Protective Advantage Research Articles

Two Cases of Dysfibrinogenemia in Childhood

IntroductionHemostasis in childhood differs from that of adults; and the hemostatic system is still developing during childhood. These differences offer a protective advantage to children with hemorrhagic and thrombotic complications. Plasma levels of coagulation factors (except for fibrinogen, factor V and factor VIII), as well as plasma levels of protein C, protein S and antithrombin are reduced. Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Clinical symptoms may vary from asymptomatic to life-threatening bleeding complications. Venous or arterial thrombotic complications occur extremely rarely during childhood. Fibrinogen, a key component in hemostasis, is a 340-kDa glycoprotein. The molecule consists of three different pairs of polypeptide chains (Aα, Bβ, and γ) each encoded by a distinct gene (FGA, FGB, and FGG). N-terminal parts of the Aα chain - fibrinopeptides A and the Bβ chain - fibrinopeptides B are situated in the central part of the molecule and block polymerization of the molecules. Conversion of fibrinogen to fibrin occurs after the cleavage of N-terminal fibrinopeptides by the serine protease thrombin. Correct conformation of fibrinopeptides is important for the cleavage by thrombin.MethodsRoutine coagulation tests were performed with citrated plasma samples on a STA-R coagulation analyzer. The functional fibrinogen level was measured by the Clauss method. Total fibrinogen level was determined by an immunoturbidimetric assay performed on a UV-2401PC spectrophotometer. Fibrin polymerization induced by either thrombin or reptilase and fibrinolysis experiments were obtained by the turbidimetrical method. Fibrinopeptide release was measured as a function of time; and the fibrinopeptides were determined by the reversed-phase, high-performance liquid chromatography (RP-HPLC) method. The purified genomic DNA was amplified by polymerase chain reaction, using specific primers; and dideoxysequencing was performed with Dye Terminator Cycle Sequencing with a Quick Start kit and a CEQ 8000 genetic analysis system).ResultsWe have examined two unrelated boys aged 4 (boy 1) and 14 (boy 2) for susp. dysfibrinogenemia. Routine coagulation tests revealed prolonged thrombin and reptilase time in both boys and also showed decreased functional fibrinogen levels in both kids (Tab. 1). Boy 1 presented with bleeding manifestation – easy bruising, epistaxis, and bleeding after tooth extraction. His 26-year-old mother presented with similar coagulation findings and with mild bleeding complications. Boy 2 was asymptomatic. Fibrin polymerization experiments carried out on plasma samples showed prolonged lag time and significantly reduced final turbidity in both cases. Measurement of kinetics of fibrinopeptide release showed a decreased amount of the released fibrinopeptide A in both patients. DNA sequencing of boy 1 revealed a point mutation in exon 2 of the FGA gene at the position 3456 G/A, which causes the substitution of Aα 16 Arg to His (fibrinogen Praha V). The boy was found to be heterozygous for the mutation as well as his mother. DNA analysis of boy 2 revealed the same point mutation in the FGAgene, causing the same substitution of Aα 16 Arg to His (fibrinogen Kralupy nad Vltavou).ConclusionIn this study we report two cases of congenital defects in the fibrinogen Aα Arg16-Gly17 bond found during childhood. It has been described earlier that the replacement of Aα 16 arginine by histidine decelerates thrombin catalyzed fibrinopeptide A release. Although both kids presented with the same genetic defect and with similar coagulation results, they have different clinical manifestation of the disease.AcknowledgmentThis work was supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.16/3.1.00/28007.Table 1:Routine coagulation test resultsBoy 1Boy 1’s motherBoy 2NormalAPTT40.1 s34.6 s35.3 s27.5 - 36.1 sProthrombin time17.5 s14.4 s16.9 s11.7 - 15.1 sThrombin time40.9 s36.2 s44.8 s17.6 - 21.6 sReptilase time58.3 s51.6 s55.7 s16.0 - 20.0 sFibrinogen (Clauss)0.51 g/l1.12 g/l0.58 g/l2.00 - 4.20 g/lFibrinogen (Immuno)2.47 g/l2.68 g/l2.31 g/l2.00 - 4.20 g/lAge42614- DisclosuresNo relevant conflicts of interest to declare.

Immune response in susceptible BALB/c mice immunized with DNA encoding Lipophosphoglycan 3 of Leishmania infantum.

Visceral leishmaniasis is a serious parasitic infection that the development of an effective vaccine is necessary to control the disease. Lipophosphoglycan 3 (LPG3) is essential for the synthesis of glycoconjugates as parasite virulence factors. In this study, we evaluated the immunogenicity of Leishmania infantum LPG3 gene as a DNA vaccine against murine visceral leishmaniasis. For this purpose, BALB/c mice were immunized subcutaneously with the DNA encoding LPG3 either alone or in combination with recombinant heat shock protein 70 (rHSP70). Next, its immunogenicity and protective efficacy were evaluated in the immunized mice. The results showed a mixed Th1/Th2 response following immunization, which was associated with the production of both IFN-γ and IL-10 by splenocytes compared with control groups but did not lead to reduction in the splenic parasite burden. Serum levels of IgG antibody isotypes indicated no significant difference between the LPG3 DNA and the empty vector. In addition, the co-administration of rHSP70 with the DNA vaccine offered no additive protective advantage on experimental infectious challenge. Thus, we propose to strengthen the immunogenic potential of L. infantum LPG3 in prime-boost approach with a powerful adjuvant to elicit a robust parasite-specific protective Th1 response.

