Culture of human ovarian tissue in xeno-free conditions using laminin components of the human ovarian extracellular matrix.

PURPOSE: Our purpose was to identify human ovarian extracellular matrix (ECM) components that would support in vitro culture of human ovarian tissue and be compatible with possible future clinical applications. We characterized ovarian expression of laminins and selected three laminin tripeptides for culture experiments to be compared with Matrigel, an undefined and animal-based mixture of ECM components. METHODS: Expression of the 12 laminin genes was determined on transcript and protein levels using cortical tissue samples (n = 6), commercial ovary RNA (n = 1), follicular fluid granulosa cells (n = 20), and single-cell RNA-sequencing data. Laminin 221 (LN221), LN521, LN511, and their mixture were chosen for a 7-day culture experiment along with Matrigel using tissue from 17 patients. At the end of the culture, follicles were evaluated by scoring and counting from serial tissue sections, apoptosis measured using in situ TUNEL assay, proliferation by Ki67 staining, and endocrine function by quantifying steroids in culture media using UPLC-MS/MS. RESULTS: Approximately half of the cells in ovarian cortex expressed at least one laminin gene. The overall most expressed laminin α-chains were LAMA2 and LAMA5, ß-chains LAMB1 and LAMB2, and γ-chain LAMC1. In culture experiments, LN221 enhanced follicular survival compared with Matrigel (p < 0.001), whereas tissue cultured on LN521 had higher proportion of secondary follicles (p < 0.001). LN511 and mixture of laminins did not support the cultures leading to lower follicle densities and higher apoptosis. All cultures produced steroids and contained proliferating cells. CONCLUSIONS: LN221 and LN521 show promise in providing xeno-free growth substrates for human ovarian tissue cultures, which may help in further development of folliculogenesis in vitro for clinical practices. The system could also be used for identification of adverse effects of chemicals in ovaries.

Dermatitis por contacto fotoalérgica a protectores solares con oxibenzona en La Plata, Argentina / Photoallergic Contact Dermatitis to Sunscreens Containing Oxybenzone in La Plata, Argentina

ANTECEDENTES: La dermatitis por contacto fotoalérgica (DCFA) a oxibenzona fue por primera vez documentada en 1980, siendo hoy el principal fotoalérgeno de Estados Unidos de América, Canadá y el cuarto en Europa. En Argentina no existen datos ni publicaciones con respecto a esta reacción cutánea. OBJETIVO: Conocer el porcentaje de pacientes con fotosensibilidad afectados con fotoalergia a oxibenzona. METODOLOGÍA: Estudio descriptivo de corte transversal. Un total de 35 pacientes con reacciones fotosensibles con prueba del fotoparche en el Centro de investigación del Hospital Público San Martín, en la ciudad de La Plata, fueron estudiados durante los años 2015 y 2016. RESULTADOS: Se observó el 17,14% de DCFA, presentando 5 (14,28%) pacientes al menos una reacción positiva a oxibenzona en el test de fotoparche, 4 pacientes solo en la zona irradiada con 5 J/cm2 (de UVA) y solo un paciente tanto en la zona irradiada como en la no irradiada. CONCLUSIONES: La DCFA a protectores solares compuestos por oxibenzona es frecuente y se presume infradiagnosticada debido a la falta de estudios confirmatorios como la prueba del fotoparche. El porcentaje de sensibilización varía de acuerdo con cada región, sobre todo por las distintas composiciones y costumbres de uso en protectores solares, cosméticos y tratamiento tópico

BACKGROUND: Photoallergic contact dermatitis (PACD) to oxybenzone was reported for the first time in 1980. Oxybenzone is the most common photoallergen in the United States and Canada and the fourth most common .in Europe. There are no studies or data on the prevalence of oxybenzone PACD in Argentina. OBJECTIVE: To determine the proportion of photosensitive patients with PACD to oxybenzone. METHODS: We conducted a descriptive cross-sectional study of 35 patients with photosensitivity reactions confirmed by photopatch testing at the Research Center of Hospital Público San Martín in La Plata, Argentina, in 2015 and 2016. RESULTS: PACD was identified in 6 patients (17.14%). Five of these (14.28%) had at least one positive reaction to oxybenzone in the photopatch test; 4 had a reaction at irradiated sites only (5 J/cm2 UVA) and one had a reaction at both irradiated and nonirradiated sites. CONCLUSIONS: PACD to sunscreens containing oxybenzone is common and is probably underdiagnosed due to a lack of confirmation by photopatch tests or other diagnostic tools. Sensitization rates vary according to region and are influenced by sunscreen ingredients and variations in the use of sunscreen products, cosmetics, and topical drugs

Photoallergic Contact Dermatitis to Sunscreens Containing Oxybenzone in La Plata, Argentina. / Dermatitis por contacto fotoalérgica a protectores solares con oxibenzona en La Plata, Argentina.

