Nucleostemin (NS), a p53-binding protein, has been shown essential for stem and cancer cell proliferation and implicated in oncogenesis. To explore potential contributions of NS to the development of clear cell renal cell carcinomas (ccRCCs), we determined NS expression in ccRCC cell lines, and in paired normal and malignant renal tissues from 31 patients with ccRCC. Nucleostemin mRNA and/or protein expression was observed in all four cell lines and 27 of 31 (87%) tumour specimens. Surprisingly, 16 of 31 (52%) adjacent normal renal samples also expressed NS mRNA and its levels in four of them were comparable with those in paired tumour tissues. Three of the patients had detectable NS mRNA in their normal renal tissues whereas lacked its expression in the matched tumours. Compared to the oncogene c-MYC expression in these same samples, NS expression showed a much less specificity for ccRCC. We further demonstrated that NS mRNA expression was closely associated with cellular proliferation in normal fibroblasts or T lymphocytes and renal cell carcinoma cell lines. Collectively, NS expression widely occurs in normal and malignant renal tissues, and is likely a proliferation marker rather than a unique regulator of cell proliferation and survival in stem and cancer cells. VHL gene) by different mechanisms is widespread and believed to be an early or a first event during the oncogenic process , the most common malignant tumour of the adult kidney in both developed and developing countries, accounts for 2–3% of all human malignancies with close to 100 000 yearly deaths worldwide  analyses of NS and c-MYC mRNA expression. To determine the relationship between NS expression and cellular proliferation, the cells were incubated in 0.2% FCS-containing medium for 72 h to induce quiescence and then re-fed with 20% FCS for 24 h. Normal T lymphocytes were isolated from the buffy-coaded blood derived from normal individuals and treated with 15 μg/ml ConA to stimulate their proliferation. Normal dermal fibroblasts were kindly provided by Dr Z. Yan .Four ccRCC cell lines, A498, KRC/Y, CAKI-2, and TK10, were maintained at 37°C in Dulbecco's modified Eagle's medium  supplemented with 10% fetal calf serum (FCS), 2 mThe ccRCC tumour samples and their adjacent normal renal tissues were obtained from 31 patients with primary ccRCC. The study was approved by the local ethics committee, and all the patients consented to the use of their renal tissues for the present investigation. The patients were treated with radical nephrectomy at Qilu Hospital, Shandong University, between 2002 and 2003. After surgery, the tumour specimens and adjacent normal renal tissues were stored at −80°C until use. For each ccRCC, tumour size (diameter), Robson stage, and regional and distant metastases at nephrectomy were evaluated by both pathologists and urologists. Patient and disease information is listed in β-Actin expression was used as an internal control and amplified with its specific primers using 25 cycles . cDNA was synthesised using random primers (N6)  and M-MLV reverse transcriptase. The polymerase chain reaction (PCR) primer sequences specific for NS mRNA (Accession number: AY825265) were: 5′-GAA ACA GAG GCT TGA AGA ACT AA (forward) and 5′-GGA GGC TTC GAT CAC CTT TTT A (reverse). Polymerase chain reaction was performed with the use of 32 amplification cycles  and the PCR product was 223 bp in length. The RT–PCR for the oncogene c-MYC mRNA (Accession number: HSMYC1) was performed as described  followed by secondary anti-rabbit horseradish peroxidase-conjugated IgG and developed with the enhanced chemiluminescent method .Western blot was performed as described  expressed detectable NS mRNA . These unexpected findings challenge the early concept of NS being a specific player for stem and cancer cell proliferation. However, one may argue that (1) tumour cell contamination in adjacent normal renal tissues may contribute to detectable NS mRNA expression, as seen here. However, these same specimens have previously been used for analyses of telomerase activity and full-length of telomerase reverse transcriptase mRNA expression, highly specific markers for transformed cells, and both markers were exclusively absent in all the studied adjacent normal tissues (in vitro cultured RCC cells undergo downregulation of NS expression in response to serum starvation. Regardless being stem cells, cancer cells, or normal differentiated cells, they may all acquire NS expression when in actively proliferating status. Therefore, NS is more like a proliferation marker rather than a molecule only involved in the cell cycle control of stem and cancer cells. This proposal provides a reasonable explanation for what have been seen in normal renal and prostate tissues: detectable NS transcript in them is virtually expressed by proliferative epithelial or other types of cells; and NS-expressing stem cells are negligible owing to their very low numbers.According to our preliminary data, NS expression is proliferation-regulated and induced in actively cycling cells such as presenescent human fibroblasts and activated T lymphocytes. On the other hand, In normal stem cells and U2OS cells, NS protein was exclusively localised in nucleus . HoweverThe oncogene c-MYC plays a key role in regulating cell proliferation and its aberrant activation contributes to the pathogenesis of many human malignancies including RCC (The study presented herein demonstrates that NS is widely expressed in both ccRCC and adjacent normal renal tissues, in sharp contrast with the recent report that NS is present in stem and cancer cells whereas absent in differentiated adult tissues . Althoug


We also quantified LOXL1 mRNA expression in the human lens capsule to examine the possible correlation between LOXL1 expression and XFG pathogenesis.We performed genetic association studies using a native Japanese population to examine the reproducibility of results of We performed a case-control study using 95 Japanese XFG patients and 190 controls. Real-time polymerase chain reaction (PCR) analysis was performed using lens capsules obtained during surgery.rs1048661 and the GG genotype in the SNP rs3825942 in exon 1 of LOXL1 were significantly associated with an increased risk of XFG under recessive models . Quantification of LOXL1 mRNA expression demonstrated no significant difference between XFG and senile cataract samples.The TT genotype in the single nucleotide polymorphism (SNP) LOXL1 SNP appear to be qualitative rather than quantitative, the amino acid substitution (R141L) caused by SNP rs1048661 is not a simple decisive factor for XFG due to the inverted allele frequency between Japanese XFG and Caucasian XFG patients. Further genetic and functional studies are essential for clarifying XFG pathogenesis.Although the functional effects of the  One of the nonsynonymous SNP (rs1048661) showed a very strong association with XFG. However, the risk allele was inverse compared to the Caucasian study. To gain further insight into the role of LOXL1 for XFG, it is important to compare the expression levels of LOXL1 mRNA in XFG eyes and non-glaucomatous eyes. We obtained anterior lens capsules during combined glaucoma-cataract surgery or during cataract surgery alone from XFG patients and non-glaucomatous senile cataract patients, respectively. We then performed a quantitative analysis of LOXL1 mRNA expression using these anterior lens capsules.Exfoliation glaucoma (XFG) is an age-related disorder associated with exfoliation syndrome (XFS), manifested by abnormal fibrillar deposits on the lens and iris epithelium . Recentl XFS/XFG . It was  XFS/XFG , therefoAll XFG patients were diagnosed by slit-lamp examination for the existence of exfoliation material on the anterior lens capsule with maximal dilation of the pupils and with glaucomatous optic neuropathy as well as visual field defect. Peripheral blood was obtained from 95 XFG patients 47–93 years of age (mean age: 75.7±8.1 years). The controls were 190 randomly-selected, population-based individuals 54–83 years of age (mean age: 65.0±6.8 years) with no glaucomatous changes or existence of exfoliation materials . All of rs1048661 and rs3825942) in the LOXL1 gene region according to the previous report [We genotyped two nonsynonymous single nucleotide polymorphisms  and 95% confidence intervals (95% CI) were also calculated.The frequencies of the genotypes were compared between XFG patients and controls in the recessive model. In this model, frequencies of the homozygous genotype for major alleles were compared using a 2x2 contingency table. Here, the association was evaluated using the χLOXL1 (Hs00173746_m1), and 18S rRNA from Applied Biosystems (Assay-on-Demand gene expression products). Real-time PCR analysis was performed on a 7500 real-time PCR system. The relative expression of LOXL1 mRNA in the anterior lens capsule was quantified by the standard curve method using 18S rRNA expression in the same cDNA as the control.Anterior capsules that were obtained during glaucoma/cataract or cataract surgery with written informed consent were immediately stored with RNAlater reagent  to protect the RNA. All procedures were approved by the ethics committee of Kyoto Prefectural University of Medicine. Total RNA was isolated with the Micro RNA extraction kit  from the anterior capsules, and then cDNA was prepared as described previously . The antrs1048661 TT genotype and the rs3825942 GG genotype in the first exon of LOXL1 were significantly associated with an increased risk of XFG under recessive models . The results obtained by the two methods were identical.We genotyped 95 XFG cases and 190 control subjects by direct sequencing methods. The rs1048661 and rs3825942 and found that those two SNPs are in a state of strong LD (Haploview [rs1048661 G/T and rs3825942 G/A), the TG haplotype showed an increased risk for XFG  in recessive models. We checked HWE among control subjects using this two-locus haplotype and found that the two-locus haplotype was concordant with HWE .First, we checked the state of linkage disequilibrium (LD) between trong LD . Next, wtrong LD  using HaLOXL1 mRNA in the lens epithelium obtained from XFG patients and senile cataract patients. (-test)cDNA was synthesized from total RNA isolated from the anterior lens capsules of patients undergoing cataract surgery. We analyzed both XFG (n=10) and senile cataract (n=10) specimens. LOXL1 SNP and XFG patients. The lysyl oxidase protein family has multiple functions including specific oxidative deamination of lysine residues and the cross-linking of elastin [LOXL1 knockout mice [LOXL1 is one of the causative genes for XFG.Similar to previous studies among Caucasian ,7,8, we  elastin . Phenotyout mice  showed tout mice . Therefors3825942 