by the Human T-cell Leukemia/Lymphoma Virus Type 1 (HTLV-1) p12 and p8 Proteins

The non-structural proteins encoded by the orf-I, II, III, and IV genes of the human T-cell leukemia/lymphoma virus type 1 (HTLV-1) genome, are critical for the modulation of cellular gene expression and T-cell proliferation, the escape from cytotoxic T-cells and natural killer cells, and virus expression. In here, we review the main functions of the HTLV-1 orf-I products. The 12kDa product from orf-I (p12) is proteolytically cleaved within the endoplasmic reticulum (ER) to generate the 8kDa protein (p8). At the steady state, both proteins are expressed at similar levels in transfected T-cells. The p12 protein remains in the ER and cis-Golgi, whereas the p8 protein traffics to the cell surface and is recruited to the immunological synapse. The p12 and the p8 proteins have seemingly opposite effects on T-cells; the ER resident p12, modulates T-cell activation and proliferation, whereas p8 induces T-cell anergy. The p8 protein also increases the formation of cellular conduits, is transferred to neighboring T-cells, and increases virus transmission. The requirement for HTLV-1 infectivity of orf-I is demonstrated by the loss of virus infectivity in macaques exposed to an engineered virus, whereby expression of orf-I was ablated. Altogether the current knowledge demonstrates that the concerted activity of p8 and p12 is essential for the persistence of virus infected cells in the host.

In vivo genetic mutations define predominant functions of the human T-cell leukemia/lymphoma virus p12I protein

The human T-cell leukemia/lymphoma virus type 1 (HTLV-1) ORF-I encodes a 99-amino acid hydrophobic membrane protein, p12(I), that affects receptors in different cellular compartments. We report here that proteolytic cleavage dictates different cellular localization and functions of p12(I). The removal of a noncanonical endoplasmic reticulum (ER) retention/retrieval signal within the amino terminus of p12(I) is necessary for trafficking to the Golgi apparatus and generation of a completely cleaved 8-kDa protein. The 8-kDa protein in turn traffics to the cell surface, is recruited to the immunologic synapse following T-cell receptor (TCR) ligation, and down-regulates TCR proximal signaling. The uncleaved 12-kDa form of p12(I) resides in the ER and interacts with the beta and gamma(c) chains of the interleukin-2 receptor (IL-2R), the heavy chain of the major histocompatibility complex (MHC) class I, as well as calreticulin and calnexin. Genetic analysis of ORF-I from ex vivo samples of HTLV-1-infected patients reveals predominant amino acid substitutions within ORF-I that affect proteolytic cleavage, suggesting that ER-associated functions of p12(I) may contribute to the survival and proliferation of the infected T cells in the host.

Humoral immune response to HTLV-1 basic leucine zipper factor (HBZ) in HTLV-1-infected individuals

Human T cell lymphotropic virus type 1 (HTLV-1) infection can lead to development of adult T cell leukemia/lymphoma (ATL) or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a subset of infected subjects. HTLV-1 basic leucine zipper factor (HBZ) gene has a critical role in HTLV-1 infectivity and the development of ATL and HAM/TSP. However, little is known about the immune response against HBZ in HTLV-1-infected individuals. In this study, we examined antibody responses against HBZ in serum/plasma samples from 436 subjects including HTLV-1 seronegative donors, asymptomatic carriers (AC), ATL, and HAM/TSP patients using the luciferase immunoprecipitation system. Immunoreactivity against HBZ was detected in subsets of all HTLV-1-infected individuals but the test did not discriminate between AC, ATL and HAM/TSP. However, the frequency of detection of HBZ-specific antibodies in the serum of ATL patients with the chronic subtype was higher than in ATL patients with the lymphomatous subtype. Antibody responses against HBZ were also detected in cerebrospinal fluid of HAM/TSP patients with anti-HBZ in serum. Antibody responses against HBZ did not correlate with proviral load and HBZ mRNA expression in HAM/TSP patients, but the presence of an HBZ-specific response was associated with reduced CD4+ T cell activation in HAM/TSP patients. Moreover, HBZ-specific antibody inhibited lymphoproliferation in the PBMC of HAM/TSP patients. This is the first report demonstrating humoral immune response against HBZ associated with HTLV-I infection. Thus, a humoral immune response against HBZ might play a role in HTLV-1 infection.

Co-dependence of HTLV-1 p12 and p8 Functions in Virus Persistence

HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4+ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8.

Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques.

The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection.

Analysis of gene expression profiles in tympanic membrane following perforation using PCR Array in rats--preliminary investigation.

OBJECTIVES The goal of this work was to identify genes, known to be involved in the skin wound healing, that express differentially in the healthy and injured tympanic membrane (TM), and designate the molecules potentially beneficial for treatment of TM perforation. The molecular mechanisms controlling the course of TM regeneration are far from being elucidated. METHODS Twenty rats had their tympanic membranes perforated, while four served as a control. Animals were sacrificed on either days 1, 2, 3, 5 and 10 post injury, and TMs were immediately dissected and frozen in liquid nitrogen. Total TM RNA was isolated and reversely transcribed. qPCR was performed using Rat Wound Healing RT(2) Profiler PCR Array (QIAGEN) containing primers for 84 genes. RESULTS Statistically significant changes in the expression of 42 genes were found in various stages of TM healing. The increased expression of genes taking part in the inflammatory reaction (interleukin 6, granulocyte and macrophage chemotactic proteins) was observed from day 2. The expression of several genes of extracellular matrix components and their remodeling enzymes was also changed. Among growth factor genes: Vegfa, Igf1 and Hbegf showed increased expression at the beginning of the healing process, while Hgf expression was highest on day 3. CONCLUSIONS Several changes in the expression of genes involved in remodeling of extracellular matrix point to important role of connective tissue in TM healing. The molecules accelerating this process, like HbEGF and HGF, seem to be good candidates for further evaluation of their possible use in clinical treatment.

