Diversity and site-specificity of the oral microflora in the elderly.

The purpose of the present study was to describe the bacterial diversity in the oral cavity of the elderly without root caries using bacterial microarrays, and to determine the site- and subject-specificity of bacterial colonization. Samples were collected from the tongue dorsum, mucosa of the buccal fold, hard palate, supragingival plaque from sound root surfaces, and subgingival plaque from the same roots. A new 16 S rRNA gene-based microarray method was used for the simultaneous detection of approximately 300 bacterial species. Overall, 175 species and clusters were detected, representing eight phyla. Species belonging to the genera Streptococcus, Veillonella, and Fusobacterium were common in all sites. The number of species per subject varied from 51 to 81. Statistical analyses revealed about 40 species or clusters with significant associations with at least one of the sites. The bacterial diversity was highest in the cheek and palate regions. Species typically associated with caries and periodontitis were detected rarely or not at all. The oral bacterial flora of the elderly appears to be diverse, and, to a large extent, site- rather than subject-specific.

Microarray analysis of the microflora of root caries in elderly.

The present study used a new 16S rRNA-based microarray with probes for over 300 bacterial species to better define the bacterial profiles of healthy root surfaces and root caries (RC) in the elderly. Supragingival plaque was collected from 20 healthy subjects (Controls) and from healthy and carious roots and carious dentin from 21 RC subjects (Patients). Collectively, 179 bacterial species and species groups were detected. A higher bacterial diversity was observed in Controls than in Patients. Lactobacillus casei/paracasei/rhamnosus and Pseudoramibacter alactolyticus were notably associated with most RC samples. Streptococcus mutans was detected more frequently in the infected dentin than in the other samples, but the difference was not significant. Actinomyces was found more frequently in Controls. Thus, species other than Actinomyces and S. mutans may play a role as pathogens of RC. The results from this study were in general agreement with those of our previous study based on 16S rRNA gene sequencing.

Effects of the medicinal mushroom Agaricus blazei Murill on immunity, infection and cancer.

Agaricus blazei Murill (AbM) is an edible, medicinal mushroom of Brazilian origin. It is used traditionally against a range of diseases, including cancer and chronic hepatitis, and has been cultivated commercially for the health food market. AbM has recently been shown to have strong immunomodulating properties, which has led to increasing scientific interest. In this article, we review current knowledge as to the immunological properties of AbM, and its possible clinical use in connection with infections and cancer. We also present some novel findings, which point to highly different biological potency between AbM extracts of different source and manufacturing.

Longitudinal observation of enterovirus and adenovirus in stool samples from Norwegian infants with the highest genetic risk of type 1 diabetes.

BACKGROUND: Enterovirus and adenovirus are common in infancy, causing mostly asymptomatic infections. However, even an asymptomatic infection may be associated with increased risk of development of certain chronic non-infectious diseases, as has been suggested for enterovirus and type 1 diabetes. Data on occurrence and course of the infections in infancy are therefore important for designing effective approaches towards study of the association. OBJECTIVES: To estimate the frequency of enterovirus and adenovirus infections in Norwegian infants, to evaluate the duration of the infections, to investigate their association with symptoms, and to establish a robust procedure that will be used to study the association between these viruses and the development of auto-immunity leading to type 1 diabetes. STUDY DESIGN: Parents of infants, recruited for a study on environmental triggers of type 1 diabetes, submitted monthly samples of infant faeces, as well as information on symptoms of infection. The samples were analysed for enterovirus and adenovirus using quantitative real-time PCR, and enterovirus-positive samples were sequenced. RESULTS: Enteroviruses were found in 142/1,255 (11.3%), and adenoviruses in 138/1,255 (11.0%) of stool samples. Approximately half of the infants were exposed to these viruses at least once during the first year of observation (period 3-14 months of age). The presence of adenovirus was associated with fever and with symptoms of cold but not with diarrhoea and vomiting. The enterovirus positivity was not associated with any symptoms. CONCLUSIONS: The prevalence of enterovirus and adenovirus in longitudinally obtained faecal samples from infants is sufficiently high to enable studies of their association with chronic diseases. The present protocol for evaluating exposure to these viruses is well suited for large-scale efforts aimed at assessing possible long-term consequences, particularly in relation to type 1 diabetes.

