Soluble α-Klotho in Liver Cirrhosis and Alcoholism.

AIMS AND BACKGROUND: Alpha Klotho is a transmembrane protein that serves as co-receptor for FGF23. Ectodomain of membrane bound α Klotho may be shed by membrane bound proteases (activated, among other factors, by tumor necrosis factor (TNF)-α) generating the soluble form of the protein (sKl) that functions as a hormone by itself. It modulates calcium influx into cells, blunts IGF-1/Insulin signaling, promotes synthesis of antioxidants, generally slows down tumor progression, delays cell senescence, is neuroprotective and promotes oligodendrocyte maturation and myelin synthesis, and muscle rejuvenation. It may be involved in inflammation and exerts antifibrogenic effects. Some of these pathways may become altered in alcoholism or liver cirrhosis, but data are scattered and scarce and an update is required. METHOD: Literature survey. RESULTS AND CONCLUSIONS: Alcohol consumption in non-alcoholics is inversely related to sKl, but alcoholic cirrhotics showed higher-than-normal sKl values in association with liver function derangement. In hepatoma cells, the intensity of Klotho staining was related to faster tumor progression and a shortened life span. Among severe alcoholic cirrhotics sKl is directly related to serum TNF-α levels, and, inversely, to brain atrophy. Given the antioxidant, anti-inflammatory, and antifibrogenic effects of Klotho, perhaps the increase in cirrhosis (and in other inflammatory conditions, such as sepsis or cancer) reflects an attempt to regulate increased inflammation, but clinical and experimental research is urgently needed in this field.

The Unyvero P55 'sample-in, answer-out' pneumonia assay: A performance evaluation.

BACKGROUND: O'Neill's recent Review on Antimicrobial Resistance expressed the view that by 2020 high-income countries should make it mandatory to support antimicrobial prescribing with rapid diagnostic evidence whenever possible. METHODS: Routine microbiology diagnosis of 95 respiratory specimens from patients with severe infection were compared with those generated by the Unyvero P55 test, which detects 20 pathogens and 19 antimicrobial resistance markers. Supplementary molecular testing for antimicrobial resistance genes, comprehensive culture methodology and 16S rRNA sequencing were performed. RESULTS: Unyvero P55 produced 85 valid results, 67% of which were concordant with those from the routine laboratory. Unyvero P55 identified more potential pathogens per specimen than routine culture (1.34 vs. 0.47 per specimen). Independent verification using 16S rRNA sequencing and culture (n = 10) corroborated 58% of additional detections compared to routine microbiology. Overall the average sensitivity for organism detection by Unyvero P55 was 88.8% and specificity was 94.9%. While Unyvero P55 detected more antimicrobial resistance markers than routine culture, some instances of phenotypic resistance were missed. CONCLUSIONS: The Unyvero P55 is a rapid pathogen detection test for lower respiratory specimens, which identifies a larger number of pathogens than routine microbiology. The clinical significance of these additional organisms is yet to be determined. Further studies are required to determine the effect of the test in practise on antimicrobial prescribing and patient outcomes.

'Sample-in, answer-out'? Evaluation and comprehensive analysis of the Unyvero P50 pneumonia assay.

This study aimed to evaluate the performance of the Unyvero P50 pneumonia assay, the first 'sample-in, answer-out' system for rapid identification of pathogens and antibiotic resistance markers directly from clinical specimens. Overall, Unyvero P50 displayed very good sensitivity (>95%); however, specificity was low (33%) mainly because 40% of the specimens were reported as normal flora. Specifically, one or more pathogens were identified in 28 of them. From a detailed analysis of 42 specimens selected at random, 76% of the additionally reported pathogens were confirmed present in primary specimens. Detection of selected resistance markers was compared to routine phenotypic susceptibility testing, supplemented with Checkpoints microarray system, PCR and sequencing. Concordance was mixed, primarily due to issues with panel's choice of markers and detection of some intrinsic beta-lactamases. Finally, we offer a critical analysis of the assay's microbial panel and resistance markers and provide suggestions for improvement.

Detection of the human herpesvirus 8-encoded cyclin protein in primary effusion lymphoma-derived cell lines.

The human herpesvirus 8 (HHV8/KSHV), along with certain other herpesviruses, encodes a gene with cyclin homology. Although the functional significance of the encoded cyclin is not clear at present, various lines of evidence propose a role for this cyclin in latently infected cells and possibly in the induction of tumors that arise in HHV8-infected individuals. We provide evidence here that the cyclin protein is expressed in HHV8 positive primary effusion lymphoma (PEL)-derived cell lines and that its level of expression varies greatly between different lines. Our analysis indicates that the level of cyclin protein expression in different PEL cell lines may correlate with the level of transcript expression during latency but not in cells induced to undergo lytic replication. In highly expressing BC-3 cells the cyclin is complexed with cdk6, cdk4, cdk2, and cdk5 under both latent and lytic conditions, although subtle changes in the level of cdk association are seen after induction of the lytic cycle. Altogether our findings support the notion that the cyclin is a latency-associated gene product expressed in PEL tumor cells. They furthermore indicate that after lytic cycle induction, the level of cyclin transcript expression may not be a reliable indicator for the level of cyclin protein expression.

Latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus interacts with RING3, a homolog of the Drosophila female sterile homeotic (fsh) gene.

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is the likely infectious cause of Kaposi's sarcoma, primary effusion lymphoma, and some cases of multicentric Castleman's disease. Its latent nuclear antigen (LANA) is expressed in the nuclei of latently infected cells and may play a role in the persistence of episomal viral DNA in dividing cells. Here we report that LANA interacts with RING3, a nuclear protein and member of the Drosophila fsh (female sterile homeotic) family of proteins, some of which have previously been implicated in controlling gene expression. Binding of RING3 to LANA involves the ET domain, characteristic of fsh-related proteins, suggesting that this highly conserved region is involved in protein-protein interactions. The interaction between RING3 and LANA results in phosphorylation of serine and threonine residues located between amino acids 951 and 1107 in the carboxy-terminal region of LANA. However, RING3 is not itself a kinase but appears to recruit an as yet unidentified serine/threonine protein kinase into the complex which it forms with LANA.

The 222- to 234-kilodalton latent nuclear protein (LNA) of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) is encoded by orf73 and is a component of the latency-associated nuclear antigen.

Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (KSHV/HHV8) is the likely cause of KS and primary effusion lymphomas or body cavity-based lymphomas (BCBLs). A latency-associated nuclear immunofluorescence antigen (LANA) (D. H. Kedes, E. Operskalski, M. Busch, R. Kohn, J. Flood, and D. Ganem, Nat. Med. 2:918-924, 1996; S. J. Gao, L. Kingsley, M. Li, W. Zheng, C. Parravicini, J. Ziegler, R. Newton, C. R. Rinaldo, A. Saah, J. Phair, R. Detels, Y. Chang, and P. S. Moore, Nat. Med. 2:925-928, 1996) and a 222- to 234-kDa nuclear protein (LNA) (S. J. Gao, L. Kingsley, D. R. Hoover, T. J. Spira, C. R. Rinaldo, A. Saah, J. Phair, R. Detels, P. Parry, Y. Chang, and P. S. Moore, N. Engl. J. Med. 335:233-241, 1996) have previously been described in BCBL cell lines by immunofluorescence and Western blotting techniques, respectively. To identify the viral gene(s) encoding this antigen(s) we screened a cDNA library from HBL-6 cells, a B-cell lymphoma cell line persistently infected with KSHV/HHV8, with KS patient sera. One set of positive clones contained the 3' end of orf73, as well as the complete orf72 and orfK13, and another set contained the 5' end of orf73. Comparison of cDNA sequences with the KSHV/HHV8 genomic sequence revealed a splice event, occurring upstream of orf73. Immunoaffinity purified antibodies to a recombinant carboxy-terminal fragment of the orf73-encoded protein showed the characteristic speckled nuclear immunofluorescence pattern of LANA and reacted with the 222- to 234-kDa LNA on Western blots. Expression of full-length orf73 in bacteria and COS7 cells reproduced the LNA banding pattern. Immunohistochemistry on cases of nodular KS revealed that orf73/LNA is expressed in the nucleus of KS spindle cells. These findings demonstrate that orf73 encodes the 222- to 234-kDa LNA, is a component of LANA, and is expressed in KS tumor cells.

Isolation of a Schizosaccharomyces pombe gene which in high copy confers resistance to the nucleoside analogue 5-azacytidine.

Treatment of Schizosaccharomyces pombe with the C5 DNA methyltransferase (C5Mtase) inhibitor 5-azacytidine (5-azaC) has previously been shown to induce G2 checkpoint-dependent cell cycle arrest. S. pombe strains defective in both the checkpoint control pathways and in DNA repair processes are sensitive to 5-azaC. Here we describe the isolation of azr1+, as a multi-copy suppressor of the 5-azaC sensitivity of G2 checkpoint and DNA repair-deficient strains. azr1+ encodes a putative 25 kDa protein with limited homology to a Saccharomyces cerevisiae open reading frame of unknown function. The azr1+ gene is not essential and the null mutant shows no alteration in either DNA repair or checkpoint properties. We also report the sequence of the putative fission yeast cytidine deaminase gene, designated pcd1+, which lies immediately adjacent to azr1+ but which plays only a moderate role in suppression of 5-azaC sensitivity.