Epistasis between polymorphisms in COMT, ESR1, and GCH1 influences COMT enzyme activity and pain

Abnormalities in the enzymatic activity of catechol-O-methyltransferase (COMT) contribute to chronic pain conditions, such as temporomandibular disorders (TMD). Thus, we sought to determine the effects of polymorphisms in COMT and functionally related pain genes in the COMT pathway (estrogen receptor 1 [ESR1], guanosine-5-triphosphate cyclohydrolase 1 [GCH1], methylenetetrahydrofolate reductase [MTHFR]) on COMT enzymatic activity, musculoskeletal pain, and pain-related intermediate phenotypes among TMD cases and healthy control subjects. Results show that the COMT rs4680 (val158met) polymorphism is most strongly associated with outcome measures, such that individuals with the minor A allele (met) exhibit reduced COMT activity, increased TMD risk, and increased musculoskeletal pain. Epistatic interactions were observed between the COMT rs4680 polymorphism and polymorphisms in GCH1 and ESR1. Among individuals with the COMT met allele, those with 2 copies of the GCH1 rs10483639 minor G allele exhibit normalized COMT activity and increased mechanical pain thresholds. Among individuals with the COMT val allele, those with 2 copies of the ESR1 rs3020377 minor A allele exhibit reduced COMT activity, increased bodily pain, and poorer self-reported health. These data reveal that the GCH1 minor G allele confers a protective advantage among met carriers, whereas the ESR1 minor A allele is disadvantageous among val carriers. Furthermore, these data suggest that the ability to predict the downstream effects of genetic variation on COMT activity is critically important to understanding the molecular basis of chronic pain conditions.

Disparities in reported and substantiated infant maltreatment by maternal Hispanic origin and nativity: a birth cohort study.

We followed Latino infants prospectively through age 1 to determine whether maternal foreign-born status conferred a protective advantage against reported and substantiated maltreatment across Hispanic-origin groups, and whether the likelihood an infant was reported or substantiated for maltreatment varied by Hispanic origin. We drew data for all Latino infants born in California between 2000 and 2006 (N = 1,909,155) from population-based birth records linked to child protective services data. We used χ(2) tests to assess distributional differences in covariates and utilized generalized linear models to estimate the adjusted relative risk of report and substantiation in models stratified by nativity. We observed significant health advantages in reported and substantiated maltreatment for infants of foreign-born mothers within every Hispanic-origin group. Risks of report and substantiation among infants of Mexican and Central/South American mothers were consistently lower than Puerto Rican and Cuban mothers despite socioeconomic disadvantage. The presence of disparities among Hispanic-origin groups in child maltreatment report and substantiation during infancy has implications for the health of Latinos across the life course. Further research is warranted to unravel the complex processes underlying observed relationships.

Frontal sinus volume predicts incidence of brain contusion in patients with head trauma

The function of the paranasal sinuses is a topic of debate. One hypothesis suggested has been that sinuses act as a "crumple zone," serving to protect the contents of the head from impact. In this study, we examine the interactions of the frontal sinus and the brain in the setting of head trauma. Our formal hypothesis is that frontal sinus volume is less in head trauma patients with contusion than in head trauma patients without contusion. Computed tomographic (CT) scans of patients who sustained blunt head trauma performed at the University of Pittsburgh Medical Center from 2007 to 2012 were reviewed retrospectively. Inclusion criteria were presence of blunt trauma and complete brain and maxillofacial CT scan. Exclusion criteria included patients with incomplete imaging, imaging unrelated to trauma, previous cranial injury, and penetrating trauma.With the use of maxillofacial CT scans, height, depth, and width were measured, and a cubical approximation of volume was determined for each frontal sinus. Presence of frontal sinus fracture was considered an indicator of high-impact trauma. Grading of brain contusion severity was performed using head CT scans. A scale of 0 to 5 was used based on the Marshall Criteria.Categorical data were analyzed using the χ(2) or Fisher's exact test. Continuous data were analyzed using Student's t test or analysis of variance. p < 0.05 was considered statistically significant. Among patients with frontal sinus fracture, the average sinus volume of those without contusion was 32.72 mL, while the average sinus volume of those with brain contusion was 21.85 mL (p = 0.023). Thus, the volume of the frontal sinuses was 33% less in patients with contusion than in patients without contusion. Our study supports the theory that the frontal sinuses impart a protective advantage against frontal brain contusion. The dynamics of head injury and force distribution during trauma may be the basis for more advanced protective devices. Prognostic study, level III.