BACKGROUND: Photoallergic contact dermatitis (PACD) to oxybenzone was reported for the first time in 1980. Oxybenzone is the most common photoallergen in the United States and Canada and the fourth most common .in Europe. There are no studies or data on the prevalence of oxybenzone PACD in Argentina. OBJECTIVE: To determine the proportion of photosensitive patients with PACD to oxybenzone. METHODS: We conducted a descriptive cross-sectional study of 35 patients with photosensitivity reactions confirmed by photopatch testing at the Research Center of Hospital Público San Martín in La Plata, Argentina, in 2015 and 2016. RESULTS: PACD was identified in 6 patients (17.14%). Five of these (14.28%) had at least one positive reaction to oxybenzone in the photopatch test; 4 had a reaction at irradiated sites only (5 J/cm2 UVA) and one had a reaction at both irradiated and nonirradiated sites. CONCLUSIONS: PACD to sunscreens containing oxybenzone is common and is probably underdiagnosed due to a lack of confirmation by photopatch tests or other diagnostic tools. Sensitization rates vary according to region and are influenced by sunscreen ingredients and variations in the use of sunscreen products, cosmetics, and topical drugs.

Prenatal viral exposure followed by adult stress produces glucose intolerance in a mouse model.

AIMS/HYPOTHESIS: It has been suggested that the uterine environment may influence metabolic disease occurring later in adult life, and that adult stress may promote disease outcome. Using a mouse model, we tested whether in utero exposure to Ljungan virus (LV) followed by adult exposure to stress produces diabetes. The influence of the timing of viral exposure over the course of pregnancy was also tested. MATERIALS AND METHODS: Pregnant CD-1 mice were exposed i.p. to LV on pregnancy days 4, 8, 12 or 17. Adult male mice from these pregnancies were stressed by being kept in shared cages. Stress only, LV exposure in utero only, and no-stress/no virus exposure groups were also followed. Outcome variables included bodyweight, epididymal fat weight, baseline glucose, glucose tolerance tests (60 and 120 min) and serum insulin. RESULTS: We demonstrated that male mice developed a type 2-like diabetes, including obesity, as adults if infected during pregnancy with LV. Diabetes at the age of 11 weeks was more severe in mice whose mothers were infected earlier than in those whose mothers were infected later in pregnancy. Only animals infected in utero and kept under stress developed diabetes; infection or stress alone did not cause disease. CONCLUSIONS/INTERPRETATION: This work demonstrates that a type 2 diabetes-like disease can be virus-induced in a mouse model. Early in utero viral insults can set the stage for disease occurring during adult life, but the final manifestation of diabetes is dependent on the combination of early viral exposure and stress in adult life.

Epidemiology of community-acquired pneumonia in adults: a population-based study.

In this prospective study, the authors assessed the incidence, aetiology, and outcome of patients with community-acquired pneumonia in the general population. From December 1993 to November 1995, a study was performed in a mixed residential-industrial urban population of the "Maresme" region in Barcelona, Spain. All subjects > or =14 yrs of age (annual average population size 74,368 inhabitants) with clinically suspected community-acquired pneumonia were registered. All cases were re-evaluated by chest radiographs on the 5th day of illness and at monthly intervals until complete recovery. Urine and blood samples were obtained for culture and antigen detection. When lower respiratory tract secretions were obtained, these were also cultured. There were 241 patients with community-acquired pneumonia, with an annual incidence rate of 1.62 cases (95% confidence interval, 1.42-1.82) per 1,000 inhabitants. Incidence rates increased by age groups and were higher in males than in females. Of 232 patients with aetiological data, 104 had an identifiable aetiology. A total of 114 pathogens were found (single pathogen 94, two pathogens 10). There were 81 episodes of bacterial infection and 33 of viral infection. The most common pathogens were Streptococcus pneumoniae, Chlamydia pneumoniae, and influenza A and B viruses. No case of Hantavirus infection was found. The rate of hospital admission was 61.4% with a mean+/-SD length of 11.7+/-10.1 days, a mean period of 23.0+/-14.3 days inactivity, and an overall mortality rate of 5%. The high rate of hospital admission, prolonged stay in hospital, and long period of inactivity all continue to constitute a social and health care burden of community-acquired pneumonia.

