Effects of the probiotic Lactobacillus animalis in murine Mycobacterium avium subspecies paratuberculosis infection.

BACKGROUND: MAP is a suspected zoonotic pathogen and the causative agent of Johne's Disease in cattle and other ruminant animals. With over $1 billion dollars in loss to the dairy industry due to Johne's Disease, efforts to eliminate or reduce MAP from cattle are of importance. The purpose of this study was to determine if daily intake of probiotics could eliminate or reduce Johne's Disease associated symptoms and pathogenesis by MAP. Post infection, animals are often asymptomatic carriers with limited shedding of the pathogen, proving early detection to be difficult. Disease and symptoms often appear 3-4 years after infection with antibiotic treatment proving ineffective. Symptoms include chronic gastrointestinal inflammation leading to severe weight-loss from poor feed and water intake cause a wasting disease. These symptoms are similar to those found in individuals with Crohn's Disease (CD); MAP has been implicated by not proven to be the causative agent of CD. Probiotics administered to livestock animals, including dairy and beef cattle have demonstrated improvements in cattle performance and health. Our objectives included determining the benefits of Lactobacillus animalis (strain name: NP-51) in MAP infected BALB/c mice by evaluating systemic and gastrointestinal response by the host and gut microbiota. Male and female animals were fed 1×106 CFU/g probiotics in sterile, powdered mouse chow daily and infected with 1 × 107 CFU/ml MAP and compared to controls. Animals were evaluated for 180 days to assess acute and chronic stages of disease, with sample collection from animals every 45 days. MAP concentrations from liver and intestinal tissues were examined using real time-PCR methods and the expression of key inflammatory markers were measured during MAP infection (interferon-gamma [IFN-Υ], Interleukin-1α, IL-12, IL-10, IL-6, and Tumor necrosis factor alpha [TNF-α]). RESULTS: Our results demonstrate administration of probiotics reduces production of IFN-Υ and IL-6 while increasing TNF-α and IL-17 in chronic disease; healthful immune responses that reduce chronic inflammation associated to MAP infection. CONCLUSIONS: We observed that the immune system's response in the presence of probiotics to MAP contributes towards host health by influencing the activity of the immune system and gut microbial populations.

Wound Healing Finally Enters the Age of Molecular Diagnostic Medicine.

BACKGROUND: Many wounds are difficult to heal because of the large, complex community of microbes present within the wound. THE PROBLEM: Classical laboratory culture methods do not provide an accurate picture of the microbial interactions or representation of microorganisms within a wound. There is an inherent bias in diagnosis based upon classical culture stemming from the ability of certain organisms to thrive in culture while others are underrepresented or fail to be identified in culture altogether. Chronic wounds also contain polymicrobial infections existing as a cooperative community that is resistant to antibiotic therapy. BASIC/CLINICAL SCIENCE ADVANCES: New methods in molecular diagnostic medicine allow the identification of nearly all organisms present in a wound irrespective of the ability of these organisms to be grown in culture. Advances in DNA analyses allow absolute identification of microorganisms from very small clinical specimens. These new methods also provide a quantitative representation of all microorganisms contributing to these polymicrobial infections. CLINICAL CARE RELEVANCE: Technological advances in laboratory diagnostics can significantly shorten the time required to heal chronic wounds. Identification of the genetic signatures of organisms present within a wound allows clinicians to identify and treat the primary organisms responsible for nonhealing wounds. CONCLUSION: Advanced genetic technologies targeting the specific needs of wound care patients are now accessible to all wound care clinicians.

Green tea polyphenols avert chronic inflammation-induced myocardial fibrosis of female rats.

OBJECTIVE: Green tea proposes anti-inflammatory properties which may attenuate chronic inflammation-induced fibrosis of vessels. This study evaluated whether green tea polyphenols (GTP) can avert fibrosis or vascular disruption along with mechanisms in rats with chronic inflammation. TREATMENTS: Forty 3-month-old female rats were assigned to a 2 (placebo vs. lipopolysaccharide, administration) × 2 (no GTP vs. 0.5% GTP in drinking water) factorial design for 12 weeks. METHODS: Masson's trichrome staining evaluated myocardial fibrosis in coronary vessels and surrounding myocardium. Whole blood specimens were counted for differentials. Spleen tumor necrosis factor-α (TNF-α) mRNA expression was determined by real-time RT-PCR. Data were analyzed by two-way analysis of variance (ANOVA) followed by mean separation procedures. RESULTS: After 12 weeks, lipopolysaccharide administration induced myocardial fibrosis in vessels and surrounding myocardium, spleen TNF-α mRNA expression, and leukocytes, while GTP supplementation in drinking water significantly averted such observation. CONCLUSIONS: GTP attenuates myocardial fibrosis through a suppression of chronic inflammation and innate immune responses.

Green tea polyphenols mitigate bone loss of female rats in a chronic inflammation-induced bone loss model.

