Human papillomavirus genotype distribution and risk factors for infection in women from a small municipality in north east Brazil.

In order to assess the prevalence of human papillomavirus (HPV) infection, the HPV genotypes and factors associated with infection, we conducted a population-based survey in a small municipality in north east Brazil among women aged between 12 and 49 years. A questionnaire regarding socioeconomic variables, reproductive life and sexual behaviour was used, and women were examined gynaecologically, followed by collection of vaginal lavage with saline solution for HPV DNA determination. HPV DNA was detected by the Digene(®) SHARP Signal(TM)-System, and further genotyped by INNO-LiPA Genotyping System(®). Of 579 women, HPV infection was present in 68 (prevalence: 11.7%; 95% CI: 9.3-14.7). The most common HPV types were 16, 31 and 74, each accounting for 14.7% of infections. Of all HPV-positive women, 35.3% showed multiple HPV genotypes. Variables independently associated with HPV infection were: ≥3 partners in life (adjusted OR [aOR]: 3.06; 95% CI: 1.68-5.60) and the use of oral contraception in the last 12 months (aOR: 2.39; 95% CI: 1.33-4.30). Previous participation in a cervical cancer screening programme was protective (aOR: 0.28; 95% CI: 0.13-0.60). HPV infection is common among women from rural Brazil, and HPV genotypes identified indicate that immunization could be an important preventive measure in this population.

[Puumala virus infection (nephropathia epidemica) as different diagnosis of acute renal failure]. / Puumala-Virus Infektion (Nephropathia epidemica) als Differenzialdiagnose der akut aufgetretenen Niereninsuffizienz.

UNLABELLED: Puumala virus infection (nephropathia epidemica) as different diagnosis of acute renal failure. HISTORY: A 34-year old patient presented in reduced status with a sudden onset of fever, headache, backpain, abdominal pain, mild diarrhea, nausea with vomiting, and blurred vision. Within a few days an acute renal failure developed. INVESTIGATIONS: On admittance there was thrombocytopenia of 27/nl, CRP of 109 mg/l and proteinuria of 5 g/l, moderate glucosuria and erythrocyturia of 250/microliter. Renal biopsy showed acute hemorrhagic interstitial nephritis. DIAGNOSIS, THERAPY AND FOLLOW UP: Diagnosis of nephropathia epidemica was proven by puumala-virus IgM- and later IgG-antibodies. Hantaan-antibodies were negative. Maximum serum creatinine of 640 micromol/l and urea of 30.5 mmol/l developed on the 5(th) day after admission. Without specific therapy the patient recovered fast and there were no persisting abnormalities during a 2-year follow up. CONCLUSION: In young patients with acute renal failure of unknown origin with the above symptoms hantavirus-infection with the subtypes puumala and dobrava should be considered in Central Europe.

Fatal cytomegalovirus pneumonia after preemptive antiviral therapy in a renal transplant recipient.

Cytomegalovirus (CMV) infections occur with an incidence of up to 70% in renal transplant patients and mortality is low due to effective antiviral drugs. We report here the case of a patient who suffered from an uncommonly severe and therapy-resistant pulmonary CMV infection. During the disease course, CMV-PCR from alveolar cells and lung biopsy material was repeatedly negative despite high CMV pp65 antigenemia. CMV pneumonia was finally diagnosed from a biopsy obtained by open thoracotomy revealing positive CMV immunostaining of lung tissue. The patient died of respiratory failure though double-treatment using both ganciclovir and foscavir was administered. Post mortem, the clinically suspected resistance to both antiviral drugs, but not to cidofovir, could be proven by bioassay testing of in vitro growth responses using viral cultures. CMV pneumonia may thus not be diagnosed by standard PCR techniques in rare cases and may be resistant to the available antiviral therapy. Severe CMV pneumonia may benefit from novel antiviral drugs such as cidofovir, which is currently used in the treatment of CMV retinitis in HIV patients.

[Hepatitis C virus-associated dermatoses: a review]. / Hepatitis-C-virus-assoziierte Dermatosen.