PYK2 Mediates Microenvironment-Specific Myeloma Survival

Multiple myeloma remains an incurable bone marrow resident malignancy due to the eventual development of drug resistance and minimal residual disease linked to both acquired genetic changes and the dynamic influences of the microenvironment. We and others have shown that adhesion of tumor cells to the extracellular matrix component fibronectin (FN) via integrins leads to cell cycle arrest and protection from chemotherapy or Cell Adhesion-Mediated Drug Resistance (CAM-DR). The pleiotropic cytokine IL-6 has also been shown to mediate drug resistance in both solid and hematopoietic tumors [1]. The influence of these two effectors on drug resistance has primarily been studied separately; however, within the context of the bone marrow, myeloma cells are influenced by both soluble and physical effectors simultaneously. Using a reductionist model of the multivariate microenvironment, we demonstrated that unique collaborative signaling between FN-adhered cells and IL-6 leads to increased proliferation of protected tumor cells and a more malignant phenotype [2]. Our results demonstrate the FN-adhesion mediated cell-cycle arrest of myeloma cells was reversed following stimulation of adhered cells with IL-6 and drug resistance was maintained. This phenotype was associated with a novel amplification of IL-6-induced STAT3 activation in adhered cells [2]. We have since validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in myeloma patient specimens by flow cytometry. To further characterize post-translational events induced by beta1 integrin mediated adhesion and under multivariate conditions, we performed immune-affinity phosphotyrosine proteomic screening in RMPI8226 myeloma cells maintained in suspension or adhered to FN with or without IL-6 stimulation. Screening identified 179 differentially tyrosine phosphorylated peptides among four conditions (Suspension or FN, +/- IL-6). Among these were proteins involved in signal transduction, cytoskeleton assembly, survival, and metabolic pathways. Among 67 peptides upregulated by adhesion to FN alone, proline-rich tYrosine Kinase 2 (PYK2/FAK2) was highly phosphorylated at Y580 in RPMI8226 myeloma cells, and focal adhesion formation in adhered cells was validated by phosphorylation of paxillin at pY118 and p130CAS at pY128/pY249. PYK2 is a downstream intermediate of integrin signaling and has been demonstrated to amplify EGFR and cSrc-induced STAT3 activation [3]. PYK2 has also been linked to downstream signaling events of JAK-dependent cytokine receptors, but not to a STAT3 amplification mechanism within the Type I or Type II cytokine receptor families [4-7]. As such, we hypothesized that PYK2 may be an important modulator of the enhanced STAT3 activation following multivariate signaling between beta1 integrin and the IL-6 signal transducer, gp130. In addition to enhanced phosphorylation of PYK2 at Y580 identified by proteomic screening, western blot analysis demonstrated PYK2 autophosphorylation of Y402 following myeloma cell adhesion to FN correlating with the amplification of IL-6-induced STAT3 and JAK1. Targeting PYK2 with RNA interference attenuated the adhesion-associated amplification of STAT3 and JAK1 phosphorylation, but did not influence the limited STAT3 activation in cells grown in suspension. PYK2 siRNA did not inhibit myeloma cell adhesion (n=4, p-value &gt;0.05). We have also demonstrated that STAT3 activation is markedly enhanced in myeloma cell lines adhered to patient bone marrow stroma (BMS), but not cells grown in transwell coculture (no myeloma cell-BMS contact). The protective advantage afforded myeloma cells in coculture was attenuated knockdown of PYK2 or STAT3 expression in myeloma cell lines and patient specimens using novel antisense oligonucleotides (ISIS Pharmaceuticals, n=4, p-value &lt; 0.05). Critically, inhibition of PYK2 with nanomolar amounts of the focal adhesion kinase inhibitor PF562.271 had a similar impact only on myeloma cells directly adhered to BMS, but did not impact BMS viability. These data demonstrate a novel PYK2-mediated JAK1/STAT3 signaling cascade within the context of multivariate stimulation. Moreover these data identify a novel PYK2-mediated survival pathway activated only within the context of multivariate stimulation in the bone marrow microenvironment, and suggests that PYK2 represents a MRD-specific target. Disclosures: No relevant conflicts of interest to declare.

Bone Marrow Endothelial Cells Protect Acute Myeloid Leukemia From Chemotherapy By Direct Contact: The BCAM/Laminin/VLA5 Axis As a Potential Therapeutic Target

Causes of refractory acute myeloid leukemia (AML) are unknown, but may be related to the bone marrow (BM) vascular network given the close relationship between hematopoiesis and the vasculature. We hypothesized that endothelial cells (ECs) provide a protective advantage to AML cells. To test this hypothesis, we first cultured human AML cells with and without primary bone marrow endothelial cells (BMECs). Co-cultures of AML cells and BMECs were then exposed to increasing doses of cytarabine chemotherapy. There was a 2-fold decrease in leukemia cell death of AML cells when adhered to BMECs compared to non-adhered (30% vs. 60%, P < 0.0001). Even irradiated BMECs protected AML cells from cytarabine chemotherapy. To identify adhesion molecules mediating this protective effect, we analyzed cell membranes and supernatants of the cytarabine-treated co-cultures using protein microarrays. After cytarabine exposure, the Lutheran blood group glycoprotein basal cell adhesion molecule (BCAM) was upregulated in both human AML cells and BMECs. Prior work has shown BCAM as a receptor for Laminin and VLA5. As AML cells are known to express VLA5, we hypothesized that blocking BCAM may represent a novel therapeutic strategy. Blocking BCAM with neutralizing antibodies resulted in a 75% increase in non-adherent AML cells. Together, these in vitro results support the concept that ECs may be a protective reservoir for AML cells, at a minimum by means of adhesion molecules. BCAM represents a viable target. Because of the complex nature of the leukemia microenvironment, we sought to test this concept in vivo. Prior intravital efforts have focused on calvaria bone, which may over-represent the endosteal niche and under-represent the vascular niche due to the very close approximation of bone surfaces. Therefore, we created an intravital animal model of human AML to track single AML cells in the bone marrow of mouse long bones. In brief, we irradiated NOD/scid/IL2Rγnull (NSG) mice, drilled a window on the tibia surface, xenotransplanted fluorescently tagged human AML cells via IV injection, and then analyzed the tibias by fluorescent microscopy for the presence of AML cells at various time points after transplant. Initially the AML cells homed to endosteal surfaces of the bone marrow as early as one day after transplant. Over time, the AML cells lining the endosteum remained as single cells or very small clusters of a few cells. However, in the central marrow region the AML cells proliferated into massive clusters around blood vessels. Ongoing experiments are being performed to determine the disruption and resurgence of AML cells after chemotherapy and blockade of adhesion molecules. In sum, ECs protect human AML cells from chemotherapy by direct contact and the BCAM/Laminin/VLA5 axis may be a therapeutic target. Using our unique intravital imaging model of bone marrow in long bones, the endosteal niche appears to be the first site of homing and engraftment while the vascular niche appears to be the site for leukemia proliferation/progression. Disclosures:No relevant conflicts of interest to declare.

