Genetic diversity does not explain variation in extra-pair paternity in multiple populations of a songbird.

Many songbirds are socially monogamous but genetically polyandrous, mating with individuals outside their pair bonds. Extra-pair paternity (EPP) varies within and across species, but reasons for this variation remain unclear. One possible source of variation is population genetic diversity, which has been shown in interspecific meta-analyses to correlate with EPP but which has limited support from intraspecific tests. Using eight populations of the genetically polyandrous red-winged blackbird (Agelaius phoeniceus), including an island population, we investigated whether population-level differences in genetic diversity led to differences in EPP. We first measured genetic diversity over 10 microsatellite loci and found, as predicted, low genetic diversity in the island population. Additional structure analyses with multilocus genotypes and mtDNA showed the island population to be distinct from the continental populations. However, the island population's EPP rate fell in the middle of the continental populations' distribution, whereas the continental populations themselves showed significant variation in EPP. This result suggests that genetic diversity by itself is not a predictor of EPP rate. We discuss reasons for the departure from previous results, including hypotheses for EPP that do not solely implicate female-driven behaviour.

ITPKC and CASP3 polymorphisms and risks for IVIG unresponsiveness and coronary artery lesion formation in Kawasaki disease.

Functional single-nucleotide polymorphisms (SNPs) in inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) (rs28493229) and caspase-3 (CASP3) (rs113420705; formerly rs72689236) are associated with susceptibility to Kawasaki's disease (KD). To evaluate the involvement of these 2 SNPs in the risk for intravenous immunoglobulin (IVIG) unresponsiveness, we investigated 204 Japanese KD patients who received a single IVIG dose of 2 g kg(-1) (n=70) or 1 g kg(-1) daily for 2 days (n=134). The susceptibility allele of both SNPs showed a trend of overrepresentation in IVIG non-responders and, in combined analysis of these SNPs, patients with at least 1 susceptible allele at both loci had a higher risk for IVIG unresponsiveness (P=0.0014). In 335 prospectively collected KD patients who were treated with IVIG (2 g kg(-1)), this 2-locus model showed a more significant association with resistance to initial and additional IVIG (P=0.011) compared with individual SNPs. We observed a significant association when all KD patients with coronary artery lesions were analyzed with the 2-locus model (P=0.0031). Our findings strongly suggest the existence of genetic factors affecting patients' responses to treatment and the risk for cardiac complications, and provide clues toward understanding the pathophysiology of KD inflammation.

Hepatocellular uptake index obtained with gadoxetate disodium-enhanced magnetic resonance imaging in the assessment future liver remnant function after major hepatectomy for biliary malignancy.

BACKGROUND: Functional assessment of the future liver remnant (FLR) after major hepatectomy is essential but often difficult in patients with biliary malignancy, owing to obstructive jaundice and portal vein embolization. This study evaluated whether a novel index using gadoxetate disodium-enhanced MRI (EOB-MRI) could predict posthepatectomy liver failure (PHLF) after major hepatectomy for biliary malignancy. METHODS: The remnant hepatocellular uptake index (rHUI) was calculated in patients undergoing EOB-MRI before major hepatectomy for biliary malignancy. Receiver operating characteristic (ROC) curve analyses were used to evaluate the accuracy of rHUI for predicting PHLF grade B or C, according to International Study Group of Liver Surgery criteria. Multivariable logistic regression analyses comprised stepwise selection of parameters, including rHUI and other conventional indices. RESULTS: This study included 67 patients. The rHUI accurately predicted PHLF (area under the curve (AUC) 0.896). A cut-off value for rHUI of less than 0.410 predicted all patients who developed grade B or C PHLF. In multivariable analysis, only rHUI was an independent risk factor for grade B or C PHLF (odds ratio 2.0 × 103, 95 per cent c.i. 19.6 to 3.8 × 107; P < 0.001). In patients who underwent preoperative portal vein embolization, rHUI accurately predicted PHLF (AUC 0.885), whereas other conventional indices, such as the plasma disappearance rate of indocyanine green of the FLR and FLR volume, did not. CONCLUSION: The rHUI is potentially a useful predictor of PHLF after major hepatectomy for biliary malignancy.