Effects on gene expression and viral load of a medicinal extract from Agaricus blazei in patients with chronic hepatitis C infection.

Extracts from the mushroom Agaricus blazei Murill (AbM) are used extensively as a non-prescription remedy against cancer and infections, including hepatitis. We previously demonstrated a potent immunomodulating effect of a particular preparation on monocytes in vitro, and a protective effect on bacterial infections in mice. Here we report the effect on gene expression in peripheral blood cells from four chronic hepatitis C patients, using global (29 k) oligo-based, single channel microarrays. The viral load was slightly, but not significantly, decreased after 1 week of AbM treatment. The cytokine genes most strongly induced in vitro were not induced in vivo. The more notable changes in mRNA levels were related to genes involved in the G-protein coupled receptor signalling pathway, in cell cycling, and in transcriptional regulation. The results suggest that the beta-glucans of the extract, which presumably are responsible for cytokine induction, did not readily enter the blood, while other components, such as substances proposed to have anticancer effects, were active in the blood.

Effect of a medicinal extract from Agaricus blazei Murill on gene expression in a human monocyte cell line as examined by microarrays and immuno assays.

Extracts from the edible mushroom Agaricus blazei Murill (AbM) are used extensively as a non-prescription remedy against cancer, infections, and immune related diseases. The presumed effect is to activate certain parts of the immune system. In order to investigate the effect, we examined the changes of gene expression caused by the extract on a human monocyte cell line (THP-1). Changes in the levels of mRNA transcripts were measured using 35 k microarrays, and the changes in select cytokine gene products by immuno assays. Lipopolysaccharide (LPS) was included for comparison. Both AbM and LPS had drastic effects on gene expression. Genes related to immune function were selectively up-regulated, particularly proinflammatoric genes such as the interleukins IL1B and IL8. Although most genes induced by AbM were also induced by LPS, AbM produced a unique profile, e.g., as to a particular increase in mRNA for the cytokines IL1A, CXCL1, CXCL2 and CXCL3, as well as PTGS2 (cyclooxygenase2).

Enteric viruses in inlet and outlet samples from sewage treatment plants.

Samples collected every two weeks from the inlet and outlet of three sewage treatment plants were screened for the presence of noro-, rota-, astro-, adeno-, hepatitis A- and circoviruses by (RT)-nested PCR, and for F-specific bacteriophages by isolation in Escherichia coli Famp. Plants A and B were secondary treatment plants and plant C used primary treatment. Noroviruses were detected in 43%, 53% and 24% of the inlet samples and 26%, 40% and 21% of the outlet samples from plants A, B and C, respectively. Astroviruses, rotaviruses and adenoviruses were more prevalent. Adenoviruses were detected in 96% of inlet and 94% of outlet samples, supporting the potential of these viruses as indicators of viral contamination from sewage. Hepatitis A virus and circoviruses were found only rarely. Reduction of infective viral particles during sewage treatment was evaluated using F-specific bacteriophages. The phages were reduced by, respectively, 99%, 87% and 0% in plants A, B and C, which corresponded to the observed differences in reduction of norovirus positive samples between the same plants. The study shows that the high viral load in sewage results in a discharge to the environment of a large amount of virus despite sewage treatment. On the other hand, the advantage of a more advanced treatment is demonstrated.

The thiol proteinase inhibitors, Z-Phe-PheCHN2 and Z-Phe-AlaCHN2, inhibit lysosomal protein degradation in isolated rat hepatocytes.

The effects on protein metabolism of Z-Phe-PheCHN2 and Z-Phe-AlaCHN2 were examined in isolated rat hepatocytes. The two thiol proteinase inhibitors caused a drastic reduction in the degradation of both endogenous and endocytosed (asialo-fetuin) protein. The inhibition was not additive to that of the lysosomotropic base methylamine, indicating that Z-Phe-PheCHN2 and Z-Phe-AlaCHN2 only affect lysosomal degradation. At high concentrations (0.1-1 mM) both inhibitors reduced protein synthesis strongly. This finding indicates non-specific/toxic effects, which may limit the usefulness of the inhibitors.