A new picornavirus isolated from bank voles (Clethrionomys glareolus).

A previously unknown picornavirus was isolated from bank voles (Clethrionomys glareolus). Electron microscopy images and sequence data of the prototype isolate, named Ljungan virus, showed that it is a picornavirus. The amino acid sequences of predicted Ljungan virus capsid proteins VP2 and VP3 were closely related to the human pathogen echovirus 22 (approximately 70% similarity). A partial 5' noncoding region sequence of Ljungan virus showed the highest degree of relatedness to cardioviruses. Two additional isolates were serologically and molecularly related to the prototype.

Hantavirus Dobrava infection with pulmonary manifestation.

Dobrava virus infection was diagnosed serologically by enzyme-linked immunosorbent and immunofluorescence assays. To determine which hantavirus serotype was involved, sera were analyzed by a focus reduction neutralization test. The clinical data indicated that only pulmonary manifestation was present. Our data support the presence of Dobrava virus infection outside the Balkan region. In conclusion, a previously healthy adult with unexplained pulmonary perfusion failure should be investigated for hantavirus infection.

Could myocarditis, insulin-dependent diabetes mellitus, and Guillain-Barré syndrome be caused by one or more infectious agents carried by rodents?

The numbers of small rodents in northern Sweden fluctuate heavily, peaking every 3 or 4 years. We found that the incidence of Guillain-Barré syndrome and insulin-dependent diabetes mellitus, as well as the number of deaths caused by myocarditis, followed the fluctuations in numbers of bank voles, although with different time lags. An environmental factor, such as an infectious agent, has been suggested for all three diseases. We hypothesize that Guillain-Barré syndrome, myocarditis, and insulin-dependent diabetes mellitus in humans in Sweden are caused by one or more infectious agents carried by small rodents. Also, a group of novel picornaviruses recently isolated from these small rodents is being investigated as the possible etiologic agent(s).

Establishing a European network for the diagnosis of "imported" viral diseases (ENIVD).

The epidemics in recent years of Ebola haemorrhagic fever in Zaire and Gabon acted as a reminder that dangerous infections can be imported very quickly into Europe. Meetings on emerging and re-emerging pathogens organised by the World Health Organization.

Establishing a European network for the diagnosis of.

The epidemics in recent years of Ebola haemorrhagic fever in Zaire and Gabon acted as a reminder that dangerous infections can be imported very quickly into Europe. Meetings on emerging and re-emerging pathogens organised by the World Health Organization

Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica).

Nephropathia epidemica (NE), Puumala (PUU) virus infection, is a febrile disease which is commonly associated with acute renal impairment. To differentiate NE from other acute febrile illnesses, a rapid and reliable serological diagnosis is important, and a number of different protocols have recently been introduced. In the present report we describe a comparative evaluation of six PUU virus immunoglobulin M (IgM) and seven IgG enzyme-linked immunosorbent assay (ELISA) protocols based on native, Escherichia coli-expressed, or baculovirus-expressed nucleocapsid protein (N). Neutralization and immunofluorescence assays were included for comparison. Equally high sensitivities and specificities were obtained with three mu-capture-based IgM ELISAs using native, baculovirus-expressed, and E. coli-expressed N antigens, respectively, and by an ELISA based on purified E. coli-expressed full-length N adsorbed to solid phase. The assays based on truncated amino-terminal N proteins, including a commercially available PUU virus IgM ELISA, all showed lower sensitivities. For detection of PUU virus-specific IgG, ELISAs based on monoclonal antibody-captured native or baculovirus-expressed N antigens showed optimal sensitivities and specificities, while the assays based on E. coli-expressed N did not detect all PUU virus IgG-positive serum samples. A commercially available PUU virus IgG ELISA based on E. coli-expressed amino-terminal N showed a significantly lower sensitivity than those of all other IgG assays.

Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment.

