MicroRNA 146-5p, miR-let-7c-5p, miR-221 and miR-345-5p are differentially expressed in Respiratory Syncytial Virus (RSV) persistently infected HEp-2 cells.

Many viruses can establish non-cytolytic, chronic infections in host cells. Beyond the intrinsically interesting questions of how this long-term parasitism is achieved, persistently infected cells can be useful to study virus-host interactions. MicroRNAs (miRNAs) are a class of noncoding RNAs transcribed from the genomes of all multicellular organisms and some viruses. Individual miRNAs may regulate several hundred genes. In this research we have studied the expression of a selective group of host-cell encoded miRNAs, as expressed in a Respiratory Syncytial Virus (RSV) persistently infected HEp-2 cell line (HEp-2 + RSV-GFP). The RSV is a virus that does not encode miRNAs in its genome. Our study shows that Dicer is down regulated, miRNA's 146a-5p is strongly up-regulated and miRNAs 345-5p, let-7c-5p and miRNA's-221 are down-regulated in HEp-2 + RSV-GFP cells. Correspondingly, changes in the miRNA 146a-5p and he sequences of the reference genes are miRNA 345-5p respective miRNAs target proteins: HSP-70 and p21, were observed. Thus, RSV persistent viral infection induces unique patterns of miRNA's expression with relevance to how the virus regulates the host cell response to infection.

Pooled nasopharyngeal and oropharyngeal samples for the identification of respiratory viruses in adults.

A pooled sample of oropharyngeal swabs, nasopharyngeal swabs and nasopharyngeal washings, taken from each of 1,000 subjects, was compared to separate specimens from the same sampling. Multiplex real-time polymerase chain reaction (mqRT-PCR) was used to identify 12 respiratory viruses. Two hundred and forty-three (97%) of the 251 viruses identified in the separate samples were also identified in the mixed samples. The sensitivity rate was identical at 100% for all virus groups except coronaviruses. This sensitivity rate clearly justifies the use of pooled samples instead of separate samples for clinical and epidemiological purposes. The reduction in costs attained from the use of pooled samples may represent a critical advantage when considering its use in extensive clinical and epidemiological studies.

ITO pattern fabrication of glass platforms for electropolymerization of light sensitive polymer for its conjugation to bioreceptors on a micro-array.

We present herein an effective and versatile method to fabricate a micro-patterned structure of conductive polymer, poly(pyrrole-benzophenone), on Indium Tin Oxide (ITO) glass chips for the subsequent photo-immobilization of various bioreceptor, antigens. Such methodologies are based on photolithography of ITO pattern fabrication on non-conductive surfaces, glass slides, and on a photo-active electrogenerated polymer films. The photo-active polymer serves as a substrate platform for the photo-immobilization of the bioreceptor reagents used for subsequent immunoreactions. We were able to show the resolution of electropolymerization on an ITO pattern as well as immobilization of more than one bioreceptor for the simultaneous detection of several analytes. The antigen micro-arrays were tested for sensitivity, specificity, and overall practicality for the simultaneous detection of analyte anti-Cholera Toxin B, anti-Hepatitis B virus surface and core protein antibodies. In addition we used our pattern ITO-poly(pyrrole-benzophenone) micro-array for the detection of serum samples of Hepatitis B virus patients previously screened by a standard hospital detection method.

Detection of HCV salivary antibodies by a simple and rapid test.

Hepatitis C (HCV) is common in developing countries, where blood sampling and expensive sophisticated methods for detection are less available. Hemodialysis patients have high prevalence of HCV and may resemble sick populations in developing countries in relation to immunosuppression and antibodies production. For these reasons anti-HCV antibodies were assayed in saliva of hemodialysis patients by ImmunoComb II assay that is less laborious, relatively inexpensive and easy to perform If the findings are confirmed by larger studies this method may be useful especially in developing countries. Serum and saliva samples were obtained from 37 hemodialysis patients and assayed by ImmunoComb II kit. In positive PCR patients the saliva test had 100% sensitivity, which was as good as serum anti-HCV Axsym testing. Saliva testing had a similar or better specificity than the serum method.

Optical fiber immunosensor based on a poly(pyrrole-benzophenone) film for the detection of antibodies to viral antigen.