Acute infection with hepatitis C virus (HCV) is often clinically inapparent, but may affect several organ systems in its chronic course. In dermatology, common diseases such as lichen planus, cryoglobulinemic vasculitis and porphyria cutanea tarda have been described in association with HCV infection. A number of other dermatologic disorders, e.g., psoriasis, chronic urticaria, chronic pruritus, pseudo-kaposi sarcoma, necrolytic migratory erythema and Behçet disease, have been associated in case reports with HCV-induced liver disease. In this study we summarize the recent literature reports, present three patients observed by our group and update the topic.

Pseudotumour of the tongue caused by herpes simplex virus type 2 in an HIV-1 infected immunosuppressed patient.

An HIV-1 infected immunosuppressed patient (CD4+ cell counts: 382 cells/microL; viral load 94,000 copies/mL) with recurrent perianal herpes simplex virus type 2 (HSV-2) infections is described, showing an unusual exophytic tumour resembling a squamous cell carcinoma in the lateral part of the tongue. He also had persistent facial herpes infection, oral candidosis, oral hairy leukoplakia and lymphadenopathy. The presence of HSV-2 was detected by polymerase chain reaction both in smears and in a tissue biopsy taken from the involved tongue area. Treatment with brivudin, a new oral virustatic drug, led to rapid regression of the tumour.

Detection of antibodies against viral capsid proteins of human herpesvirus 8 in AIDS-associated Kaposi's sarcoma.

Sequences of a new herpesvirus with homology to gammaherpesvirinae were recently identified in AIDS-associated Kaposi's sarcoma (KS). Subsequently this novel virus, called KS-associated virus (KSHV) or human herpesvirus (HHV) 8 was detected in classical KS and AIDS-associated body cavity based lymphomas by polymerase chain reaction. In this report major and minor capsid proteins of HHV-8 were molecularly cloned and produced as recombinant proteins in Escherichia coli. Sera from 69 HIV-1 infected patients with KS, 30 HIV-1 infected patients without KS and 106 control individuals were tested by enzyme-linked immunosorbent assay for anti-HHV-8 capsid IgM and IgG antibodies. Sera from four patients were tested over periods ranging from 18 months to 6 years. IgG antibodies directed against HHV-8 capsid antigens were detected in patients with AIDS-associated KS and in some AIDS patients without KS. Seroconversion with IgM and IgG antibodies directed against HHV-8 capsid proteins occurred more than 1 year prior to diagnosis of KS. In a considerable portion of KS patients no IgM or IgG antibodies against HHV-8 capsid proteins were detected. In these patients there was an inverse relationship between antibodies against HHV-8orf26 and the CD4/CD8 ratio, suggesting that the inconsistency of anti-HHV-8orf26 antibodies is due at least partly to an impaired immune response. No reactivity against HHV-8 capsid antigens was detected in the vast majority of sera from HIV-negative control individuals. Our findings indicate that a specific humoral immune response against capsid proteins is raised in HHV-8 infected individuals, and that anti-capsid antibodies can be used to diagnose HHV-8 infection. The correlation between occurrence of anti-HHV-8 antibodies and KS supports the hypothesis of a causative role of HHV-8.

Stool viruses, coinfections, and diarrhea in HIV-infected patients. Berlin Diarrhea/Wasting Syndrome Study Group.

To examine the prevalence of stool viruses and their role in the pathogenesis of diarrhea in HIV infection, we evaluated biopsies and repeated stool samples of 256 HIV-infected patients undergoing diagnostic endoscopy because of diarrhea (n = 136) or other symptoms (n = 120) for bacterial, protozoal, and viral enteropathogens. In 70% of the patients with diarrhea, at least one potential enteropathogen was detected. Stool virus was detected by electron microscopy in 17% (44 of 256), adenovirus in 6.6% (17 of 256), and coronavirus in 11.3% (29 of 256) of the patients. Adenovirus and coronavirus were detected more frequently in patients with diarrhea than in patients without diarrhea [adenovirus 10% (13 of 136) vs. 3.3% (4 of 120), p = 0.0129; coronavirus 15% (21 of 136) vs. 6.6% (8 of 120), p = 0.0142]. Sixty-one percent of patients harboring stool virus were coinfected by another enteropathogen. Pathogens other than stool virus were detected more frequently in patients harboring adenovirus (82%) than in patients without stool virus (48%, p < 0.025). Adenovirus and coronavirus are frequently detected in stools of HIV- infected patients and may contribute to diarrhea. Adenovirus infection may facilitate the occurrence of other intestinal pathogens. Due to frequent coinfections, detection of stool viruses reduces the rate of diarrhea of unknown origin only by approximately 5%.