Human genetic polymorphisms in the Knops blood group are not associated with a protective advantage against Plasmodium falciparum malaria in Southern Ghana

BackgroundThe complex interactions between the human host and the Plasmodium falciparum parasite and the factors influencing severity of disease are still not fully understood. Human single nucleotide polymorphisms SNPs associated with Knops blood group system; carried by complement receptor 1 may be associated with the pathology of P. falciparum malaria, and susceptibility to disease.MethodsThe objective of this study was to determine the genotype and haplotype frequencies of the SNPs defining the Knops blood group antigens; Kna/b, McCoya/b, Swain-Langley1/2 and KCAM+/- in Ghanaian patients with malaria and determine possible associations between these polymorphisms and the severity of the disease. Study participants were patients (n = 267) admitted to the emergency room at the Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana during the malaria season from June to August in 1995, 1996 and 1997, classified as uncomplicated malaria (n = 89), severe anaemia (n = 57) and cerebral malaria (n = 121) and controls who did not have a detectable Plasmodium infection or were symptomless carriers of the parasite (n = 275). The frequencies were determined using a post-PCR ligation detection reaction-fluorescent microsphere assay, developed to detect the SNPs defining the antigens. Chi-square/Fisher’s exact test and logistic regression models were used to analyse the data.ResultsAs expected, high frequencies of the alleles Kna, McCb, Sl2 and KCAM- were found in the Ghanaian population. Apart from small significant differences between the groups at the Sl locus, no significant allelic or genotypic differences were found between the controls and the disease groups or between the disease groups. The polymorphisms define eight different haplotypes H1(2.4%), H2(9.4%), H3(59.8%), H4(0%), H5(25.2%), H6(0.33%), H7(2.8%) and H8(0%). Investigating these haplotypes, no significant differences between any of the groups were found.ConclusionThe results confirm earlier findings of high frequencies of certain CR1 alleles in Africa; and shed more light on earlier conflicting findings; the alleles McCb, Sl2, Knb and KCAM- or combined haplotypes do not seem to confer any protective advantage against malaria infection or resulting disease severity. Based on these findings, in a very well-characterized population, malaria does not seem to be the selective force on these alleles.

The Buffering Effect of Social Support on Hypothalamic-Pituitary-Adrenal Axis Function During Pregnancy

Recent studies suggest that effective social support during pregnancy may buffer adverse effects of maternal psychological distress on fetal development. The mechanisms whereby social support confers this protective advantage, however, remain to be clarified. The aim of this study was to assess whether individual differences in social support alter the covariation of psychological distress and cortisol during pregnancy. Eighty-two pregnant women's psychological distress and cortisol were prospectively assessed in all three trimesters using an ecological momentary assessment strategy. Appraisal of partner social support was assessed in each trimester via the Social Support Effectiveness questionnaire. In multilevel analysis, ambulatory assessments of psychological distress during pregnancy were associated with elevated cortisol levels (unstandardized β = .023, p < .001). Consistent with the stress-buffering hypothesis, social support moderated the association between psychological distress and cortisol (unstandardized β = -.001, p = .039), such that the covariation of psychological distress and cortisol increased with decreases in effective social support. The effect of social support for women with the most effective social support was a 50.4% reduction in the mean effect of distress on cortisol and a 2.3-fold increase in this effect for women with the least effective social support scores. Pregnant women receiving inadequate social support secrete higher levels of cortisol in response to psychological distress as compared with women receiving effective social support. Social support during pregnancy may be beneficial because it decreases biological sensitivity to psychological distress, potentially shielding the fetus from the harmful effects of stress-related increases in cortisol.

Diabetic Nephropathy

Genetic susceptibility contributes to the overall risk of nephropathy among individuals with diabetes. In this issue of JASN , Sandholm et al. [1][1] present the results of a sex-specific genome-wide association study aimed at identifying genetic factors for ESRD in type 1 diabetes. In identifying

Site Selectivity of the Pit Oichnus excavatus Donovan and Jagt Infesting Hemipneustes striatoradiatus (Leske) (Echinoidea) in the Type Maastrichtian (Upper Cretaceous, The Netherlands)

Two tests of the holasteroid echinoid Hemipneustes striatoradiatus (Leske) from the type area of the Maastrichtian Stage (Upper Cretaceous) bear a varied infestation of episkeletozoans (oysters, bryozoan colony, and serpulids), borings (probable Caulostrepsis isp., Oichnus simplex Bromley), surface abrasion (Gnathichnus? isp.), and pits (O. excavatus Donovan and Jagt). Only O. excavatus represents a premortem infestation. In one specimen, the four individual pits of this ichnospecies are each associated with a different ambulacrum and pore pairs that, in life, bore respiratory tube feet; the anterior ambulacrum, of different gross morphology, is not infested. In the second test, three out of four of the same ambulacra are infested, although there are also O. excavatus in the interambulacra. The association between O. excavatus and the ambulacra of the echinoid, and thus its tube feet, is open to several plausible explanations, but most likely provided some form of feeding or protective advantage to the pit-forming organism.

