Septin-regulated actin dynamics promote Salmonella invasion of host cells.

Actin nucleators and their binding partners play crucial roles during Salmonella invasion, but how these factors are dynamically coordinated remains unclear. Here, we show that septins, a conserved family of GTP binding proteins, play a role during the early stages of Salmonella invasion. We demonstrate that septins are rapidly enriched at sites of bacterial entry and contribute to the morphology of invasion ruffles. We found that SEPTIN2, SEPTIN7, and SEPTIN9 are required for efficient bacterial invasion. Septins contributed to the recruitment of ROCK2 kinase during Salmonella invasion, and the downstream activation of the actin nucleating protein FHOD1. In contrast, activation of the ROCK2 substrate myosin II, which is known to be required for Salmonella enterica serovar Typhimurium invasion, did not require septins. Collectively, our studies provide new insight into the mechanisms involved in Salmonella invasion of host cells.

Salmonella exploits host Rho GTPase signalling pathways through the phosphatase activity of SopB.

Salmonella uses Type 3 secretion systems (T3SSs) to deliver virulence factors, called effectors, into host cells during infection. The T3SS effectors promote invasion into host cells and the generation of a replicative niche. SopB is a T3SS effector that plays an important role in Salmonella pathogenesis through its lipid phosphatase activity. Here, we show that SopB mediates the recruitment of Rho GTPases (RhoB, RhoD, RhoH, and RhoJ) to bacterial invasion sites. RhoJ contributes to Salmonella invasion, and RhoB and RhoH play an important role in Akt activation. R-Ras1 also contributes to SopB-dependent Akt activation by promoting the localised production of PI(3,4)P2 /PI(3,4,5)P3 . Our studies reveal new signalling factors involved in SopB-dependent Salmonella infection.

Pediatric reference intervals for 1,25-dihydroxyvitamin D using the DiaSorin LIAISON XL assay in the healthy CALIPER cohort.

BACKGROUND: 1,25-dihydroxyvitamin D (1,25(OH)2D), the biologically active vitamin D metabolite, plays a critical role in calcium and phosphate homeostasis. 1,25(OH)2D is measured to assess calcium and phosphate metabolism, particularly during periods of profound growth and development. Despite its importance, no reliable pediatric reference interval exists, with those available developed using adult populations or out-dated methodologies. Using the fully automated chemiluminescence immunoassay by DiaSorin, we established 1,25(OH)2D pediatric reference intervals using healthy children and adolescents from the CALIPER cohort. METHODS: Serum samples from healthy subjects (0 to <19 years) were analyzed for 1,25(OH)2D using the DiaSorin LIAISON XL assay and age-specific reference intervals were established. The Mann-Whitney U-test was used to determine seasonal differences. Pooled neonatal and infantile samples were quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine if elevated concentrations during the first year of life may be attributed to cross-reacting moieties. RESULTS: Three reference interval age partitions were required with highest levels in subjects 0 to <1 year (77-471 pmol/L), which declined and narrowed after 1 year (113-363 pmol/L) and plateaued at 3 years (108-246 pmol/L). 1,25(OH)2D concentration was not significantly affected by seasonal variation or sex. Elevated 1,25(OH)2D concentrations in neonatal and infantile samples may be the result of an interfering substance. The absence of 3-epi-1,25-dihydroxyvitamin D in the pooled samples makes it unlikely to be the interfering moiety. CONCLUSIONS: Pediatric reference intervals for 1,25(OH)2D were established to improve test result interpretation in children and adolescents. 1,25(OH)2D is elevated in a proportion of neonates and infants, which may be the result of a cross-reacting moiety.

Bacterial subversion of host cytoskeletal machinery: hijacking formins and the Arp2/3 complex.

The host actin nucleation machinery is subverted by many bacterial pathogens to facilitate their entry, motility, replication, and survival. The majority of research conducted in the past primarily focused on exploitation of a host actin nucleator, the Arp2/3 complex, by bacterial pathogens. Recently, new studies have begun to explore the role of formins, another family of host actin nucleators, in bacterial pathogenesis. This review provides an overview of recent advances in the study of the exploitation of the Arp2/3 complex and formins by bacterial pathogens. Secreted bacterial effector proteins seem to manipulate the regulation of these actin nucleators or functionally mimic them to drive bacterial entry, motility and survival within host cells. An enhanced understanding of how formins are exploited will provide us with greater insight into how a fundamental eurkaryotic cellular process is utilized by bacteria and will also advance our knowledge of host-pathogen interactions.

