Anthrax in animals and a farmer in Alto Adige, Italy.
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Agriculture↗
Biomedical subjects
H P Huemer
Publications and source records attributed to H P Huemer.
Prevalence of respiratory viruses, including newly identified viruses, in hospitalised children in Austria.
The aim of this epidemiological study was to determine the prevalence of respiratory viruses, including new viruses, in hospitalised children in Austria. Two hundred fourteen nasopharyngeal samples from hospitalised children were tested for the presence of viruses using cell culture and PCR and/or viral antigen assays. The results revealed a parainfluenza virus 1 (PIV1) outbreak that ended right before the onset of the influenza season, with nearly no overlapping, moderate respiratory syncytial virus (RSV) activity, and only a few adenoviruses. Human metapneumovirus (hMPV) was present in 14.5% of the total samples but was detected in combination with other viruses in only five cases: with PIV1 in three cases and with RSV in two cases. There were no cases of dual infection with hMPV and flu or adenovirus. This suggests that hMPV alone is a leading cause of hospitalisation in children under 1 year of age. Interestingly, hMPV, in contrast to RSV, coincided with PIV1 but was absent during the community outbreak of the flu. Samples were also tested for Mimiviridae, a group of newly described DNA viruses that are similar to Legionella spp., replicate in water amoebae, and also have been found in alveolar cells. However, mimivirus was detected neither in respiratory samples nor in amoebae-containing water samples, indicating that this particular type of virus is either not abundant or does not contribute to paediatric respiratory illnesses.
Animals↗
Generalized cowpox infection probably transmitted from a rat.
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Adolescent↗
Visceral leishmaniasis in an AIDS patient on successful antiretroviral therapy: failure of parasite eradication despite increase in CD4+ T-cell count but low CD8+ T-cell count.
An unusual cutaneous relapse of visceral leishmaniasis (initially mistaken for eruptive histiocytomas) was seen in an AIDS patient despite good virological and CD4+ T-cell responses to highly active antiretroviral therapy. Splenectomy and the patient's low CD8+ T-cell count are discussed as possible causes of failed disease control.
AIDS-Related Opportunistic Infections↗
Use of apathogenic vaccinia virus MVA expressing EHV-1 gC as basis of a combined recombinant MVA/DNA vaccination scheme.
The nonreplicating chicken adapted vaccinia virus strain MVA was used in a combined vaccine scheme. Using the equine herpesvirus type 1 (EHV-1) encoded complement-receptor glycoprotein C as antigen, only poor antibody response was induced by exclusive vaccination with DNA plasmids. The administration of recombinant MVA followed by plasmid immunization elicited both humoral and cellular immune responses in hamster comparable to EHV-1 full virus vaccines. Our results indicate that recombinant constructs based on MVA represent a safe and efficient way to overcome problems of poor immunogenicity of certain antigens upon intramuscular DNA vaccination, thus replacing sophisticated adjuvants or application methods, which are not readily applicable in routine practice.
Animals↗
Induction of recombinant gene expression in stably transfected cell lines using attenuated vaccinia virus MVA expressing T7 RNA polymerase with a nuclear localisation signal.
There are major drawbacks using vaccinia virus (VV) expressing T7 polymerase for eukaryotic expression. VV is infectious for humans and due to cytosolic replication of Poxviridae, transient transfection of T7 promoter containing plasmids is necessary, which varies in efficiency. Several improvements have been introduced to this system to enhance expression of herpes viral glycoproteins. Stably transfected cell lines were generated with an EBV-based episomal plasmid vector which can be pushed to increasing copy numbers under selective pressure. The avirulent vaccine MVA strain was adopted to generate a safe laboratory vector for inserting the bacteriophage T7 RNA polymerase gene with (+) or without (-) a nuclear localisation signal. Constructs were designed for recombination into the VV haemagglutinin gene as recombinants could not be isolated successfully when inserting into the MVA thymidine kinase locus. Both T7 MVA recombinants induced foreign protein expression in transiently transfected cells but only the T7-/+ MVA induced target protein expression in stably transfected cells. The level of protein expression by this induction mechanism was comparable to, or superior to levels obtained with VV recombinants expressing the gene under control of the VV 11 k IE promoter. The results suggests that the T7+ MVA virus can be used to induce gene expression in stable recombinant cell lines and offers an attractive and safe alternative to other inducible eucaryotic expression systems.