genotype, our results are consistent with those of Thorleifsson et al. [rs3825942 G is consistently higher among Caucasian and Japanese XFG patients so it is reasonable to conclude that rs3825942 A is a protective allele against the occurrence of XFG.For the rs1048661-“T” among the Japanese population and rs1048661-“G” among the Caucasian population. We double-checked these genotype results (rs3825942 and rs1048661) by both direct sequencing and the TaqMan genotyping assay because the genotype results of rs1048661 within control subjects were not concordant with HWE. The results obtained from two different methods were in complete agreement. Therefore non-concordance with HWE is not due to genotyping errors. We further checked the two-locus haplotype and found that the haplotype was concordant with HWE. Since the two-locus, rs1048661 and rs3825942, was in the state of linkage disequilibrium (D’=1.0), it was appropriate to check the HWE by haplotype.On the other hand, the risk allele for XFG occurrence is rs1048661 G/T genotype may have a protective role against XFG occurrence, or (3) there might be another causative polymorphism in a state of linkage disequilibrium with rs1048661 G/T. If the last possibility is the case, there might be a historical recombination between the SNP rs1048661 and the causative polymorphism.Inverted genotypes of XFG patients between the Japanese and Caucasian population suggests the following possibilities: (1) The 141st amino acid substitution (R141L) does not have a dominant role for the pathophysiology of XFG, (2) the heterozygote for the LOXL1 mRNA expression in anterior lens capsules and found that there was no significant difference between XFG and senile cataract controls. These results are different from the previous study by Thorleifsson et al. [LOXL1 mRNA expression with the TT genotype (the protective genotype against XFG in the Caucasian population) than those with the TG or GG genotype using adipose tissues. We hypothesize that the difference might be due to the mixed LOXL1 SNP genotype of our senile cataract surgery samples, which are not able to clarify genotype due to ethical procedure reasons. However, our LOXL1 mRNA expression analysis reflects a direct pathological site within the ocular tissue and better represents LOXL1 expression status in the affected eyes than that of adipose tissue [LOXL1 mRNA is not a direct pathogenetic cause of XFG.As a next step, we quantified relative n et al. , which se tissue . TherefoLOXL1 for XFG, we are now performing extensive SNP discovery around LOXL1 to find out other possible causative polymorphisms. In addition, functional analysis of LOXL1-fibulin5 protein interaction [LOXL1 and its mixture to gain further insight into our genetic association results showing that the rs1048661 G/T heterozygote is protective against XFG. It is also important to investigate the protective role of LOXL1 protein variants that possess the 153D amino acid, which is indicated by multiple genetic association studies including our own [To further clarify the pathophysiological role of eraction  using tweraction ,12, we a our own ,8 as wel


The LIM domain protein Fhl5 was previously found to interact with CREM, a DNA binding transcriptional regulator necessary for spermiogenesis in mammals. Co-transfection experiments using heterologous promoter constructs indicated a role for Fhl5 in transcriptional up-regulation of CREM-dependent testicular genes. Male mice lacking Fhl5 were reported to be fertile but displayed partially abnormal sperm maturation and morphology.To identify Fhl5 testicular target genes we carried out two whole-genome expression profiling experiments using high-density oligonucleotide microarrays and total testis samples from Fhl5 wild-type versus homozygous mutant mice first in different and then in isogenic strain backgrounds.Weak signal differences were detected in non-isogenic samples but no statistically significant expression changes were observed when isogenic Fhl5 mutant and wild-type samples were compared.The outcome of these experiments suggests that testicular expression profiling is extremely sensitive to the genetic background and that Fhl5 is not essential for testicular gene expression to a level detected by microarray-based measurements. This might be due to redundant function of the related and similarly expressed protein Fhl4. Sexual reproduction of male mammals requires genes involved in meiosis, sperm formation and maturation as well as fertilization many of which are controlled by developmental stage-specific transcription factors ,2. InducWhile CREM is essential for spermatogenesis, Fhl5 is not because mutant male mice were found to be fertile. However, they displayed partially abnormal sperm maturation and morphology which was suggested to be a consequence of impaired CREM/Fhl5-dependent post-meiotic gene expression . Three tTo identify Fhl5 target genes, a whole-genome expression profiling experiment using total testicular samples and high-density oligonucleotide microarrays (GeneChips) containing probes for all known mouse protein-coding genes was carried out. We report that deletion of Fhl5 does not have a measurable effect on testicular gene expression at the level of sensitivity reached by microarray analysis when wild-type and Fhl5 mutant mice are examined in an isogenic background.The initial experiment was carried out with Fhl5-/- mice from a mixed genetic background (C57BL/6 and 129/SvPas)  as compaGenotyping was done using allele specific primers as in reference .Decapsulated total testis samples were prepared from adult mice at the age of 9 weeks (C57BL/6 and 129/SvPas) as well as 9 and 14 weeks (129/SvPas) using a protocol as described [Total RNA preparation, cRNA target synthesis and raw data production using MG430 2.0 GeneChips (Affymetrix) was done as previously published ,21. TotaData analysis was essentially done as published using tools implemented in AMEN ,22. The In the second experiment 23921 probe sets were selected for which signal intensities >5.5 (median) were observed. Among those 21 showed a fold-change ≥ 2 but none was identified as reproducibly differentially expressed using LIMMA .Raw data CEL files are available for downloading via the EBI's ArrayExpress public repository  under thIt was previously reported that deletion of Flh5 in a mixed genetic background (C57BL/6 & 129/SvPas) did not impair fertility but resulted in reduced sperm counts, altered sperm motility and partially abnormal morphology . To exclTo identify Flh5 target genes we first obtained decapsulated total testicular samples from mutant mice (C57BL/6 & 129/SvPas) whose genotype was verified by PCR and Western blotting (not shown). For practical purposes and cost-effectiveness cRNA targets were synthesized from the mutant mice testes, hybridized to MG430 2.0 GeneChips and compared to testicular and cell-type specific data from B6129SF2/J wild-type mice already available in our laboratory . We deteper se, total RNA was prepared from back-crossed mice's total testis samples at 9-15 weeks of age. Strikingly, no differentially expressed genes were detected and as a consequence none of the genes first identified as up- or down-regulated showed any change of transcription between wild-type and mutant samples from animals that were litter mates . For further interpretation these probe sets were grouped into two clusters using a k-means algorithm that distinguished transcripts for which signals were either stronger (149) or weaker (63) in the mutant as compared to the wild-type strain. No Gene Ontology terms  were fouWe next investigated the expression patterns of genes previously identified as being differentially expressed in testis (DET) using enriched Sertoli cells, spermatogonia, spermatocytes and spermatids . None ofFhl5 has initially been identified as a CREM-binding factor required for gene activation in the male germline. It was our aim to verify the hypothesis that abnormal sperm maturation and morphology observed in mutant mice lacking Fhl5 was due to impaired CREM-dependent gene expression. To this end we carried out two independent profiling experiments using total testicular samples and MG430 2.0 GeneChips covering all known protein-coding genes of the mouse genome.The first experiment was based on total testicular samples from adult nine week-old Fhl5-/- mice produced in a mixed genetic background (C57BL/6 and 129/SvPas)  that werin vivo role as a transcriptional regulator is too subtle to be detected by whole-genome profiling using total testicular samples and that highly enriched populations of round spermatids would have to be used instead. We consider this unlikely, however, since Fhl5 was reported to be a strong activator and approximately 60% of the adult testicular cell mass consists of spermatids [The most obvious explanation of these results is that the loci identified in the first experiment showed distinct expression patterns because we compared samples from different genetic backgrounds while the second experiment based on isogenic litter mates revealed that Fhl5 is not essential for testicular gene expression at the level of sensitivity achieved with microarrays and whole gonad samples. This result is in keeping with a previous report that the absence of Fhl5 has no effect on the expression of known CREM targets such as TNP1, PRN1 or PRN2 as assayed by PCR . It alsoermatids .An alternative explanation is that a related factor to a large degree compensates the deletion of Fhl5 such that transcriptional effects are masked while sperm maturation and morphology remain partially abnormal. One likely candidate is the LIM-domain protein Fhl4 which was initially shown to be expressed in testis ,28 and lThe corollary of our work is that deletion of Fhl5 does not cause any significant changes in testicular expression at the genome-wide level at a level detectable by high-density oligonucleotide microarray expression profiling when compared to an isogenic wild-type sample. We speculate that this is the case because Fhl5 is redundant with Fhl4, a structurally related factor showing a similar testicular expression pattern. If this idea were correct, mice lacking Fhl4 should display a phenotype reminiscent of the one reported for Fhl5 mutants while deletion of both genes should disrupt spermatogenesis at the post-meiotic stage because of impaired CREM-dependent gene expression.The authors declare that they have no competing interests.AL processed and interpreted the data and contributed to the manuscript. FC contributed to data analysis. PD processed the samples and produced the raw microarray data. PSC and MP designed research and MP interpreted the data and wrote the paper. All authors have read and approved the final manuscript.