Formin-mediated actin polymerization promotes Salmonella invasion.

Salmonella invade host cells using Type 3 secreted effectors, which modulate host cellular targets to promote actin rearrangements at the cell surface that drive bacterial uptake. The Arp2/3 complex contributes to Salmonella invasion but is not essential, indicating other actin regulatory factors are involved. Here, we show a novel role for FHOD1, a formin family member, in Salmonella invasion. FHOD1 and Arp2/3 occupy distinct microdomains at the invasion site and control distinct aspects of membrane protrusion formation. FHOD1 is phosphorylated during infection and this modification is required for promoting bacterial uptake by host cells. ROCK II, but not ROCK I, is recruited to the invasion site and is required for FHOD1 phosphorylation and for Salmonella invasion. Together, our studies revealan important phospho-dependent FHOD1 actin polymerization pathway in Salmonella invasion.

Establishing preanalytical stability of vitamin A and vitamin E.

Vitamin A and Vitamin E, a group of lipid-soluble vitamins, can be degraded by photolysis and photooxidation after exposure to light. As an essential area of the preanalytical stage, inappropriate storage conditions of patient samples could lead to inaccurate results. In this study, we evaluated three of the most common preanalytical storage conditions (RT, +4°C, and -20 °C) in the workflow in the clinical laboratory setting using both clear and amber Eppendorf tubes. Analyte stability in each storage condition was tested with five patient pools. After 24 hrs at RT, a significant decrease in vitamin A and E was observed for samples stored in clear Eppendorf tubes (19.2 % and 17.9 %, for vitamin A and E, respectively), compared with samples stored in amber Eppendorf tubes (9.1 % and 7.3 %), respectively. After the specimen was stored at 4 °C for 7 days, vitamin A concentrations decreased in clear tubes by 14.7 % and in amber tubes by 12.7 %. Similarly, vitamin E concentrations decreased in both clear and amber tubes by 11.2 % and 13.4 %, respectively. Both vitamin A and vitamin E demonstrated acceptable stability in clear and amber Eppendorf tubes at -20 °C for up to 22 days. It is preferable to use amber tubes to protect vitamin A and E from light during short term storage. Storage of vitamin A and E specimens beyond 7 days should be at -20 °C.

CLSI-based transference and verification of CALIPER pediatric reference intervals for 29 Ortho VITROS 5600 chemistry assays.

BACKGROUND: Evidence-based reference intervals (RIs) are essential to accurately interpret pediatric laboratory test results. To fill gaps in pediatric RIs, the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) project developed an age- and sex-specific pediatric RI database based on healthy pediatric subjects. Originally established for Abbott ARCHITECT assays, CALIPER RIs were transferred to assays on Beckman, Roche, Siemens, and Ortho analytical platforms. This study provides transferred reference intervals for 29 biochemical assays for the Ortho VITROS 5600 Chemistry System (Ortho). METHODS: Based on Clinical Laboratory Standards Institute (CLSI) guidelines, a method comparison analysis was performed by measuring approximately 200 patient serum samples using Abbott and Ortho assays. The equation of the line of best fit was calculated and the appropriateness of the linear model was assessed. This equation was used to transfer RIs from Abbott to Ortho assays. Transferred RIs were verified using 84 healthy pediatric serum samples from the CALIPER cohort. RESULTS: RIs for most chemistry analytes successfully transferred from Abbott to Ortho assays. Calcium and CO2 did not meet statistical criteria for transference (r2<0.70). Of the 32 transferred reference intervals, 29 successfully verified with approximately 90% of results from reference samples falling within transferred confidence limits. Transferred RIs for total bilirubin, magnesium, and LDH did not meet verification criteria and are not reported. CONCLUSIONS: This study broadens the utility of the CALIPER pediatric RI database to laboratories using Ortho VITROS 5600 biochemical assays. Clinical laboratories should verify CALIPER reference intervals for their specific analytical platform and local population as recommended by CLSI.

Pediatric Reference Intervals for Biochemical Markers: Gaps and Challenges, Recent National Initiatives and Future Perspectives.

