Propionibacterium species and follicular keratinocyte activation in acneic and normal skin.

BACKGROUND: The pathogenesis of acne vulgaris is multifactorial with increased sebum production, alteration in the quality of sebum lipids, dysregulation of the hormone microenvironment, follicular hyperkeratinization and Propionibacterium acnes-driven inflammation as major contributory factors. Hyperproliferation of keratinocytes is believed to contribute to hypercornification and eventually leads to comedone development. While the distribution of P. acnes is relatively well documented in acneic and healthy skin, little is known about P. granulosum and P. avidum. OBJECTIVES: To visualize directly the three major Propionibacterium in 117 control and 26 acneic skin samples. In addition, keratinocyte proliferation was evaluated. METHODS: Propionibacteria were visualized by immunofluorescence microscopy, and keratinocyte proliferation was assessed by Ki67, keratin (K) 16 and p63 immunochemistry. RESULTS: P. acnes was identified in 68 samples (48%), while P. granulosum was identified in 12 (8%) samples; P. avidum was not detected at all. Unexpectedly, acne samples did not show higher keratinocyte proliferation than controls, nor was there any association between bacterial colonization and expression of Ki67/K16/p63. CONCLUSIONS: Our findings do not support earlier notions of follicular keratinocyte hyperproliferation as a cause of ductal hypercornification in acneic facial skin. Further studies on the mechanisms underlying hypercornification in acne pathogenesis are needed.

Sampling and detection of skin Propionibacterium acnes: current status.

A connection between acne vulgaris and Propionibacterium acnes has long been suggested. Over the years, several human skin microbiota sampling methods have been evolved and applied, e.g. swab, scrape, extraction techniques including cyanoacrylate gel sampling as well as punch biopsy. Collected samples have been processed following various methodologies ranging from culture studies to probe labelling and molecular analysis. Direct visualization techniques have recently shown the existence of anatomically distinct skin P. acnes populations: epidermal and follicular. P. acnes biofilms appear to be a common phenomenon. Current sampling approaches target different skin populations of P. acnes and the presence of microbial biofilms can influence the retrieval of P. acnes. The anatomical considerations must be taken into account while interpreting microbiological data.

An increased incidence of Propionibacterium acnes biofilms in acne vulgaris: a case-control study.

BACKGROUND: Acne vulgaris is a disorder of the sebaceous follicles. Propionibacterium acnes can be involved in inflammatory acne. OBJECTIVES: This case-control study aimed at investigating the occurrence and localization of P. acnes in facial biopsies in acne and to characterize the P. acnes phylotype in skin compartments. METHODS: Specific monoclonal and polyclonal antibodies were applied to skin biopsies of 38 patients with acne and matching controls to localize and characterize P. acnes and to determine expression of co-haemolysin CAMP factor, a putative virulence determinant. RESULTS: Follicular P. acnes was demonstrated in 18 (47%) samples from patients with acne and eight (21%) control samples [odds ratio (OR) 3·37, 95% confidence interval (CI) 1·23-9·23; P = 0·017]. In 14 (37%) samples from patients with acne, P. acnes was visualized in large macrocolonies/biofilms in sebaceous follicles compared with only five (13%) control samples (OR 3·85, 95% CI 1·22-12·14; P = 0·021). Macrocolonies/biofilms consisting of mixed P. acnes phylotypes expressing CAMP1 were detected in both case and control samples. Only four samples tested positive for the presence of Staphylococcus spp. and fungi were not observed. CONCLUSIONS: We have for the first time visualized different P. acnes phylotypes in macrocolonies/biofilms in sebaceous follicles of skin biopsies. Our results support the hypothesis that P. acnes can play a role in the pathogenesis of acne as acne samples showed a higher prevalence of follicular P. acnes colonization, both in terms of follicles containing P. acnes and the greater numbers of bacteria in macrocolonies/biofilms than in control samples.

No link between viral findings in the prostate and subsequent cancer development.

In an investigation of 201 prostate tissue samples from patients with benign prostate hyperplasia that later progressed to prostate cancer and 201 matched controls that did not, there were no differences in the prevalence of adenovirus, herpesvirus, papilloma virus, polyoma virus and Candida albicans DNA.