Ciliary neurotropic factor, interleukin 11, leukemia inhibitory factor, and oncostatin M are growth factors for human myeloma cell lines using the interleukin 6 signal transducer gp130.

Interleukin 6 (IL-6) is a major growth factor for tumor plasma cells involved in human multiple myeloma (MM). In particular, human myeloma cell lines (HMCL), whose growth is completely dependent on addition of exogenous IL-6, can be obtained reproducibly from every patient with terminal disease. Four cytokines, ciliary neurotropic factor (CNTF), IL-11, leukemia inhibitory factor (LIF), and oncostatin M (OM), use the same transducer chain (signal transducer gp130) as IL-6 and share numerous biological activities with this IL. We found that these four cytokines stimulated proliferation and supported the long-term growth of two out of four IL-6-dependent HMCL obtained in our laboratory. Half-maximal proliferation was obtained with cytokine concentrations ranging from 0.4 to 1.2 ng/ml for IL-11, LIF, and OM. CNTF worked at high concentrations only (90 ng/ml), but addition of soluble CNTF receptor increased sensitivity to CNTF 30-fold. The growth-promoting effect of these four cytokines was abrogated by anti-gp130 antibodies, contrary to results for anti-IL-6 receptor or anti-IL-6 antibodies. No detectable changes in the morphology and phenotype were found when myeloma cells were cultured with one of these four cytokines instead of IL-6. Concordant with their IL-6-dependent growth, the four HMCL expressed membrane IL-6R and gp130 detected by FACS analysis. LIF-binding chain gene (LIFR) was expressed only in the two HMCL responsive to LIF and OM.

Analysis of interleukin 6 receptor and gp130 expressions and proliferative capability of human CD34+ cells.

We recently demonstrated that stimulation of gp130 by a combination of soluble interleukin 6 receptor (sIL-6R) and IL-6 but not IL-6 alone significantly stimulates the ex vivo expansion of primitive hematopoietic progenitors and the generation of erythroid cells from human CD34+ cells in the presence of stem cell factor (SCF). Here, we show that gp130 is found low positively on most CD34+ cells, whereas IL-6R is expressed on only 30-50% of these cells. Although most of the colonies generated from FACS-sorted CD34+IL-6R+ cells were granulocyte/macrophage (GM) colonies, CD34+IL-6R- cells gave rise to various types of colonies, including erythroid bursts, GM, megakaryocytes, and mixed colonies in methylcellulose culture with a combination of IL-6, sIL-6R, and SCF. Similar results were obtained in culture supplemented with a combination of IL-3, IL-6, SCF, granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. A limiting dilution analysis of long-term culture-initiating cells (LTC-IC) showed that the CD34+IL-6R- cells contained a larger number of LTC-IC than did the CD34+IL-6R+ cells. In a serum-free suspension of CD34+IL-6R- cells, the addition of sIL-6R to the combination of IL-6 and SCF dramatically increased the total and multipotential progenitors, whereas CD34+IL-6R+ cells failed to do so under the same conditions. These results indicate that most of the erythroid, megakaryocytic, and primitive human hematopoietic progenitors are included in the IL-6R- populations, and the activation of gp130 on these progenitors can be achieved by a complex of IL-6-sIL-6R, but not by IL-6 alone. The present culture system using IL-6, sIL-6R, and SCF may provide a novel approach for ex vivo expansion of human primitive hematopoietic progenitors.

Erythropoietin-independent erythrocyte production: signals through gp130 and c-kit dramatically promote erythropoiesis from human CD34+ cells.

