Characterization of gene expression associated with the adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity.

Oxygen (O2) is a double-edged sword to cells, for while it is vital for energy production in all aerobic animals and insufficient O2 (hypoxia) can lead to cell death, the reoxygenation of hypoxic tissues may trigger the generation of reactive oxygen species (ROS) that can destroy any biological molecule. Indeed, both hypoxia and hypoxia-reoxygenation (H/R) stress are harmful, and may play a critical role in the pathophysiology of many human diseases, such as myocardial ischemia and stroke. Therefore, understanding how animals adapt to hypoxia and H/R stress is critical for developing better treatments for these diseases. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from H/R stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to Pseudomonas aeruginosa pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Membrane-bound globin X protects the cell from reactive oxygen species.

Globin X (GbX) is a member of the globin family that emerged early in the evolution of Metazoa. In vertebrates, GbX is restricted to lampreys, fish, amphibians and some reptiles, and is expressed in neurons. Unlike any other metazoan globin, GbX is N-terminally acylated and anchored in the cell membrane via myristoyl and palmitoyl groups, suggesting a unique function. Here, we compared the capacity of GbX to protect a mouse neuronal cell line from hypoxia and reactive oxygen species (ROS) with that of myoglobin. To evaluate the contribution of membrane-binding, we generated a mutated version of GbX without acyl groups. All three globins enhanced cell viability under hypoxia, with myoglobin having the most pronounced effect. GbX but not myoglobin protected the cells from hydrogen peroxide (H2O2)-induced stress. Membrane-bound GbX was significantly more efficient than its mutated, soluble form. Furthermore, myoglobin and mutated GbX increased production of ROS upon H2O2-treatment, while membrane-bound GbX did not. The results indicate that myoglobin enhances O2 supply while GbX protects the cell membrane from ROS-stress. The ancient origin of GbX suggests that ROS-protection reflects the function of the early globins before they acquired a respiratory role.

Cytoglobin is expressed in hepatic stellate cells, but not in myofibroblasts, in normal and fibrotic human liver.

Cytoglobin (CYGB) is ubiquitously expressed in the cytoplasm of fibroblastic cells in many organs, including hepatic stellate cells. As yet, there is no specific marker with which to distinguish stellate cells from myofibroblasts in the human liver. To investigate whether CYGB can be utilized to distinguish hepatic stellate cells from myofibroblasts in normal and fibrotic human liver, human liver tissues damaged by infection with hepatitis C virus (HCV) and at different stages of fibrosis were obtained by liver biopsy. Immunohistochemistry was performed on histological sections of liver tissues using antibodies against CYGB, cellular retinol-binding protein-1 (CRBP-1), α-smooth muscle actin (α-SMA), thymocyte differentiation antigen 1 (Thy-1), and fibulin-2 (FBLN2). CYGB- and CRBP-1-positive cells were counted around fibrotic portal tracts in histological sections of the samples. The expression of several of the proteins listed above was examined in cultured mouse stellate cells. Quiescent stellate cells, but not portal myofibroblasts, expressed both CYGB and CRBP-1 in normal livers. In fibrotic and cirrhotic livers, stellate cells expressed both CYGB and α-SMA, whereas myofibroblasts around the portal vein expressed α-SMA, Thy-1, and FBLN2, but not CYGB. Development of the fibrotic stage was positively correlated with increases in Sirius red-stained, α-SMA-positive, and Thy-1-positive areas, whereas the number of CYGB- and CRBP-1-positive cells decreased with fibrosis development. Primary cultured mouse stellate cells expressed cytoplasmic CYGB at day 1, whereas they began to express α-SMA at the cellular margins at day 4. Thy-1 was undetectable throughout the culture period. In human liver tissues, quiescent stellate cells are CYGB positive. When activated, they also become α-SMA positive; however, they are negative for Thy-1 and FBLN2. Thus, CYGB is a useful marker with which to distinguish stellate cells from portal myofibroblasts in the damaged human liver.

Fecal immunochemical tests compared with guaiac fecal occult blood tests for population-based colorectal cancer screening.