Detectable shifts toward higher saturations were evident in all tumours (n = 16) immediately after the onset of carbogen breathing. The SO2 reached a new equilibrium within 1 min and remained approximately constant during 200–300 s of administration. The return to baseline saturation was more gradual when carbogen delivery was stopped. The degree to which carbogen increased SO2 was variable among tumours, with a tendency for tumours with lower initial SO2 to exhibit larger changes. Tumour haemoglobin concentrations at the time of peak enhancement were also variable. In the majority of cases, haemoglobin concentration decreased in response to carbogen, indicating that increased tumour blood volume was not responsible for the observed elevation in SO2. We observed no apparent relationship between the extent of the change in tumour haemoglobin concentration and the magnitude of the change in the saturation. Near infrared diffuse reflectance spectroscopy provides a rapid, non-invasive means of monitoring spatially averaged changes in tumour haemoglobin oxygen saturation induced by oxygen modifiers. © 1999 Cancer Research CampaignWe have evaluated the ability of steady-state, radially-resolved, broad-band near infrared diffuse reflectance spectroscopy to measure carbogen-induced changes in haemoglobin oxygen saturation (SO


Renal carriage and shedding of leptospires is characteristic of carrier or maintenance animal hosts. Sporadic reports indicate that after infection, humans may excrete leptospires for extended periods. We hypothesized that, like mammalian reservoir hosts, humans develop asymptomatic leptospiruria in settings of high disease transmission such as the Peruvian Amazon.Leptospira. Approximately one-third of the 314 asymptomatic participants had circulating anti-leptospiral antibodies. Among enrolled participants, 189/314 (59%) had evidence of recent infection (microscopic agglutination test (MAT0 ≥1∶800 or ELISA IgM-positive or both). The proportion of MAT-positive and high MAT-titer (≥1∶800) persons was higher in men than women (p = 0.006). Among these people, 13/314 (4.1%) had Leptospira DNA-positive urine samples. Of these, the 16S rRNA gene from 10 samples was able to be sequenced. The urine-derived species clustered within both pathogenic (n = 6) and intermediate clades of Leptospira (n = 4). All of the thirteen participants with leptospiral DNA in urine were women. The median age of the DNA-positive group was older compared to the negative group (p≤0.05). A group of asymptomatic participants  of enrolled individuals) without serological evidence of recent infection was identified; within this group, 6/102 (5.9%) excreted pathogenic and intermediate-pathogenic Leptospira (75–229 bacteria/mL of urine).Using a cross-sectional study design, we used a combination of epidemiological data, serology and molecular detection of the leptospiral 16S rRNA gene to identify asymptomatic urinary shedders of Leptospira can persist as renal colonization in humans. The pathogenic significance of this finding remains to be explored but is of fundamental biological significance.Asymptomatic renal colonization of leptospires in a region of high disease transmission is common, including among people without serological or clinical evidence of recent infection. Both pathogenic and intermediate  Leptospira are classified as pathogenic, intermediately pathogenic, or saprophytic  based on their ability to cause disease and on genetic information. Unique among the spirochetes that infect humans, Leptospira live both in the environment (in surface waters and moist soils), and in mammals, where they cause chronic infection by colonizing kidney tubules. Infected animals are the source of human infection, but humans have not been systematically studied as chronic Leptospira carriers. In our study, we found that more than 5% of people  in a rural Amazonian village, without clinical evidence of infection by Leptospira, were chronically colonized by the bacteria. Chronic infection was not associated with a detectable immune response against the spirochete. Pathogenic and intermediately pathogenic Leptospira caused asymptomatic, chronic kidney infections. Future work is needed to determine whether such chronic infection can lead to human-to-human transmission of leptospirosis, and whether subtle measures of kidney disease are associated with asymptomatic, long-term leptospiral infection.Leptospirosis is a bacterial disease commonly transmitted from animals to humans. The more than 200 types of spiral-shaped bacteria (spirochetes) in the genus  Leptospira. Found worldwide, leptospirosis is more common in tropical and sub-tropical areas where environmental and socioeconomic conditions favor its transmission. It has been identified in recent years as a global public health problem because of its increased mortality and morbidity. The disease is principally transmitted to humans indirectly by contact with water or soil contaminated with the urine of domestic and wild animals with persistent renal infection by LeptospiraLeptospirosis is a zoonotic disease caused by spirochetes of the genus Leptospira-infected animals Leptospira species, L. licerasiaeLeptospira is common in daily life in this tropical setting The tropical climate of the Peruvian Amazon region of Iquitos is ideal for the maintenance and transmission of leptospirosis. In developing countries, impoverished populations typically live either in rural areas or under highly crowded conditions in urban slums. These factors increase the risk of human exposure to the urine of Leptospira develop sub-clinical disease or have very mild symptoms, and thus do not seek medical attention Leptospira remains: are some serovars are more likely than others to establish asymptomatic renal infection in man?Leptospirosis in humans is frequently misidentified because of several factors: 1) variable and nonspecific clinical presentation; 2) lack of awareness of the disease among clinicians; and 3) difficulty in access to reliable and rapid diagnostic tests. Clinical manifestations, when present, vary from a mild ‘flu-like’ febrile illness to a severe disease variably including jaundice, renal failure, pulmonary hemorrhage, refractory shock and other grave manifestations. However, many if not most people infected by Leptospira species, but rarely develop symptoms and are not impaired by the infection of their kidneys. After infection, humans can also excrete leptospires into the urine transiently for weeks or, more rarely, months or more Renal colonization and persistent shedding of leptospires is characteristic of carrier or maintenance animal hosts Leptospira such as L. interrogans and intermediate pathogens such as the newly discovered L. licerasiaeLeptospira directly in the urine of healthy ambulatory people. If found, we reasoned that the high prevalence of asymptomatic urinary infection might provide fundamental insights into the nature of Leptospira-human interactions, where humans are considered to be accidental hosts. Such a finding would also provide the basis for understanding mechanisms of naturally acquired immunity in human leptospiral infection.We hypothesized that like mammalian reservoir hosts, humans develop asymptomatic leptospiruria, including pathogenic This study was approved by the Human Subjects Protection Program, University of California San Diego, and the Ethical Committees of Asociacion Benefica PRISMA, Lima, Peru, and Universidad Peruana Cayetano Heredia, Lima, Peru. All human subjects provided written informed consent before being enrolled in the study.LeptospiraThis study was carried out in the village of Padrecocha, a rural community near Iquitos, located north of the city along the Nanay River, a tributary that branches from the Amazon River 15 km downstream from Iquitos. The climate is tropical: rainfall averages 300 mm per year and temperatures range from 21.8°C to 31.6°C; the village is surrounded by a vast expanse of humid tropical rainforest. The population of this village is approximately 1,500. Most inhabitants live in brick houses, and their water supply comes from wells and local streams. These water sources harbor pathogenic and intermediate-pathogenic Using a whole-village canvassing strategy to develop a set of candidate houses from which to randomly select asymptomatic inhabitants of the rural village Padrecocha of age ≥5 years for enrollment. Subjects were excluded if they had fever within the previous 2 weeks or if they declined participation . All parVenous blood samples were drawn into tubes without anticoagulant  and transported to the study laboratory within 4 hr at ambient temperature. Serum was separated, frozen in 1 mL aliquots at −20°C, and transported on dry ice to the National Leptospirosis Reference Laboratory at the Instituto Nacional de Salud (INS) in Lima, where the presence of anti-leptospiral antibodies was determined. An ELISA incorporating 6 pathogenic serovars (strains)–Icterohaemorrhagiae (RGA), Australis , Bratislava (Jez Bratislava), Ballum (MUS127), Canicola (Hond Utretch IV), Cynopteri (3522 C), and Grippotyphosa (Moskva V)–was used to detect anti-leptospiral IgM antibodies. An ELISA IgM result of 11.0 IU/mL or more was considered to be positive Urine samples were collected in 50 mL sterile polypropylene centrifuge tubes and centrifuged at 3,000 g for 30 min at room temperature. DNA from the pellet was extracted using the QIAamp® DNA Mini Kit  following manufacturer's directions. DNA was extracted from samples on-site in the Iquitos laboratory the same day of collection and stored at −20°C.Leptospira using a published qPCR TaqMan assay targeting the leptospiral 16S ribosomal gene µM, primers at a final concentration of 0.5 µM, and a 20 µL reaction volume Leptospira interrogans serovar Copenhageni strain M20. Standards were prepared as follows. Leptospires were counted using a Petroff-Hauser counting chamber  and serially diluted with sterile double-distilled H2O to 108 to 100 leptospires/ml. Genomic DNA was subsequently prepared using the DNeasy Tissue Kit . Standards were run in triplicate to generate a standard curve with each run. A negative result was assigned where no amplification occurred before 40 cycles. Controls lacking template were extracted and added to qPCR master mix to detect the presence of contaminating DNA.All urine samples collected were screened for the presence of pathogenic and intermediate-pathogenic µg/mL ampicillin. Individual clones were grown overnight in LB broth containing 100 µg/mL ampicillin. Plasmid DNA from these clones was purified using the QIAprep Spin Miniprep Kit  and cycle sequenced. Sequencing was performed on an ABI 3100 automated sequencer  using the M13 forward and reverse primers . Reaction conditions were according to manufacturer's directions.To address potential limitations of sensitivity, a nested PCR strategy was used using the general eubacterial outer primers fD1 and rD1 Atopobium vaginae, an organism that has been recently recognized in the microflora associated with bacterial vaginosis Leptospira shared sufficient sequence homology with the Atopobium 16S rDNA gene that under the conditions described these primers generated amplicons for both Leptospira (∼1327 bp) and the Atopobium (∼1345 bp) species, making it difficult to distinguish this amplicon by agarose gel electrophoresis alone. To overcome this problem we developed a dot blot hybridization assay using a digoxigenin (DIG)-labeled DNA probe. Briefly, a 111bp DIG-labeled DNA probe (Lepto_DIG_probe) was synthesized by PCR-labeling with the PCR DIG Probe Synthesis Kit . Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 genomic DNA was used as template; primers lepto16S620f (5′-GAGTTTGGGAGAGGCAAGTGGAATTC-3′) and lepto16S730r (5′-GTGCCTCAGCGTCAGTTTTAGGCC-3′) for the probe synthesis. These primers were selected from conserved regions of the 16S rDNA genes of pathogenic and intermediate-pathogenic leptospiral species, but were absent from the saprophytes (http://rdp.cme.msu.edu) and aligned using clustalW 1.83  were used. The PCR reaction mix was prepared following the manufacturer's directions with the primers at a final concentration of 0.4 µM and a 1∶12 ratio of DIG-dUTP:dTTP nucleotides. The synthesis was conducted in a DNA Engine PCT-200 Peltier Thermal Cycler (MJ Research). The amplification protocol consisted of 95°C for 2 min, followed by 30 cycles of amplification, each cycle consisting of 95°C for 10 s, 64°C for 30 s, and 72°C for 2 min.After the initial evaluation of the DNA sequences of the first PCR-positive samples we observed a high rate of false positivity for the nested PCR in urine samples . The 16S ribosomal RNA gene sequences of the organisms present in the urine of the false positive PCR samples were frequently identical to the 16S ribosomal RNA gene sequence of the bacterium rophytes . PublishGenomic DNA from urine samples and controls were PCR-amplified using the aforementioned nested PCR assay, and the PCR products were separated by agar electrophoresis and purified from the agarose gels. PCR products were then denatured by boiling for 10 min in a denaturing solution , then cooled on ice and spotted into an Immobilon-Ny+ Charged Nylon Transfer Membrane  using a 96-well vacuum manifold . After spotting the samples, the membrane was air-dried and the DNA was fixed by UV cross-linking using the Stratalinker UV Crosslinker 2400 . The spotted membrane was hybridized overnight at 56°C with 5 pmol of Lepto_DNA_probe in 3.5 mL of DIG Easy Hyb hybridization buffer  according to manufacturer's directions. After hybridization and stringency washes, the blot was developed using a chemiluminescent assay with Anti-Digoxigenin-AP and CPD-Star  according to manufacturer's directions. For detection of the chemiluminescent signal, X-ray film was exposed to the blot for 1 minute.Atopobium DNA (data not shown). Thus to efficiently distinguish clones containing leptospiral 16S rDNA amplicons for sequencing, we developed a colony hybridization assay using the Lepto_DIG_probe whereby we could screen all the transformants on a plate simultaneously. Briefly, 16S ribosomal RNA genes previously PCR-amplified using the nested PCR assay from dot blot-positive samples (n = 13) were cloned using the PCR2.1-TOPO vector in E. coli as previously described. Transformed bacteria were grown at 37°C in Petri dishes (LB agar with 100 µg/mL ampicillin) overnight. Colonies bearing the cloned 16S rDNA gene were adsorbed onto positively charged circular nylon membranes  and lifted from the agar plate. After adsorption, the bacteria were denatured in the membranes and their DNA fixed to them by UV cross-linking. A mean of 4 Petri dishes (or ∼200 colonies) with transformed bacteria bearing the 16S ribosomal RNA gene per urine sample were screened with this method. Membranes were hybridized with the Lepto_DIG_probe and developed as described above. Positive colonies in this assay were handpicked and grown overnight. Plasmid DNA was purified and cycle sequenced as described previously to identify the present leptospires to the species level.The dot-blot positive samples contained a high proportion of contaminating http://pbil.univ-lyon1.fr/cap3.php) then aligned using CLUSTALW v1.83 with default parameters. Leptospira 16S ribosomal RNA gene sequences from GenBank were used as controls in the tree (http://mrbayes.csit.fsu.edu/) running for0 3,000,000 generations. The data were analyzed using the GTR+I+G nucleotide substitution model with gamma-distributed rates and proportion of invariant sites. The resulting data sets were then analyzed using flat priors for the substitution rate parameters DNA sequences were assembled (using 4- to 6-times coverage per nucleotide base) using the program CAP3  with a significance level (α) of 0.05.The proportion of the seropositivity rate (at any MAT titer) and distribution of the demographic variables were compared between the subjects with and without leptospiruria using the chi-square test and Mann-Whitney p = 0.051).In the pre-study census and sampling period, 1320 people in 225 houses were identified in the Peruvian Amazon village of Padre Cocha near Iquitos. The study enrolled 354 participants of age ≥5 years from 175 households randomly picked from a census map. Of those 354, 40 participants were excluded since 19 presented with fever within 2 weeks of enrollment and 21 did not provide urine samples (patient enrollment diagrammed in Leptospira) and high MAT-titer  persons was higher in men  than women (24.9% (p = 0.006) and 2.2% (p = 0.006)). The difference stayed significant after adjusting for the age.Blood samples were available from 282 of 314 participants (89.8%). Of these, 97 were from males and 185 from females. Circulating anti-leptospiral antibodies were found, by either IgM ELISA or MAT or both, in 108 (38%) of the 281 subjects for whom serological data were available . The mosAtopobium DNA  positive samples. Further evaluation of these samples with the nested PCR assay confirmed their positivity. Among these 63 PCR-positive samples, a newly designed dot-blot assay, designed to exclude false-positive samples containing only bium DNA , identifbium DNA ; sequencLeptospira (n = 4)  and intermediate clades of  (n = 4) . Althoug (n = 4)  had seroLeptospira DNA positive urine . The median age of the DNA positive group  = 9−58) was older compared to the women in the negative group . The difference stayed significant if the men were included in the negative group  years, p = 0.011). Univariate analysis did not show significant association between other epidemiological factors and leptospiral DNA positivity in urine (data not shown).All thirteen participants who had leptospiral DNA in their urine were women and the proportion  of the women was significantly higher compared to that in the leptospiral-DNA-negative group ; we call them “long-term asymptomatic individuals.” Within this group, six (5.9%) excreted pathogenic and intermediate-pathogenic Leptospira  were confirmed to excrete positive . After cLeptospira but did not have serological or clinical evidence of recent infection. Third, we found that both pathogenic and intermediately pathogenic Leptospira persistent infected the renal tubules of humans. Such observations have not been reported previously and are particularly notable because they demonstrate that inapparent leptospiral infection is common and frequently leads to shedding of organisms in urine. The long-term clinical significance of this finding remains to be determined.This study has several important findings. First, asymptomatic individuals living in a region hyperendemic for leptospirosis had a high rate of seropositivity (at any level) for leptospiral infection (38% of 314 participants). Almost 60% of the seropositive individuals had evidence of recent sub-clinical infection, as indicated by MAT titer ≥1/800. Second, and of unique interest, a novel 16S rDNA hybridization assay used to screen urine samples for the presence of leptospiral DNA found that almost 5% of healthy people living in a rural Amazonian community were urinary shedders of Leptospira, but the data presented here do not rule out this possibility.The occurrence of leptospirosis, and indeed many infectious diseases, depends on several interacting variables. These include favorable environmental conditions, the density of local reservoir host populations, the type and frequency of exposure, exposure to infectious doses of the etiologic agent, the virulence of the infecting strain, and the lifestyle preferences and susceptibility of individuals within the exposed human population Leptospira in this rural Amazonian study population was common  with many subjects having evidence of recent sub-clinical infection. However, the serological data presented here need to be interpreted with caution: in an endemic setting, a high individual MAT titer (≥1∶800) and/or IgM positivity are not reliable indicators of recent or current infection as antibodies may persist for prolonged periods Exposure to Leptospira for periods exceeding a year; extending previous understandings of the carrier state. Ten asymptomatic individuals without clinical (no febrile episodes in more than a year) or serological evidence of recent exposure were found to be shedding either pathogenic or intermediately pathogenic Leptospira in their urine. Although these persons may have been recently sub-clinically infected and either failed to produce anti-leptospiral antibodies or all produced ‘false-negative’ serology, these explanations seem unlikely. It is more likely that they represent long-term renal asymptomatic shedders of Leptospira, regardless of whether patients were subclinically infected or had acute illness. However, a prospective study would be needed to assess this possibility. Nonetheless, prospective observational studies of such patients are required to confirm this hypothesisIt is generally accepted that humans can excrete leptospires from weeks to months after infection Leptospira than are men; with a significant increase in incidence with age in women, possibly reflecting increased exposure with age or alternatively increased susceptibility. It is possible that the conclusion that women are more likely to be long-term asymptomatic urinary shedders than men may reflect a bias in the study, considering its relative underrepresentation of men. However, this observation may also reflect increased susceptibility of women to persistent leptospiral kidney infections; the reasons for this are unclear. However in our study population, the MAT titer was significantly lower in women than in men perhaps indicating that men are able to mount a more effective immune response than are women. Alternatively, men may have persistently higher antibody titers as a result of more frequent exposure due to work or recreational practices. Such possibilities require prospective study to address.Our data also suggest that women  are more likely to develop long-term renal carriage of Leptospira need to be explored, in particularly the effect of persistent infection on renal function and electrolyte balance. Moreover, the nature of the infecting strains needs to be more carefully explored as some strains may be more likely than others to result in persistent renal infections in humans. Although we have identified the species of the infecting strains in the present study, other methods that are able to identify serovars, particularly isolation, will be more informative.The long-term consequences of human renal infection by Leptospira for their families and explore human-human transmission more carefully. Infection in carrier animals is usually acquired at an early age, and the prevalence of chronic excretion in the urine increases with age; we observed a similar trend in this population. Of note, none of the long-term urinary shedders had circulating anti-leptospiral antibodies; this is in accordance with early observations in Leptospira-carrier mammals, where chronic urinary carriage was associated with low seropositivity to urinary culture rates in asymptomatic well-established serovar-specific carriers While humans are considered to be exclusively incidental hosts, animals can be maintenance and/or incidental hosts; maintenance hosts are defined as species in which infection is endemic, of low or no pathogenicity and (as a key factor) transmitted directly to the same species Leptospira-positive clones; we detected both Leptospira and Atopobium DNA in multiple samples, making the selection of colonies harboring the leptospiral 16S gene less efficient, in some instances, several hundred colonies had to be screened; we were unable to sequence the infecting strain in three enrollees due these technical limitations. Though unlikely, it is also possible that in these three instances the dot-blot gave false positive results. A third limitation of this study is that culture isolation of leptospires from urine was not attempted. Future work will be needed to further validate the molecular results presented here, and will use the PCR method to screen patients who then would have urine cultured for Leptospira. Nonetheless, the deployment of a valid molecular tool to detect leptospiruria represents a new approach to assessing chronic asymptomatic infections in humans without the need for obtaining isolates. Finally, because L. licerasiae serovar Varillal The study design had several limitations. First, we relied only on the recall of participants to define absence of fever in 1 year. Men were underrepresented; fewer men were recruited because of a lack of availability at the time of recruitment (most were away working). Thus, no leptospiruric males were detected. This observation suggests that we may have underestimated the overall number of asymptomatic shedders, as men have been typically associated with a higher risk of exposure due to work-related contact and behavioral practices. Another limitation was the initial non-efficient PCR screening strategy. The presence of other bacterial DNA hindered the identification of eptospira-specific PCR have been applied in clinical or field settingsFew of the published LLeptospira or the pathogenesis of leptospirosis remains limited. Naturally acquired immunity that protects against re-infection by Leptospira does occur and has been shown in animal models. It has been assumed that naturally acquired immunity is humorally-mediated particularly by antibodies against oligosaccharides of leptospiral LPS. Evidence also suggests that antibodies specific to Leptospira membrane-associated proteins may play a role in host defense Leptospira and high prevalence of seropositivity, the prevalence of severe disease is low Leptospira and asymptomatic infection could induce protective acquired immunity. Longitudinal studies are needed to test this hypothesis.Current understanding of host immune responses to Leptospira. The health implications of long-term renal colonization and whether antibiotic treatment of such patients is required remain to be determined.In conclusion, we have identified a long-term renal shedder group among persons asymptomatically infected with pathogenic and intermediately pathogenic Alternative Language Abstract S1Japanese translation of the abstract by MS.(0.03 MB DOC)Click here for additional data file.Alternative Language Abstract S2Spanish translation of the abstract by CAG.(0.03 MB DOC)Click here for additional data file.Checklist S1STROBE checklist.(0.09 MB DOC)Click here for additional data file.


