Detection of HHV-6 Virus in specimen of a ductal pancreatic adenocarcinoma with comparison in tumor and normal tissue.

AIMS: The association of human herpesvirus 6 (HHV-6) species with pancreatic cancer is controversially discussed. The aim of this study was to further investigate the postulated association and to identify the basis of HHV-6 DNA positivity reported for pancreatic cancer tissue. METHODS: All samples of patients with pancreatic cancer (cancer and surrounding tissue) were analyzed for presence of HHV-6 DNA by PCR and then selected cases by immunohistochemistry. RESULTS: Sixty eight per cent (68% = 52/77) of all patients were HHV-6 DNA positive in any of the samples, 49% (38/77) were positive in tumor tissue. Specimens of just one patient were HHV-6A DNA positive, all other patients were positive for HHV-6B. Immunohistochemical analysis of HHV-6 DNA positive samples did not reveal any specific HHV-6B protein positive tumor cell. In contrast, supposed immune cells presented intra- and peritumorally expressed HHV-6B-protein. The cause of presence of these cells in the tumor stroma is unknown, as of yet. CONCLUSIONS: HHV-6 DNA-positivity of pancreatic cancer tissue described by us and others is probably not due to the infection of pancreatic cells by HHV-6, but rather due to the migration of HHV-6 positive immune cells into the pancreas. Based on our data, we suppose that there is no direct evidence for HHV-6 as a causative agent of pancreatic cancer, but further in-depth studies (including investigation of immune status of patients) are necessary to make definitive conclusions.

Humoral and cellular immune response to tick-borne-encephalitis (TBE) vaccination depends on booster doses in patients with Juvenile Idiopathic Arthritis (JIA).

Juvenile Idiopathic Arthritis (JIA) patients living in areas with high prevalence of tick-borne-encephalitis-virus-(TBEV)-infection are recommended for administration of inactivated TBE-vaccination. However, there are serious concerns regarding protective vaccine-induced immune responses against TBEV in immunocompromised patients. The present study aimed to analyze the humoral and cellular immune response to TBE-vaccination in previously TBE-vaccinated JIA patients compared to healthy controls (HC) including investigation of IgG-anti-TBEV avidity, neutralization capacity, cellular reactivity by IFNgamma-ELISPOT and cytokine secretion assays. Similar IgG-anti-TBEV antibody concentrations, neutralization titers and cellular reactivity were found between JIA and HC. The number and the early timing of booster vaccinations after primary vaccination had the most prominent effect on neutralizing antibodies in JIA and on IgG-anti-TBEV concentrations in both JIA and HC. Administration of booster vaccinations made it more likely for JIA patients to have IgG-anti-TBEV concentrations ≥165 VIEU/ml and avidities >60%. TNF-alpha inhibitors had a positive and MTX administration a negative effect on humoral immune responses. In conclusion, irrespective of having JIA or not, vaccinated children showed similar humoral and cellular immunity against TBEV several years after primary TBE-vaccination. However, in JIA, booster vaccinations mounted a significantly higher humoral immune response than in JIA without boosters. Our results highlight the need for timely administration of boosters particularly in JIA. Although immunosuppressive treatment at vaccinations in diagnosed JIA had a negative effect mainly on TBEV-specific cellular immunity, most JIA patients mounted a favorable humoral immune response which was maintained over time. Thus, successful TBE-vaccination seems highly feasible in JIA patients with immunosuppressive regimens.

Expanding the spectrum of neurological disease associated with Epstein-Barr virus activity.

The purpose of this study was to delineate the spectrum of neurological diseases attributed to Epstein-Barr virus (EBV) activity. The approach was a retrospective study on patients with EBV activity proven by a positive EBV antibody-specific index (AI) and/or cerebrospinal fluid (CSF) PCR. One hundred six children and adults (AI positive = 77, AI + PCR positive = 3, PCR positive = 26) were identified, most with reactivated infections. Twenty-eight showed typical EBV-related diseases (encephalitis, neuritis, meningitis), 19 further infections (HSV encephalitis, neuroborreliosis, HIV infection, bacterial meningitis), nine immune-mediated disorders (multiple sclerosis, optic neuritis), and 50 further diseases not typical for EBV. The highest AI values occurred in patients with encephalitis. No relationship between disease category or AI values and viral loads was found. Additional reanalysis of 1,500 consecutive CSF EBV PCR studies revealed the highest positive rates among patients with further infections (n = 18/227, 7.9%) but lower rates among patients with typical EBV-related disorders (5/395; 1.3%), immune-mediated disorders (n = 2/174; 1.1%) and other conditions (n = 4/704; 0.6%). Intrathecal EBV activity is not restricted to typical EBV-related disorders, unexpectedly frequent in further CNS infections and also present in non-inflammatory conditions. Prospective studies should assess the pathogenic role of EBV in these different diseases.

