Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7 adhesion to epithelial cells

Adherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface-layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S-layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp-2 and T84) cells were treated with S-layer protein extract alone, infected with E. coli O157:H7, or pretreated with S-layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S-layer protein extracts were characterized by phase-contrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre-treatment of host epithelial cells with S-layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching-effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non-viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.     

Effects of Expanded Coverage for Chiropractic Services on Medicare Costs in a CMS Demonstration

Background

Moderately convincing evidence supports the benefits of chiropractic manipulations for low back pain. Its effectiveness in other applications is less well documented, and its cost-effectiveness is not known. These questions led the Centers for Medicaid and Medicare Services (CMS) to conduct a two-year demonstration of expanded Medicare coverage for chiropractic services in the treatment of beneficiaries with neuromusculoskeletal (NMS) conditions affecting the back, limbs, neck, or head.

Methods

The demonstration was conducted in 2005–2007 in selected counties of Illinois, Iowa, and Virginia and the entire states of Maine and New Mexico. Medicare claims were compiled for the preceding year and two demonstration years for the demonstration areas and matched comparison areas. The impact of the demonstration was analyzed through multivariate regression analysis with a difference-in-difference framework.

Results

Expanded coverage increased Medicare expenditures by $50 million or 28.5% in users of chiropractic services and by $114 million or 10.4% in all patients treated for NMS conditions in demonstration areas during the two-year period. Results varied widely among demonstration areas ranging from increased costs per user of $485 in Northern Illinois and Chicago counties to decreases in costs per user of $59 in New Mexico and $178 in Scott County, Iowa.

Conclusion

The demonstration did not assess possible decreases in costs to other insurers, out-of-pocket payments by patients, the need for and costs of pain medications, or longer term clinical benefits such as avoidance of orthopedic surgical procedures beyond the two-year period of the demonstration. It is possible that other payers or beneficiaries saved money during the demonstration while costs to Medicare were increased.     

Relative dental development of Upper Pleistocene hominids compared to human population variation

The relative development of permanent teeth in samples of Neandertal/archaic Homo and Early Modern/Upper Paleolithic hominids is compared to the range of variability found in three recent human samples. Both fossil hominid samples are advanced in relative M2 and M3 development compared to white French-Canadians, but only the Neandertal/archaic Homo M3 sample is advanced when compared to black southern Africans. Both fossil hominid samples are delayed in relative I1 and P3 development compared to the recent human samples. Two hypotheses concerning the significance of the advanced M3 and M2 development found in both hominid groups and southern Africans compared to French-Canadians are discussed. The first postulates that the differences in relative molar development are due simply to variation in tooth/jaw size relationships. The second postulates that the relatively advanced M3 and M2 development found in the fossil hominids and southern Africans is a correlate of their potential for advanced skeletal maturation compared to French-Canadians and other European-derived populations. It appears that dental development patterns have continued to evolve from the Upper Pleistocene to present times, and that Neandertals and Early Moderns shared similar patterns of relative dental development. © 1996 Wiley-Liss, Inc.     

Developmental mutants isolated from wild-caught Xenopus laevis by gynogenesis and inbreeding

Xenopus laevis obtained from indigenous African populations are a rich source of mutants affecting development. Gynogenesis and inbreeding were used to isolate mutants affecting development from wild-caught Xenopus laevis females. Fourteen mutants were recovered from eight females tested. One mutant was recovered from each of two females. This load of 1.875 developmental mutants per female is similar to that found in the axolotl (Ambystoma mexicanum), a urodele amphibian, and is only slightly less than the load of mutants with major developmental effects found in Drosophila and man. These results suggest that the anuran amphibian Xenopus laevis, an ancestrally tetraploid species, has undergone extensive diploidization of developmentally important loci and that gynogenesis and inbreeding of wild-caught animals can provide adequate mutants at diploid loci for developmental genetic studies.     

