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PubMed2007

Molecular features of astrovirus associated with a gastroenteritis outbreak in an aged-care centre.

The study presented here was conducted in order to gain a better understanding of the role of astroviruses (AsVs) in outbreaks of gastroenteritis among the elderly. This report is the first to provide detailed information on the molecular characteristics of an AsV causing an outbreak in an aged-care centre and is the first to clearly establish that individuals infected in such an outbreak were, in fact, elderly. The outbreak under investigation took place in Victoria, Australia, in October 2005. Twelve individuals (mean age +/- standard deviation [SD] 85.5 +/- 12.3 years) became ill during the outbreak from a total population of 86 susceptible residents. The mean duration (+/-SD) of illness was 2.3 +/- 1.6 days; symptoms included diarrhoea, abdominal pain, nausea and headache. No bacterial pathogens were detected. AsV was identified in five faecal specimens using electron microscopy and reverse transcription-polymerase chain reaction methodologies; no other gastroenteritis virus was detected. Nucleotide sequence analysis indicated the AsV identified could be assigned to the 1d lineage of AsV serotype 1 and that the AsV was not a recombinant form. The findings, taken together with previous work, indicate the AsV serotype most commonly associated with gastroenteritis outbreaks among the elderly is serotype 1 AsV.

Aged, 80 and over↗

PubMed2005

An electrokinetically-controlled immunoassay for simultaneous detection of multiple microbial antigens.

An electrokinetically-controlled heterogeneous immunoassay microchip for multiple analyte detection was developed in this study. Numerical simulation was employed to study the transport process in a microfluidic network (microFN). The operation parameters obtained from numerical simulation was then applied to immunoassay experiment. The effectiveness of the automatic electrokinetic control was demonstrated in a separate experiment using fluorescein dye. The immunoassay microchip was made of poly(dimethylsiloxane)(PDMS)/PDMS-coated glass using soft lithography and replica molding. Multi-antigen immobilization was accomplished by adsorbing the antigen molecules onto a PDMS-coated glass slide and by using a microFN. Immobilized lysate antigen of Escherichia coli O157: H7 at different concentrations was assayed and the lower detect limit was 3 microg/mL. The assay also displayed very good specificity, when different microbial lysate antigens were immobilized, including Escherichia coli and Helicobacter pylori, and the primary and secondary antibodies were mixtures of different species. The time required for the immunoassay, from antigen coating to signal detection, was only one hour. While still an un-optimized prototype, this automatic-operating, high-throughput immunoassay microchip shows a great potential in detecting multiple pathogenic infections efficiently for clinical applications.

Antigens, Bacterial↗

PubMed2005

Self-actuated, thermo-responsive hydrogel valves for lab on a chip.

An easy to fabricate, thermally-actuated, self-regulated hydrogel valve for flow control in pneumatically driven, microfluidic systems is described. This microvalve takes advantage of the properties of the hydrogel, poly(N-isopropylacrylamide), as well as the aqueous fluid itself to realize flow control. The valve was designed for use in a diagnostic system fabricated with polycarbonate and aimed at the detection of pathogens in oral fluids at the location of the sample collection. The paper describes the construction and characterization of the hydrogel valves and their application for flow control, sample and reagent metering, sample distribution into multiple analysis paths, and the sealing of a polymerase chain reaction (PCR) reactor to suppress bubble formation. The hydrogel-based flow control is electronically addressable, does not require any moving parts, introduces minimal dead volume, is leakage and contaminant free, and is biocompatible.

Acrylic Resins↗

PubMed2003

Herpes simplex viral pneumonia after coronary artery bypass grafting.

We report a case of herpes simplex viral (HSV) pneumonia as a post-CABG pulmonary complication in a 70-year-old man. Chest radiography on postoperative day 9, showed a glass-like shadow and pleural effusion in the left lung field, and the man's condition began deteriorating rapidly. Bronchofiberscopy to detect the pathogen and a bronchoalveolar lavage with polymerase chain reaction (PCR) yielded a definitive diagnosis of HSV pneumonia. Once therapy with acyclovir was begun, his condition improved markedly. Our case suggests that 1 viral pulmonary infection should be considered as a possible cause in postoperative cardiac patients with unexplained progressive pulmonary infiltrates, and 2 DNA amplification using PCR is rapid--it can be completed within 1 day--and sensitive and specific in diagnosing such infections.

Acyclovir↗

PubMed1985

Hybridization on squashed flies: a method to detect gene sequences in individual Drosophila.

Specific gene sequences can be detected by DNA hybridization to individual Drosophila squashed on cellulose or nylon filters. This "squash-blot" method permits the rapid survey of DNA polymorphism in large Drosophila population samples. It could also be useful for studying chromosome aberrations, departure from diploidy, and detection of pathogenic agents in vector insects.