CP55,940 attenuates spatial memory retrieval in mice

BACKGROUND Cannabinoids constitute a varied group of lipophilic substances able to infiltrate the blood-brain barrier and influence neuronal processes. Clinical observations supported by experimental data have revealed that these compounds exert a deleterious effect on cognitive processes. The present study was carried out to determine the influence of a single systemic administration of CP55,940, a potent synthetic agonist of cannabinoid receptors, on spatial memory retrieval assessed in a Morris water maze. METHODS C57BL/6J male mice were submitted to three consecutive days of training to find a hidden platform in the water maze. CP55,940 was given intraperitoneally once, at doses of 0.025, 0.125 or 0.25mg/kg on the fourth day, 30min before testing memory retrieval, and in separate groups before testing psychomotor activity and anxiety level in a hole-board test. RESULTS CP55,940 only at the highest dose of 0.25mg/kg significantly altered all parameters used to assess spatial memory. It increased the latency in the first crossing of the former platform location (target area), decreased the number of target area crossings and shortened the time spent in the target quadrant. Moreover, CP55,940 at doses of 0.25 and 0.125mg/kg attenuated motor and exploratory activity in hole-board test. CONCLUSION Since the attenuated psychomotor activity after a dose of 0.125mg/kg did not interfere with memory retrieval, we assume that the impairment of spatial memory observed after the highest dose of CP55,940 (0.25mg/kg) was exerted by its influence on cognitive processes, however, the impact on locomotion could not be excluded.

CCN1 expression in interleukin-6 deficient mouse kidney in experimental model of heart failure

Chronic heart failure often leads to worsening of the renal function. Mediators of this process include inflammatory and neuroendocrine factors. CCN1 (Cyr 61), a member of growth factor-inducible immediate early genes, which modulates inflammation and fibrogenesis, is excreted with urine in the early phase of acute renal injury and may be involved in the pathogenesis of the cardiorenal syndrome. The aim of the study was to evaluate CCN1 protein abundance and localization in the kidney of IL-6-deficient C57BL/6J (IL-6 KO) mice and respective wild-type (WT) animals in basal conditions and in animals with chronic heart failure twelve weeks after myocardial infarction. Age- and sex-matched mice from both strains subjected to sham operation served as controls. One group of WT animals subjected to myocardial infarction was treated with antagonist of AT1 receptor telmisartan over 12 weeks. Abundance and localization of CCN1 protein in kidney were assessed with Western blotting and immunohistochemistry, respectively. In all groups the strongest immunohistochemical reaction for CCN1 was observed in distal convoluted tubules and in smaller arteries, however, the total expression of CCN1 protein was lower in IL-6 KO mice in comparison to WT animals. The main difference in CCN1 distribution between the examined genotypes was lack of reaction in internal renal medulla and very weak reaction in proximal convoluted tubules in IL-6 KO mice. Experimental heart failure only slightly attenuated the expression of CCN1 protein in the kidney of WT mice and had no effect in IL-6 KO mice. Although, blockade of AT1 receptor did not alter CCN1 protein expression in kidneys of WT mice after myocardial infarction, it significantly changed its CCN1 distribution in the renal tubular system.

Transcriptional and post-transcriptional regulation of CCN genes in failing heart

BACKGROUND CCN family of proteins has been implicated in various processes in cardiovascular physiology and pathology, including angiogenesis, regeneration and fibrosis. In this study we assessed long term changes of CCN1 and CCN2 gene products abundance in the failing ventricular myocardium. METHODS Male, 12-14-weeks-old C57BL6/J and C57BL6/J (IL-6-/-) mice were used. To assess short term changes, a transient reversible ischemia model was utilized. Heart failure was caused by ligation of anterior descending coronary artery. The presence of systolic dysfunction was confirmed by echocardiography and left ventricular ANP RNA expression. Molecular analysis was performed on left ventricular samples from animals sacrificed 12-14 weeks after infarction. Western blotting and QT-PCR were used to investigate abundance of CCN proteins and RNAs, respectively. RESULTS Short ischemia resulted in marked increase of CCN1 transcript. However, three months after myocardial infarction (MI), remote myocardium showed a markedly increased expression of CCN1 protein, but not RNA. In the case of CCN2, the RNA was distinctly up-regulated, whereas the protein presented only modest, non-significant increase in failing myocardium. Expression of CCN2 RNA closely correlated with expression of ANP. Long-term telmisartan administration after infarction decreased the expression of CCN1 protein. Interleukin 6 (IL-6) deficiency caused increased CCN2 protein abundance in control animals, but the difference was absent after MI. Infarction did not increase CCN1 protein in the hearts of IL-6 deficient mice. CONCLUSION CCN genes are activated in heart failure. Their regulation is multidimensional both transcriptional and posttranscriptional. The involved pathways include angiotensin II and IL-6.