A genome-wide deletion mutant analysis in budding yeast reveals genes required for respiratory growth, mitochondrial genome maintenance and mitochondrial protein synthesis. The mitochondrial respiratory chain produces metabolic energy by oxidative phosphorylation. Biogenesis of the respiratory chain requires the coordinated expression of two genomes: the nuclear genome encoding the vast majority of mitochondrial proteins, and the mitochondrial genome encoding a handful of mitochondrial proteins. The understanding of the molecular processes contributing to respiratory chain assembly and maintenance requires the systematic identification and functional analysis of the genes involved.RRG1 through RRG10). Systematic functional analysis of 319 respiratory-deficient mutants revealed 16 genes essential for maintenance of the mitochondrial genome, 88 genes required for mitochondrial protein translation, and 10 genes required for expression of specific mitochondrial gene products. A group of mutants acquiring irreversible damage compromising respiratory capacity includes strains defective in assembly of the cytochrome c oxidase that were found to be particularly sensitive to aging.We pursued a systematic, genome-wide approach to define the sets of genes required for respiratory activity and maintenance and expression of the mitochondrial genome in yeast. By comparative gene deletion analysis we found an unexpected phenotypic plasticity among respiratory-deficient mutants, and we identified ten previously uncharacterized genes essential for respiratory growth (These data advance the understanding of the molecular processes contributing to maintenance of the mitochondrial genome, mitochondrial protein translation, and assembly of the respiratory chain. They revealed a number of previously uncharacterized components, and provide a comprehensive picture of the molecular processes required for respiratory activity in a simple eukaryotic cell. Mutants with lesions in the mitochondrial genome are referred to as cytoplasmic petite, whereas respiratory-deficient strains carrying mutations in the nuclear genome are referred to as nuclear petite or pet mutants  mtDNA  mtDNA . However+] mtDNA . To testr et al.  and Luba  . Strains were grown to logarithmic growth phase in medium containing fermentable carbon sources, before cytosolic translation was stopped by the addition of cycloheximide. Newly synthesized mitochondrial proteins were labeled with pet309 was observed to be completely translation-inactive here, whereas mitochondrial translation products could be observed when this mutant was constructed in the W303 genetic background  mitochondrial genome, as indicated by the mating experiment. In addition, three independently performed cytoduction experiments suggest that replenishment of cytoplasmic material reproducibly restores and maintains respiratory growth, at least for a few generations. These observations point to the possibility that respiratory competence may involve acquired properties that are not strictly linked to the nuclear or mitochondrial genotype. In order to corroborate this assumption, we tested whether cytoduction with a [rho0] donor strain would also restore respiratory growth. Rescue was observed in 11 strains , suggesting that, at least in some cases, cytoplasmic components other than mtDNA are able to improve respiratory functions. We hypothesize that respiratory deficiency may be an acquired phenotype that does not exclusively depend on the genotype.The respiratory-deficient phenotype of 23 coq5, Δcoq10, Δcox10, Δcox16, Δcox19, Δmct1, Δmss2, Δnfu1, Δslm3, and Δsom1) are four mutants that are specifically defective in the assembly of the cytochrome c oxidase (COX complex). Cox10 is required for the synthesis of the heme A cofactor  genome and wild-type-like mitochondrial protein translation activity. Although a mitochondrial location of Rrg3 has not been shown experimentally, the Mitoprot program  genome. The RRG10 gene encodes a small mitochondrial protein [Δ protein ,35 of onpet phenotype when different versions of the yeast deletion library are screened for growth on non-fermentable carbon sources. While some differences can be ascribed to wrong deletions present in the library, most of the variations are likely due to intrinsic properties of the mutant strains. We present four lines of evidence suggesting that the plasticity of pet phenotypes is much greater than previously anticipated. First, several deletions produce different phenotypes in different versions of the deletion library  that are obligatorily required for respiratory metabolism in yeast. These include ten largely uncharacterized genes, RRG1 through RRG10. Remarkably, almost all of these highly penetrant mutants (95%) have been reported to show decreased fitness on non-fermentable carbon sources when the whole-genome pool of yeast deletion mutants was analyzed [et al. [et al. study [pet genes . Our present work suggests that 165 of these originally identified mutants do not reproducibly give rise to a pet phenotype and should be considered as important but not essential for respiratory growth of yeast. Thus, Figure et al. [rrg2 and Δrrg6 mutants are respiratory deficient, and that the Δrrg3 mutant shows an increased petite frequency [RRG genes were only found by the comparative gene deletion analysis described here, demonstrating the value of our approach.Comparative gene deletion analysis enabled us to define by stringent criteria a set of 163 protein-coding genes . Plasmid pRS416/MSS2 was constructed by PCR amplification of the MSS2 gene using primers 5' AAA GGA TCC GAT TTT ATG TGT GGA ATG CTA ACG ATG AAC and 5' AAA CTC GAG CTC TAA CAG TAT TTC CTA ATT ATT TCA TAG GTA AC and subcloning into the BamHI and XhoI sites of vector pRS416 [COX16 was constructed by PCR amplification of the COX16 gene using primers 5' AAA GGA TCC AAT ATT ACC GTG AAT ATC GCG AGC TAC and 5' AAA CTC GAG AGG TAT TTA CAA TCA TTT CCT AGA CAT TCT and subcloning into the BamHI and XhoI sites of vector pRS416. For complementation tests, yeast strains were transformed with plasmids pG12/T4 [COX10, pRS416/COX16, pG188/T1 [COX19, or pRS416/MSS2.Yeast strains used in this study were isogenic to BY4741, BY4742 and BY4743 , with th library  and its  library  was obtar pRS416 . Plasmid pG12/T4  expressipG188/T1  expressiPCR analyses to confirm the identity of deletion mutants were performed in a way that one primer was homologous to a sequence within the coding region or within the deletion marker cassette, respectively, whereas the other primer was homologous to a sequence outside the deleted part of the gene. Thus, a PCR product can be generated only if the correct allele corresponding to the primer combination is present. Primers used to confirm the identity of yeast deletion mutants are listed in Additional data file 10.S. cerevisiae was cultivated and manipulated according to standard procedures [et al. study [ocedures . For scrl. study . Respiramip1, yeast deletion strains were transferred with a sterile pinning tool from 96-well plates to a lawn of MATa Δmip1 cells on YPD plates. After incubation overnight to allow for mating, cells were replica-plated two times on plates containing minimal SD medium selective for diploid cells. Then, growth on YPG plates was determined as above. Cytoduction was performed as described [For high-throughput complementation tests with Δescribed . Cytoducin vivo was performed essentially as described [Labeling of mitochondrial translation products escribed  with the2 size. Plates were incubated for 14 to 28 days at room temperature to allow chronological aging. Then, a small amount of aged cells (or young cells taken directly from glycerol stocks as a control) was spread on a fresh YPD plate, incubated overnight at 30°C, and transformed according to the rapid transformation protocol described by Truong and Gietz [Using sterile toothpicks, cells were taken from glycerol stocks and spread on YPD plates as patches of about 2 cmnd Gietz . Using sStaining of mtDNA with DAPI in methanol-fixed cells was as described . For theGAL4 forward, 5' TTT CTC CTG GCT CAG TAG GGC; GAL4 reverse, 5' AGT TAC GAG AGG GTG GAC GGT; COX3 forward, 5' ATT GAA GCT GTA CAA CCT ACC GAA TT; COX3 reverse, 5' CCT GCG ATT AAG GCA TGA TGA. Data were analyzed with Sequence Detection Software Version 1.2.3 7000 System SDS software Core Application (Applied Biosystems) and calculated according to the 2C-ΔΔT-method [Yeast strains were grown overnight in liquid YPD medium and DNA was extracted using the YeaStar™ Genomic DNA Kit  according to the manufacturer's instructions. PCR reactions were performed in 20 μl volume in 96-well plates using Maxima™ SYBR Green qPCR Master Mix (2×)  according to the manufacturer's instructions in an ABI PRISM 7000 Sequence Detection System . The following primers were used: T-method .c oxidase; DAPI: 4',6-diamidino-2-phenylindole; DHR: dihydrorhodamine 123; ER: endoplasmic reticulum; GFP: green fluorescent protein; mtDNA: mitochondrial DNA; ORF: open reading frame; PPR: pentatricopeptide; ROS: reactive oxygen species.COX: cytochrome SM performed the experiments, SM and BW conceived the study, analyzed the data and wrote the manuscript.pet genes isolated from the MATa deletion library (Additional data file pet genes unique to this study (Additional data file pet genes grouped according to their occurrence in pet screens and localization and function of the encoded gene products (Additional data file pet genes producing growth defects only on specific carbon sources (Additional data file pet genes (Additional data file pet mutants (Additional data file pet genes dispensable for respiration (Additional data file pet mutants that show a wild-type pattern of mitochondrial translation products (Additional data file The following additional data are available with the online version of this paper: a table listing pet genes isolated from the MATα deletion library.Click here for filepet genes unique to this study.Click here for filepet genes grouped according to their occurrence in pet screens and localization and function of the encoded gene products.Click here for filepet genes producing growth defects only on specific carbon sources.Click here for filepet genes.Mutants belonging to four classes of Click here for filepet mutants.Quantification of loss of mtDNA in class IV Click here for filepet genes dispensable for respiration.Click here for filepet mutants that show a wild-type pattern of mitochondrial translation products.Genes required for respiratory activity in class I and III Click here for fileGenes possibly affecting mitochondrial function in combination with acquired defects.Click here for filePrimers used to confirm the identity of yeast deletion mutants.Click here for file


We report 2 cases of Sin Nombre virus (SNV) infection in field workers, possibly contracted through rodent bites. Screening for antibodies to SNV in rodents trapped in 2 seasons showed that 9.77% were seropositive. Quantitative real-time PCR showed that 2 of 79 deer mice had detectable titers of SNV RNA. Peromyscus maniculatus) is its main reservoir. Since the first reported outbreak of the disease in 1993 in the southwestern United States, rodent serologic surveys have confirmed that SNV is present through most of the range where deer mice exist, including Colorado (Hantaviruses are rodent-borne viruses that in the Americas have been implicated as the causative agents of hantavirus cardiopulmonary syndrome (HCPS)  but without cardiorespiratory compromise. A strip immunoblot assay identified immunoglobulin (Ig) M and IgG against SNV N and Gn proteins. On July 6, 2005, a 22-year-old IgM- and IgG-seropositive woman was admitted to Boulder Community Hospital with fever and dyspnea; she subsequently experienced bilateral lung infiltrates and thrombocytopenia . She required oxygen supplementation but recovered almost completely by July 11. She reported performing fieldwork in the same period as did patient 1 but with no overlap among the sites (the distances between sampling sites where the 2 field workers most probably contracted their infections ranged from 6.4 to 9.8 km). Both patients engaged in field activities involving manipulating traps and rodents in areas where deer mice were seropositive for SNV .