First report of an autochthonous hepatitis E virus genotype 3 infection in a 5 month old female child in Germany.

Hepatitis E virus (HEV) is well-known to cause endemic outbreaks of hepatitis in tropical countries, mostly caused by HEV genotypes 1 or 2 and transmitted from humans to humans via the fecal-oral route. In contrast, HEV genotypes 3 or 4 are commonly encountered as sporadic cases in a non-endemic setting; these autochthonous cases are transmitted from animals to humans and commonly affect elderly male subjects. We report a five-month-old caucasian girl presenting with diarrhea, emesis, and elevated ALT. Surprisingly, acute infection with Hepatitis E virus (HEV) genotype 3 was laboratory-confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing. Thirteen months later, RT-PCR for HEV from stool tested negative whereas anti-HEV IgG in serum tested positive. Neither HEV RNA nor anti-HEV antibodies could be detected in stool or serum of the parents. To our knowledge, this is the first pediatric case of a HEV infection in Germany. Thus, HEV should be included into the differential diagnosis of pediatric infectious liver and bowel disease.

Impact of herpes simplex virus detection in respiratory specimens of patients with suspected viral pneumonia.

BACKGROUND: Respiratory infection and failure is a commonly encountered problem in intensive care unit (ICU) patients. However, despite the accumulating body of evidence to suggest that herpes simplex virus type 1 (HSV-1) is associated with pneumonia, the exact role played by this virus in this process is still not fully understood. Therefore, to identify patients at risk, we have conducted a case-control study to characterize patients with HSV-1-positive pneumonia. PATIENTS AND METHODS: Between 2007 and 2009, all patients with suspected viral pneumonia were tested for the presence of herpes viruses using a PCR assay approach with respiratory specimens. To identify possible associations, risk factors, and impact of HSV, HSV-1-positive ICU patients (n = 51) were compared to age-, gender-, and department- and season-matched HSV-negative patients (n = 52). RESULTS: HSV-positive patients differed significantly from the HSV-negative ones only in terms of time of mechanical ventilation (13 vs. 6 days, respectively; p = 0.002). Subgroup analysis in the patients aged >60 years and in those without bacterial detection revealed a similar trend (p = 0.01 and p = 0.004, respectively). Mortality did not differ between the groups or between the HSV-1-positive patients treated with aciclovir and those who were not. A viral load >10E+05 geq/ml was associated with mechanical ventilation (20/21 vs. 17/29; p = 0.004), acute respiratory distress syndrome (ARDS; 19/21 vs. 18/29; p = 0.005), sepsis (18/21 vs. 14/29; p = 0.008), detection of a bacterial pathogen in the same specimen (10/21 vs. 4/29; p = 0.01) and longer ICU stay (25 vs. 30 days; p = 0.04). CONCLUSION: Despite several associations with high viral load, the clinical outcome of HSV-1-positive ICU patients did not differ significantly from the clinical outcome of HSV-negative patients. This finding indicates that HSV-1 viral loads in respiratory specimens are a symptom of a clinically poor condition rather than a cause of it. Longitudinal and therapy studies are therefore needed to distinguish between HSV-1 as a causative pathogen and HSV-1 as a bystander of pneumonia/ARDS.

[The impact of viruses in lower respiratory tract infections of the adult. Part III: therapy and prevention]. / Die Rolle von Viren bei tiefen Atemwegsinfektionen des Erwachsenen. Teil 3: Therapie und Prävention.