Selective inhibition of lysosomal protein degradation by the thiol proteinase inhibitors E-64, Ep-459 and Ep-457 in isolated rat hepatocytes.

The effects on protein degradation of the thiol proteinase inhibitor E-64 of fungal original, and its two synthetic analogs Ep-459 and Ep-475, were examined, using isolated rat hepatocytes. All three inhibitors were found to act selectively on lysosomal protein degradation. i.e., their effects were not additive to the lysosomotropic weak base propylamine. Such weak bases appear to be relatively complete and selective inhibitors of lysosomal protein degradation. Ep-475 and E-64 were found to be the most potent of the three, inhibiting as much as 50% of the total degradation (i.e., approx. 70% of the lysosomal degradation) at concentrations at which they did not disturb protein synthesis. Their lack of additivity to the lysosomotropic weak base propylamine further testifies to the usefulness of weak bases differentiating between lysosomal and non-lysosomal protein degradation.

Inhibition of autophagy by benzyl alcohol.

Benzyl alcohol caused a rather complete and selective inhibition of the methylamine sensitive (i.e., the putative lysosomal) pathway of protein degradation in isolated rat hepatocytes. The effect was found to be entirely reversible within 30 min of removing the agent. A morphometric examination of electron micrographs revealed that the inhibition of lysosomal protein degradation coincided with a block in the formation of autophagic vacuoles. The number of acidic vacuoles (i.e., vacuoles induced to swell by adding methylamine) was not drastically reduced.

Differential effects of proteinase inhibitors and amines on the lysosomal and non-lysosomal pathways of protein degradation in isolated rat hepatocytes.

Ammonia, which like other lysosomotropic amines inhibits protein degradation in isolated rat hepatocytes by 70---80%, was utilized as a diagnostic tool to distinguish between the relative effects of various proteinase inhibitors on the lysosomal and non-lysosomal pathways of intracellular protein degradation. Leupeptin was found to inhibit lysosomal protein degradation by 80---85%, and non-lysosomal degradation by about 15%. Antipain had a similar, but somewhat weaker effect. Pepstatin, bestatin and aprotinin (Trasylol) produced minor inhibitory effects (possibly on both degradation pathways), whereas bacitracin and soybean trypsin inhibitor were ineffective. Chymostatin inhibited lysosomal protein degradation by about 45%, whereas the non-lysosomal pathway was inhibited by more than 50%. Chymostatin was unique among the inhibitors tested in causing such a pronounced effect on non-lysosomal protein degradation, and appeared to selectively inhibit the energy-dependent portion of this pathway. The effects of the various inhibitors were additive to the extent expected on the basis of their known actions only sosomal and non-lysosomal protein degradation. Thus, a combination of methylamine, leupeptin and chymostatin inhibited overall protein degradation by about 90%, resulting in a substantial improvement of the cellular nitrogen balance. The degradation inhibitors caused a partial inhibition of protein synthesis, apparently mainly by shutting down the supply of amino acids from the lysosomes. The inhibitory effects of leupeptin and antipain were completely reversed by amino acid addition, whereas some inhibition remained in the case of chymostatin and the lysosomotropic amines, possibly reflecting a certain nonspecific toxicity.

Effects of amino acid analogues on protein degradation in isolated rat hepatocytes.

Analogues and derivatives of six of the amino acids which most effectively inhibit protein degradation in isolated rat hepatocytes (leucine, asparagine, glutamine, histidine, phenylalanine and tryptophan) were investigated to see if they could antagonize or mimic the effect of the parent compound. No antagonists were found. Amino alcohols and amino acid amides tended to inhibit protein degradation strongly, apparently by direct lysosomotropic effect as indicated by their ability to cause lysosomal vacuolation. Amino acid alkyl esters and dipeptides inhibited degradation to approximately the same extent as did their parent amino acids, possibly by being converted to free amino acids intracellularly. Of several leucine analogues tested, four (L-norleucine, L-norvaline, D-norleucine and L-allo-isoleucine) were found to be as effective as leucine in inhibiting protein degradation. None of the analogues had any effect on protein synthesis. Since leucine appears to play a unique role as a regulator of bulk autophagy in hepatocytes, the availability of active leucine agonists may help to elucidate the biochemical mechanisms for control of this important process.