Erythropoietin (EPO) is the primary humoral regulator of erythropoiesis and no other factor has previously been reported to support proliferation and terminal maturation of erythroid cells from hemopoietic stem cells. Here we show that stimulation of glycoprotein (gp130) by a combination of recombinant human soluble interleukin 6 receptor (sIL-6R) and IL-6 but not sIL-6R or IL-6 alone can support proliferation, differentiation, and terminal maturation of erythroid cells in the absence of EPO from purified human CD34+ cells in suspension culture containing stem cell factor (SCF). A number of erythroid bursts and mixed erythroid colonies also developed in methylcellulose culture under the same combination. The addition of anti-gp130 monoclonal antibodies but not anti-EPO antibody to the same culture completely abrogated the generation of erythroid cells. These results clearly demonstrate that mature erythroid cells can be emerged from hemopoietic progenitors without EPO in vitro. Together with the previous reports that human sera contain detectable levels of sIL-6R, IL-6, and SCF, current data suggest that gp130 signaling in association with c-kit activation may play a role in human erythropoiesis in vivo.

Reduced susceptibility to amoxicillin of oral streptococci following amoxicillin exposure.

As antibiotic pressure often triggers bacterial resistance, the use of short-duration therapies is increasingly recommended. The objective of the present study was to evaluate both the clinical efficiency and the impact on oral streptococci of a 3 day versus a 7 day amoxicillin therapy for odontogenic infection requiring tooth extraction. On day 0, patients were randomly assigned to a 3 day or 7 day amoxicillin treatment. The tooth was extracted on day 2 and the post-operative follow-up was carried out on day 9. Oral flora was collected on days 0, 9 and 30, and the susceptibility of the streptococci to amoxicillin was determined. The results showed that treatment with amoxicillin for 3 or 7 days had a similar clinical efficiency, and also induced similar selection of oral streptococci with reduced susceptibility to amoxicillin, suggesting that the selection of strains with reduced susceptibility to amoxicillin is a rapid phenomenon, appearing even with short-duration therapies.

IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase.

The biological functions of interleukin-6 (IL-6) are mediated through a signal-transducing component of the IL-6 receptor, gp130, which is associated with the ligand-occupied IL-6 receptor (IL-6R) protein. Binding of IL-6 to IL-6R induced disulfide-linked homodimerization of gp130. Tyrosine kinase activity was associated with dimerized but not monomeric gp130 protein. Substitution of serine for proline residues 656 and 658 in the cytoplasmic motif abolished tyrosine kinase activation and cellular responses but not homodimerization of gp130. The IL-6-induced gp130 homodimer appears to be similar in function to the heterodimer formed between the leukemia inhibitory factor (LIF) receptor (LIFR) and gp130 in response to the LIF or ciliary neurotrophic factor (CNTF). Thus, a general first step in IL-6-related cytokine signaling may be the dimerization of signal-transducing molecules and activation of associated tyrosine kinases.

Association of transcription factor APRF and protein kinase Jak1 with the interleukin-6 signal transducer gp130.

Interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, interleukin-11, and ciliary neurotrophic factor bind to receptor complexes that share the signal transducer gp130. Upon binding, the ligands rapidly activate DNA binding of acute-phase response factor (APRF), a protein antigenically related to the p91 subunit of the interferon-stimulated gene factor-3 alpha (ISGF-3 alpha). These cytokines caused tyrosine phosphorylation of APRF and ISGF-3 alpha p91. Protein kinases of the Jak family were also rapidly tyrosine phosphorylated, and both APRF and Jak1 associated with gp130. These data indicate that Jak family protein kinases may participate in IL-6 signaling and that APRF may be activated in a complex with gp130.

Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor.

Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.

Effects of prolonged exposure to CO2 on behaviour, hormone secretion and respiratory muscles in young female rats.