Animals↗

PubMed1985

Changes in tyrosine hydroxylase, glutamic acid decarboxylase and choline acetyltransferase after local microinoculation of scrapie agent into the nigrostriatal system of the golden hamster.

A microinjection of a homogenate of scrapie agent-infected brain (strain 263 K) into the nigrostriatal system in the golden hamster is followed by the progressive development of the disease which terminates by the death of animals around the 4th month postinoculation. These intracerebral inoculations induce more rapid changes in neuronal activity which can be revealed by the assessment of the specific synthesizing enzymes of neurotransmitter systems. The microinoculation of a homogenate of an infected brain unilaterally into the substantia nigra (SN) provokes a decrease in tyrosine hydroxylase (TH), the synthesizing enzyme for dopamine in the dopaminergic neurones, in the striatum ipsilateral to the injected SN. This biochemical response, specifically induced by the active pathogen, is detectable as soon as the 5th day postinoculation and is detectable towards the 80th day. A return of TH levels to control values is detected after this period. At the end of the incubation period and towards the death of the animals, TH is not different from control TH measured from intact animals. The decrease in TH is concomitant with an increase in striatal glutamic acid decarboxylase (GAD), the synthesizing enzyme for gamma-aminobutyric acid (GABA), measured 20 days postinoculation with no change in choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Studies of the biochemical responses associated locally to the scrapie agent inoculation have been performed at the striatal level. The intrastriatal administration of the infective agent induces 20 days postinoculation an increase in GAD with no change in TH and ChAT. Ninety days postinoculation, a decrease in GAD was detected associated with an increase in TH with no change in ChAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

A two-year study of enteric infections associated with diarrhoeal diseases in children in urban Somalia.

A hospital-based systematic sample of 1667 children with severe diarrhoeal disease was studied in Mogadishu, Somalia, throughout 1983 and 1984. One or more enteric pathogens were found in 61% of the patients. Rotavirus (25%), enterotoxigenic Escherichia coli (11%), Shigella spp. (9%), Aeromonas hydrophila (9%), Giardia lamblia trophozoites (8%), Campylobacter jejuni (8%), and Vibrio cholerae non-O1 (6%) were the most frequently identified pathogens. Age-specific detection rates of enteric pathogens and helminths, seasonal patterns, and relationship of some specific infections with feeding status and main clinical features have been defined for all the sample examined.

Adolescent↗

PubMed1996

Quantitative assessment of murine retrovirus LP-BM5 infection in MAIDS by PCR and anion exchange HPLC.

In vitro amplification of DNA by PCR is a powerful tool to detect small amounts of DNA. It is now widely used for detection of pathogenic agents from extracellular fluids and organs. The use of anion exchange HPLC to quantify the PCR product resulting from the specific amplification of the DNA from the replicative-defective viral DNA responsible for MAIDS is described. This technique allows precise quantification of MAIDS virus DNA in different organs and circumvents the use of radioactivity and gel electrophoresis.

Animals↗

PubMed1996

Isolation of Escherichia coli O157:H7 from some Egyptian foods.

A survey was done in middle Egypt to determine if Escherichia coli O157:H7 was present in 175 samples of raw ground beef, chicken, lamb and unpasteurized milk. The pathogen was detected in 3 of 50 (6%) beef samples, 2 of 50 (4%) chicken samples, 1 of 25 (4%) lamb samples and 3 of 50 (6%) milk samples obtained from slaughterhouses, supermarkets and farmer's homes.

Animals↗

PubMed1995

Current techniques in cell and molecular biology.

Recent advances in the field of molecular biology have revolutionized our understanding of the functioning of living organisms and facilitated the development of robust tools for both diagnosis and treatment of diseases. With particular reference to the field of critical care medicine, development of molecular biology techniques have aided in the following: (1) rapid and highly specific detection of pathogenic infectious agents (eg, Mycobacterium tuberculosis, Pneumocystis carinii, cytomegalovirus, Legionella); (2) development of assays for measurement of circulating cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1 that has helped our understanding of the pathogenesis of the sepsis syndrome; (3) administration of antibodies or soluble receptors to attempt to prevent untoward effects of cytokines such as TNF or IL-1; and (4) the administration of recombinant deoxyribonucleic acid (DNA) or proteins to patients in an attempt to alter the course of a disease such as antioxidant enzymes (superoxide dismutase). The rapidity of progress in this field has been staggering, which necessitates frequent updating of our knowledge for clinicians to put these molecular tools to their best use. This brief review attempts to explain the basic principles of commonly used techniques in molecular biology including recombinant DNA, polymerase chain reaction, DNA libraries, gene therapy, and protein biochemistry in a manner that is understandable to those without an in-depth knowledge of the field.