This paper reports the establishment of a model for hantavirus host adaptation. Wild-type (wt) (bank vole-passaged) and Vero E6 cell-cultured variants of Puumala virus strain Kazan were analyzed for their virologic and genetic properties. The wt variant was well adapted for reproduction in bank voles but not in cell culture, while the Vero E6 strains replicated to much higher efficiency in cell culture but did not reproducibly infect bank voles. Comparison of the consensus sequences of the respective viral genomes revealed no differences in the coding region of the S gene. However, the noncoding regions of the S gene were found to be different at positions 26 and 1577. In one additional and independent adaptation experiment, all analyzed cDNA clones from the Vero E6-adapted variant were found to carry substitutions at position 1580 of the S segment, just 3 nucleotides downstream of the mutation observed in the first adaptation. No differences were found in the consensus sequences of the entire M segments from the wt and the Vero E6-adapted variants. The results indicated different impacts of the S and the M genomic segments for the adaptation process and selective advantages for the variants that carried altered noncoding sequences of the S segment. We conclude that the isolation in cell culture resulted in a phenotypically and genotypically altered hantavirus.

Puumala and Dobrava viruses cause hemorrhagic fever with renal syndrome in Bosnia-Herzegovina: evidence of highly cross-neutralizing antibody responses in early patient sera.

Hantavirus infection was diagnosed serologically by mu-capture IgM and IgG ELISAs in hemorrhagic fever with renal syndrome (HFRS) patients admitted to Tuzla Hospital, Bosnia-Herzegovina. The results indicated that more than one hantavirus caused the outbreak. To address the question of which hantavirus serotypes were involved, sequentially drawn sera were analyzed by focus reduction neutralization test (FRNT) for antibodies against Puumala, Hantaan, Dobrava, and Seoul hantaviruses. The data revealed that acute- or early convalescent-phase sera, even when drawn as late as 3 weeks after the onset of disease, could not be used for typing of the causative hantavirus; a significant number of these samples showed similar reactivity of neutralizing antibodies to several different hantavirus serotypes. Moreover, although several acute-phase sera showed the highest FRNT titer to Hantaan virus, convalescent sera from these patients in all cases showed high specificity for Puumala or Dobrava viruses. This phenomenon, interpreted as a cross-neutralizing primary antibody response, makes several earlier reports concerning causative agents of HFRS questionable. Serological examination of small rodents trapped in the endemic area identified Puumala- and Dobrava-like virus infections. RT-PCR and sequencing of rodent lung samples identified Dobrava virus in one yellow-necked field mouse (Apodemus flavicollis). Cross-FRNT data, using polyclonal rabbit antibodies, clearly confirmed Dobrava virus as a unique hantavirus serotype. In conclusion, the results revealed that both Puumala- and Dobrava-like viruses caused HFRS in Bosnia-Herzegovina, whereas no signs of Hantaan or Seoul virus involvement were found.

Puumala hantavirus genome in patients with nephropathia epidemica: correlation of PCR positivity with HLA haplotype and link to viral sequences in local rodents.

Reverse transcription-PCR was used to analyze specimens from 20 Finnish nephropathia epidemica (NE) patients hospitalized during the period from October 1994 to January 1995. Blood and/or urine sediment specimens from seven patients were found to be positive for the genome sequences of Puumala hantavirus (PUU). PCR positivity of the specimens from the patients correlated well with the HLA-DRB1*0301 and HLA B8 alleles, which previously were shown to associate with severe courses of NE. Genetic analysis of the partial M-and/or S-segment sequences obtained from three severely ill NE patients revealed three PUU strains related to but distinct from previously reported strains from Finland. The M-segment sequence of PUU from bank voles trapped near the probable site of infection for one of the patients showed 98.2% identity to that of the PUU strain obtained from the patient, suggesting a link between wild-type PUU from the natural focus and the NE case. The S-segment sequences from the patient and the bank voles, however, showed substantially lower identity (95.8%). As this difference in diversity for M and S genes (1.8 and 4.2%) is atypical for PUU genetic drift, one possibility is that the strain acquired at the putative place of infection is a reassortant one.

Contact allergy to the UV absorber Tinuvin P in a dental restorative material.

A case of contact allergy to the UV absorber Tinuvin P is described in a patient with gingivitis who was previously treated with a dental restorative material. With high-performance liquid chromatography analysis, the presence of Tinuvin P could be shown in the restorative material.

Characterization of Tula virus antigenic determinants defined by monoclonal antibodies raised against baculovirus-expressed nucleocapsid protein.