Atmospheric CO2 concentrations increased significantly over the last century and continuing increases are expected to have significant effects on current ecosystems. This study evaluated the behavioural and physiological (hormone status, muscle structure) effects of prolonged CO2 exposure in young female Wistar rats exposed at 700ppm of CO2 during 6h a day for 15days. Prolonged CO2 exposure, though not continuous, produced significant disturbances in behaviour with an increase in drinking, grooming and resting, and a reduction in rearing, jumping-play and locomotor activity. Furthermore, CO2 exposure was accompanied by increased plasma levels of corticosterone, suggesting that prolonged exposure to CO2 was stressful. The muscular structure can also be modified also when respiratory working conditions change. The expression of myosin heavy chain was significantly affected in the diaphragm and oral respiratory muscles: Masseter Superficialis and Anterior Digastric. Modified behaviour and hormonal changes both appear to be at the origin of the observed muscular adaptation.

A human monocyte growth factor produced by lung cancer cells.

Human lung cancer cell line, T3M-30, has been shown to produce a growth factor that stimulates proliferation of peripheral blood monocytes. In the presence of this factor, human circulating monocytes were able to proliferate in vitro. Gel exclusion chromatography of the conditioned medium revealed a single peak of monocyte growth-promoting activity at an apparent molecular weight of 16,000. The growth-promoting activity was adsorbed to an anion-exchange column, Mono Q, and eluted with a salt gradient as a single peak of bioactivity at 300 mM NaCl. When the sample was applied to a Vydac C4 column, a reverse-phase high-performance liquid chromatography column, a single peak of activity was observed at a concentration of 76% acetonitrile in 0.1% trifluoroacetic acid. The monocyte growth-promoting activity was heat stable at 56 degrees C. It was partially destroyed by trypsin. The activity was lost after treatment with 2-mercaptoethanol.

Purification and characterization of a cell growth factor from a human leukemia cell line: immunological identity with ferritin.

We have succeeded in long-term cultivation of a human erythroleukemia cell line, K-562-T1 (T. Okabe, M. Fujisawa, and F. Takaku, Proc. Natl. Acad. Sci. USA, 81: 453-455, 1984). The cells grown in a protein-free chemically defined medium have been shown to produce cell growth factors (A. Mihara et al., In Vitro Cell. Dev. Biol., 23: 317-322, 1987). In this study, we have purified a cell growth factor from the conditioned medium that stimulates the proliferation of human leukemia cells, HL-60. In the purified factor, two major protein bands of 24 kDa and 22 kDa were identified on a sodium dodecyl sulfate-polyacrylamide gel. The 22 kDa protein was stained with a monoclonal antibody to the light chain of ferritin. The growth-promoting activity of the purified factor was coprecipitated with a monoclonal antibody to the light chain or heavy chain of human ferritin. These results suggest that K-562-T1 cells produce a cell growth factor that is related to ferritin.

Proton nuclear magnetic resonance study of human interleukin 6: chemical modifications and partial spectral assignments for the aromatic residues.

Partial assignments for the 1H-NMR resonances of the aromatic residues in human interleukin 6 (IL-6) are reported. The homonuclear Hartmann-Hahn spectrum clearly shows all connectivities for the histidine, tyrosine and tryptophan residues that exist in IL-6. Using a deuterium exchange method, the imidazole proton resonances of His-16 and His-165 have been assigned. Iodination of the tyrosine residues led to the assignment of Tyr-32. Photo-chemically induced dynamic nuclear polarization data have shown that His-16, Tyr-32 and Trp-158 are exposed to solvent, whereas His-165, Tyr-98 and Tyr-101 are buried. Iodination of Tyr-32 gave no significant effect on IL-6 activity, suggesting that Tyr-32 is not responsible for IL-6 activity.

Alleviation of myocardial ischemia after Kawasaki disease by heparin and exercise therapy.