Helicobacter pylori Peptidoglycan Modifications Confer Lysozyme Resistance and Contribute to Survival in the Host

The prominent host muramidase lysozyme cleaves bacterial peptidoglycan (PG), and the enzyme is abundant in mucosal secretions. The lytic enzyme susceptibility of Gram-negative bacteria and mechanisms they use to thwart lytic enzyme activity are poorly studied. We previously characterized a Helicobacter pylori PG modification enzyme, an N-deacetylase (PgdA) involved in lysozyme resistance. In this study, another PG modification enzyme, a putative PG O-acetyltransferase (PatA), was identified. Mass spectral analysis of the purified PG demonstrated that a patA strain contained a greatly reduced amount of acetylated muropeptides, indicating a role for PatA in H. pylori PG O-acetylation. The PG modification mutant strains (pgdA, patA, or pgdA patA) were more susceptible to lysozyme killing than the parent, but this assay required high lysozyme levels (up to 50 mg/ml). However, addition of host lactoferrin conferred lysozyme sensitivity to H. pylori, at physiologically relevant concentrations of both host components (3 mg/ml lactoferrin plus 0.3 mg/ml lysozyme). The pgdA patA double mutant strain was far more susceptible to lysozyme/lactoferrin killing than the parent. Peptidoglycan purified from a pgdA patA mutant was five times more sensitive to lysozyme than PG from the parent strain, while PG from both single mutants displayed intermediate sensitivity. Both sensitivity assays for whole cells and for purified PGs indicated that the modifications mediated by PgdA and PatA have a synergistic effect, conferring lysozyme tolerance. In a mouse infection model, significant colonization deficiency was observed for the double mutant at 3 weeks postinoculation. The results show that PG modifications affect the survival of a Gram-negative pathogen.Importance Pathogenic bacteria evade host antibacterial enzymes by a variety of mechanisms, which include resisting lytic enzymes abundant in the host. Enzymatic modifications to peptidoglycan (PG, the site of action of lysozyme) are a known mechanism used by Gram-positive bacteria to protect against host lysozyme attack. However, Gram-negative bacteria contain a thin layer of PG and a recalcitrant outer membrane permeability barrier to resist lysis, so molecular modifications to cell wall structure in order to combat lysis remain largely unstudied. Here we show that two Helicobacter pylori PG modification enzymes (PgdA and PatA) confer a clear protective advantage to a Gram-negative bacterium. They protect the bacterium from lytic enzyme degradation, albeit via different PG modification activities. Many pathogens are Gram negative, so some would be expected to have a similar cell wall-modifying strategy. Understanding such strategies may be useful for combating pathogen growth.

Pyk2 Mediates Enhanced STAT3 Phosphorylation Following Collaborative Signaling Between gp130 and beta1 Integrins in Adhered Myeloma Cells

AbstractAbstract 4021Multiple myeloma remains an incurable bone marrow resident malignancy, in part, due to the eventual development of drug resistance linked to both acquired genetic changes and the dynamic influences of the microenvironment. We and others have shown that adhesion of tumor cells to the extracellular matrix component fibronectin (FN) via integrins leads to cell cycle arrest and protection from chemotherapy or Cell Adhesion-Mediated Drug Resistance (CAM-DR). The pleiotropic cytokine IL-6 has also been shown to mediate drug resistance in both solid and hematopoietic tumors [1]. The influence of these two effectors on drug resistance has primarily been studied separately; however, within the context of the bone marrow myeloma cells are influenced by both soluble and physical effectors simultaneously. Using a reductionist model of the multivariant microenvironment, we demonstrated that unique collaborative signaling between FN-adhered cells and IL-6 leads to increased proliferation of protected tumor cells and a more malignant phenotype [2]. Our results demonstrate the FN-adhesion mediated cell-cycle arrest of myeloma cells was reversed following stimulation of adhered cells with IL-6 and drug resistance was maintained. This phenotype was associated with a novel amplification of IL-6-induced STAT3 activation in adhered cells [2]. We have since validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in myeloma patient specimens by flow cytometry. To further characterize post-translational events induced under multivariant conditions, we performed immune-affinity phosphotyrosine proteomic screening in RMPI8226 myeloma cells maintained in suspension or adhered to FN with or without IL-6 stimulation. Screening identified 338 differentially tyrosine phosphorylated peptides. Among these were proteins involved in signal transduction, cytoskeleton assembly, survival, and metabolic pathways. Proline-rich tYrosine Kinase 2 (Pyk2/FAK2) was highly phosphorylated at Y597 in FN-adhered RPMI8226 myeloma cells. Eighty-three phosphorylation events were up-regulated by adhesion, including paxillin at pY118 and p130CAS at pY249 indicating focal adhesion formation. Pyk2 is a downstream intermediate of integrin signaling and has been demonstrated to amplify EGFR and cSrc-induced STAT3 activation. As such, we hypothesized that Pyk2 may be an important modulator of the enhanced STAT3 activation following multivariant signaling between beta1 integrins and gp130 [3]. In addition to enhanced phosphorylation of Pyk2 at Y579 identified by proteomic screening, western blot analysis demonstrated Pyk2 autophosphorylation of Y402 following myeloma cell adhesion to FN correlating with the amplification of IL-6-induced STAT3 and JAK1. Targeting Pyk2 with RNA interference attenuated the adhesion-associated amplification of STAT3 and JAK1 phosphorylation, but did not influence the limited STAT3 activation in cells grown in suspension. Pyk2 siRNA did not inhibit myeloma cell adhesion (n=4, p-value >0.05). Our previous results demonstrated that that the enhanced STAT3 signaling involved a FN-adhesion specific binding of unphosphorylated STAT3 with gp130 (independent of IL-6 stimulation). To determine if Pyk2 was similarly recruited to gp130 under co-stimulatory conditions we have used proximal ligation assay (PLA). Confocal imaging of the PLA reaction with antisera to Pyk2 and gp130 demonstrated colocalization of the two effectors upon adhesion of patient myeloma cells to FN, but not in those grown in suspension. We have also demonstrated that STAT3 activation is markedly enhanced in myeloma cell lines adhered to patient bone marrow stroma (BMS), but not cells grown in transwell coculture (no myeloma cell-BMS contact). Critically, the protective advantage afforded myeloma cells in coculture was attenuated only by dual knockdown of STAT3 and Pyk2 using novel antisense oligonucleotides (ISIS Pharmaceuticals, n=4, p-value 0.027). These data demonstrate a novel Pyk2-mediated JAK1/STAT3 signaling cascade within the context of multivariant stimulation. Moreover, these data suggest that strategies targeting of both lateral and vertical signaling (Pyk2 & STAT3) may be required to overcome therapy resistance conferred to tumor cells by the multivariant bone marrow niche. Disclosures:No relevant conflicts of interest to declare.