Tula virus was recently discovered by RT-PCR in lung samples from European common voles (Microtus arvalis and M. rossiaemeridionalis). Since virus isolation attempts had been unsuccessful, no antigen was available for analysis or for use in immunoassays. To circumvent this, complete Tula virus nucleocapsid protein (bac-TUL-N) was expressed in recombinant baculovirus. Rodent antibody end-point titers to bac-TUL-N and to truncated N fragments indicated that the NH2-terminal region is the major antigenic target and revealed a high cross-reactivity to Puumala virus N. Immunizations with crude bac-TUL-N preparations evoked high antibody responses to native hantavirus N in Balb/c mice and six monoclonal antibodies (Mabs) were generated. Epitope mapping of the Mabs, based on a competitive assay, reactivities to truncated recombinant N fragments, and reactivity patterns to different hantavirus strains, identified five recognition sites on Tula virus N. One epitope, which was identified as specific for Tula virus, was located in a region of N which is highly variable among the hantaviruses (aa 226-293), and four epitopes were mapped to the NH2-terminal region of the protein (aa 1-61). One epitope was expressed only in Tula and Prospect Hill viruses, one epitope in Tula, Prospect Hill, Khabarovsk, and Sin Nombre viruses, while two epitopes were conserved in all examined hantaviruses carried by rodents within the subfamily Arvicolinae of the Muridae family.

Distribution and genetic heterogeneity of Puumala virus in Sweden.

Small mammals trapped in Sweden were analysed for specific antibody responses against three hantavirus serotypes and for the presence of viral antigen. To determine the genetic identity of viral RNA in lungs of seropositive bank voles (Clethrionomys glareolus), polymerase chain reactions and subsequent partial sequencing of both the M and S segments were employed. The sequences obtained were all identified as Puumala (PUU) virus, with a high degree of heterogeneity between the different geographical localities. Alignment of nucleotide and deduced amino acid sequences, together with phylogenetic analysis, showed that PUU viruses circulating in central Sweden were distinct from those in the northern region. The localization of the two distinct PUU virus genotypes was shown to correlate with the postglacial recolonization of Sweden by bank voles.

Biotin-labeled antigen: a novel approach for detection of Puumala virus-specific IgM.

A novel enzyme-linked immunosorbent assay, BLA IgM-ELISA, based on baculovirus-expressed Puumala (PUU) virus nucleocapsid protein, was developed for rapid diagnosis of nephropathia epidemica. The recombinant antigen (bac-PUU-N) was purified to homogeneity by HPLC and conjugated to biotin. The biotin-streptavidin system, in combination with the mu-capture technique, rendered the BLA IgM-ELISA a sensitivity similar to or higher than that of PUU virus IgM mu-capture ELISA based on native antigen. The assay was shown to be highly specific when evaluated using a panel of 160 patient and control sera.

[The antigenic differentiation of hantaviruses by using monoclonal antibodies]. / Antigennaia differentsiatsiia khantavirusov s pomoshch'iu monoklonal'nykh antitel.

85 hantavirus (HV) strains from Russia and other counties were examined by indirect fluorescent antibody test using monoclonal antibodies (MAbs) prepared against hantaviruses: Hantaan (Hantaan 76-118, A-9 strains), Seoul (SR-1, R-22 strains) and Puumala (Hallnas 83-223 strain). 68 HV strains were differentiated into five virus type groups: Hantaan, Puumala, Seoul, Prospect Hill and Belgrade-Dobrava. 17 strains were found to be antigenically closely related but distinct from Hantaan type (4 strains), Puumala (6 strains) and Prospect Hill (7 strains). The antigenic characteristics of these 17 strains suggested two supplementary antigenic subgroups of the Hantaan, two of Puumala and two of Prospect Hill.

Khabarovsk virus: a phylogenetically and serologically distinct hantavirus isolated from Microtus fortis trapped in far-east Russia.

Two hantavirus strains, MF43 and MF113, isolated from Microtus fortis trapped in the Khabarovsk region of far-eastern Russia, were analysed by direct nucleotide sequencing of PCR generated fragments of the M and S segments, by immunofluorescence and by focus reduction neutralization tests (FRNT). The nucleotide sequences revealed that the two isolates were closely related to each other but distinct from all other hantaviruses. Phylogenetic analysis of the M and S segments showed that the MF strains form a separate branch in the Hantavirus tree, positioned between the branches of Prospect Hill and Puumala viruses. The strains were shown to be serologically distinct from the other hantavirus serotypes by FRNT using immune rabbit sera. Puumala virus was the closest relative, both genetically and serologically. We propose that this new hantavirus serotype should be named Khabarovsk (KBR).