Animals↗
Influence of viral infection on expression of cell surface antigens in human retinal pigment epithelial cells.
BACKGROUND: Subacute viral infection is known to change the phenotype of infected cells, thereby causing immune-mediated tissue damage. The aim of this study was to investigate the expression of different cell surface molecules on human retinal pigment epithelial cells (RPEC) following viral infection, with special emphasis on those having immune-regulatory functions. METHODS: Cultured RPEC were infected with cytomegalovirus (CMV), coxsackie-virus B3 (CVB) or herpes simplex virus type I (HSV). Double-staining fluorescence technique was used for visualization of virus infection and cell surface markers in the same cells by laser microscopy. RESULTS: CMV downregulated MHC class I antigens on RPEC, whereas CVB and HSV did not alter MHC class I antigen expression. No induction of class II antigens was observed in RPEC infected with CVB, HSV or CMV. The intercellular adhesion molecule ICAM-1 (CD54) was strongly expressed in uninfected RPEC, and a slight increase was observed after virus infection. Vascular cell adhesion molecule 1 (VCAM-1) was expressed in low amounts in both uninfected and infected RPEC. No expression of intercellular adhesion molecule 2 (ICAM-2), E-selectin ELAM-1 or lymphocyte-function-associated antigen 1 (LFA-1) was observed on RPEC before or after virus infection. CONCLUSION: Downmodulation of immune-regulating cell surface antigens has been suggested to provide a means of long-term survival of viruses in the infected cell, favoring establishment of persistent infection. Our observation in cultured human RPEC indicates that this mechanism might indeed contribute to the development of disease affecting retinal tissue.
Animals↗
Epstein-Barr virus-associated persistent polyclonal B-cell lymphocytosis with a distinct 69-base pair deletion in the LMP1 oncogene.
Epstein-Barr virus (EBV) genomes have been detected in peripheral blood lymphocytes (PBL) of patients with persistent polyclonal B-cell lymphocytosis (PPBL). This is consistent with the hypothesis that latent EBV infection is involved in the pathogenesis of this disorder. Two EBV-encoded proteins expressed in viral latency are the latent membrane proteins 1 and 2A (LMP1 and LMP2A). We have studied the LMP1 oncogene and the LMP2A gene in a female patient with PPBL and her five siblings. A cell line derived from peripheral blood lymphocytes (PBL) of the patient was also analyzed. A distinct 69-base pair deletion was identified within the carboxy terminal NF-kappa B activation domain of the LMP1 oncogene in PBL of the patient and in the cell line, whereas none of the siblings harbored this deletion. The tyrosine-signaling motif and the HLA A2.1 epitope of the LMP2A gene were wild type in the patient and all siblings. The presence of a 69-base pair deletion variant of the LMP1 oncogene within the lymphocytes of a PPBL patient but absence of this deletion variant in the unaffected siblings suggests a direct implication of altered LMP1 oncoprotein-dependent function in the pathogenesis of PPBL.
Amino Acid Sequence↗
Susceptibility of human retinal pigment epithelial cells to different viruses.
BACKGROUND: Different viruses have been reported to be involved in retinal diseases in animal systems. In humans, herpes simplex virus and cytomegalovirus have been found to cause retinal disease. Most of the studied viruses are neurotropic. In this study, the in vitro susceptibility of human retinal pigment epithelial cells (RPEC) to representative members of different groups of human pathogenic viruses was investigated. METHODS: Early cultures of RPE C - after two or three passages - were infected with the following viruses: herpes simplex virus (HSV) type 1, human herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus types 1 and 7, measles virus, parainfluenza virus and coxsackie virus B3. RESULTS: Cultures of RPE C could be infected with neurotropic viruses like HSV or measles virus as well as with typical respiratory viruses like parainfluenza or adenoviruses. Coxsackievirus, an enterovirus, replicated as well as human CMV, whereas EBV and HHV-6, two lymphotropic viruses, failed to infect RPE. CONCLUSION: These findings suggest that a variety of viruses, including those causing rather common illnesses, might be capable of inducing retinal lesions under certain circumstances due to haematogenous spread during the course of viraemia.