We describe herein a newly developed optical microbiosensor for the diagnosis of hepatitis C virus (HCV) by using a novel photoimmobilization methodology based on a photoactivable electrogenerated polymer film deposited upon surface-conductive fiber optics, which are then used to link a biological receptor to the fiber tip through light mediation. This fiber-optic electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide on the silica surface of the fiber optics. Monomers are then electropolymerized onto the conductive metal oxide surface; thereafter, the fibers are immersed in a solution containing HCV-E2 envelope protein antigen and illuminated with UV light (wavelength approximately 345 nm). As a result of the photochemical reaction, a thin layer of the antigen becomes covalently bound to the benzophenone-modified surface. The photochemically modified fiber optics were tested as immunosensors for the detection of anti-E2 protein antibody analyte that was measured through chemiluminescence reaction. The biosensor was tested for sensitivity, specificity, and overall practicality. Our results suggest that the detection of anti-E2 antibodies with this microbiosensor may enhance significantly HCV serological standard testing especially among patients during dialysis, which were diagnosed as HCV negative, by standard immunological tests, but were known to carry the virus. If transformed into an easy to use procedure, this assay might be used in the future as an important clinical tool for HCV screening in blood banks.

Measles virus: evidence of an association with Hodgkin's disease.

The quest for an infectious agent that may account for cases of Hodgkin's disease (HD) especially in young adults has proven vain until lately. We have recently reported findings that suggested the presence of measles virus (MV) antigens and MV RNA in the tissues of patients with HD. Support for an association between MV and HD has been provided by recent epidemiological findings relating the occurrence of HD to exposure to measles in pregnancy and the perinatal period. We now present further evidence of this putative association based on immunohistochemical, reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridisation studies (ISH) on HD tissues. Biopsies from 82 (54.3%) of our cohort of 154 patients showed a positive immunostain with at least two of the anti-measles antibodies used. Latent membrane protein-1 immunostaining for Epstein-Barr virus was positive in 46 (31.1%) of the patients examined. Reverse transcriptase-PCR and ISH for measles RNA were positive in seven and 10 of 28 patients, respectively. Preliminary clinicopathological associations between MV and HD are noted in this study, but no causal relationship can be claimed at this stage.

New candidate virus in association with Hodgkin's disease.

Epidemiologic and molecular investigations of Hodgkin's disease (HD) suggest a strong infectious association. The Epstein-Barr virus (EBV), together with its viral proteins, is expressed in Hodgkin-Reed-Sternberg (HRS) cells in the lymph nodes involved by HD. EBV is more likely to be related to childhood and older adult cases of HD and is much less frequently expressed in young adult HD patients, the group most expected to be associated with an infectious agent. In addition, the "hit and run" theory of EBV infection remains speculative and no other lymphotropic viruses studied to date seem to satisfy the quest for a new candidate virus in young adults with HD. We have recently found preliminary evidence suggesting a possible association between the measles virus (MV) and HD. This evidence is the subject of the present review.

Hepatitis C virus infection in renal failure patients in the absence of anti-hepatitis C virus antibodies.

The magnitude and clinical significance of Hepatitis C virus (HCV) infection in dialysis patients is controversial and underestimated. This study was conducted in order to evaluate the correlation between HCV replication and antibody response to HCV in dialysis patients. HCV infection in dialysis patients was evaluated over a period of 3 years and compared to HCV infection in Liver Clinic patients. Sera were collected from 310 dialysis patients and tested for anti-HCV and HCV-RNA. In addition, HCV genotype and HCV viral load were determined in HCV-RNA-positive sera. Anti-HCV was detected in 43 (14%) of the dialysis patients. Of these, 37 (86%) were HCV-RNA-positive. Among the 267 HCV-seronegative dialysis patients, 25 (9%) were found to be HCV-RNA-positive in more than one sample during the study. These patients were characterized by low viral load; at least two orders of magnitude lower than in the group of HCV-seropositives. In contrast, in the Liver Clinic patients, HCV-RNA was found exclusively in HCV-seropositive patients. Comparison of the genotype pattern in the two groups did not reveal a difference. Our results suggest that HCV infection in dialysis units may be underestimated due to cases of low viral load, depending on the method of RNA extraction and sensitivity of the test used. Low viral load might contribute to the lack of humoral immune response seen in some dialysis patients.

Salivary HCV-antibodies; a follow-up cohort study of liver disease patients.