Involvement of skeletal muscle gene regulatory network in susceptibility to wound infection following trauma

Despite recent advances in our understanding the pathophysiology of trauma, the basis of the predisposition of trauma patients to infection remains unclear. A Drosophila melanogaster/Pseudomonas aeruginosa injury and infection model was used to identify host genetic components that contribute to the hyper-susceptibility to infection that follows severe trauma. We show that P. aeruginosa compromises skeletal muscle gene (SMG) expression at the injury site to promote infection. We demonstrate that activation of SMG structural components is under the control of cJun-N-terminal Kinase (JNK) Kinase, Hemipterous (Hep), and activation of this pathway promotes local resistance to P. aeruginosa in flies and mice. Our study links SMG expression and function to increased susceptibility to infection, and suggests that P. aeruginosa affects SMG homeostasis locally by restricting SMG expression in injured skeletal muscle tissue. Local potentiation of these host responses, and/or inhibition of their suppression by virulent P. aeruginosa cells, could lead to novel therapies that prevent or treat deleterious and potentially fatal infections in severely injured individuals.     

High dynamic range characterization of the trauma patient plasma proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this "divide-and-conquer" strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3,654 different proteins with 1,494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 "classic" cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2,910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1,553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.     

Molecular mechanism(s) of burn-induced insulin resistance in murine skeletal muscle: role of IRS phosphorylation

Hyperglycemia, glucose intolerance and elevated insulin levels frequently occur in burned patients; however, the mechanism(s) for this insulin resistance has not been fully elucidated. One possible mechanism could involve alterations in the phosphorylation of serine 307 of the insulin receptor substrate-1 (IRS-1) via activation of stress kinase enzymes, including SAPK/JNK. In the present study we examined the time course of the effect of burn injury to mice on: levels of IRS-1 protein, phosphorylation of serine 307 of IRS-1, SAPK/JNK kinase levels and activity and Akt kinase activity in hind limb skeletal muscle. Burn injury produced a reduction in hind limb muscle mass 24 h after injury, and, which persisted for 168 h. At 24 h after injury, there was a dramatic ( approximately 9-fold) increase in phosphorylation of IRS-1 serine 307 followed by a more moderate elevation thereafter. Total IRS-1 protein was slightly elevated at 24 h after injury and decreased to levels below sham treated animals at the later times. Burn injury did not appear to change total SAPK/JNK protein content, however, enzyme activity was increased for 7 days after injury. Akt kinase activity was decreased in skeletal muscle following burn injury; providing a biochemical basis for burn-induced insulin resistance. These findings are consistent with the hypothesis that burn-induced insulin resistance may be related, at least in part, to alterations in the phosphorylation of key proteins in the insulin signaling cascade, including IRS-1, and that changes in stress kinases, such as SAPK/JNK produced by burn injury, may be responsible for these changes in phosphorylation.     

Fibrinolytic activity associated with cultured human neoplastic and normal cells

Summary Fibrinolytic activity was studied in a number of different established as well as secondary human cell cultures derived from both malignant and normal tissues. The ability to degrade [¹²⁵I]-labeled fibrin was found to be characteristic of some malignant cultures as well as some normal cultures, and to be dependent upon the presence of serum. For the most part, this activity was detected in cultures with a relatively shortin vitro passage history (<30 passages). Low passaged colon and rectal carcinoma cells, HCT-8 and HRT-18, as well as normal rectal, colon and foreskin fibroblasts were positive for fibrinolytic activity, while long established (>100 passages) cultures of malignant cells (colon carcinoma, HeLa, Hep-2, KB) as well as normal cells (HEI, AV₃) were negative. It is proposed that although some normal cells synthesize plasminogen activators, the fibrinolytic capability of both malignant and normal cells may be lost on prolongedin vitro cultivation.     

Cytohesin-associated scaffolding protein (CASP) is a substrate for granzyme B and ubiquitination

Natural killer (NK) cells are a sub-population of cytotoxic lymphocytes that can kill tumor or infected cells without prior exposure, by secreting the contents of preformed cytotoxic vesicles, containing perforin and granzymes, at the immune synapse. Cytohesin-associated scaffolding protein (CASP) is an adaptor molecule uniquely expressed in lymphocytes that forms complexes with both vesicle-initiating and sorting proteins, and has roles in NK cell migration, cytotoxicity, and cytokine secretion. In this study, we show that CASP contains a conserved granzyme B cleavage site that could modify its intracellular localization and interaction with sorting nexin 27. We also provide evidence that CASP is modified by ubiquitination. Both of these post-translational modifications could rapidly modify CASP function and highlight the dynamic regulatory mechanisms that direct its role in NK cell functions.