In industrialized countries respiratory tract infections are one of the most common reasons for medical consultations. It is assumed that almost one third of these infections include the lower respiratory tract (LRTI), e. g. acute bronchitis, acute exacerbation of chronic obstructive pulmonary disease (COPD), community- or hospital-acquired pneumonia and influenza. Due to a lack of sufficient and valid investigations to prove the presence of respiratory viruses their impact in the pathogenesis of lower respiratory tract infection has probably been underestimated for a long time. Therefore, there might have been many cases of unnecessary antibiotic treatment, especially in cases of acute bronchitis or acute exacerbations of COPD, because of an assumed bacteriological cause. With the introduction of more sensitive investigational procedures, such as polymerase chain reaction, it is possible to sufficiently prove respiratory viruses and therefore illuminate their role in the pathogenesis of lower respiratory tract infections of the adult. We have reviewed the current literature on the impact of viruses in lower respiratory tract infections to elucidate the role of viruses in the pathogenesis of lower respiratory tract infections. The preceding parts of this series provided an introduction to the frequently found viruses, pathogenesis, and diagnostic procedures (part I) as well as common viral infections of the lower respiratory tract (part II). The present 3 (rd) part deals with therapy for and prevention of viral LRTI.

[The impact of viruses in lower respiratory tract infections of the adult. Part II: acute bronchitis, acute exacerbated COPD, pneumonia, and influenza]. / Die Rolle von Viren bei tiefen Atemwegsinfektionen des Erwachsenen. Teil 2: Akute Bronchitis, exazerbierte COPD, Pneumonie und Influenza.

In industrialized countries respiratory tract infections are one of the most common reasons for medical consultations. It is assumed that almost one third of these infections affect the lower respiratory tract (LRTI), e. g. acute bronchitis, acute exacerbation of chronic obstructive pulmonary disease (COPD), community- or hospital-acquired pneumonia and influenza. Due to a lack of sufficient and valid investigations on the epidemiology of respiratory viruses, their impact on the pathogenesis of LRTI has probably been underestimated for a long time. Therefore, there might have been many cases of needless antibiotic treatment, particularly in cases of acute bronchitis or acute exacerbations of COPD, because of an assumed bacteriological aetiology. Following the introduction of diagnostic procedures with increased sensitivity, such as polymerase chain reaction, it is possible to reliably detect respiratory viruses and to illuminate their role in the pathogenesis of LRTI of the adult. We have reviewed the current literature to elucidate the role of viruses in the pathogenesis of LRTI. The first part of this series described frequent viral pathogens, pathogenesis of viral LRTI, and diagnostic procedures. In this 2 (nd) part the aetiological role of viruses in the most frequent forms of LRTI will be highlighted, and the third and last part will provide an overview of therapeutic and preventive options.

[The impact of viruses in lower respiratory tract infections of the adult. Part I: Pathogenesis, viruses, and diagnostics]. / Die Rolle von Viren bei tiefen Atemwegsinfektionen des Erwachsenen. Teil 1: Erreger, Pathogenese und Diagnostik.

In industrialised countries respiratory tract infections are one of the most common reasons for medical consultations. It is assumed that almost one third of these infections include the lower respiratory tract (LRTI), e. g. acute bronchitis, acute exacerbation of chronic obstructive pulmonary disease (COPD), community- or hospital-acquired pneumonia and influenza. Due to a lack of sufficient and valid investigations on the epidemiology of respiratory viruses, their impact on the pathogenesis of LRTI has probably been underestimated for a long time. Therefore, there might have been many cases of needless antibiotic treatment, particularly in cases of acute bronchitis or acute exacerbations of COPD, because of an assumed bacteriological aetiology. Following the introduction of diagnostic procedures with increased sensitivity, such as polymerase chain reaction, it is possible to reliably detect respiratory viruses and to illuminate their role in the pathogenesis of LRTI of the adult. We have reviewed the current literature to elucidate the role of viruses in the pathogenesis of LRTI. The first part of this series deals with the relevant pathogens, pathogenesis, and diagnostic procedures. In the subsequent 2 parts of this series a review will be given on the most common variants of viral LRTI (part II), and therapeutic and preventive options (part III).

[Human Bocavirus-infection (HBoV): an important cause of severe viral obstructive bronchitis in children]. / Infektionen mit dem humanen Bocavirus (hBoV): Eine wichtige Ursache schwerer viraler Atemwegsinfektionen im frühen Kindesalter.