Colorectal cancer (CRC) is the second most common cause of cancer deaths in Canadian men and women - accounting for almost 12% of all cancer deaths. In Ontario, it is estimated that 8100 persons were diagnosed with CRC in 2011, and 3250 died from the disease. CRC incidence and mortality rates in Ontario are among the highest in the world. Screening offers the best opportunity to reduce this burden of disease. The present report describes the findings and recommendations of Cancer Care Ontario's Fecal Immunochemical Tests (FIT) Guidelines Expert Panel, which was convened in September 2010 by the Program in Evidence-Based Care. The purpose of the present guideline is to evaluate the existing evidence concerning FIT to inform the decision on how to replace the current guaiac fecal occult blood test with FIT in the Ontario ColonCancerCheck Program. Eleven articles were included in the present guideline, comprising two systematic reviews, five articles reporting on three randomized controlled trials, and reports of four other studies. Additionally, one laboratory study was obtained that reported on several parameters of FIT tests that helped to inform the present recommendation. The performance of FIT is superior to the standard guaiac fecal occult blood test in terms of screening participation rates and the detection of CRC and advanced adenoma. Given greater specimen instability with the use of FIT, a pilot study should be undertaken to determine how to implement the FIT in Ontario.

A membrane-bound vertebrate globin.

The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2)-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O(2) binding with a variable affinity (P(50)∼1.3-12.5 torr), depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.

Neuroglobina: nuevo miembro de la familia de las globinas / Neuroglobin: a novel member of the globin family

Durante mucho tiempo se asumió que la hemoglobina y la mioglobina eran las únicas globinas de los vertebrados. En el año 2000 se descubrió un tercer tipo de globina, que sobre la base de su ubicación preferencial en el sistema nervioso fue denominada neuroglobina. Aunque aún se desconoce su función específica, se han planteado varias hipótesis entre las que se destaca la que sugiere que puede destoxificar las especies reactivas del oxígeno y el nitrógeno. Otros estudios proponen que es parte de una cadena de transducción de señales que transmite el estado redox de la célula o que inhibe la apoptosis. Aunque algunas funciones son más probables que otras, aún no se ha establecido definitivamente cuál es la función fisiológica de la neuroglobina en los vertebrados. No obstante, no hay dudas de que esta globina tiene una función esencial, conservada y que es beneficiosa para las neuronas(AU)

For a long time, it was taken for granted that hemoglobin and mioglobin were the only vertebrate globins. In 2000, a third type of globins was discovered on the basis of its preferential location in the nervous system and it was called neuroglobin. Although its specific function is still unknown, a number of hypotheses has been put forward, mainly the one suggesting that it may detoxify the reactive oxygen species and the nitrogen. On the other hand, other studies state that neuroglobin is part of a signal transduction chain that transmits the redox state of the cell or inhibits apoptosis. Though some functions are more probable than others, the real physiological function of neuroglobin in vertebrae has not been finally established. Nevertheless, this globin has undoubtedly an essential preserved function that is useful for neurons(AU)

Neuroglobina: nuevo miembro de la familia de las globinas / Neuroglobin: a novel member of the globin family

Durante mucho tiempo se asumió que la hemoglobina y la mioglobina eran las únicas globinas de los vertebrados. En el año 2000 se descubrió un tercer tipo de globina, que sobre la base de su ubicación preferencial en el sistema nervioso fue denominada neuroglobina. Aunque aún se desconoce su función específica, se han planteado varias hipótesis entre las que se destaca la que sugiere que puede destoxificar las especies reactivas del oxígeno y el nitrógeno. Otros estudios proponen que es parte de una cadena de transducción de señales que transmite el estado redox de la célula o que inhibe la apoptosis. Aunque algunas funciones son más probables que otras, aún no se ha establecido definitivamente cuál es la función fisiológica de la neuroglobina en los vertebrados. No obstante, no hay dudas de que esta globina tiene una función esencial, conservada y que es beneficiosa para las neuronas

For a long time, it was taken for granted that hemoglobin and mioglobin were the only vertebrate globins. In 2000, a third type of globins was discovered on the basis of its preferential location in the nervous system and it was called neuroglobin. Although its specific function is still unknown, a number of hypotheses has been put forward, mainly the one suggesting that it may detoxify the reactive oxygen species and the nitrogen. On the other hand, other studies state that neuroglobin is part of a signal transduction chain that transmits the redox state of the cell or inhibits apoptosis. Though some functions are more probable than others, the real physiological function of neuroglobin in vertebrae has not been finally established. Nevertheless, this globin has undoubtedly an essential preserved function that is useful for neurons

Deviations from Hardy-Weinberg proportions for multiple alleles under viability selection.