Albinism and disease causing pathogens in Tanzania: Are alleles that are associated with OCA2 being maintained by balancing selection?

Oculocutaneous albinism type 2 (OCA2) is present at significantly higher frequencies in sub-Saharan African populations compared to populations in other regions of the world. In Tanzania and other sub-Saharan countries, most OCA2 is associated with a common 2.7kb deletion allele. Leprosy is also in high prevalence in sub-Saharan African populations. The infectious agent of leprosy, Mycobacterium leprae, contains a gene, 38L, that is similar to OCA2. Hypopigmented patches of skin are early symptoms that present with infection of leprosy. In consideration of both the genetic similarity of OCA2 and the 38L gene of M. leprae and the involvement of pigmentation in both disorders, we hypothesized that the high rates of OCA2 may be due to heterozygote advantage. Hence, we hypothesized that carriers of the 2.7kb deletion allele of OCA2 may provide a protective advantage from infection with leprosy. We tested this hypothesis by determining the carrier frequency of the 2.7kb deletion allele from a sample of 240 individuals with leprosy from Tanzania. The results were inconclusive due to the small sample size; however, they enabled us to rule out a large protective effect, but perhaps not a small advantage.Mycobacterium tuberculosis is another infectious organism prevalent in sub-Saharan Africa that contains a gene, arsenic-transport integral membrane protein that is also similar to OCA2. Interestingly, chromosomal region 15q11-13, which also contains OCA2, was reported to be linked to tuberculosis susceptibility. Although variants within OCA2 were tested for association, the 2.7kb deletion allele of OCA2 was not tested. This led us to hypothesize that the deletion allele may confer resistance to susceptibility. Confirmation of our hypothesis would enable development of novel pharmocogenetic therapies for the treatment of tuberculosis, which in turn, may enable development of drugs that target other pathogens that utilize a similar infection mechanism as M. tuberculosis. From an evolutionary perspective, confirmation of our hypothesis may provide another example of heterozygote advantage.

Detrimental effects of prolonged warm renal ischaemia‐reperfusion injury are abrogated by supplemental hydrogen sulphide: an analysis using real‐time intravital microscopy and polymerase chain reaction