The estimated impact of the CCR-5 delta32 gene deletion on HIV disease progression varies with study design. Oslo HIV Cohort Study Group.

OBJECTIVES: To study the impact of the genotype CCR-5 wild-type +/A32 on the progression rate to AIDS and death, and to discuss sources of bias according to study design. METHODS: A prospective study of 310 HIV-positive subjects with follow-up time from study entry (prevalent cohort), and a prospective study of 105 HIV-positive subjects with well-defined time of HIV seroconversion, with follow-up time from the retrospectively assessed date of HIV seroconversion (retrospective incident cohort). RESULTS: Slower progression to AIDS among subjects with CCR-5 +/delta32 than those with CCR-5 +/+ genotype was estimated in the prevalent cohort (P=0.07, log-rank test). Slower progression to death from any cause was also estimated for subjects with CCR-5 +/delta32 (P < 0.05, log-rank test). No differences in survival after AIDS diagnosis were seen (P=0.89, log-rank test). No differences in the progression rate to AIDS (P=0.82, log-rank test) or death (P=0.78, log-rank test) were estimated in the retrospective incident cohort. CONCLUSIONS: The varying estimates of the impact of CCR-5 genotype on progression to AIDS in this and other studies, may be real and reflect differences in the dependence of HIV on the CCR-5 receptor, or may be due to systematic errors caused by study design. Several methodological difficulties occur when the factor studied, such as CCR-5 genotype, is associated both with the risk of being HIV-infected and the progression of disease.

Evolution of mouse mammary tumor virus-related sequences in the human genome.

The human genome contains numerous copies of elements with sequence homology to the mouse mammary tumor virus (MMTV). We have been interested in whether these elements are still actively transposing in the germ lines, and investigated this question by looking at restriction fragment patterns. Genomic DNA from humans and selected animals were digested with three different restriction enzymes and hybridized with five probes representative of five subgroups of the human MMTV-related elements. Two polymorphisms as well as two cases of sexual dimorphism were discovered in human DNA. The latter indicate the presence of at least two elements of the MMTV family on the Y chromosome. DNA samples from non-primate mammals were all unreactive. Both chimpanzee and rhesus monkey had restriction patterns of roughly the same number of bands and total intensity as humans. The number of differences in patterns between humans, and between humans and chimpanzee or rhesus monkey, were compatible with being caused by point mutations. The data indicate that these elements were actively spreading in the genome some time before the split between apes and Old World monkeys, while being relatively inactive, at least as to transpositions in the germ line, in later ages.

The proteinase inhibitor pepstatin A inhibits formation of reverse transcriptase in H9 cells infected with human immunodeficiency virus 1.

Retroviruses depend on a virus-encoded proteinase. As this enzyme is an interesting target for antiviral therapy, we examined the effect of various low-molecular-weight proteinase inhibitors, as well as a few oligopeptides related to the proteolytic cleavage sites, on the replication of HIV-1 in H9 cells. The increase in reverse transcriptase activity during incubation was assumed to reflect viral replication. Cellular DNA synthesis was measured to quantitate the adverse effects of the inhibitors on the cells. Only one of the substances tested, pepstatin A, had an appreciable selective effect on viral replication. Substances that decreased DNA synthesis generally caused an equally large decrease in reverse transcriptase activity.

Intersubtype recombinant HIV type 1 involving HIV-MAL-like and subtype H-like sequence in four Norwegian cases.