BACKGROUND: Apart from established pathogens of lower respiratory tract infections, such as respiratory syncytial virus (RSV), an increasing number of additional agents has been identified in recent years. In 2005 the human bocavirus (hBoV) has been isolated from respiratory tract samples and has been reported worldwide with frequencies ranging from 1.5 to 18.3% in respiratory samples from children with airway infections. PATIENTS: We investigated 173 specimens of a total number of 162 children who were inpatients with severe respiratory tract infections most of whom required oxygen therapy. METHOD: We analyzed respiratory tract samples (83% nasopharyngeal washes, 15% tracheal secretions, 2% bronchoalveolar lavages) for adenoviruses, influenza A und B viruses, parainfluenzaviruses types 1 to 3 and RSV using antigen-specific immunofluorescence assays. Additionally we tested human metapneumovirus (hMPV) and hBoV using a PCR assay. MAIN RESULTS: 35.8% specimens were negative in all assays, 54.3% were positive for RSV and 9.8% were positive for adeno-, influenza-, parainfluenzaviruses or hMPV. HBoV could be detected in 17 specimens (9.8%), defining HBoV to be the second most frequent pathogen. Nine of these patients showed a coinfection with RSV, one with parainfluenza virus. Viral loads did range from 2x10 (2) to 5.6x10 (10) genome equivalents/ml with higher viral loads being observed in the first days after disease onset. Most children were infected in the months between December and April. Half of the patients with isolated HBoV infection showed rhinopharyngitis, a third suffered from pulmonary obstruction and nearly every second required oxygen therapy. However, no HBoV-specific symptoms were found. CONCLUSION: HBoV is a common pathogen causing viral respiratory tract infection in infants and young children. Among the here reported patients HBoV was the second most frequent identified pathogen. X-ray studies frequently revealed peribronchial and pneumonic infiltrates with only moderately elevated laboratory inflammatory markers. So far, no HBoV-specific clinical symptoms are known. Additional questions for example related to the way of transmission and optimal treatment remain to be investigated in prospective studies.

Application of virus-specific immunoglobulin M (IgM), IgG, and IgA antibody detection with a polyantigenic enzyme-linked immunosorbent assay for diagnosis of Epstein-Barr virus infections in childhood.

The Enzygnost anti-Epstein-Barr virus enzyme-linked immunosorbent assay (ELISA) system, which is based on a defined antigen mixture and on detection of antibodies of the immunoglobulin G (IgG), IgM, and IgA classes, was evaluated for its reliability in diagnosing Epstein-Barr virus infections in childhood. With samples from 66 children, the Epstein-Barr virus status and the infection phase were defined by indirect immunofluorescence and anticomplement fluorescence assays: 11 children were seronegative, 8 had a primary infection, 20 had a recent primary or past infection, and in 27 a reactivated Epstein-Barr virus infection was diagnosed. When applying the Enzygnost ELISAs, 15 serum samples (22.7%) were not interpretable due to indeterminate results in at least one of the assays used and were therefore excluded from further evaluation. The respective sensitivities and specificities for the diagnosis of seronegativity were 100 and 100%, those for the diagnosis of primary infection were 100 and 97%, those for the diagnosis of recent primary or past infection were 100 and 52%, and those for the diagnosis of reactivated infection were 10 and 100%. This poor performance of the Enzygnost system with reactivated infections is due to the prerequisite of an IgG antibody value of >650 IU/ml for the diagnosis of viral activity, which was fulfilled in only two of the children. Despite the high rate of indeterminate results, the Enzygnost system is useful in diagnosing acute and past Epstein-Barr virus infection in childhood. For serological diagnosis of viral activity in childhood, a supplementary assay is necessary.

Cloning and expression of the human single copy homologue of the mouse zinc finger protein zfr.