Cell Physiological Phenomena↗

PubMed1992

Polymerase chain reaction with additional primers allows identification of amplified DNA and recognition of specific alleles.

The use of additional primers in the standard two primer polymerase chain reaction (PCR) is described. This modification allows detection of a target gene in a single reaction, and identification of the amplification product obtained or recognition of a specific allele. The oligonucleotides used are internal to the original amplification primers and amplification-compatible with one of the original primers. Annealing of an additional primer to the target gene as well as to the primary amplification product will lead to the appearance of an additional smaller amplification fragment upon agarose gel electrophoresis of PCR products. Use of one or more allele-specific oligonucleotides as additional primers, in addition to two gene-specific primers, will allow recognition of different alleles of the target gene in a single PCR without further analysis except gel electrophoresis. The general applicability of the method was determined with several PCR assays for the detection of pathogenic bacteria.

Alleles↗

PubMed1991

T cell receptor beta-chain usage in experimental autoimmune uveoretinitis.

Genomic rearrangements to the T-cell receptor (TCR) V beta 8 gene locus were examined in T cells derived from the lymph nodes of Lewis rats immunized with either S-Antigen or peptides derived from interphotoreceptor retinoid binding protein (IRBP). The cells used in these studies are from T-cell lines that have been selected by several cycles of antigen/IL-2 stimulations, or clones isolated from these lines. No apparent rearrangement of the V beta 8 gene was observed by Southern analysis, suggesting that if indeed there are T cells using V beta 8 gene elements they represent small proportions of the cells in these T-cell lines that induce EAU (uveitogenic T cells) and that the lines may consist of large numbers of clones. On the other hand, we have demonstrated V beta 8 gene expression in uveitogenic T-cell populations by Northern analysis and by polymerase chain reaction (PCR). Although V beta 8 gene transcripts were detectable in pathogenic, but not in non-pathogenic, T-cell lines using a V beta 8 cDNA probe, RNA from pathogenic T cell lines did not hybridize to another probe specific for rat V beta 8.2. Taken together, these results suggest that, unlike the T-cell lines that mediate experimental allergic encephalomyelitis (EAE), some T-cell lines that induce EAU do not predominantly express V beta 8.2 gene but other member(s) of the V beta 8 family.

Animals↗

PubMed2004

Class switch recombination in B lymphopoiesis: a potential pathway for B cell autoimmunity.

Pathogenic autoantibodies detected in autoimmune diseases are predominantly IgG isotypes, reflecting the generation and activation of an autoimmune memory B cell repertoire. It is not completely understood how such autoreactive cells are generated and escape central and/or peripheral tolerance mechanisms, and several models to explain this have been proposed. It is generally thought that B lymphocytes utilize IgM receptors for development and tolerance establishment, whereas IgG receptors are primarily used to promote memory formation and signal for memory-type responses. In here we review recent findings suggesting that spontaneously occurring class switch recombination in B lymphopoiesis confer B lymphocytes with a novel developmental pathway that is driven by non-IgM receptors. The physiological relevance of this developmental pathway in generating an autoimmune memory repertoire, as well as a Fas-dependent mechanism regulating it, is discussed.

Animals↗

PubMed2006

The transmembrane domain directs TLR9 to intracellular compartments that contain TLR3.

Toll-like receptors (TLRs) are type I transmembrane (TM) proteins indispensable for sensing microbial and viral infection. Despite their conserved primary structures, some TLRs that detect pathogen-derived nucleic acids (TLR3, TLR7, TLR8, and TLR9) are retained in the cytoplasm. The intracellular localization of TLR9 is important for its ability to discriminate self- and non-self DNA, but the mechanism by which it is retained in the cytoplasm is unclear. In the present study, we found that the TM domain of TLR9 directs its intracellular localization. The TM domain of TLR9 also targets CD25, a heterologous type I TM protein, to intracellular compartments that contain TLR9. We also found that TLR9 generally co-localizes with TLR3, although its linker region, not its TM domain, directs intracellular localization of TLR3. These data demonstrate that the TM domain of TLR9 is a critical regulatory element that targets TLR9 to its intracellular location.

Animals↗

PubMed2006

DNA biosensor based on the electrochemiluminescence of Ru(bpy)3(2+) with DNA-binding intercalators.