High-level sensitivity of quantitative pp65 cytomegalovirus (CMV) antigenemia assay for diagnosis of CMV disease in AIDS patients and follow-up.

Cytomegalovirus (CMV) antigenemia was evaluated in 174 patients positive for human immunodeficiency virus. Antigenemia could be detected in 96.7% of patients with CMV disease, 76.9% of patients suffering from a relapse of the disease, and 11.4% of asymptomatic patients with CD4 levels of < 100 cells per microliter. No antigenemia was detected in patients with CD4 levels of 250 to 500 cells per microliter. Specificity and the positive predictive value for CMV disease were increased only if more than 5 positive cells per slide were considered. However, CMV disease may also occur in patients with low-grade antigenemia.

Multicentre study for diagnostic evaluation of an assay for simultaneous detection of antibodies to HIV-1, HIV-2 and HIV-1 subtype 0 (HIV-0).

The aim of the study was to evaluate a new ELISA for detection of HIV-1, HIV-2 and HIV-1 subtype 0 (HIV-0) antibodies. The assay format is based on the antigen sandwich principle. To enable specific detection of HIV-0 antibodies, in addition to HIV-1 and HIV-2 antigens HIV-0 antigen is used for coating the solid phase and for the conjugate. The results show that all 12 HIV-0 samples tested were detected with a high degree of reactivity, as were all the 1,144 anti-HIV-1 and 424 anti-HIV-2 positive samples. The capacity of the test to enable early detection of seroconversions is equivalent to that of other sandwich ELISAs. The specificity of the assay was determined to be 99.89/99.94% (initial/after retest) using 58,366 samples, which is superior to the other ELISAs used for comparison. Even with difficult samples (i.e. samples of African origin, samples known to cause false-positive reactivity in different ELISAs, or samples containing potential interference factors) there were very few false-positive reactions. Therefore, the new assay is well suited for screening blood donations as well as for evaluating samples from patients of different geographic origin.

Low prevalence of hepatitis C virus infection in porphyria cutanea tarda in Germany.

Previous studies from Spain, Italy, and France have demonstrated a high prevalence (71% to 91%) of antibodies against hepatitis C virus in patients with porphyria cutanea tarda (PCT). To determine the role of hepatitis C virus (HCV) in PCT in Germany, we have assessed the prevalence of antibodies against HCV and hepatitis B virus (HBV) in 106 patients (mean age, 60 +/- 14 years) with the disease. Eight of 106 patients (8%) were positive for HCV antibodies and HCV RNA using second-generation enzyme-linked immunosorbent assay (ELISA), recombinant immunoblot assay, and polymerase chain reaction. Antibodies against HBV core antigen were found in 14 patients (13%). Of the patients with antibodies against HCV alanine transaminase (ALT) (aspartate transaminase [AST]) levels above normal occurred in 71% (86%). Because elevated ALT (AST) levels were also found in 51% (64%) of 88 patients without markers of HCV or HBV, we suggest that liver damage in PCT may exist in absence of these viruses. This is supported by the finding that in patients without HCV or HBV markers, higher serum ALT and AST activities were found in patients with overt disease or relapse (ALT, 59 +/- 44 U/L; AST, 37 +/- 21 U/L), whereas patients in remission displayed significantly lower serum enzyme activities (ALT, 16 +/- 8 U/L; AST, 16 +/- 7 U/L), (P < .001). These results indicate that HCV infection does not play a major role in the pathogenesis of PCT in Germany.

The binding site of ribosomal protein L10 in eubacteria and archaebacteria is conserved: reconstitution of chimeric 50S subunits.