A human homologue of the murine zinc finger protein zfr is transcriptionally induced in the Epstein-Barr virus-positive Burkitt lymphoma cell line Raji upon treatment with the granulocyte/macrophage lineage ganglioside IV(3)NeuAc-nLcOse(4)Cer. The gene was cloned by a rapid amplification of cDNA ends approach based on a cDNA clone. The resulting hzfr sequence is 3393 base pairs in length coding for a protein of 1057 amino acids. Sequence alignments between hzfr and zfr reveal an identity of 92% on the nucleotide level and an identity of 96.4% on the amino acid level, respectively. Based on Southern blot data hzfr can be addressed as a single copy gene. Tissue-specific expression was determined by semi-quantitative PCR of normalized cDNA populations from various human tissues with glyceraldehyde-3-phosphate dehydrogenase as an internal control. Highest levels of transcripts were found in brain. hzfr transcripts could not be detected in skeletal muscle.

Detection of cytomegalovirus DNA in human specimens by LightCycler PCR.

Detection of human cytomegalovirus (CMV) DNA in clinical specimens is considered a cornerstone in the diagnosis of CMV disease. The aim of this study was to evaluate a newly designed LightCycler-based quantitative CMV PCR. Specimens of human origin (n = 200) were tested using the LightCycler PCR, the quantitative COBAS AMPLICOR CMV MONITOR (CACM) assay, and a qualitative in-house PCR assay for the presence of CMV DNA. Samples that were reactive in at least two of the three assays were considered CMV DNA positive (n = 95 [47. 5%]), while samples that were nonreactive in two of the three assays were considered CMV DNA negative (n = 105 [52.5%]). Using the LightCycler assay, CMV DNA was detected in 91 of the 95 CMV DNA-positive human specimens (sensitivity, 95.8%; 95% confidence interval [CI], 89.6 to 98.8) and in 1 of the CMV DNA-negative specimens (specificity, 99%; 95% CI, 94.8 to 99.8). Results of CMV load determination as assessed by both quantitative test systems were correlated (r = 0.73; P < 0.0001; 95% CI, 0.61 to 0.81). Results for undiluted samples containing a high CMV load were more accurate with the LightCycler test than were results obtained with the CACM test, which underestimated the viral load of samples containing high DNA copy numbers. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler technology are favorable for the use of this system in the detection of CMV DNA in clinical specimens.

Enhanced transcription of the s-adenosylhomocysteine hydrolase gene precedes Epstein-Barr virus lytic gene activation in ganglioside-stimulated lymphoma cells.

Stimulation of Epstein-Barr virus (EBV) genome-positive Burkitt lymphoma cells with the ganglioside IV3NeuAc-nLcOse4Cer leads to the induction of cell differentiation processes and activates the EBV lytic viral cycle. In cells of the Burkitt lymphoma line Raji differential expression of host cell genes was analysed in the early phase (150 min) post stimulation with the ganglioside to display the cell activities that precede the activation of the EBV lytic cycle using the differential display reverse transcription-polymerase chain reaction technique. Multiple fragment cDNAs derived from control cells and ganglioside-stimulated cells were amplified using random primers and displayed via polyacrylamide gel electrophoresis. The expression pattern of 8,400 bands was analysed. Eleven differentially expressed fragment cDNAs were reamplified and identified by nucleotide sequencing. Six of these could be identified as coding for proteins that may take part in virus reactivation and differentiation. The most striking finding was the induction of s-adenosylhomocysteine hydrolase (AHCY) expression. The cellular enzyme AHCY plays an important role in transmethylation reactions controlling the replication of several viruses. Thus. an involvement in EBV replication can be suggested.

Induction of growth rate and transcriptional modulation of growth promoters and growth inhibitors in epithelial cells by EBV-LMP1.

LMP1 is a genuine oncogene encoded by the Epstein-Barr virus (EBV). The cellular response to expression of the EBV-encoded gene LMP1 in the epithelial cell line Wish was studied. Cells were stably transfected with pCEP-LMP, an expression vector for LMP1. On transcript level a transient expression of the LMP1-gene with a maximum 2 days post transfection was observed. Six days post transfection the rate of DNA synthesis of LMP1-transfected Wish cells was increased by 80% compared to control cells, after 2 further days the number of cells was increased by 32%. A human cDNA-array was screened with probes from LMP1-transfected and control cells showing induction of transcription for proliferation associated genes and repression for growth suppressor genes.

Early steps in termination of the immortalization state in Burkitt lymphoma: induction of genes involved in signal transduction, transcription, and trafficking by the ganglioside IV(3)NeuAc-nLcOse(4)Cer.