Control of ammonia formation during Bacillus subtilis fermentation of legumes.

The control of ammonia formation during the Bacillus subtilis fermentation of autoclaved, roasted soybean cotyledons (Glycine max) and of autoclaved African locust bean cotyledons (Parkia spp.) was investigated. Addition of NaCl, 1.5 mol (kg wet cotyledons)-1, part way through the fermentation inhibited ammonia formation and softening of the cotyledons. Addition of glycerol, 1.7 mol (kg wet cotyledons)-1 part way through the fermentation inhibited alkalinisation and ammonia formation while allowing enzymic activity and softening of the cotyledons to continue. Restriction of the oxygen supply by incubating the cotyledons in a sealed container also prevented excessive ammonia production and increase in pH value. Fermentations conducted in sealed containers with an air to cotyledons ratio of approximately 130-175 ml air (g wet cotyledons)-1 supported good microbial growth and proteolysis without the formation of detectable ammonia aroma.

Individuals with a chemical-dependent family member. Does their health care use increase?

OBJECTIVE: To examine health care utilization patterns of individuals who have a chemical-dependent family member (parent, spouse, or child). DESIGN: Morbidity and health care utilization patterns were examined for a 12-month period using the claims administrative data from the Alberta Health Care Insurance Plan (AHCIP). SETTING: General medical practice specializing in the treatment of addiction disorders. PARTICIPANTS: Two hundred seventy-eight (73 male, 205 female) subjects were identified between 1987 and 1990. No members of the subject group were Natives, and none were dependent on chemicals during the study period. A control group, matched for age, sex, and family size, was randomly selected from AHCIP records. MAIN OUTCOME MEASURES: Number and type of health care services and diagnoses listed in health care claims classified using the International Classification of Diseases. RESULTS: In 91% of cases, the chemical-dependent family member was male. Members of the subject group presented more often with mental disorders, digestive system problems, obstetrical problems, injuries, and poorly defined conditions. Also, they were more likely to have nonreferred visits, to have specialist visits, and to use laboratory services than population-matched controls. The subject group's use of specialists and laboratory services amounted to twice the cost of the control group's use of these services. CONCLUSIONS: Results show that individuals who have a chemical-dependent family member use more health care services than the general population. Morbidity among the subject group appears to be similar to that among chemical-dependent individuals; diagnoses related to stress and trauma are common.

Regional variations in the prevalence rates of multiple sclerosis in the province of Alberta, Canada.

Multiple sclerosis (MS) prevalence rates were examined for the fiscal years 1984/1985-1988/1989, in the province of Alberta, Canada, by age, sex and census division. Data were derived from the health care records of individuals registered with the Alberta Health Care Insurance Plan which requires registration by all residents of the province (approximately 2.4 million). The overall crude prevalence rate was 216.7 per 100,000 (173.1 for males; 260.3 for females) population. Females had a significantly higher prevalence (p < 0.05) and the rates were highly correlated between the sexes (r = 0.94, p < 0.01). The present study confirms other studies finding a high prevalence rate within the province of Alberta. The prevalence rate for Alberta is among the highest reported in the world indicating that the province appears to be an excess risk area relative to other global locations. Also, the results indicate that MS is unevenly distributed throughout the province which offers support for the involvement of environmental factors related to the onset of this disorder.

Geographic variations in the prevalence rates of Parkinson's disease in Alberta.

Parkinson's disease prevalence rates were examined for the Province of Alberta by age, sex and census division. Using the claims administrative data from the Alberta Health Care Insurance Plan, a cohort of all registered individuals (2.4 million) was extracted and followed for the five year period, April 1, 1984 to March 31, 1989. No new members were added to the cohort and an attrition rate averaging 6% per year was observed. The overall crude prevalence rates of 248.9 and 239.8 per 100,000 population were noted for males and females respectively. Both sexes were found to have a statistically significant variation across Alberta's 19 census divisions. For males, examination of standardized morbidity ratios found a low risk of Parkinson's disease associated with five census divisions, of which two contained Alberta's two largest cities. An excess risk was associated with four primarily rural census divisions. Females, on the other hand, had a low risk associated with one rural census division and excess risk in four census divisions. The uneven distribution within Alberta offers support for an environmental theory of etiology which may be associated with rural living.

Coronary artery bypass grafting in Alberta from 1984 to 1989.