It has been shown by electron microscopy that the selective removal of the stalk from 50S ribosomal subunits of two representative archaebacteria, namely Methanococcus vaniellii and Sulfolobus solfataricus, is accompanied by loss of the archaebacterial L10 and L12 proteins. The stalk was reformed if archaebacterial core particles were reconstituted with their corresponding split proteins. Next, structurally intact chimeric 50S subunits have been reconstituted in vitro by addition of Escherichia coli ribosomal proteins L10 and L7/L12 to 50S core particles from M vaniellii or S solfataricus, respectively. In the reverse experiment, using core particles from E coli and split proteins from M vaniellii, stalk-bearing 50S particles were also obtained. Analysis of the reconstituted 50S subunits by immunoblotting revealed that E coli L10 was incorporated into archaebacterial core particles in both presence or absence of E coli L7/L12. In contrast, incorporation of E coli L7/L12 into archaebacterial cores was only possible in the presence of E coli L10. Our results suggest that in archaebacteria - as in E coli - the stalk is formed by archaebacterial L12 proteins that bind to the ribosome via L10. The structural equivalence of eubacterial and archaebacterial L10 and L12 proteins has thus for the first time been established. The chimeric reconstitution experiments provide evidence that the domain of protein L10 that interacts with the ribosomal particle is highly conserved between eubacteria and archaebacteria.

Three-dimensional localization of the NH2- and carboxyl-terminal domain of ribosomal protein S1 on the surface of the 30 S subunit from Escherichia coli.

Antibodies were raised against Escherichia coli ribosomal protein S1 and its NH2- and COOH-terminal fragments, and their specificity was demonstrated by a variety of immunological techniques. These antibodies were then used to investigate the location of protein S1 and its NH2- and COOH-terminal domains on the surface of the 30 S ribosomal subunit by immunoelectron microscopy. In order to prevent dissociation of the protein during the experiments, S1 was cross-linked to 30 S subunits with dithiobis(succinimidyl-propionate); cross-linking yield was 100%. Epitopes of the NH2-terminal domain of S1 were localized at the large lobe of the 30 S ribosomal subunit, close to the one-third/two-thirds partition on the side which in the 70 S ribosome faces the cytoplasm. Experiments with monovalent Fab fragments specific for the COOH-terminal part of S1 provide evidence that the COOH-terminal domain forms an elongated structure extending at least 10 nm from the large lobe of the small subunit into the cytoplasmic space.

Two-dimensional maps in very acidic immobilized pH gradients.

Up to the present time it has been impossible to perform two-dimensional (2-D) separations in very acidic immobilized pH gradients (IPG), due to the lack of suitable buffering acrylamido derivatives to be incorporated into the polyacrylamide matrix. The advent of the pK 3.1 buffer (2-acrylamido glycolic acid; Righetti et al., J. Biochem. Biophys. Methods 16, 1988, 185-192) allowed the formulation of such acidic gradients. We report here separations in IPG pH 2.8-5.0 intervals of polypeptide chains from total lysates of rat intestinal and liver cells and 30S and 50S ribosomal proteins from Halobacterium marismortui. Conditions are given for highly reproducible first and second dimension gels and for a proper silver staining of 2-D maps with practically no background deposition.

The ribosomal domain of the bacterial release factors. The carboxyl-terminal domain of the dimer of Escherichia coli ribosomal protein L7/L12 located in the body of the ribosome is important for release factor interaction.

1. Polyclonal antibodies (pAb 1-73 and pAb 26-120) have been raised against both an N-terminal fragment of Escherichia coli ribosomal protein L7/L12 (amino acids 1-73), and a fragment lacking part of the N-terminal domain (amino acids 26-120). 2. Only pAb 26-120 inhibited release-factor-dependent in vitro termination functions on the ribosome. This antibody binds over the length of the stalk of the large subunit of the ribosome as determined by immune electron microscopy, thereby not distinguishing between the C-terminal domains of the two L7/L12 dimers, those in the stalk or those in the body of the subunit. 3. A monoclonal antibody against an epitope of the C-terminal two thirds of the protein (mAb 74-120), which binds both to the distal tip of the stalk as well as to a region at its base, reflecting the positions of the two dimers is strongly inhibitory of release factor function. 4. A monoclonal antibody against an epitope of the N-terminal fragment of L7/L12 (mAb 1-73), previously shown to remove the dimer of L7/L12 in the 50S subunit stalk but still bind to the body of the particle, partially inhibited release-factor-mediated events. 5. The mAb 74-120 inhibited in vitro termination with a similar profile when the stalk dimer of L7/L12 was removed with mAb 1-73, indicating that the body L7/L12 dimer, and in particular its C-terminal domains, are important for release factor/ribosome interaction. 6. The two release factors have subtle differences in their binding domains with respect to L7/L12.

Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli.

We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide. The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34. The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach. Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.

Immunoblotting analysis of protein-protein crosslinks within the 50S ribosomal subunit of Escherichia coli. A study using dimethylsuberimidate as crosslinking reagent.

50S ribosomal subunits of Escherichia coli have been crosslinked with the bifunctional imidoester dimethyl-suberimidate and the protein-protein crosslinks have been analyzed by immunoblotting, using antisera specific for the individual ribosomal proteins of the large ribosomal subunit. Crosslinked protein pairs which occurred in yields higher than 5% have been unambiguously identified. Thus 13 crosslinks have been identified, namely L1-L33, L5-L7/12, L6-L19, L7/12-L10, L7/12-L11, L9-L28, L10-L11, L13-L20, L16-L27, L17-L32, L18-L22, L19-L25 and L27-L33. These data, together with the results which we will be presenting elsewhere, contribute considerably to our knowledge of the protein topography of the 50S ribosomal proteins as determined by immunoelectron microscopy. We can now propose the approximate locations of ten proteins that have not previously been localized.

Localization of proteins L4, L5, L20 and L25 on the ribosomal surface by immuno-electron microscopy.

Ribosomal proteins L4, L5, L20 and L25 have been localized on the surface of the 50S ribosomal subunit of Escherichia coli by immuno-electron microscopy. The two 5S RNA binding proteins L5 and L25 were both located at the central protuberance extending towards its base, at the interface side of the 50S particle. L5 was localized on the side of the central protuberance that faces the L1 protuberance, whereas L25 was localized on the side that faces the L7/L12 stalk. Proteins L4 and L20 were both located at the back of the 50S subunit; L4 was located in the vicinity of proteins L23 and L29, and protein L20 was localized between proteins L17 and L10 and is thus located below the origin of the L7/L12 stalk.

Protein-protein cross-linking of the 50 S ribosomal subunit of Escherichia coli using 2-iminothiolane. Identification of cross-links by immunoblotting techniques.

We have investigated the protein-protein cross-links formed within the 50 S subunit of the Escherichia coli ribosome using 2-iminothiolane as the cross-linking reagent. The members of the cross-links have been identified by immunoblotting from one-dimensional and two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gels using antisera specific for the individual ribosomal proteins. This method also allowed a quantitation of the yield of cross-linking for each cross-link. A total of 14 cross-links have been identified: L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L30, L17-L32, L19-L25, L20-L21, L22-L32, and L23-L34. Our results are compared with those of Traut and coworkers (Traut, R. R., Tewari, D. S., Sommer, A., Gavino, G. R., Olson, H. M., and Glitz, D. G. (1986) in Structure, Function and Genetics of Ribosomes (Hardesty, B. and Kramer, G., eds) pp. 286-308, Springer-Verlag, New York). Our cross-linking data allow us to propose the approximate locations of eight proteins of the 50 S ribosomal subunit that so far have not been localized by immunoelectron microscopy and they thus contribute considerably to our knowledge of ribosome structure.

A model for the spatial arrangement of the proteins in the large subunit of the Escherichia coli ribosome.

A three-dimensional model for the arrangement of 29 of the 33 proteins from the Escherichia coli large ribosomal subunit has been generated by interactive computer graphics. The topographical information that served as input in the model building process was obtained by combining the immunoelectron microscopically determined network of epitope-epitope distances on the surface of the large ribosomal subunit with in situ protein-protein cross-linking data. These two independent sets of data were shown to be compatible by geometric analysis, thus allowing the construction of an inherently consistent model. The model shows (i) that the lower third of the large subunit is protein-poor, (ii) that proteins known to be functionally involved in peptide bond formation and translocation are clustered in two separate regions, (iii) that proteins functionally interdependent during the self-assembly of the large subunit are close neighbours in the mature subunit and (iv) that proteins forming the early assembly nucleus are grouped together in a distinct region at the 'back' of the subunit.