We have recently shown in Liver Clinic patients that saliva instead of serum may be used for anti-HCV detection. As compared to blood withdrawing, saliva is easier to obtain, non invasive, especially for infants. In the present study, sequential determination of serum and salivary anti-HCV was performed in the same cohort for 36 months. Anti-HCV seropositive and seronegative patients were studied. Blood and saliva samples were obtained simultaneously. From the anti-HCV seronegative patients (n=33), 161 sequential serum and 161 matched saliva samples were obtained. All were anti-HCV negative. From the anti-HCV seropositive patients (n=35), 131 sequential serum and 131 matched saliva samples were obtained. All sequential serum samples were anti-HCV positive. Of the saliva samples 126 (96%) were anti-HCV positive and five (4%) were anti-HCV negative. These five samples were obtained from two patients with autoimmune hepatitis and HCV-RNA seronegative by PCR. The results suggest that saliva may serve as a substitute for serum for the detection of anti-HCV antibodies.

Hepatitis C virus infection and genotypes in Southern Israel and the Gaza Strip.

The Gaza Strip borders the southern part of Israel and Egypt. There is a remarkable difference in the prevalence of antibodies to hepatitis C virus (HCV) between Israel (0.5%) and Egypt (10%). A few thousand inhabitants cross the borders daily from the Gaza Strip to both countries. The objectives of this study were to investigate the prevalence of HCV infection in the Gaza Strip, an area that was not studied before, and to study HCV transmission in the Gaza Strip by characterizing the genotypes of HCV in Southern Israel and the Gaza Strip and comparing them with those found in Egypt. HCV prevalence in the Gaza Strip was found to be 2.2%, relatively higher than in Israel but lower than in Egypt. The most common genotypes found were type 1 b in Southern Israel and type 4 in the Gaza Strip, corresponding to the most prevalent genotype in Egypt. Similarity between type 4 isolates from the Gaza Strip and Egypt was illustrated further by sequence analysis of the HCV 5' noncoding region (NCR).

HCV antibodies in saliva and urine.

Infection with hepatitis C virus (HCV) is usually established by detection of serum antibodies (anti-HCV). This study was conducted in order to evaluate whether saliva and urine may substitute serum for anti-HCV detection. Serum, saliva, and urine were obtained simultaneously from 141 patients with a variety of liver diseases and from 52 patients with autoimmune diseases (systemic lupus erythematosus n = 27 and rheumatoid arthritis n = 25). The cell free fraction of saliva and urine samples was tested for anti-HCV using a modification of a serum anti-HCV kit. Western blot analysis was used as a confirmation method. Of the patients with liver diseases, 73 were anti-HCV-seropositive. Salivary and urinary anti-HCV could be detected in 66 (90%) and 36 (49%) of the anti-HCV-seropositive patients, respectively. The presence of anti-HCV in saliva or urine was not related to the severity of liver disease. All the anti-HCV-seronegative liver patients were negative for salivary anti-HCV and 22 (32%) had urinary anti-HCV. The patients with autoimmune diseases were all anti-HCV-seronegative. None had detectable salivary anti-HCV while 33 (63%) were positive for urinary anti-HCV. Western Blot analysis confirmed the presence of anti-HCV in all serum and saliva samples tested but only in 2/12 urine samples. The results suggest that saliva, but not urine, may serve as a substitute for serum for the determination of anti-HCV positivity.

Hepatitis C virus infection and extrahepatic malignancies.

An association between chronic hepatitis C (HCV) infection and non-Hodgkin's lymphoma has been reported. We carried out this study to evaluate the possibility of an association between HCV infection and other extrahepatic malignancies. The medical records of 103 unselected, consecutively chosen, anti HCV-positive and 105 hepatitis B surface antigen (HBsAg)-positive patients attending the liver clinic or hospitalized in the Department of Medicine were reviewed. Patients in whom anti-HCV positivity was detected after the malignancy was diagnosed were excluded. Malignancy rates in the general Israeli population were obtained from the Israeli cancer registry. The ages of anti-HCV-positive and HBsAg-positive patients were 54 +/- 16 (+/-SD) (range, 15-84) and 45 +/- 12 (range, 20-78) years, respectively; the male/female ratios were 50/53 and 73/32, respectively. Extrahepatic malignancies were found in 15 (14.6%) of the anti-HCV and in three (2.9%) of the HBsAg-positive patients. Thirteen of the malignancies were found among the 60 anti-HCV-positive patients aged > or =55 years old. Only one malignancy was found among the 28 HBsAg-positive patients of the same age group (p < 0.01). The rate of extrahepatic malignancies in these HCV-infected patients was significantly higher (p < 0.01) than expected in the general population. An association between HCV infection and extrahepatic malignancy may exist, but further prospective studies, including a large number of patients with HCV infection, will be necessary to define this observation.