>1 times by rodents, including both case-patients. Patient 2 was bitten twice, with 1 bite resulting in bleeding despite the worker’s use of nitrile gloves. Patient 1 reported being bitten by a vole (Microtus sp.) on June 2, and patient 2 was bitten by 2 deer mice on June 14. Their wounds were treated by immediate cleaning and bandaging. Patient 2 also applied an antimicrobial ointment before bandaging her bleeding wound.Along with the 2 patients, another 15 field workers were surveyed to assess possible exposures to SNV. Ninety-five questions were asked involving, among others, contacts with rodents and use of personal protective measures and equipment. Most  reported previous experience with rodents in the field; all workers were required to wear nitrile gloves and use a powered air-purifying respirator when handling animals. No differences in risk exposure to contract hantavirus were evident between infected and noninfected persons. Six persons reported having been bitten Cynomysludovicianus) were also trapped at a subset of these study sites during June–July 2005 with live traps . A total of 1,868 animals from 10 mammalian species were captured during both trapping periods  during August–September 2005. A total of 44 sites were sampled during both trapping periods by using live traps  in grids for 4 consecutive nights. Prairie dogs , which carry El Moro Canyon virus, had higher seroprevalence. Two (2.5%) of 81 hispid pocket mice (Chaetodipus hispidus) were also positive but are unlikely to play an epidemiologic role. Small mammal capture frequencies varied during the 2 sampling periods; seroprevalence for pocket mice, prairie voles, and harvest mice increased, and that for meadow voles (M. pennsylanicus) decreased. Seroprevalence among deer mice was higher during May–June (when the field workers contracted their infections) than in August–September.We screened blood samples by strip immunoblot assay for antibodies against SNV N protein  quantitative real-time PCR on a subset of 79 (of 187) samples from deer mice that had detectable antibodies to SNV N antigen. The samples selected for PCR analysis were those for which the volume of blood was deemed sufficient (>25 μL) to carry out a satisfactory RNA extraction. We chose 25 μL as the minimal amount for detecting SNV small segment RNA by nested reverse transcription–PCR on the basis of a spiking experiment in which 5 μL of lung homogenate from an infected deer mouse had been added to 20 μL of blood from an uninfected deer mouse, resulting in a positive finding. The equivalents of 10-μL aliquots of total blood RNA  were subjected to quantitative real-time PCR with primers, probes, and PCR conditions as described . This loThe primary mode of hantavirus transmission to humans is through rodent excreta and secretions through the aerosol route (Our results suggest that detecting SNV RNA of sufficient magnitude (>80 copies/mL) to score as positive in TaqMan assays might be uncommon in the natural reservoir. Therefore, high loads of SNV RNA might not be a major factor in virus transmission in the wild. Alternatively, SNV might cause only a brief RNA viremia in wild deer mice (


Eighty-two per cent of tumour sections from 105 patients with lung cancer showed positive immunocytochemical localization of an anti-carcinoembryonic antigen (CEA) immunoglobulin free of antibody to normal cross-reacting antigen (NCA). The highest incidence was found in adenocarcinomas, and no association between staining and disease stage was found. There was a relationship between positive-staining tumours and preoperative and postoperative serum CEA levels of greater than or equal to 20 ng/ml, but the high incidence of CEA+, less than 20 ng/ml serum patients indicated that immunocytochemical localization was of little value in selecting patients for sequential serum monitoring. Staining for CEA was not prognostic but a preoperative serum CEA levels greater than or equal to 20 ng/ml was associated with a poor prognosis in patients undergoing radical surgery for lung cancer (P = 0.043). this prognostic effect of CEA was seen mainly in patients whose tumours showed the greatest immunocytochemical localization (P = 0.017) and in Stage III patients (P = 0.04).


The "ideal" timing and modality of fracture fixation for unstable thoracolumbar spine fractures in multiply injured patients remains controversial. The concept of "damage control orthopedics" (DCO), which has evolved globally in the past decade, provides a safe guidance for temporary external fixation of long bone or pelvic fractures in multisystem trauma. In contrast, "damage control" concepts for unstable spine injuries have not been widely implemented, and the scarce literature in the field remains largely anecdotal. The current practice standards are reflected by two distinct positions, either (1) immediate "early total care" or (2) delayed spine fixation after recovery from associated injuries. Both concepts have inherent risks which may contribute to adverse outcome.We hypothesize that the concept of "spine damage control" – consisting of immediate posterior fracture reduction and instrumentation, followed by scheduled 360° completion fusion during a physiological "time-window of opportunity" – will be associated with less complications and improved outcomes of polytrauma patients with unstable thoracolumbar fractures, compared to conventional treatment strategies.We propose a prospective multicenter trial on a large cohort of multiply injured patients with an associated unstable thoracolumbar fracture. Patients will be assigned to one of three distinct study arms: (1) Immediate definitive (anterior and/or posterior) fracture fixation within 24 hours of admission; (2) Delayed definitive (anterior and/or posterior) fracture fixation at > 3 days after admission; (3) "Spine damage control" procedure by posterior reduction and instrumentation within 24 hours of admission, followed by anterior 360° completion fusion at > 3 days after admission, if indicated. The primary and secondary endpoints include length of ventilator-free days, length of ICU and hospital stay, mortality, incidence of complications, neurological status and functional recovery.A "spine damage control" protocol may save lives and improve outcomes in severely injured patients with associated spine injuries. In adocedures ,10. Thisocedures . The conocedures ,10.n = 142) versus "late" fixation . The authors found that the early fixation of thoracic spine fractures resulted in a lower incidence of pneumonia, fewer ventilator-dependent days, a shorter ICU stay, and reduced hospital charges [et al. reported data from a randomized prospective pilot study on 27 patients who underwent surgical stabilization of an unstable fracture in the thoracolumbar region from T8 to L2 [n = 12) or more than 3 days (n = 15). The authors found that those patients who underwent spine fixation within 8 hours had a significantly decreased incidence of pulmonary complications, such as pneumonia, and shorter length of ICU and hospital stay, compared to the group with delayed spine fixation [et al. analyzed all published articles in Medline and Embase databases which provided a comparison between different time-points of surgical stabilization of thoracic or lumbar spine fractures [While the concepts and timing of fracture fixation for isolated spinal injuries – with or without neurological compromise – are well defined in the pertinent literature, the question about the "ideal" time-point of spine fracture fixation in severely injured patients remains an ongoing topic of debate. Few studies have assessed the impact of timing of spine fracture fixation on non-neurological outcome and complications. In a landmark article, Croce and colleagues performed a retrospective analysis of a prospective database on 291 consecutive patients with unstable spine fractures requiring surgical fixation . Patient charges . More reT8 to L2 . Patientfixation . Althougfixation . This noHowever, due to the lack of high level scientific evidence from prospective randomized trials, a consensus on the "ideal" timing of spine fracture fixation in multisystem trauma has not yet been reached. Advocates of early spine fixation cite multiple intuitive advantages when managing severely injured patients with unstable spine fractures. Prolonged bed rest and the inability of adequate positioning and mobilization of polytrauma patients have been associated with severe posttraumatic complications. These include the development of pressure sores, pulmonary complications, and thromboembolic events. Multiply injured patients are at additional increased risk of sustaining such adverse events secondary to their profound immunological dysfunction, as outlined above ,2. PolytWe hypothesize that the concept of "spine damage control" will provide a safe and effective treatment modality for unstable thoracolumbar fractures in multiply injured patients, associated with less complications and improved outcomes compared to conventional treatment strategies. We define "spine damage control" as a staged procedure of immediate posterior fracture reduction and instrumentation within 24 hours ("day 1 surgery") ,17, follnd therapeutic option of "early total care" for femur shaft fracture fixation  "Early total care" with definitive fixation on day 1; (2) Initial non-operative management with delayed fixation; (3) "Damage control" procedure on day 1, with scheduled conversion to definitive fixation at a later time-point  are currently treated by exclusive posterior fixation in German and Austrian level 1 trauma centers [In contrast, "damage control" spine stabilization concepts in multisystem trauma have been addressed scarcely in the peer-reviewed literature and are based on limited anecdotal reviews or case reports ,17,24. T centers . The aut centers . Impresset al., unpublished observations). Thus, our preliminary data suggest that "spine damage control" represents a save and efficient modality for staged spine fixation in this selected cohort of multiply injured patients , T8/T9 (n = 2), T10/T11 (n = 3), T12/L1 (n = 6), L2 (n = 2), L3, L4. None of the patients died as a result of their injuries, or as a consequence of an overly aggressive spine fixation strategy  Immediate definitive (anterior and/or posterior) fracture fixation within 24 hours of admission(2) Delayed definitive (anterior and/or posterior) fracture fixation at > 3 days after admission;(3) "Spine damage control" procedure by posterior reduction and instrumentation within 24 hours of admission, with a staged anterior completion fusion at > 3 days, if indicated.nd intervention for anterior 360° completion fusion, as determined by biomechanical (anterior column stabilization) and/or neurological  indications. The 2nd procedure should be performed at > 3 days, in order to bridge the acute phase of hyperinflammation and to ensure adequate resuscitation from traumatic hemorrhage and coagulopathy, in order to reduce the intraoperative risk of bleeding from cancellous bone and epidural veins [For selected patients, the posterior "spine damage control" fixation may be considered the definitive treatment, e.g. in case of a pure osseous flexion/distraction type injury . All other patients may benefit from an adjunctive 2al veins .et al., unpublished observations).Performing a randomized prospective trial on this topic does not appear ethically feasible. This is based on the consideration that "spine damage control" is a safe strategy for multiply injured patients and that the allocation to a "delayed care" or "early total care" treatment arm will put the patient's safety at risk. In fact, we have implemented the "spine damage control" practice as a mandatory clinical care standard at Denver Health Medical Center in 2008. This protocol mandates the clearance from log-roll precautions in any patient with a thoracolumbar fracture within 24 hours of admission, either by clearance and mobilization of patients with stable fracture patterns, or by early posterior reduction and instrumentation on day 1. Preliminary prospective data from our institution show a significant reduction of pressure sores and pulmonary complications after implementation of this clinical care standard, suggesting that "spine damage control" is feasible and safe for the management of polytrauma patients with unstable thoracolumbar fractures  > 15 points, presence of unstable thoracolumbar fracture (AO/OTA location 52- or 53-) with or without spinal cord injury (ASIA scores A-E)."in extremis" with an unjustifiable risk of a severe complication or death related to a spine surgical procedure performed on day 1.Age < 18 years, stable thoracolumbar fracture amenable to non-operative treatment, patients Length of ventilator-free days/ICU days.Mortality; rate of complications, including ARDS, MOF, thromboembolic events, pressure sores, infections; long-term clinical outcome. Patients will be followed clinically for up to 1 year after trauma and assessed for radiographic outcome , neurological impairment (ASIA score), and functional outcome . The hypothesis may be confirmed by demonstrating a lower incidence of mortality and complications, and an improved long-term outcome in the "spine damage control" group, compared to the two conventional study groups.In summary, there is currently no consensus on the "ideal" timing and modality of spine fracture fixation in multiply injured patients. Delaying surgical fixation of unstable spine fractures in multisystem trauma has been associated with an increased risk of severe complications attributed to restrictions in mobilization by maintaining prolonged log-roll precautions. On the other hand, early spine fixation within the first 48 hours after trauma  has been associated with a significantly increased mortality , which iThe authors declare that they have no competing interests.PFS wrote the first version of the manuscript. MAF designed the graphic artwork in figures


Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. To date, the disease process is still poorly understood. In this study, brain tissue samples from animals naturally infected with BSE were analysed to identify differentially regulated genes using Affymetrix GeneChip Bovine Genome Arrays. A total of 230 genes were shown to be differentially regulated and many of these genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response and transcription. Seventeen genes are associated with the endoplasmic reticulum (ER) and 10 of these 17 genes are involved in stress related responses including ER chaperones, Grp94 and Grp170. Western blotting analysis showed that another ER chaperone, Grp78, was up-regulated in BSE. Up-regulation of these three chaperones strongly suggests the presence of ER stress and the activation of the unfolded protein response (UPR) in BSE. The occurrence of ER stress was also supported by changes in gene expression for cytosolic proteins, such as the chaperone pair of Hsp70 and DnaJ. Many genes associated with the ubiquitin-proteasome pathway and the autophagy-lysosome system were differentially regulated, indicating that both pathways might be activated in response to ER stress. A model is presented to explain the mechanisms of prion neurotoxicity using these ER stress related responses. Clustering analysis showed that the differently regulated genes found from the naturally infected BSE cases could be used to predict the infectious status of the samples experimentally infected with BSE from the previous study and vice versa. Proof-of-principle gene expression biomarkers were found to represent BSE using 10 genes with 94% sensitivity and 87% specificity. Sc) of the cellular prion protein (PrPC) and it is further thought that the accumulation of the misfolded prion protein leads to disease Sc is characterized by a high β-sheet content and resistance to protease treatment. In addition to the accumulation of PrPSc, the pathological features of prion diseases in the brain of affected subjects include neuronal cell loss and vacuolation. Prion diseases have long incubation periods prior to the onset of clinical signs.Transmissible spongiform encephalopathies (TSEs), also termed prion diseases, are fatal, neurodegenerative diseases including Creutzfeldt-Jakob disease (CJD) in humans, scrapie in goats and sheep and bovine spongiform encephalopathy (BSE) in cattle BSE was first discovered in 1986 Sc in the animal differs from that of BSE; there is less PrPSc accumulation in the brain stem and the biochemical signature of PrPSc is different.In recent years atypical bovine spongiform encephalopathy has been identified The pathogenesis of BSE is still poorly understood. In a previous gene expression study using brain tissue samples from cattle experimentally infected with BSE, we have demonstrated that the largest number of differentially regulated genes is detected at 21 months post inoculation, suggesting that there are many pathogenic processes in the animal brain even prior to the detection of infectivity in the CNS of these orally dosed cattle To further understand the pathogenesis of BSE and to explore the possibility of using gene expression profiles as biomarkers, we analysed brainstem RNA samples from confirmed naturally infected cases of BSE (field cases) in cattle and from healthy controls.The expression of genes in the brain of naturally infected BSE samples was compared with negative controls. In order to identify differentially regulated genes in BSE, the following stringent conditions were set with two filters: 2 fold change and one-way ANOVA with the p value being 0.05. 409 probe sets  were identified as differentially regulated between the BSE infected (n = 14) and negative (n = 12) samples. After removal of duplications, 230 genes were identified and these genes are listed in Only 18 genes (8%) were down-regulated and 212 (92%) genes were up-regulated after the repeated or un-annotated probe sets were removed . Each stThe largest functional group amongst the 230 identified genes was the genes involved in transport (39 genes), followed by the membrane protein group (25 genes), the metabolism group (20 genes) and the DNA and RNA binding group  and KCNB2, TNFRSF5 (down-regulated) .Clustering analysis using the 409 probe sets showed that the samples were divided into two groups, Group A contained only negative control samples, while Group B contained all the BSE infected samples plus one negative control sample . This anThere were 17 differentially regulated genes whose products are associated with the ER  is induced to restore cell function by reduction in newly translated proteins entering into the ER, by an increase in the capacity for protein folding In our previous BSE time course study, 205 differentially regulated probe sets (corresponding to 114 genes) have been used to show that preclinical animals at 45 months post inoculation (mpi) cluster with cases positive for BSE and allowed the prediction that they are indeed preclinical and close to developing BSE In a reverse analysis, the 409 probe sets identified in this study were used for clustering the samples from the time course study The analyses above indicate that either the genes from the time course study or the field case samples could be used to predict the infection status. However, it would not be practical to apply all 409 or 205 probe sets as biomarkers to represent BSE. A group of 10 genes were sought to represent BSE from these 230 genes listed in These 10 genes above were then used to classify the preclinical samples from the time course study with clustering analysis. The clustering analysis produced two groups: three negatives and three 6 mpi samples being one group and three positives and 45 mpi samples being the other with 100% (5/5) sensitivity and 100% (6/6) specificity . TherefoIn this study, 230 genes were found to be differentially regulated between BSE field cases and controls . These gTo cope with accumulation of misfolded proteins, ER stress induces ER associated protein degradation I  ERAD II is also known as autophagy. It is a pathway of self-degradation of cellular components in which autophagosomes sequester organelles or protein aggregates and fuse with lysosomes for degradation. When the production of misfolded proteins exceeds the capacity of ER chaperones and ERAD I, misfolded and aggregated proteins are targeted by the aggresome-autophagy pathway Sc. The question, therefore, is how much these ER related pathogenic events contribute to fatal prion diseases in general. When the GPI anchor of the PrP protein is removed, the transgenic mice infected with scrapie, also a prion disease, can survive up to 400–600 days post infection (dpi) without clinical scrapie, while the wild type controls develop clinical signs within 140–160 dpi Sc than clinically sick controls. The results indicate that infectivity (PrPSc accumulation) and toxicity can be uncoupled. One model to explain it is intra neuronal generation of a toxic intermediate Sc accumulation in the ER is because the ER quality control system senses the misfolded forms of PrP and ER chaperones retain them in the ER for folding or degradation by ERAD I. PrPSc is protease resistant so that the rate of removing the misfolded protein is slow; while more and more PrPC converts to PrPSc. Eventually, PrPSc accumulation causes ER stress and the subsequent activation of the UPR and ERAD I and II. Prolonged ER stress leads to cell death Sc bypasses the ER quality control? There are lines of evidence that the anchorless prion protein is not detected by the ER quality control system Sc can pass the ER efficiently, there is no toxicity to cause clinical scrapie. Since the cell is not under ER stress, the ERAD pathways are not activated. As a result, more PrPSc accumulates in the brain of transgenic mice with anchorless PrP than in the brain of the wild type controls.In this study, the analyses suggest that ER stress might be involved in BSE pathogenesis and that the UPR, ERAD I and II might all be activated in a concerted effort to rid the cell of harmful PrPBoth the current field case study and the previous time course study were carried out with brainstem tissues infected with BSE res is still the only reliable method. There are some reported potential biomarkers for the disease such as 14-3-3 protein Considerable efforts have been made to find biomarkers for the prion diseases, especially in the early stage of the incubation period. To date, the detection of PrPIn conclusion, gene expression analysis suggests that BSE infection caused ER stress and the UPR, ERAD I and II might be induced in response to ER stress. Clustering analysis showed that the differentially regulated genes could be used to predict infection status. Ten genes were selected to represent gene expression state in BSE, which might eventually be used as biomarkers.Brainstem tissues from 100 confirmed cases of BSE in cattle were supplied by the TSE archive at the Veterinary Laboratories Agency, UK. The animals were females, between 4 and 10 years old that had been diagnosed clinically and killed on farm. The major breed was Holstein/Friesian and other breeds were: Limousin Cross, Guernsey, Hereford Cross and Brown Swiss. The negative controls (100 brainstem tissue samples) were from LGC Forensics  and were comparable in breed, sex and age with the naturally infected BSE samples. Since all samples were from the Archives, approval from the Ethics Committee was not necessary.The preparations of samples and reagents were carried out according to the Affymetrix GeneChip Expression Analysis manual and as described in the previous study post hoc tests, without assume variances equal for the parametric test.Two filters were used to find differently regulated genes: 2 fold change and the one way ANOVA statistical analysis with the parameters of 0.05 for p-value cutoff, multiple testing correction and Student-Newman-Keuls for the Cell-free extracts (60 µg protein) were loaded on 12% 1-D SDS PAGE (Invitrogen) and resolved proteins from several mini-gels were transferred to the same PVDF membrane (Millipore) so that one set of samples was used to monitor protein loading using β-Actin. The blots were immuno-stained with mouse monoclonal anti-β-Actin IgG (Santa Cruz Biotech), rabbit polyclonal anti-Grp78  and rabbit polyclonal anti-Chop (BioLegend). The protein bands were visualized by using secondary antibodies, alkaline phosphatase conjugated IgGs  and the ECL developer kit (Amersham). The images were captured by Fluor-S MultiImager (Bio-Rad) and the protein bands were quantified by the Quantity One software (Bio-Rad).TCC ATT AAC GAT TCT AAA TAA AGG AAA CT, 5′-TGC TAT GGA AAA AAG CCC CC and the probe, FAM-5′-TGG TGT TGC TAT GCG TGA GAT CCT CTC C; for DNAJ, 5′-TCT GTG AAA ATA AAG CAG GAG TGA A, 5′- AGT GAG AAA CAG CCA AAA TAC TGA AC and the probe, FAM-5′- CCT TTG CAG ACT TCA GAC TGG TTG GAT TTC; for KCNB2, 5′- TGA TGA CTT CTT AGA GCT CCA GGG, 5′-CAA GCA GTT TGG GCT GGA GT and the probe, FAM-5′-AGG AGG CCG GAC AAG CAG GCA; for HS70, 5′- GGA CTT TGG TCT TGC CCT ATA TTT AC, 5′-CAC ACT CAC TAT AAC ATA CAG AAA TAA CA AAA A and the probe, FAM-5′-TGT GAT GTG TCA GTT TGT TCT ATG ATA AGG TTG TAA TCT C; for TNFRSF5, 5′-CGT GGA GAC GAT TGA TCC G, 5′- AGC ATA AGG TCT CTT GCA CCG and the probe, FAM-5′- AGG ATT TTC CCG GCC CCC ACC.The RNA samples were treated with the DNA free™ kit (Ambion) for 1 h at 37°C to remove any trace of DNA. The treated RNA was then used as a template for cDNA synthesis with the TaqMan reverse transcription kit (Applied Biosystems). The real time PCR was carried out by denaturing at 95°C for 15 s, annealing at 50°C for 2 min and extension at 60°C for 1 min for 40 cycles using an ABI Prism 7700 Sequencing Detector. The GAPDH gene was used as an internal control to normalize the expression levels of target mRNA. The primer sets were chosen by the Primer Express 1.5 for TaqMan software. The sequences of the primer sets were as following: for CD47, 5′-Figure S1Validation of microarray data by RT-PCR. 1 and 2: CD47; 3 and 4: DNAJ; 5 and 6: KCNB; 7 and 8: HS70; 9 and 10: TNFRSF5. The values of gene expression are listed at the top for comparison. No fill: negative controls; Grey: clinical BSE.(0.24 MB TIF)Click here for additional data file.Table S1Unannotated probe sets in the BSE field case study(0.18 MB DOC)Click here for additional data file.