What's known on the subject? and What does the study add? Hydrogen sulphide (H(2) S) has recently been classified as a member of the gasotransmitter family. Its physiological and pathophysiological effects are rapidly expanding with numerous studies highlighting the protective effects of H(2) S on ischaemia-reperfusion injury (IRI) in various organ systems, e.g. heart, liver, CNS and lungs. The mechanisms behind its protective effects reside in its vasodilatory, anti-inflammatory and anti-oxidant characteristics. These specific mechanistic profiles appear to be different across different tissues and models of IRI. We recently showed that supplementation of preservation solutions with H(2) S during periods of prolonged cold renal storage and subsequent renal transplantation leads to a massive and significant survival, functional and tissue protective advantage compared with storage in standard preservation solution alone. However, there have only been a few studies that have evaluated the effects of H(2) S against warm renal IRI; although these studies have focused primarily upon shorter periods of warm renal pedicle clamping, they have shown a clear survival benefit to H(2) S supplementation. The present study adds to the existing literature by evaluating the effects of H(2) S in a model of warm IRI with clinically relevant, prolonged warm ischaemia-reperfusion times (1 h ischaemia, 2 h reperfusion). We show an unprecedented view into real-time renal and hepatic perfusion with intravital microscopy throughout the reperfusion period. We show, for the first time, that supplemental H(2) S has multiple protective functions against the warm IRI-induced tissue damage, which may be clinically applicable to both donation after cardiac death models of renal transplantation, as well as to uro-oncological practices requiring surgical clamping of the renal pedicle, e.g. during a partial nephrectomy. • To determine the protective role of supplemental hydrogen sulphide (H(2) S) in prolonged warm renal ischaemia-reperfusion injury (IRI) using real-time intravital microscopy (IVM). • Uninephrectomised Lewis rats underwent 1 h of warm ischaemia and 2 h of reperfusion during intraperitoneal treatment with phosphate buffer saline (IRI, n = 10) or 150 µmol/L NaHS (IRI+H(2) S, n = 12) and were compared with sham-operated rats (n = 9). • Blood was collected for measurement of serum creatinine (Cr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). • IVM was performed to assess renal and hepatic microcirculation. • Kidneys were sectioned for histology and real-time quantitative polymerase chain reaction for markers of inflammation. • The mean (sd) Cr concentration raised to 72.8(2.5) µmol/L after IRI from 11.0 (0.7) µmol/L (sham) but was partially inhibited with H(2) S to 62.8 (0.9) µmol/L (P < 0.05). • H(2) S supplementation during IRI increased renal capillary perfusion on IVM, and improved acute tubular necrosis and apoptotic scores on histology (P < 0.05). • Supplemental H(2) S decreased expression of the pro-inflammatory markers toll-like receptor 4, tumour necrosis factor α, interleukin 8, C-C chemokine receptor type 5, interferon γ and interleukin 2 (P < 0.05). • Distant organ (liver) dysfunction after renal IRI was limited with H(2) S supplementation: blunting of the ALT and AST surge, decreased hepatic sinusoidal vasodilation, and decreased leukocyte infiltration in post-sinusoidal venules (P < 0.05). • H(2) S supplementation directly inhibited interleukin 8-induced neutrophil chemotaxis in vitro (P < 0.05). • These findings are the first to show the real-time protective role of supplemental H(2) S in prolonged periods of warm renal IRI, perhaps acting by decreasing leukocyte migration and limiting inflammatory responses. • The protective effects of H(2) S suggest potential clinical applications in both donors after cardiac death models of renal transplantation and oncological practices requiring vascular clamping.

Interspecific and intraspecific variation in selenium:mercury molar ratios in saltwater fish from the Aleutians: Potential protection on mercury toxicity by selenium

A number of factors affect the consumption risk from mercury in fish, including mercury levels, seasonal patterns of mercury concentrations, human consumption patterns, and sensitive populations (e.g. pregnant women, fetuses, young children, and yet unknown genetic factors). Recently the protective effects of selenium on methylmercury toxicity have been publicized, particularly for saltwater fish. We examine levels of mercury and selenium in several species of fish and seabirds from the Aleutians (Alaska), determine selenium:mercury molar ratios, and examine species-specific and individual variation in the ratios as a means of exploring the use of the ratio in risk assessment and risk management. Variation among species was similar for mercury and selenium. There was significant interspecific and intraspecific variation in selenium:mercury molar ratios for fish, and for birds. The mean selenium:mercury molar ratios for all fish and bird species were above 1, meaning there was an excess of selenium relative to mercury. It has been suggested that an excess of selenium confers some protective advantage for salt water fish, although the degree of excess necessary is unclear. The selenium:mercury molar ratio was significantly correlated negatively with total length for most fish species, but not for dolly varden. Some individuals of Pacific cod, yellow irish lord, rock greenling, Pacific halibut, dolly varden, and to a lesser extent, flathead sole, had selenium:mercury ratios below 1. No bird muscle had an excess of mercury (ratio below 1), and only glaucous-winged gull and pigeon guillemot had ratios between 1 and 5. There was a great deal of variation in selenium:mercury molar ratios within fish species, and within bird species, making it difficult and impractical to use these ratios in risk assessment or management, for fish advisories, or for consumers, particularly given the difficulty of interpreting the ratios.

Comparison of intensity-modulated radiation therapy and 3-dimentional conformal radiotherapy plansfor cervical carcinoma with recurrence and metastasis

Objective To compare the treatment effects,dosimetry,and toxicities to organs at risk of intensity-modulated radiation therapy (IMRT) and 3-dimentional conformal radiotherapy (3DCRT) plans for cervical carcinoma patients with recurrence and metastasis. Methods 62 cervical carcinoma patients with recurrence and metastasis were analyzed retrospectively, and divided into two groups including intensitymodulated radiation therapy group (n=29) and 3-dimentional conformal radiotherapy group (n=32).Patients were performed with 6 MV-X ray.The regimen was 1.8-2.2 Gy/f,1 f/d,18-33 times in total.Prescribed dose was 40-60 Gy and median dose was 52.8 Gy. At the same time for the IMRT group 29 cases were performed with 3DCRT, which was designed using the same prescribed dose to compare radiation dose distributed in organs at risk (OAR).Results The maximum dose of the two plans showed that bladder and small intestine in IMRT plans were lower than that in 3DCRT (P＜0.05), showing the IMRT's protective advantage. The maximum dose of PTV in IMRT plans were significantly higher than 3DCRT (P＜0.05). In the group of IMRT plans, the overall 1-,2-,and 3-year survival rates were 65.5 ％(19/29), 42.1％(8/19), and 25.0 ％(2/8),respectively,the median survival time was 19 months,of 28 deaths,21 patients died of tumor progression,7 patients died of distant metastases.In the group of 3DCRT plans,the overall 1-,2-,and 3-year survival rates were 60.6 ％(20/33),35.0 ％(7/20),and 14.3 ％(1/7),respectively,the median survival time was 17 months,of 32 deaths, 24 patients died of tumor progression, 8 patients died of distant metastasis. There were no significant differences in overall 1-, 2-, and 3-year survival rates between the two groups (P＞0.05). The incidence rate of toxicity in the IMRT plans was significantly lower than that in the 3DCRT plans, especially for patients with Ⅰ level and Ⅱ level.Conclusion The treatment effects of the IMRT plans are better than the 3DCRT plans for cervical carcinoma patients with recurrence and metastasis. IMRT plans can improve radiation dose in tumors and reduce the dose distributed in normal issue and reduce the incidence of the side effect. Key words: Uterine cervical neoplasms; Recurrence; Neoplasm metastasis; Radiotherapy,intensity-modulated; Radiotherapy, conformal