Antibodies, Monoclonal↗
Pathogenesis of chronic Epstein-Barr virus infection: detection of a virus strain with a high rate of lytic replication.
In rare cases Epstein-Barr virus (EBV) leads to chronic active infection (CAEBV) which is characterized by persistant symptoms of infectious mononucleosis. Previously we described a case of persisting polyclonal B-cell lymphocytosis (PPBL) that was associated with CAEBV. Using reverse transcription and polymerase chain reaction we showed that in late passages of a spontaneous cell line, SM, latent EB viral genes such as EBNA1, EBNA2, EBNA3A/3B/ 3C, LMP1 and LMP2A were active. The master gene of the lytic cycle, BZLF1, was silent. This indicated that there was no general defect in immortalization and establishing latency by this CAEBV isolate SM. We obtained virus from the standard immortalizing strain B95-8 and the CAEBV strain SM from latently infected LCL quantified the number of virus particles by competitive PCR and demonstrated that the impaired capacity to immortalize umbilical cord blood lymphocytes was a virus strain-specific property, and was not due to an incapability to infect purified CD19+ B lymphocytes. Transcription of latency- and immortalization-associated genes such as EBNA1, EBNA2 and LMP2A was reduced, in contrast to a strongly enhanced activity of the master gene of the lytic cycle, BZLF1. A scenario for an antagonistic regulation of lytic and latent cycle genes is presented and a role for the pathogenesis of CAEBV is discussed.
B-Lymphocytes↗
gp13 (EHV-gC): a complement receptor induced by equine herpesviruses.
Equine herpesviruses type 1 (EHV-1) and type 4 (EHV-4) induce a complement receptor protein on the surface of infected cells capable of binding to the third component of complement (C3). The protein mediating the binding to the C3 component of complement was identified as glycoprotein 13 (gp13, EHV-gC), as expression of the cloned viral gene under the control of a CMV promoter induced C3 binding activity at the transfected cell surface. Comparable to glycoprotein C (gC) from herpes simplex virus type 1 (HSV-1-gC), glycoprotein III from pseudorabiesvirus (gIII, PRV-gC) and bovine herpesvirus-1 (gIII, BHV-1-gC), gp13 derived from EHV-infected cell lysates bound to C3 fixed to solid phase, showing preferential binding to the appropriate host complement component. Similar to wild-type isolates, a highly attenuated vaccine EHV-1 strain also displayed complement receptor activity despite apparent differences of the gp13 gene in restriction enzyme digest pattern and reactivity with monoclonal antibodies. In addition, other structural proteins were altered in the vaccine strain as compared to wild-type strains, which might contribute to its attenuated phenotype. In contrast to the situation observed with HSV-1-gC, the interaction of gp13 (EHV-gC) with horse complement was not inhibited by polyanionic substances like heparin or dextran sulfate. These results suggest structural differences in the particular binding mechanism of the respective viral envelope proteins.
Animals↗
Chronic active Epstein-Barr virus disease in a case of persistent polyclonal B-cell lymphocytosis.
Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare haematological disorder. It is characterized by activated and morphologically atypical B lymphocytes and polyclonal IgM production and has been associated with female sex, cigarette smoking, and HLA-DR7 expression. We report a case of PPBL with intermitting symptoms compatible with a chronic fatigue syndrome, recurrent erythema nodosum and multiforme. Serological findings suggested a chronic active Epstein-Barr virus (EBV) infection. Messenger RNA of EBV immediate early gene transactivation BZLF1 was detected in peripheral blood lymphocytes by reverse transcriptase PCR indicating a persistent replication of the virus. Over 2 years of observation we detected varying numbers of atypical lymphocytes. These cells hybridized with a probe specific for the EBV internal repeat region (BamHI W) which indicates a productive infection. Of interest, no reaction was observed with a probe specific for the latency-associated small RNAs (EBERs). The immunological phenotype of the polyclonal B cells was similar to B-cell lines immortalized by EBV in vitro, expressing a number of activation molecules (CD23, CD25, CD54) and the bcl-2 protein. In summary, our findings suggest that persistent EBV replication might be crucial in the development of lymphoproliferative disorders such as PPBL.