OBJECTIVE: To examine the geographic and temporal trends in the performance of coronary artery bypass grafting (CABG) in the province of Alberta. This examination was designed to improve understanding of the geographic distribution and temporal changes in the incidence of the procedure. DESIGN: A cohort of all individuals (2.4 million) registered with the Alberta Health Care Insurance Plan (AHCIP) was followed for the five-year period from April 1, 1984 to March 31, 1989. No new members were admitted to the cohort and an attrition rate averaging 6% per year was observed. SETTING: Data from the claims administrative database of the AHCIP were used. The AHCIP includes the records of all registered residents of the province and is virtually complete. PARTICIPANTS: Participants included all 2963 members of the cohort (2413 males, 550 females) who underwent CABG over the study period. MAIN RESULTS: The overall mean surgical rate was 28.8 per 100,000 (46.7 for males, 10.8 for females; chi 2 = 1172.8, df = 1, P < 0.0001). The mean age at surgery was 59.6 years (57.9 for males, 61.4 for females), with 70 and 76% of procedures occurring between the ages of 60 and 70 years for males and females, respectively. The largest increases in incidence were in males (223%) and females (148%) aged 70 years or older. Both males (chi 2 linearity = 25.95, df = 3, P < 0.01) and females (chi 2 linearity = 8.34, df = 3, P < 0.05) displayed an increasing trend over the study period. Data were aggregated across the five-year study period to assess geographic variations. No difference was noted across census divisions for either sex. CONCLUSIONS: The similarity in rates across census divisions implies consistent use of criteria for the performance of this procedure. Also, the results imply that research, in Alberta, should focus more on indications for the procedure and its outcomes, than on geographic disparities.

Pathophysiology of shock and hemorrhage in a fulminating viral infection (Ebola).

Eleven rhesus monkeys were monitored intensively during experimental infection with Ebola virus. Prominent neutrophilia with left shift and lymphopenia were the earliest abnormalities and were statistically significant by day 4 (P less than .02 and P less than .01, respectively). By day 4 falls in platelet counts were not statistically significant, whereas in vitro platelet aggregation was markedly depressed, progressing rapidly to complete failure by the time of maximum illness. Intraplatelet protein studies suggested this event was the result of in vivo activation and degranulation. Coagulation cascade defects were mainly in the intrinsic system and were surprisingly mild, with no evidence of selective consumption or production deficit of factor VII or VIII. When the possibility of indirectly mediated damage to endothelium possibly by a nonspecific immune response was examined, weight loss was less severe in drug-treated monkeys, and all had detectable plasma prostacyclin metabolites, but there was no improvement in survival.

Preliminary report on arbovirus-antibody prevalence among patients in Kuwait: evidence of Congo/Crimean virus infection.

From two major hospitals in Kuwait 502 sera were randomly selected from patients during the period December 1979 to October 1982. Serological investigations demonstrated Flavivirus activity in the area and antibody to Congo/Crimean haemorrhagic fever virus was found in 4% of the samples. Clinical data indicate that some cases may have been due to recent Congo/Crimean virus infection.

Haematological and biochemical monitoring of Ebola infection in rhesus monkeys: implications for patient management.

Patients with severe viral infections such as Lassa or Ebola may be denied adequate laboratory investigations because of justifiable fears among laboratory staff. This study in monkeys was designed to provide comprehensive haematological and biochemical monitoring in a contained environment during all stages of Ebola infection. Marked neutrophilia, depletion of lymphocytes, and early failure of platelet aggregation preceded a consumption coagulopathy with a microangiopathic haemolytic anaemia, thrombocytopenia, and failure of prostacyclin production by vascular endothelium. Liver dysfunction was moderate but in conjunction with the dehydration and hypoalbuminaemia could be expected to precipitate renal failure and shock. It seems reasonable to anticipate successful patient support with a patient management isolator and treatment with platelet transfusions, fresh frozen plasma, and possibly prostacyclin when haemostasis is defective during this otherwise self-limiting illness.

The susceptibility of Culicoides variipennis Coq. (Diptera: Ceratopogonidae) to laboratory infection with Rift Valley fever virus.

The vector potential of Culicoides variipennis for Rift Valley fever virus (RVFV) was investigated. Insects from a colony maintained at the Animal Virus Research Institute, Pirbright, were fed through a membrane on a mixture of mouse blood and RVFV (virus concentration of blood meal 7 X 95 log10 MLD50ml). Engorged insects were maintained at 25 degrees C +/- 1 degrees C. Samples of insects were tested at daily intervals to determine their virus content. Four of the five females tested immediately after feeding contained virus. The mean virus concentration of these infected flies was 2 X 7 log10MLD50. The virus concentration per fly decreased to 0 by day 2 post infection. On day 3, a virus concentration of 2 X 4 log10 MLD50 per fly was recorded from a pool of 17 flies but between day 4 and day 12 when the experiment terminated no virus was detected in any of the 135 females tested. Because of the pathogenic nature of RVFV, this work was carried out under the stringent security regulations at the PHLS Centre for Applied Microbiology and Research, Porton Down. The problems arising from experiments requiring the handling and infection of insects under such conditions are described.