The Effect of Bilingualism on Amnestic Mild Cognitive Impairment

Previous reports have found that lifelong bilingualism is associated with a delay in the onset of dementia, including Dementia of the Alzheimer's Type (DAT). Because amnestic mild cognitive impairment (aMCI) is often a transition stage between normal aging and DAT, our aim in this paper was to establish whether this delay in symptom onset for bilinguals would also be seen in the onset of symptoms of aMCI and whether this delay would be consistent in different subtypes of aMCI. We examined the effect of bilingualism on the age of diagnosis in individuals with single- or multiple-domain aMCI who were administered a battery of neuropsychological tests and questionnaires about their language and social background. Our results showed an interaction between aMCI type and language history. Only individuals diagnosed with single-domain aMCI demonstrated a later age of diagnosis for bilinguals (M = 79.4 years) than monolinguals (M = 74.9 years). This preliminary evidence suggests that the early protective advantage of bilingualism may be specific to single-domain aMCI, which is the type of aMCI most specifically associated with progression to DAT.

A morpho‐anatomical characterisation of Myrothamnus moschatus (Myrothamnaceae) under the aspect of desiccation tolerance

Morpho-anatomical traits of the rarely studied dicotyledonous desiccation-tolerant shrub Myrothamnus moschatus were examined and compared for the first time to Myrothamnus flabellifolius under the aspect of desiccation tolerance. Both species almost exclusively occur on rock outcrops and differ mainly in their geographic range and leaf morphology (fan-shaped in M. flabellifolius, lanceolate in M. moschatus) but have a very similar leaf and wood anatomy, except for the lack of hydathodes in M. moschatus. Both species adopt the parallel leaf venation of monocots, although this is more pronounced in M. moschatus. This provides a mechanical and protective advantage over the net venation pattern of most dicots and facilitates the reversible, drought-induced, accordion-like leaf contraction. The sclerenchyma, as a stabilising tissue, is mainly confined to vascular bundles in leaves of both species. Here, mechanical support seems to be less crucial for survival in long periods of drought than other morpho-anatomical traits (e.g. parallel leaf venation).

Editorial: Molecular Endocrinology Articles in the Spotlight for January 2012

Beginning with this issue of Molecular Endocrinology, we’ll be highlighting three articles that should be of special interest to our readers. Although all manuscripts accepted for publication in Molecular Endocrinology have a high degree of scientific merit, the three selected for an editorial highlight are meant to showcase particularly novel and significant findings reflective of others that can be found throughout the issue. This first issue of 2012 includes the following articles that are excellent examples of the relevance of basic science findings to clinical management of endocrine disorders. The first, “Reduced bone turnover in mice lacking the P2Y13 receptor of ADP” by Alison Gartland and colleagues, addresses the issue of osteoporosis and the need for drugs that provide alternative treatment for osteoporosis. The authors show that deletion of the P2Y13 receptor, a G protein-coupled receptor for extracellular ADP, leads to a reduction in trabecular bone mass, reduction in osteoblast and osteoclast numbers and an overall reduction in the rate of bone remodeling in mice. They also find that P2Y13R( / ) mice are protected from ovariectomyinduced bone loss. Therefore, the P2Y13 receptor may provide a new therapeutic target that confers a protective advantage in conditions of accelerated bone turnover such as estrogen deficiency-induced osteoporosis. This would be a tremendous benefit to the one third of all women and one fifth of all men over fifty who will experience an osteoporotic fracture in their lifetime. “FXR protects lung from LPS-induced acute injury”, by Wendong Huang and colleagues, is another article in the spotlight this month. This research addresses acute lung injury, which is associated with 17,000–43,000 deaths each year. In one of the important findings of this study, Huang et al. demonstrate that expression of a constitutively active FXR repressed the expression of proinflammatory genes and improved lung permeability and lung regeneration in FXR / mice. Therefore, FXR may be an ideal target for drug development as novel and safe FXR ligands may be effective for the treatments of lung injury and other related diseases. Finally, Signe Altmae and colleagues bring us a Research Resource contribution, “Interactome of human embryo implantation: identification of gene expression pathways, regulation, and integrated regulatory networks.” This study helps us to better understand the complex genetic network that leads to successful embryo implantation and will be relevant to the development of future strategies to alleviate human infertility. The authors are the first to perform thorough computational analysis to identify the potential molecular interactions between implantation-stage endometrium and the embryo. I hope you enjoy these articles, as well as the rest of the exceptional articles in this issue. With best wishes for 2012— Donald B. DeFranco, Editor-in-Chief