Antigens, CD↗
Role of Epstein-Barr virus and soluble CD21 in persistent polyclonal B-cell lymphocytosis.
The expression of EBV proteins and immunological properties were studied in the first stable cell line (SM) established from a patient presenting with persistent polyclonal B-cell lymphocytosis (PPBL). SM cells which represent a small population of EBV-positive atypical cells found in the peripheral blood of the patient express the KI-1 antigen (CD30) as well as the proto-oncogene bcl-2 product and cell surface markers of mature activated B lymphocytes. The cells harbour an EBV subtype A genome and contain EBNA2 protein. This argues against a transformation-incompetent virus as the main cause of the chronic active EBV infection observed in our patient. Latent membrane protein (LMP1) was weakly expressed and found predominantly in a perinuclear localization, a location which could lead to decreased immunogenicity in vivo. Similar to the EBV-transformed marmoset cell line B95-8, SM cells were in part productively infected as transcription of the immediate early gene BZLF1 could be shown and in some cells high levels of EBV-genome were detected by in situ hybridization with a BamH1 W-probe. Comparable to the atypical cells in the peripheral blood of the patient. EBV small RNAs were not detected with EBER-specific probes. Of interest, we noticed a markedly increased production of soluble CD21 (sCD21) antigen by SM cells as compared to LCL-type Burkitt's lymphoma cell lines. This could explain the elevated sCD21 levels observed in the serum of our PPBL patient and confirms our previous findings in patients with acute EBV infection. It also suggests a possible role of sCD21 in EBV-mediated regulation of the immune response and provides a possible explanation for the dysregulation of the humoral immune system observed in PPBl patients.
B-Lymphocytes↗
Expression of properdin in human monocytes.
Properdin is the only known positive regulator of the alternative pathway of complement activation. Northern blot analysis of cell lines derived from fibroblasts, B-cells, hepatoma cells, and cells of the monocyte-macrophage lineage revealed properdin expression only in the myelomonocytic cell line HL-60, in the monoblastic cell line U-937 and in the monocytic line Mono Mac 6. Culture of Mono Mac 6 cells for 24 h with phorbol 12-myristate 13-acetate, bacterial lipopolysaccharide and the cytokines interleukin-1 beta and tumour necrosis factor-alpha enhanced mRNA abundance, with the strongest effect (tenfold) being observed with the lipopolysaccharide. In contrast, recombinant interferon-gamma consistently halved properdin mRNA abundance. The same pattern was found for the secretion of properdin as detected by ELISA of Mono Mac 6 supernatants. The suppressive effect of interferon-gamma on properdin mRNA abundance was also demonstrated for primary blood monocytes. The data suggest that the expression and secretion of this complement regulatory protein by monocytes is differentially regulated by cytokines and link the immune response with alternative pathway activation.
Adult↗
Properdin, a positive regulator of complement activation, is expressed in human T cell lines and peripheral blood T cells.
Properdin plays an important regulatory role in the activation of the complement system. Here we report the biosynthesis of properdin in four different human T cell lines and in T cells purified from peripheral blood. Cell sorting experiments, in conjunction with Northern blotting, showed that both CD4- and CD8-bearing populations of T cells have the potential to synthesize properdin. The functional activity of properdin secreted from these T cell lines was determined in a hemolytic assay. In view of the numerous ways in which complement activation may influence cellular immune response, the present results indicate, for the first time, a possible interaction between the complement and the T cell system.
Adult↗
Species selective interaction of Alphaherpesvirinae with the "unspecific" immune system of the host.