This paper reports a novel detection method for DNA hybridization based on the electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) with a DNA-binding intercalator as a reductant of Ru(bpy)(3)(3+). Some ECL-inducible intercalators have been screened in this study using electrochemical methods combined with a chemiluminescent technique. The double-stranded DNA intercalated by doxorubicin, daunorubicin, or 4',6-diamidino-2-phenylindole (DAPI) shows a good ECL with Ru(bpy)(3)(2+) at +1.19 V (versus Ag/AgCl), while the non-intercalated single-stranded DNA does not. In order to stabilize the self-assembled DNA molecules during ECL reaction, we constructed the ECL DNA biosensor separating the ECL working electrode with an immobilized DNA probe. A gold electrode array on a plastic plate was assembled with a thru-hole array where oligonucleotide probes were immobilized in the side wall of thru-hole array. The fabricated ECL DNA biosensor was used to detect several pathogens using ECL technique. A good specificity of single point mutations for hepatitis disease was obtained by using the DAPI-intercalated Ru(bpy)(3)(2+) ECL.

Biosensing Techniques↗

PubMed2005

Effectiveness of hydroxyapatite-vancomycin bone cement in the treatment of Staphylococcus aureus induced chronic osteomyelitis.

In the field of local application of antimicrobials, a number of novel drugs and/or new drug delivery systems have been developed in recent years. The present study aimed to investigate hydroxyapatite cement (HAC) as a carrier for vancomycin in the treatment of chronic osteomyelitis due to Staphylococcus aureus strains with various mechanisms of resistance. The release of vancomycin from standard test cylinders was determined in vitro and the efficacy of the delivery system was measured in vivo using a rabbit model of chronic osteomyelitis. First, powdered HAC was mixed with vancomycin at 80, 160 and 240 mg/g. After hardening, formed cylinders were eluted in phosphate buffer and antibiotic release was measured by agar diffusion. High levels of release (1512+/-318 to 1937+/-336 microg/ml) were obtained for 12 to 20 days depending on the dosage of vancomycin. Additionally, bone infection was induced in the tibia of 30 New Zealand white rabbits by injecting either a methicillin-resistant S. aureus strain (MRSA) or a S. aureus strain with a small colony variant (SCV) phenotype. After 3 weeks (chronic infection), all animals were treated by debridement. Moreover, group 1 (challenged with SCVs) and group 2 (challenged with MRSA) were treated by filling the marrow with HAC alone, whereas in groups 3 (SCVs) and 4 (MRSA) the marrow was filled with HAC/vancomycin (160 mg/g). After 6 weeks all animals were sacrificed. At 3 weeks, pathogens were detected in 24 of 30 animals. All swabs of the control groups, positive for S. aureus on day 21, were also positive on day 42 and S. aureus strains recovered were shown to be clonal to the strains used for induction of osteomyelitis. By contrast, no growth was found in the treatment group following 7 days of incubation in BHI bouillon. HAC/vancomycin-treated animals showed no histological evidence of infection on day 42. In the other groups, different stages of chronic osteomyelitis were found histologically. No local or systemic side effects due to HAC or vancomycin were seen. HAC is an effective carrier material for antibiotic compounds even in refractory infections due to MRSA or S. aureus SCVs.

Animals↗

PubMed2005

Rapid PCR confirmation of E. coli O157:H7 after evanescent wave fiber optic biosensor detection.

Escherichia coli O157:H7 is an enteric pathogen of public health importance, which is monitored by several government agencies. Many rapid detection tests have been developed to identify foodstuff and water supplies contaminated by E. coli O157:H7. However, these methods can be time consuming (24-48 h) due to the need to culture the bacteria to confirm detection results. Fiber optic biosensors can rapidly detect pathogens from complex matrices, yet confirmation tests can take up to 10h to complete. In addition, fiber optic biosensors can also be used to reduce the impact of PCR inhibitors present in complex matrices such as food and water. This paper presents methodologies to reduce the time necessary for confirmation from 10 to about 2 h, by direct PCR of bacteria from the fiber optic waveguides without the need for culture or enrichment steps.

Antibodies, Bacterial↗

PubMed2005

Two Pseudomonas syringae type III effectors inhibit RIN4-regulated basal defense in Arabidopsis.

Plant cells have two defense systems that detect bacterial pathogens. One is a basal defense system that recognizes complex pathogen-associated molecular patterns (PAMPs). A second system uses disease-resistance (R) proteins to recognize type lll effector proteins that are delivered into the plant cell by the pathogen's type III secretion system. Here we show that these two pathways are linked. We find that two Pseudomonas syringae type III effectors, AvrRpt2 and AvrRpm1, inhibit PAMP-induced signaling and thus compromise the host's basal defense system. RIN4 is an Arabidopsis protein targeted by AvrRpt2 and AvrRpm1 for degradation and phosphorylation, respectively. We find that RIN4 is itself a regulator of PAMP signaling. The R proteins, RPS2 and RPM1, sense type III effector-induced perturbations of RIN4. Thus, R proteins guard the plant against type III effectors that inhibit PAMP signaling and provide a mechanistic link between the two plant defense systems.

Arabidopsis↗