Prevalence of specific IgA and IgM anti-HBc antibodies compared with HBV DNA in the sera of HBsAg chronic carriers.

OBJECTIVE: We evaluated the significance of IgA antibodies directed against the hepatitis B virus core antigen (IgA anti-HBc) as a marker for viral replication. STUDY DESIGN/METHODS: Serum samples of 143 hepatitis B surface antigen (HBsAg) carriers and 189 HBsAg-negative subjects were studied. Hepatitis B virus (HBV) DNA was detected by polymerase chain reaction. IgA anti-HBc was determined by a capture enzyme-linked immunosorbent assay developed in our laboratory. The results were compared with those for IgM anti-HBc, which were determined by a commercially available method. RESULTS: IgA anti-HBc was detected in 57 (40%) and HBV DNA in 38 (27%) of the HBsAg carriers. Among the HBsAg-negative subjects, IgA anti-HBc and HBV DNA were detected simultaneously in four samples. All 42 HBV DNA-positive samples were IgA anti-HBc positive. IgM anti-HBc was detected in 27 (64%) of them. CONCLUSIONS: IgA anti-HBc is a sensitive marker for HBV replication, and its absence may exclude HBV replication. The role of IgA anti-HBc in monitoring response to therapy and predicting clinical course is being evaluated.

Chlamydia pneumoniae-induced ciliostasis in ciliated bronchial epithelial cells.

The ciliary activity of ciliated bronchial epithelial cells was studied after infection with Chlamydia pneumoniae (strain TW-183) and Chlamydia trachomatis (biovar L2 [434/Bu]). C. pneumoniae, known to cause respiratory infections, had a marked ciliastatic effect, completely aborting ciliary motion within 48 h. This effect was not inhibited by inactivation of C. pneumoniae elementary bodies (EBs) by UV irradiation. In contrast, inactivation of EBs by heat (56 degrees C, 30 min) or by rabbit-specific immune sera to TW-183 antigen blocked the ciliastatic effect induced by C. pneumoniae infection. The effect of C. pneumoniae was specific. C. trachomatis, which causes sexually transmitted disease, did not block ciliary motion by 48 h after infection. A decrease in ciliary activity of ciliated bronchial cells produced by C. pneumoniae can contribute to both initiation and pathogenesis of respiratory infections induced by this pathogen.

Self-association of the "death domains" of the p55 tumor necrosis factor (TNF) receptor and Fas/APO1 prompts signaling for TNF and Fas/APO1 effects.

Signaling by the p55 tumor necrosis factor (TNF) receptor and by the structurally related receptor Fas/APO1 is initiated by receptor clustering. Data presented here and in other recent studies (Wallach, D., Boldin, M., Varfolomeev, E. E., Bigda, Y., Camonis, H.J. and Mett, I. (1994) Cytokine 6, 556; Song, H.Y., Dunbar, J.D., and Bonner, D.B. (1994) J. Biol. Chem. 269, 22492-22495) indicate that part of that region within the intracellular domains of the two receptors that is involved in signaling for cell death, as well as for some other effects (the "death domain", specifically self-associates. We demonstrate also the expected functional consequence of this association; a mere increase in p55 TNF receptor expression, or the expression just of its intracellular domain, is shown to trigger signaling for cytotoxicity as well as for interleukin 8 gene induction, while expression of the intracellular domain of Fas/APO1 potentiates the cytotoxicity of co-expressed p55 TNF receptor. These findings indicate that the p55 TNF and Fas/APO1 receptors play active roles in their own clustering and suggest the existence of cellular mechanisms that restrict the self-association of these receptors, thus preventing constitutive signaling.

Serum reactivity to Chlamydia trachomatis and C. pneumoniae antigens in patients with documented infection and in healthy children by microimmunofluorescence and immunoblotting techniques.