During evolution Herpesviridae have developed glycoproteins, which interact with essential components of the immune system. Besides immunoglobulin-binding proteins (= Fc-receptors), expressed by several members of the herpesfamily, the interaction with the complement system plays a role in the pathogenicity of herpes simplex virus. Here we report that the ability to interact with the third complement component (C3), the central mediator of complement activation, was also found among several animal alphaherpesviruses. This interaction appeared to be species-selective as the viral proteins preferentially bound to the C3 originated from the respective host. That could provide a possible explanation for the evolution of a variety of herpesviruses as the species tropism observed among Herpesviridae may be influenced by specific adaptation of protective virus-proteins to the immune system of the different hosts. The data have critical implications for the studies of virus host interactions in heterologous systems and support a role for the C3-binding proteins in pathogenesis. Since the C3-binding proteins are conserved among different herpesviruses they could serve as suitable subunit-vaccine candidates.
Ambystoma↗
Determination of soluble CD21 as a parameter of B cell activation.
In this study we established a novel solid-phase immunoassay for CD21 using the time-resolved fluorescence of lanthanide chelates. The capture assay was able to detect concentrations of as low as 100 pg of CD21 antigen per millilitre of sample and was used for quantitative determination of CD21 in lysates of different cell lines as well as in patient serum specimens. CD21 was measured in lysates of tonsils and cell lines of B, T cell and myelomonocyte lineage, and appeared to consist of monomeric antigen under the detergent conditions used. Elevated levels of soluble CD21 were observed in serum of patients with Epstein-Barr virus (EBV) infection, a disease known to be associated with polyclonal B cell activation, and in infection with the lymphotropic rubella virus. Significantly increased levels were also found in malignancies which are associated with EBV. In patients with nasopharyngeal carcinoma (NPC), a correlation with the titre of EBV-specific IgA was observed, thus supporting a possible role of soluble CD21 as a marker for disease activity in certain malignancies. Our data suggest that measurement of soluble CD21 could serve as a marker for activation of the immune system and diseases involving the B cell lymphoid system. Possible mechanisms and functions of soluble CD21 are discussed.
B-Lymphocytes↗
Herpes simplex virus glycoprotein C: molecular mimicry of complement regulatory proteins by a viral protein.
Herpes simplex virus (HSV) encodes a protein, glycoprotein C (gC), which binds to the third complement component, the central mediator of complement activation. In this study the structural and functional relationships of gC from HSV type 1 (HSV-1) and known human complement regulatory proteins factor H, properdin, factor B, complement receptor 1 (CR1) and 2 (CR2) were investigated. The interaction of gC with C3b was studied using purified complement components, synthetic peptides, antisera against different C3 fragments and anti-C3 monoclonal antibodies (mAb) with known inhibitory effects on C3-ligand interactions. All the mAb that inhibited gC/C3b interactions, in a differential manner, also prevented binding of C3 fragments to factors H, B, CR1 or CR2. No blocking was observed with synthetic peptides representing different C3 regions or with factor B and C3d, whereas C3b, C3c and factor H were inhibitory, as well as purified gC. There was no binding of gC to cobra venom factor (CVF), a C3c-like fragment derived from cobra gland. Purified gC bound to iC3, iC3b and C3c, but failed to bind to C3d. Glycoprotein C bound only weakly to iC3 derived from bovine and porcine plasma, thus indicating a preference of the viral protein for the appropriate host. Binding of gC was also observed to proteolytic C3 fragments, especially to the beta-chain, thus suggesting the importance of the C3 region as a binding site. Purified gC from HSV-1, but not HSV-2, inhibited the binding of factor H and properdin but not of CR1 to C3b. The binding of iC3b to CR2, a molecule involved in B-cell activation and binding of the Epstein-Barr virus, was also inhibited by the HSV-1 protein. As factor H and properdin, the binding of which was inhibited by gC, are important regulators of the alternative complement pathway, these data further support a role of gC in the evasion of HSV from a major first-line host defence mechanism, i.e. the complement system. In addition, the inhibition of the C3/CR2 interaction may suggest a possible immunoregulatory role of HSV glycoprotein C.
Antibodies, Monoclonal↗