Association between the presence of bacterial 16S RNA in prostate specimens taken during transurethral resection of prostate and subsequent risk of prostate cancer (Sweden).

OBJECTIVE: To study bacterial 16S RNA in archival prostate samples from 352 patients with benign prostate hyperplasia (BPH) and evaluate whether the presence of bacterial DNA was different in those who later developed prostate cancer (n = 171) and in the matched controls that did not progress to cancer (n = 181). METHODS: 16S DNA PCR followed by cloning and sequencing the positive samples. RESULTS: In 96/352 (27%) of the prostate tissue specimens 16S RNA were detected. Sequence analysis revealed Propionibacterium acnes as the predominant microorganism (23% of 16S RNA positive patients). The second most frequent isolate-Escherichia coli was found in 12 (12%) patients. The other isolates included Pseudomonas sp. (3 patients), Actinomyces sp. (2), Streptococcus mutans (1), Corynebacterium sp. (2), Nocardioides sp. (1), Rhodococcus sp. (1) Veillonella sp. (2). In P. acnes positive samples 62% exhibited severe histological inflammation versus 50% in the bacteria-negative group (p = 0.602). The presence of P. acnes in the prostate was associated with prostate cancer development (OR 2.17, 95% CI 0.77-6.95). CONCLUSIONS: This study has revealed P. acnes as the most common bacteria in the prostate in BPH. Further studies are needed to clarify its role in contributing to the development of prostatic inflammation and prostate cancer.

Recent high incidence of fulminant hepatitis in Samara, Russia: molecular analysis of prevailing hepatitis B and D virus strains.

Until 1991, the Russian city of Samara was largely isolated from other parts of Russia and the rest of the world. Very recently, Samara has seen an alarming increase in the incidence of hepatitis. The proportion of fulminant cases is unusually high. We wanted to assess the roles of hepatitis B virus (HBV) and hepatitis D virus (HDV) in acute viral hepatitis in this region by analyzing the prevailing strains of both and by determining their genotypes and possible origin. Serum samples were screened for different serological markers and by PCR followed by direct sequencing. Of the 94 HBV-positive samples (80% of which were acute infections), 37 (39%) were also HDV positive. Sixty-seven percent of the patients had anti-HCV antibodies. Twenty-five percent of all patients in the study had fulminant hepatitis. Statistically significant sex differences were found among fulminant cases. For HBV, the core promoter sequences of 62 strains were determined and all but one were found to be of genotype D. None of these had any deletions. Only one strain, from a patient with fulminant fatal hepatitis, showed multiple mutations. The pre-S2 region sequences of 31 HBV strains were also compared. Phylogenetically, these fell into two distinct groups within genotype D, suggesting different origins. For HDV, part of the region encoding the delta-antigen was sequenced from four strains. All proved to be of genotype I and were similar to Far Eastern and Eastern European strains. The contribution of intravenous drug use to the sharp increase in viral hepatitis in this unique setting is discussed.

A minority of seropositive wild bank voles (Clethrionomys glareolus) show evidence of current Puumala virus infection.

Bank voles (Clethrionomys glareolus) serve as the reservoir for Puumala (PUU) virus, the aetiologic agent of nephropathia epidemica. The animals are believed to be persistently infected and the occurrence of serum antibodies is usually taken as an evidence of active infection. We found serum antibodies to PUU virus in 42 of 299 wild bank voles captured in a PUU virus endemic area. PUU virus RNA was demonstrated in lung specimens of 11 of these 42 animals and in 2 of them antigen was also found. Thus in the lungs of 31 of 42 seropositive animals neither PUU virus RNA nor antigen was detected. In 2 of 257 seronegative animals, lung specimens showed presence of PUU virus antigen and RNA. Isolation of PUU virus from lung tissue was successful in all 4 antigen-positive bank voles but in none of 16 tested antigen-negative animals. In conclusion, only a minority of bank voles with serum antibodies to PUU virus showed evidence of current infection.