The geometric mean serum HI antibody titers to swine influenza viruses were also significantly higher (p<0.001) among the farm participants. Swine virus seropositivity was significantly (p<0.05) associated with being a farm owner or a farm family member, living on a farm, or entering the swine barn >4 days/week. Because pigs can play a role in generating genetically novel influenza viruses, swine farmers may represent an important sentinel population to evaluate the emergence of new pandemic influenza viruses.We evaluated seropositivity to swine and human H1 influenza viruses in 74 swine farm owners, employees, their family members, and veterinarians in rural south-central Wisconsin, compared with 114 urban Milwaukee, Wisconsin, residents. The number of swine farm participants with positive serum hemagglutination-inhibition (HI) antibody titers  Infections with influenza viruses that circulate within the human population are a common and important cause of respiratory disease in people and result in an average of approximately 20,000 deaths and 114,000 hospitalizations per year in the United States alone . Influen The occurrence of H5N1 and H9N2 virus infections among people in Asia in 1997–1999 ,9 highli Zoonotic infections of humans with swine influenza viruses, first confirmed by isolation of swine influenza viruses from both pigs and their caretaker on a farm in southern Wisconsin in November 1976  Names and contact information for swine farmers living in rural areas of south-central Wisconsin were provided by area swine veterinarians. We contacted these farmers initially by telephone and then, if they were interested in participating, one of the study directors met with them to explain the project’s objectives and procedures. To take part in the study, persons were required to allow home health nurses to collect two blood samples for influenza virus serologic testing and to complete a questionnaire regarding their general health and the nature of their contact with pigs. Participation was also extended to other employees on the farm, spouses and children >7 years of age, and farm veterinarians. A total of 79 participants were initially enrolled, including 76 persons from 22 farms, as well as 3 farm veterinarians. All participants who completed the study were compensated by payment of a $100 honorarium. We chose the time period of this study to correspond with the seasonality of swine influenza. In the northern midwestern United States, swine influenza activity is maximal in the late fall and winter  Farm participants were questioned as to their age and sex, their overall health, the nature of their contact with swine, and their influenza virus vaccination history. The specific questions asked of each participant are listed in the questionnaire . Each participant was assigned an ID number so that laboratory samples could be assayed without knowledge of personal identifier information. The human serum samples were treated with receptor-destroying enzyme  at 37°C for 18 h to eliminate nonspecific inhibitors of hemagglutination, after which the samples were tested for HA-specific antibodies using a standard hemagglutination-inhibition (HI) assay >40 to either swine virus were compared among the preseason farm participant samples versus the urban control samples by chi-square analysis. The GMTs of the samples from preseason farm participants were compared to the GMTs of the urban control sera by using Wilcoxon rank sum analysis with normal approximation. We also evaluated associations between preseason seropositivity to swine influenza viruses at HI titers >40 or >80 among the farm participants and specific aspects of swine exposure or other variables. These associations were then examined for statistical significance by chi-square or two-sided Fisher’s exact analyses. Multivariate analysis was not done because of the small numbers of participants with elevated preseason titers to swine influenza viruses . The numbers of sera with an HI titer  Seventy-nine swine farming participants were initially enrolled, including 20 farm owners (ages 28–59 years), 14 spouses of farm owners (34–57 years), 12 children of farm owners (7–21 years), 21 farm employees (19–43 years), 9 spouses of farm employees (21–39 years), and 3 veterinarians (29–54 years). In total, the farm participants included 44 males of median age 37 (range 13–59 years) and 35 females of median age 34 (range 7–57 years). Of these participants, two people moved out of the area during the study and did not participate further, and three people chose to withdraw from the study before their second blood samples were collected. The preseason samples from these five participants were not included in the analyses. Two participants (both with elevated titers to swine viruses) did not complete the questionnaire. Serologic data from these two participants were included in the comparison of preseason titers of farm participants versus controls but could not be used to assess variables associated with seropositivity to swine viruses. The control sera were from 54 males of median age 32 (range 2–58 years) and 60 females of median age 34 (range 4–54 years). To interpret the HI titers as showing differences in exposure to swine versus human influenza viruses, we first had to demonstrate that no minimal serologic cross-reactivity in HI assays between the human and swine reference strains existed. We compared HI titers by using virus-specific sheep and ferret reference sera  and clea>40 (titer range 40–160) against either A/NEB or Sw/IND; 15/17 were seropositive to both swine viruses. These participants included seven farm owners (range 41–55 years), seven family members of farm owners (range 7–54 years), a 33-year-old farm employee, a 38-year-old family member of a farm employee, and a 43-year-old veterinarian. In contrast, only 1/114 of the urban control serum samples (from a 41-year-old) had a positive HI titer against a swine virus (HI titer=40 against only A/NEB). The difference in the number of seropositive samples between the farm participant and urban control cohorts was statistically significant (p<0.001). Similarly, the GMTs of the preseason serum samples from the farm participants to both swine-lineage viruses (A/NEB and Sw/IND) were significantly higher (p<0.001) than the GMTs of the samples from the urban control participants . In contrast, the farm participants’ GMTs to the reference human H1 (A/JOH) and H3 (A/NAN) viruses were not significantly different from those of the urban control samples  was associated with swine virus seropositivity; having received the swine flu vaccine in 1976–77 or having ever received any influenza virus vaccine was also associated with swine virus seropositivity.  Each of the variables on the questionnaire  was inve Although zoonotic infections with swine influenza viruses have been documented previously ,15, the >50 years of age was associated with swine virus seropositivity. In an earlier study, Schnurrenberger et al. >50 years of age were farm owners or farm family members. However, several factors indicate that, although age may play a role in seropositivity to swine viruses, exposure to swine is a more dominant factor. Farmers and their family members were significantly more likely than employees and their family members to have elevated titers to swine viruses; farmers and their families were also more likely to have exposure to swine and to be exposed over a longer period of time. Specifically, 88% of the farm owners and their families lived on the swine farm, compared to 7% of the employees and their families. Furthermore, of the farm owners and their families who lived on the farm, 77% had lived there >11 years. Farm owners had significantly more years in swine production than their employees . Among younger study participants (<50 years of age), 21% and 18% of the farm family members had titers of >40 and >80 to swine viruses, respectively, compared to only 7% and 4% for employees and their family members. Although these differences among the younger participants were not statistically significant (p=0.09 to 0.14), they are consistent with the pattern of elevated titers seen among those with a higher level of exposure to pigs. Finally, our control population was of the same overall age distribution as our farm participants, yet only a single 41-year-old person among these controls was seropositive to a swine virus. Two factors not directly related to swine contact were also statistically associated with seropositivity to swine viruses in our study. First, being >40, but not at >80. Although we do not have historical data for our urban control samples, we have no a priori reason to suspect that these people would have had substantially different vaccination histories. However, only 1/114 of these participants demonstrated a titer >40 to a swine virus. Likewise, the proportions of employees and their family members who received the swine influenza vaccine (4%) or other influenza vaccines (32%) were not significantly different from the proportions of farm family members who received the swine influenza vaccine (6%) or other influenza vaccine (22%), but farm family members were significantly more likely to have elevated titers to swine viruses. Regarding having received the 1976–77 swine influenza vaccine, antibodies produced against that vaccine would not likely be present at detectable levels 20 years later. However, studies have shown that vaccination with more recent human influenza A (H1N1) viruses can boost titers to swine-like viruses in those previously exposed  A second factor unrelated to swine contact significantly associated with swine virus seropositivity was having received either the swine influenza virus human vaccine in 1976–77 or ever having received any human influenza virus vaccine. However, vaccination status alone most likely did not determine seropositivity to swine viruses among our farm participants. Vaccination was only associated with seropositivity at a titer  Because a relatively small number of zoonotic swine influenza virus infections have been documented by virus isolation, whether infections with swine influenza viruses are clinically different than infections with routine human influenza viruses remains unclear. Our data suggest that aggressively pursuing virus isolation when people involved in swine farming have influenza-like illnesses would be valuable. In this way, retrospective studies of the clinical appearance of a larger number of zoonotic swine influenza cases may be possible. Our findings suggest a second issue. Pigs may serve as hosts for the adaptation of avian viruses to replication in mammalian species