BACKGROUND: Heparin promotes angiogenesis. We evaluated the effects of combined treatment with heparin and exercise on myocardial ischemia in the chronic stage of Kawasaki disease. METHODS AND RESULTS: This study was conducted in 7 patients (aged 6 to 19 years) who had a totally occluded coronary artery and stress-induced myocardial ischemia in the collateral-dependent areas. Twice-daily exercise using a bicycle ergometer was performed with increments of 0.5 W/kg every 3 minutes up to maximal exertion for 10 days. Heparin, which immediately increased circulating hepatocyte growth factor, was given intravenously 10 minutes before each exercise period. Newly developed myocardial infarction, ventricular tachyarrhythmia, anginal attack, or hemorrhagic complication was not observed in any patient. Dipyridamole-loading single photon emission computed tomography documented improved myocardial perfusion in the collateral-dependent areas and a significant reduction in total defect scores in all patients after the completion of 20 sessions (P=0.01). In control patients who did not receive the heparin-exercise therapy, however, stress defect scores remained unchanged (n=1) or increased (n=2) during follow-up. Computerized quantitative coronary angiography provided evidence that the heparin-exercise therapy increased the diameter of the occluded artery to which collaterals terminated (P=0.001) but not that of the reference artery with which collaterals were not connected (P=0.96). CONCLUSIONS: The findings suggest that a series of heparin and exercise treatments over 10 days may have a dramatic effect on the alleviation of myocardial ischemia in collateral-dependent regions. This may be a safe, noninvasive revascularization therapy for patients with coronary artery occlusion in the chronic stage of Kawasaki disease.

Maintenance of the pluripotential phenotype of embryonic stem cells through direct activation of gp130 signalling pathways.

Propagation of the undifferentiated pluripotential phenotype of embryonic stem (ES) cells is dependent on the cytokine differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF). The DIA/LIF receptor complex is a heterodimer of DIA/LIF receptor (DIA/LIF-R) and gp130. The latter is also a component of the interleukin-6 (IL-6) receptor complex. We report that a combination of IL-6 and soluble IL-6 receptor (sIL-6R), which can induce homodimerisation of gp130 and activation of signalling processes, sustains self-renewal of pluripotential ES cells. Our findings indicate that the IL-6/sIL-6R complex acts on ES cells through gp130 alone, bypassing DIA/LIF-R, and therefore implicate gp130 as the key component in the signalling pathway responsible for stem cell renewal.

Characterization of soluble gp130 released by melanoma cell lines: A polyvalent antagonist of cytokines from the interleukin 6 family.

gp130 acts as a common transducing signal chain for all receptors belonging to the interleukin (IL)-6 receptor family. The IL-6-related cytokines [IL-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin] often modulate tumor phenotype and control the proliferation of many tumor cell lines. We demonstrate that melanoma cell lines release, in vitro and in vivo (when transplanted in nude mice), soluble gp130 (sgp130), a potential antagonist of cytokines from the IL-6 family. Biochemical analysis revealed that sgp130 derived from melanoma patients' sera or from culture supernatants of melanoma cell lines is a Mr 104,000 protein that resolved after deglycosylation as a Mr 58,000 protein. PCR and Northern blot analysis only identified one gp130 membrane mRNA, suggesting that the soluble form of gp130 is generated by proteolytic cleavage. OSM reproducibly increases sgp130 released by melanoma cell lines, whereas leukemia inhibitory factor stimulates the production of sgp130 in only one of three cell lines tested. This tumor-derived sgp130 is functional because it binds in solution to the IL-6-soluble IL-6 receptor (gp80) complex. Recombinant sgp130 inhibits the growth inhibitory activity of the IL-6-soluble IL-6 receptor complex and OSM on some melanoma cell lines. Therefore, this soluble gp130 represents a natural antagonist of cytokines from the IL-6 family.

Dramatic decrease of circulating levels of monocyte chemoattractant protein-1 in Kawasaki disease after gamma globulin treatment.