Chlamydia trachomatis and C. pneumoniae are both important human pathogens. Antigenic cross-reactivity between the two species may complicate serologic diagnosis of infection with one or the other agent. In this study we examined sera of persons with chlamydia infections and of healthy children by microimmunofluorescence (MIF) against C. trachomatis L2 antigen and against C. pneumoniae TW-183 antigen to explore the degree of cross-reactivity found by these two methods. Likewise, the cross-reactivity seen by immunoblot with sera of rabbits immunized against one of the antigens when tested on the other was examined. While among healthy children stratified by age, MIF seropositivity to C. pneumoniae TW-183 increased with age, no such trend was observed with respect to seropositivity to C. trachomatis L2 antigens, and 81% of children seropositive to TW-183 did not react on L2 antigen. Moreover, 27% of those positive on L2 antigen were negative on TW-183. Immunoblot analysis showed much greater antibody cross-reactivity than that detected by MIF. The immunoblot cross-reactivity was probably not attributable solely to double infection since sera of rabbits immunized to only one species of chlamydia reacted strongly with both chlamydiae in immunoblot analysis. The data presented need to be taken into account in the development of serologic tests based on a small number of antigens or on partially denatured antigens.

Variation in serum levels of the soluble TNF receptors among healthy individuals.

Soluble forms of the two receptors for tumor necrosis factor (TNF) are present in human sera at concentrations that increase greatly in various disease states as well as varying among healthy individuals. Measurements of the soluble TNF receptor (sTNF-R) concentrations in healthy individuals at time lapses of 3 months (17 individuals) or 1 year (51 individuals) showed a significant correlation between the first and the second measurements from each individual, implying that individual differences are stable. Since the sTNF-Rs are believed to function as physiological attenuators of TNF activity, these steady individual differences may contribute to differences in the severity of the harmful effects of TNF in disease states.

Implications for persistent chlamydial infections of phagocyte-microorganism interplay.

In vitro models of Chlamydia trachomatis inhibition by cytokines, human-monocyte derived macrophages (HMDM) and human polymorphonuclear leukocytes (HPMN) are discussed in an attempt to delineate the molecular basis of parasite-host cell interplay in persistent and chronic chlamydial infection. Interferon gamma (IFN) has been found to reversibly inhibit chlamydial growth at an early stage in the replicative cycle, while tumor necrosis factor (TNF) has a more profound effect on chlamydial growth resulting in production of aberrant reticulate bodies and enhancement of production of prostaglandin E2 (PGE2). Chlamydia trachomatis (serovar L2) replicate in HMDM while serovar K has been found to be restricted in these cells. Chlamydiae are killed by HPMN but the cell walls persist undegraded, inducing production of oxygen radicals which can be demonstrated to induce DNA strand scissions in HeLa target cells. Evidence is accumulating that chlamydia specific serum IgA antibodies may serve as a noninvasive serological marker for diagnosis of a number of acute and persistent Chlamydia trachomatis infections.

Inhibition of growth of Chlamydia trachomatis by tumor necrosis factor is accompanied by increased prostaglandin synthesis.

Development of Chlamydia trachomatis (L2/434/Bu) in HEp-2 cells was inhibited by treatment of the cells with recombinant human alpha tumor necrosis factor (TNF). In the infected cultures that were treated with TNF, high concentrations of prostaglandin E2(PGE2) were detected, exceeding by far the concentrations found in TNF-treated but uninfected cells or in infected cells that were not treated with TNF. PGE2 levels increased gradually for 2 days after infection. Raising the tryptophan concentration in the culture medium, which reversed the inhibition of chlamydial replication by TNF, also blocked the increase in PGE2 formation. However, neutralizing antibodies to beta interferon, which also interfered with the antichlamydial effect of TNF, did not decrease PGE2 formation. Excessive formation of PGE2 by cells infected with chlamydiae and treated by TNF might be related to some of the complications associated with chlamydial infection.

Inhibition of tumor necrosis factor-induced cell killing by tryptophan and indole.

Cells sensitive to the cytocidal effect of tumor necrosis factor (TNF) were protected against this effect when growth in the presence of elevated concentrations of tryptophan. Several other indole derivatives also provided protection against TNF cytotoxicity. Most effective were indole itself and its monomethyl derivatives, providing a degree of protection greatly exceeding that observed with tryptophan. Protection was also observed against the cytocidal effect of TNF applied in the presence of a protein synthesis inhibitor. The protective effect of tryptophan was largely dependent on preexposure of the cells, for several hours, to a high concentration of this amino acid. On the other hand, indole was protective also when applied to cells together with TNF, or even two hours after TNF application. The inhibition of the cytotoxicity of TNF by tryptophan and other indole derivatives may serve as a useful experimental tool in exploring the mechanisms and the physiological implications of TNF cytotoxicity.