Reference intervals provide valuable information to medical practitioners in their interpretation of quantitative laboratory test results, and are critical in the assessment of patient health and in clinical decision-making. The reference interval serves as a health-associated benchmark with which to compare an individual test result. While the concept of reference intervals and their utility appear straightforward, the process of establishing accurate and reliable reference intervals is considerably complex and involved. Currently, many pediatric laboratory tests are inappropriately interpreted using reference intervals derived from either adult populations, hospitalized pediatric populations, or from outdated and/or inaccurate technology. Thus, many pediatric reference intervals used in diagnostic laboratories are incomplete and may be inappropriate for clinical use. The use of inappropriate reference intervals impacts clinical decision-making and has potential detrimental effects on the quality of patient healthcare including misdiagnosis, delayed diagnosis, inappropriate treatments, and patient risk. These are critical gaps in pediatric healthcare and it is imperative to update and establish appropriate reference intervals for pediatric populations based on specific age- and sex-stratifications. In the present review, specific issues, challenges and deficiencies in pediatric reference intervals for biochemical markers will be discussed. Early studies using hospitalized patients will be examined, followed by a review of recent national and global initiatives on establishing reference intervals from healthy pediatric population. We will highlight the achievements and milestones of the Canadian CALIPER project, including the establishment of a comprehensive biobank and database which has addressed several of these critical gaps. CALIPER's mandate is to establish and provide comprehensive, up-to-date pediatric reference intervals to all biochemical markers of pediatric disease. CALIPER has also begun knowledge translation initiatives to disseminate its data via peer-reviewed publication, an online database, and a smartphone application to allow greater access to CALIPER pediatric reference interval data. Finally, limitations, future perspectives and harmonization of pediatric reference intervals to improve pediatric diagnostics in Canada and worldwide will be discussed.

Normalization of obesity-associated insulin resistance through immunotherapy.

Obesity and its associated metabolic syndromes represent a growing global challenge, yet mechanistic understanding of this pathology and current therapeutics are unsatisfactory. We discovered that CD4(+) T lymphocytes, resident in visceral adipose tissue (VAT), control insulin resistance in mice with diet-induced obesity (DIO). Analyses of human tissue suggest that a similar process may also occur in humans. DIO VAT-associated T cells show severely biased T cell receptor V(alpha) repertoires, suggesting antigen-specific expansion. CD4(+) T lymphocyte control of glucose homeostasis is compromised in DIO progression, when VAT accumulates pathogenic interferon-gamma (IFN-gamma)-secreting T helper type 1 (T(H)1) cells, overwhelming static numbers of T(H)2 (CD4(+)GATA-binding protein-3 (GATA-3)(+)) and regulatory forkhead box P3 (Foxp3)(+) T cells. CD4(+) (but not CD8(+)) T cell transfer into lymphocyte-free Rag1-null DIO mice reversed weight gain and insulin resistance, predominantly through T(H)2 cells. In obese WT and ob/ob (leptin-deficient) mice, brief treatment with CD3-specific antibody or its F(ab')(2) fragment, reduces the predominance of T(H)1 cells over Foxp3(+) cells, reversing insulin resistance for months, despite continuation of a high-fat diet. Our data suggest that the progression of obesity-associated metabolic abnormalities is under the pathophysiological control of CD4(+) T cells. The eventual failure of this control, with expanding adiposity and pathogenic VAT T cells, can successfully be reversed by immunotherapy.

Islet glia, neurons, and beta cells.

Type 1 diabetes (T1D) is caused by autoimmune beta cell destruction. The early events triggering T1D and the forces that keep diabetic autoimmunity pancreas specific have been unclear. Our discovery that autoimmune islet destruction is not beta-cell-exclusive but includes cytotoxic T cell targeting of peri-islet glia, evoked the possibility that T1D pathogenesis may involve neuronal elements beyond beta cell/immune interactions. Recently, we have found that sensory afferent neurons are a critical component in prediabetes initiation, promoting islet inflammation through altered glucose homeostasis and progressive beta cell stress. These factors orchestrate a catastrophic cascade culminating in insulin insufficiency mediated by an autoimmune-prone host. This neuro-immuno-endocrinological triad explains diabetic inflammation as a consequence of local neuropeptide deficiency, leading to an innovative concept of disease pathogenesis with novel therapeutic implications.