Central nervous system and ophthalmic involvement in nephropathia epidemica (European type of haemorrhagic fever with renal syndrome).

Central nervous system (CNS) - related symptoms occur in haemorrhagic fever with renal syndrome (HFRS). To study the CNS and ophthalmic involvement in nephropathia epidemica (NE), the European type of HFRS, we included 26 patients in a prospective study. Most common CNS-related symptoms were headache (96%), insomnia (83%), vertigo (79%), nausea (79%), and vomiting (71%). Ophthalmic symptoms were reported by 82% of patients; 41% had photophobia and 50% had impaired vision. A transient loss of vision was recorded in one patient, who also had a generalized seizure. Minor white matter lesions were found in about half of the patients investigated with brain magnetic resonance imaging (MRI). Electroencephalography (EEG) showed severe alterations in only one patient, and slight and reversible patterns in another two patients. Neopterin, interleukin-6 and interferon-gamma levels in the cerebrospinal fluid (CSF) were elevated, which may indicate immune activation. However, we found no evidence of intrathecal NE virus replication. We conclude that CNS-related symptoms are common in NE, and transient ophthalmic involvement can be demonstrated in about half of the patients.

The first human isolate of Puumala virus in Scandinavia as cultured from phytohemagglutinin stimulated leucocytes.

A virus isolate was recovered from blood leucocytes of a patient with nephropathia epidemica (NE). Leucocytes were isolated from EDTA-blood by dextran sedimentation and cultured on monolayers of Vero E6 cells in the presence of phytohemagglutinin (PHA) in roller tubes during the first 72 hours of incubation followed by rolling culture for three weeks in total. Thereafter the first subculture was done in a plastic flask and afterward at at least 6 week intervals. Antigen was first detected after 6 months and 2 weeks of culture. When tested by monoclonal antibodies and patient sera the isolate had the characteristics of a PUU virus. PCR amplification using PUU-specific primers and subsequent partial sequencing of the S and M segments revealed that the Umeå/305/human/95 virus differs from the Finnish PUU Sotkamo rodent prototype virus and is similar but not identical to rodent strains of PUU virus acquired from the same region as the patient isolate. It is we concluded that the first human isolate of the etiologic agent of NE in Scandinavia was recovered from blood leucocytes stimulated with PHA by long-term culture in Vero E6 cells. The isolate belongs to the PUU serotype of hantaviruses as shown by its serologic profile and partial sequencing data.

Increased plasma levels of soluble CD23 in haemorrhagic fever with renal syndrome; relation to virus-specific IgE.

In 15 consecutive patients hospitalized with nephropathia epidemica, a European form of haemorrhagic fever with renal syndrome (HFRS) caused by Puumala virus, plasma concentrations of soluble CD23 (sCD23) and Puumala virus-specific IgE were determined. In the acute phase of illness, 11/15 patients had increased sCD23 levels (> 91 U/ml), whereas in convalescence, values of 8/10 patients were normalized. Maximal sCD23 values were correlated to maximal concentrations of Puumala virus-specific serum IgE (r = 0.597; P = 0.025). The results are compatible with a known ability of sCD23 to augment IgE production.

High prevalence of hantavirus antibodies in bank voles (Clethrionomys glareolus) captured in the vicinity of households afflicted with nephropathia epidemica.

Puumala virus, the causative agent of nephropathia epidemica (NE), occurs endemically in Europe and is spread mainly by the bank vole (Clethrionomys glareolus). In the vicinity of each of four households afflicted with NE, we studied rodents with regard to population density and prevalence of Puumala virus-specific antibodies. For each case area, a control area was randomly selected 10 km away, without regard to the presence of human settlement. During 6,000 trap nights, 328 rodents were caught, of which 299 were C. glareolus. The mean rodent densities of case and control areas were 6.6 and 3.7 animals per 100 trap nights (P < 0.001). The prevalence of serum antibodies was 15.9% in case areas compared with 5.6% in control areas (P < 0.05). In three of the case areas, where NE had occurred 3-10 weeks before trapping, the rodent density and seroprevalence were much higher than in the fourth area, where NE occurred 38 weeks before trapping. In conclusion, C. glareolus seropositive for Puumala virus occurred more frequently near households afflicted with NE than in control areas 10 km away.