Suspected epidemiological links between three cases of human immunodeficiency virus type 1 (HIV-1) infection were verified by the finding of a shared unique virus genotype. A probable male index case was not available for testing. Case 1 was a female sexual partner of the index case. Case 2 was an adult son of case 1. Case 3 was a female sexual partner of case 2. The link to the index case was substantiated by the subsequent finding of another female sexual contact of the index case, harboring the same HIV-1 genotype as the three other cases. To characterize the genotype further, the complete provirus nucleotide sequence was obtained directly from blood cell DNA of case 3. HIV cultivated from case 3 demonstrated CCR5 dependence, an extreme slow-low phenotype, and some genotypic features not present in its directly sequenced counterpart. Most of the gag, pol, and vif genes of these viruses clustered with one of the earliest African HIV-1 strains, MAL, previously classified as a recombinant between the subtypes A, D, and I. Most of the rest of the genome was related to subtype H, albeit with less than 90% identity in most regions. These viruses are the only ones shown to display extensive similarity with MAL in the gag-pol region and among the first HIV-1 recombinants described involving subtype H. We postulate that the gag-pol genes of MAL and these viruses are derived from a common ancestor that is not necessarily intersubtype recombinant in the pol region.

Detecting inhibition of coxsackievirus replication by measuring DNA synthesis--effect of proteinase inhibitors.

The replication of picornaviruses can be monitored by microscopic examination of the cytopathogenic effect. We found that measuring cellular DNA synthesis gave a more objective and reliable estimate of the viral effect on the cells. Furthermore, by simultaneously measuring DNA synthesis in uninfected cells, we obtained a sensitive indication of whether the agents tested influenced cellular activity. The method was employed to investigate the effect of various proteinase inhibitors on the replication of coxsackie-B3 virus in HEp2 cells. Certain inhibitors of metallo-proteinases had a limited, but consistent, protective effect against viral activity.

Molecular epidemiology of viral infections. How sequence information helps us understand the evolution and dissemination of viruses.

Viruses evolve much faster than cellular organisms. Together with recent advances in nucleic acid sequencing and biocomputing, this allows us to distinguish between related strains of viruses, and to deduce the relationships between viruses from different outbreaks or individual patients. Databases of nucleotide sequences contain a large number of viral sequences with which novel sequences from local outbreaks can be compared. In this way the dissemination of viruses can be followed both locally and globally. We here review the biological and technological background to the use of virus nucleic acid sequences in epidemiological studies, and provide examples of how this information can be used to monitor human viruses. Molecular studies are particularly valuable for understanding the dissemination and evolution of viruses. The knowledge obtained is useful in epidemiological reconstructions, in real-time surveillance, and may even enable us to make predictions about the future developments of viral diseases.

Prevalence of human papillomavirus in cervical scrapes, as analyzed by PCR, in a population-based sample of women with and without cervical dysplasia.

HPV is suspected of being a major cause of cancer of the uterine cervix. To understand the risk of disease in the general population of women, it is important to estimate the prevalence of HPV infection in a random population-based sample of women without disease. In this study, a total of 231 randomly selected women without dysplasia (controls) were examined, and compared with 103 women with histologically confirmed CIN II-III (patients). The prevalence of HPV DNA in cervical scrapes was determined by general nested PCR, which was expected to detect any relevant HPV type commonly found in cervical samples. The nested positive samples were typed with type-specific PCR. In the general nested PCR, 15% of the controls were positive, compared to 91% of the patients. In the population-based sample, 2.2% had HPV types 6 and 11 and 10% had types 16, 18, 31, and 33. In both groups, HPV DNA was observed less frequently in women above than below the age of 30. The results are among the few population-based figures on the prevalence of HPV in women, and provide a baseline for understanding the risk of developing cancer of the uterine cervix, and determining the proportion of women to be included in intervention studies.

A lysozyme isolated from rainbow trout acts on mastitis pathogens.

The antibacterial effects of two lysozymes purified from rainbow trout kidney (type I and II) were tested on eight bacterial strains isolated from cases of clinical mastitis (staphylococci, streptococci and coliforms). Three other lytic agents were included in the experiments as controls: hen egg-white lysozyme, lysostaphin and mutanolysin. Proliferating bacteria were incubated with the various lytic agents, either in hearts infusion broth or in milk. The type II rainbow trout lysozyme decreased the number of live bacteria (colony forming units) of all the strains tested, but was most efficient against staphylococci. The other two lysozymes had little effect.