Stimulation by the ganglioside IV(3)NeuAc-nLcOse(4)Cer leads to growth arrest in the Burkitt lymphoma cell line Raji. In order to analyze the primary response of Raji cells to that stimulus, a cDNA array screen and a suppression subtractive hybridization-PCR approach were performed. Twenty-four genes with assigned functions were confirmed to be induced by the ganglioside in reverse Northern blot experiments covering e.g. protein kinase B, phospholipase C, the MAP-kinase ERK3, the transcription factors YY1, DR1 and NSEP, the membrane traffic protein TAP, and the nuclear export protein CRM1. Most of the genes identified are involved in signal transduction, transcription, and cell trafficking. For selected genes, the induction of expression was quantified by semiquantitative RT-PCR.

GB virus C/hepatitis G virus infections in traumatologic outpatients, chronic non-A-E hepatitis and extrahepatic malignancies.

GB virus C/hepatitis G virus (GBV-C/HGV) is a recently discovered flavivirus of still unknown pathogenic relevance. We examined traumatologic outpatients to determine GBV-C/HGV viremia for further epidemiological studies, as blood donors hitherto used as controls represent healthy individuals without risk factors. Anti-GBV-C/HGV antibodies were detectable in 13.2% (95% confidence interval [CI] 9.3-18.2) and GBV-C/HGV RNA was detectable in 4.5% (95% CI 2.4-8.2) of the outpatients. In chronic non-A-E hepatitis patients GBV-C/HGV viremia was detectable at a significantly higher level of 16.1% (95% CI 6.1-34.5), while the prevalence of anti-GBV-C/HGV antibodies was 12.9% (95% CI 4.2-30.8). The rate of GBV-C/HGV viremia in patients with malignant diseases (different types of tumors, blood recipients were excluded) was 12.5% (95% CI 8.4-18.1), a significant elevation compared to traumatologic outpatients. The seroprevalence in the tumor group was 22.1% (95% CI 16.7-28.6), also significantly elevated. Thus, there are two messages. Firstly, testing for GBV-C/HGV may be a useful extension of the diagnostic procedure of viral hepatitis. Secondly, common risk factors or etiologic relations of GBV-C/HGV and extrahepatic malignancies should be discussed.

Isolation and expression analysis of two stress-responsive sucrose-synthase genes from the resurrection plant Craterostigma plantagineum (Hochst.).

The enzyme sucrose synthase (UDP-glucose: D-fructose 2alpha-glucosyltransferase, EC 2.4.1.13) is a key enzyme in carbohydrate metabolism, catalyzing the reversible conversion of sucrose uridine-diphosphate into fructose and UDP-glucose. We report the molecular characterization of two classes of cDNA and genomic clones encoding sucrose synthase from Craterostigma plantagineum Hochst., a resurrection plant in which the turnover of sucrose is considered to have an important role in the unique phenomenon of surviving desiccation. Sucrose-synthase transcript and protein levels are modulated by dehydration and rehydration. In-situ hybridization revealed that transcripts preferentially accumulate in phloem tissues. Promoter analysis underlined a role for class-I sucrose-synthase genes in dehydration stress and in response to cis-abscisic acid. A DNA sequence motif common to class-I sucrose-synthase and sucrose-phosphate-synthase genes was discovered.

Characterization of cytostatically active glycosphingolipids isolated from thioglycollate-elicited murine macrophages.

Two acidic glycosphingolipids (gangliosides) derived from mouse macrophage membranes and separated by thin-layer chromatography have a strong cytostatic effect on human and mouse tumor cells. The structure of the two gangliosides, named M phi G1 and M phi G2, was elucidated by application of physicochemical and immunochemical methods. Gas chromatography and mass spectrometry of M phi G1 and M phi G2 classified them as isomeric monosialogangliosides with ceramide moieties composed of sphingosine as the long-chain base, C16 and C18 fatty acids, respectively, and a lacto-tetraose backbone. For M phi G1, additional immunochemical findings led to the proposed structure IV3NeuAc-nLcOse4Cer. The immunochemical reactions of M phi G2 suggest a branched structure for the oligosaccharide moiety.