Serological diagnosis of hantavirus infections by an enzyme-linked immunosorbent assay based on detection of immunoglobulin G and M responses to recombinant nucleocapsid proteins of five viral serotypes.

Worldwide, hantaviruses cause more than 100,000 human infections annually. Rapid and accurate methods are important both in monitoring acute infections and for epidemiological studies. We and others have shown that the amino termini of hantavirus nucleocapsid proteins (Ns) are sensitive tools for the detection of specific antibodies in hantavirus disease. Accordingly, we expressed truncated Ns (amino acids 1 to 117) in Escherichia coli from the five hantaviruses known to be pathogenic to man; Hantaan (HTN), Seoul (SEO), Dobrava (DOB), Sin Nombre (SN), and Puumala (PUU) viruses. In order to obtain pure antigens for use in an enzyme-linked immunosorbent assay (ELISA), the recombinant proteins were purified by polyhistidine-metal chelate affinity chromatography. Polyclonal animal antisera and a panel of serum specimens from hantavirus-infected individuals from Scandinavia, Slovenia, Russia, Korea, China, and the United States were used to evaluate the usefulness of the method. With both human and animal sera, it was possible to designate the antibody response into two groups: those with HTN, SEO, and DOB virus reactivity on the one hand and those with SN and PUU virus reactivity on the other. In sera from Scandinavia, European Russia, and the United States, the antibody response was directed mainly to the PUU and SN virus group. The sera from Asia reacted almost exclusively with the HTN, SEO, and DOB types of viruses. This was true for both the immunoglobulin M (IgM) and IgG antibody responses, indicating that this type of discrimination can be done during the acute phase of hantavirus infections. Both the HTN, SEO, and DOB virus and the PUU and SN virus types of antibody response patterns were found in patients from the Balkan region (Solvenia).

Pathogenetic and clinical aspects of the renal involvement in hemorrhagic fever with renal syndrome.

Hemorrhagic fever with renal syndrome is the most common clinical manifestation of hantavirus infection. The main target organ is the kidney, resulting in an interstitial hemorrhagic nephritis and sometimes acute tubular necrosis. The pathogenesis is still largely unknown, but several recent studies indicate an important role for immune mechanisms including increased expression of cytokines, for example, tumor necrosis factor. Immunohistochemical studies of kidney biopsies have revealed deposits of IgG, IgM, and C3, but deposits were significantly less numerous than in chronic immune complex disease. Since hantaviruses are not cytolytic, a direct detrimental effect of the infecting virus is less likely. The long-term prognosis of hemorrhagic fever with renal syndrome seems to be favorable, but there are reports that previous hantavirus infection is associated with an increased risk of hypertensive renal disease. Prospective longitudinal studies addressing this issue are underway.

A major antigenic domain for the human humoral response to Puumala virus nucleocapsid protein is located at the amino-terminus.

Nephropathia epidemica (NE), the major form of hemorrhagic fever with renal syndrome in Europe, is caused by the hantavirus serotype Puumala (PUU). The PUU virus nucleocapsid protein (N) has been shown to be highly immunogenic both in laboratory animals and in man. We aimed to locate domains important in humoral immune reactivity and to use this information to develop a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of NE. Escherichia coli poly-histidine fusion protein expression vectors containing over-lapping gene segments encoding the PUU virus N (PUU rN) were constructed. The resulting gene products were examined by immunoblots and ELISA with polyclonal and monoclonal antibodies. The dominating antigenic region of PUU rN was located between amino acids (aa) 7 and 94. A recombinant fusion protein containing aa 7-137 of PUU virus N (PUU rN delta 5) was used for the detection of specific IgG and IgM responses in NE. ELISA based on PUU rN delta 5 was found to have equal sensitivity and specificity as compared to the full length recombinant PUU rN by ELISA, for both acute serological diagnosis of NE and for seroepidemiological screening purposes. Furthermore, this protein is easier to handle than full length PUU rN due to its higher solubility in aqueous solutions.