The purpose of this study was to explore the bioavailability, efficacy and molecular mechanisms of green tea polyphenols (GTP) related to preventing bone loss in rats with chronic inflammation. A 2 [placebo vs. lipopolysaccharide (LPS)]×2 (no GTP vs. 0.5% GTP in drinking water) factorial design enabled the evaluation of effects of LPS administration, GTP levels, and LPS×GTP interaction. Urinary GTP components and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels were determined by high-pressure liquid chromatography for bioavailability and molecular mechanism, respectively. Efficacy was evaluated by examining changes in femoral mineral content (BMC) and density (BMD) using dual-energy X-ray absorptiometry, and bone turnover biomarkers [osteocalcin (OC) and tartrate-resistant acid phosphatase (TRAP)] using respective ELISA kits. The mRNA expression of tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) in spleen was determined by real-time RT-PCR. Neither LPS administration nor GTP levels affected body weight and femoral bone area throughout the study period. Only GTP supplementation resulted in increased urinary epigallocatechin and epicatechin concentrations. LPS administration led to a decrease in femur BMC and BMD, and serum OC levels, but an increase in serum TRAP, urinary 8-OHdG and spleen mRNA expression of TNF-α and COX-2 levels. GTP supplementation resulted in higher values for femur BMC, BMD and serum OC, but lower values for serum TRAP, urinary 8-OHdG and spleen mRNA expression of TNF-α and COX-2 levels. We conclude that GTP mitigates bone loss in a chronic inflammation-induced bone loss model by reducing oxidative stress-induced damage and inflammation.

Effect of long-chain n-3 polyunsaturated fatty acid on inflammation mediators during osteoblastogenesis.

This study examined the effects of eicosapentaenoic acid (EPA) and arachidonic acid (AA) on inflammation mediators during osteoblastogenesis, in terms of modulation of the cyclooxygenase (COX)-2 and the inducible nitric oxide (NO) synthase (iNOS) pathways. We hypothesized that n-3 polyunsaturated fatty acid (PUFA) would reduce the production of inflammation mediators, including prostaglandin E(2) (PGE(2)) and NO, and related mRNA gene expression during osteoblastogenesis. Mouse bone marrow stromal cells (ST-2) were treated with 40 microM ethanol (as a control), 40 microM AA, or 40 microM EPA in osteogenic medium for 7, 14, 21, or 28 days. Prior to harvest, cells were treated with respective treatments along with cytokine mixtures for an additional 24 hours, and then cells were harvested for mRNA expression. In addition, cells were also treated with respective treatments along with the same cytokine mixtures for an additional 48 hours for experiment measuring PGE(2) and NO production using conditioned culture medium and protein expression using cells. Except for 7 days of culture, AA treatment resulted in the highest value for PGE(2) production throughout 28 days of culture. AA treatment also enhanced COX-2 mRNA expression up to 21 days. AA treatment resulted in a higher value for NO production after 7 days, while EPA treatment yielded a higher value for NO production relative to those receiving AA treatment after 14 and 21 days. Our investigation has corroborated that the protective action of EPA on osteoblastogenesis was mediated by the modulation of PGE(2) and the NO pathway.

Forensic aspects of DNA-based human identity testing.

The forensic applications of DNA-based human identity laboratory testing are often underappreciated. Molecular biology has seen an exponential improvement in the accuracy and statistical power provided by identity testing in the past decade. This technology, dependent upon an individual's unique DNA sequence, has cemented the use of DNA technology in the forensic laboratory. This paper will discuss the state of modern DNA-based identity testing, describe the technology used to perform this testing, and describe its use as it relates to forensic applications. We will also compare individual technologies, including polymerase chain reaction (PCR) and Southern Blotting, that are used to detect the molecular differences that make all individuals unique. An increasing reliance on DNA-based identity testing dictates that healthcare providers develop an understanding of the background, techniques, and guiding principles of this important forensic tool.

Survey of candidate genes for autism susceptibility.

The causes of autism are unknown, and there is an increase over recent years in the diagnosis of children with this condition. Several chromosome regions have been evaluated to find linkages to the susceptibility for autism. Linkage has been found on chromosomes 2, 4, 6, 7, 10, 15, 16, 17, 19, 21, and 22. The genes of interest still need to be evaluated and experiments will continue to look for more candidate genes.

A molecular phylogenetic analysis of the family Rhacophoridae with an emphasis on the Asian and African genera.

Using characters from mitochondrial DNA to construct maximum parsimony and maximum likelihood trees, we performed a phylogenetic analysis on representative species of 14 genera: 12 that belong to the treefrog family Rhacophoridae and two, Amolops and Rana, that are not rhacophorids. Our results support a phylogenetic hypothesis that depicts a monophyletic family Rhacophoridae. In this family, the Malagasy genera Aglyptodactylus, Boophis, Mantella, and Mantidactylus form a well-supported sister clade to all other rhacophorid genera, and Mantella is the sister taxon to Mantidactylus. Within the Asian/African genera, the genus Buergeria forms a well-supported clade of four species. The genera, except for Chirixalus, are generally monophyletic. An exception to this is that Polypedates dennysii clusters with species of Rhacophorus, suggesting that the taxonomy of the rhacophorids should be revised to reflect this relationship. Chirixalus is not monophyletic. Unexpectedly, there is strong support for Chirixalus doriae from Southeast Asia forming a clade with species of the African genus Chiromantis, suggesting that Chiromantis dispersed to Africa from Asia. Also, there is strong support for the sister taxon relationship of Chirixalus eiffingeri and Chirixalus idiootocus apart from other congeners.