Kawasaki disease (KD) is a systemic vasculitis preferentially affecting coronary arteries. Extensive monocytes/macrophages infiltrate in the vascular lesions, implying the involvement of a chemotactic cytokine in their recruitment. We investigated the role of monocyte chemoattractant protein-1 (MCP-1, also termed monocyte chemotactic and activating factor) in KD. In the immunohistochemical studies using the cardiac tissues of patients with fatal KD, MCP-1 but not interleukin (IL) -8 or macrophage inflammatory protein-1alpha was localized at the extracellular matrix associated with mononuclear cellular infiltration. The sites of MCP-1 expression correlated with the distribution of the acute inflammation, including early coronary vasculitis. In prospectively studied patients with KD, circulating levels of MCP-1, IL-8, tumor necrosis factor alpha (TNF-alpha), and IL-1alpha were elevated in 73, 77, 57, and 0% of samples before gamma globulin (GG) treatment (400 mg/kg x 5 days = total 2 g/kg), respectively, compared with respective control values. GG treatment correlated with a rapid decrease in the circulating levels of MCP-1 (P = 0.001) but not IL-8 (P = 0.19) or TNF-alpha (P = 0.33). In the sensitive Western blotting, MCP-1 bound to GG. Furthermore, GG inhibited the MCP-1-induced Ca2+ influx in a human monocytic cell line in vitro. These findings suggest a role of MCP-1 in KD, and indicate that GG treatment may block MCP-1 activity, thus alleviating KD vasculitis.

WT1 expression level and clinical factors in multiple myeloma.

Although Wilm's Tuomor gene (WT1) was first identified as a tumor suppressor gene for Wilm's tumor, WT1 overexpression has been detected in different malignant cell types including leukemia. Increased expression of WT1 in acute leukemia is potentially used as a marker of minimal residual disease. However, the significance of the gene for multiple myeloma is still not clear. To determine the clinical relevance of WT1 expression in multiple myeloma, we examined the association of clinical parameters and WT1 expression in bone marrow for 17 newly diagnosed multiple myeloma patients. WT1 was assessed by real-time quantitative polymerase chain reaction (RQ-PCR) and calculated standardized WT1 expression level per 100 plasma cells in the bone marrow specimen as "corrected WT1". The expression of standardized WT1 and corrected WT1 in myeloma was 59 to 1,600 copies/microg RNA and 0.05 to 406.3 copies/microg RNA/100 plasma cells, respectively, lower than in leukemia. WT1 transcripts increased when clinical factors worsen, including the stage, amount of M protein, Hb, platelet count, blood urea nitrogen (BUN), creatinine, serum alkaline phosphatase (ALP), calcium, beta2-microglobulin, thymidine kinase activity (TK), and C-reactive protein (CRP). In conclusion, the expression level of WT1 could be an additional marker to the standard parameters considered in risk assessment for multiple myeloma.

Identification of a 130-kilodalton tyrosine-phosphorylated protein induced by interleukin-11 as JAK2 tyrosine kinase, which associates with gp130 signal transducer.

Interleukin-11 (IL-11) shares the common signal transducer gp130 with IL-6, leukemia inhibitory factor (LIF), and oncostatin M (OSM) and triggers activation of unknown tyrosine kinases as the early steps of signal transduction pathway. Here we identify a 130-kilodalton tyrosine-phosphorylated protein induced by IL-11 in 3T3-L1 cells as JAK2 tyrosine kinase. We further show that the in vitro kinase activity of JAK2 is greatly enhanced following stimulation with IL-11 in 3T3-L1 cells and TF-1 cells. Furthermore, we demonstrate that JAK2 physically associates with the signal transducer gp130. Similar results were observed following stimulation with IL-6, LIF, and OSM. However, we were unable to show that JAK1 is tyrosine phosphorylated and activated by IL-11 under identical conditions. These results suggest that JAK2 tyrosine kinase is one of the tyrosine kinases involved in signal transduction mediated by IL-11, IL-6, LIF, and OSM.