Departures of genotype frequencies from Hardy-Weinberg proportions (HWP) for a single autosomal locus due to viability selection in a random mating population have been studied only for the two-allele case. In this article, the analysis of deviations from HWP due to constant viability selection is extended to multiple alleles. The deviations for an autosomal locus with k alleles are measured by means of k fii fixation indices for homozygotes and k(k-1)/2 fij fixation indices for heterozygotes, and expressions are obtained for these indices (FIS statistics) under the multiallele viability model. Furthermore, expressions for fii and fij when the multiallele polymorphism is at stable equilibrium are also derived and it is demonstrated that the pattern of multiallele Hardy-Weinberg deviations at equilibrium is characterized by a global heterozygote excess and a deficiency of each of the homozygotes. This pattern may be useful for detecting whether a given multiallelic polymorphism is at stable equilibrium in the population due to viability selection. An analysis of Hardy-Weinberg deviations from published data for the three-allele polymorphism at the beta-globin locus in human populations from West Africa is presented for illustration.

An alteration in the levels of populations of CD4+ Treg is in part responsible for altered cytokine production by cells of aged mice which follows injection with a fetal liver extract.

We have shown previously that a fetal sheep liver extract (FSLE) containing significant quantities of fetal ovine gamma globin chain (Hbgamma) and LPS injected into aged (>20 months) mice could reverse the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFNgamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. The mechanism(s) behind this change in cytokine production were not previously investigated. We report below that aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+) Treg and so-called Tr3 (CD4(+)TGFbeta(+)), and that their number/function is restored to levels seen in control (8-week-old) mice by FSLE. In addition, on a per cell basis, CD4(+)CD25(-)Treg from aged mice were >4-fold more effective in suppression of proliferation and IL-2 production from ConA-activated lymphoid cells of a pool of CD4(+)CD25(-)T cells from 8-week-old mice than similar cells from young animals, and this suppression by CD25(-)T cells was also ameliorated following FSLE treatment. Infusion of anti-TGFbeta and anti-IL-10 antibodies in vivo altered Treg development following FSLE treatment, and attenuated FSLE-induced alterations in cytokine production profiles.

Identification of an immunogenic CD8+ T-cell epitope derived from gamma-globin, a putative tumor-associated antigen for juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder. Although allogeneic stem cell transplantation can induce long-term remissions, relapse rates remain high and innovative approaches are needed. Since donor lymphocyte infusions have clinical activity in JMML, T-cell-mediated immunotherapy could provide a nonredundant treatment approach to compliment current therapies. Gamma-globin, an oncofetal protein overexpressed by clonogenic JMML cells, may serve as a target of an antitumor immune response. We predicted 5 gamma-globin-derived peptides as potential human leukocyte antigen (HLA)-A2 restricted cytotoxic T lymphocyte (CTL) epitopes and showed that 4 (g031, g071, g105, and g106) bind A2 molecules in vitro. Using an artificial antigen-presenting cell (aAPC) that can process both the N- and C-termini of endogenously expressed proteins, we biochemically confirmed that g105 is naturally processed and presented by cell surface A2. Furthermore, g105-specific CD8(+) CTLs generated from A2-positive healthy donors were able to specifically cytolyze gamma-globin(+), but not gamma-globin(-) JMML cells in an A2-restricted manner. These results suggest that this aAPC-based approach enables the biochemical identification of CD8(+) T-cell epitopes that are processed and presented by intact cells, and that CTL immunotherapy of JMML could be directed against the gamma-globin-derived epitope g105.

[Preparation and identification of monoclonal antibodies against different epitopes on human zeta globin chain].

OBJECTIVE: To prepare and identify the monoclonal antibodies (mAb) against human zeta globin chain. METHODS: BALB/c mice were immunized with purified recombinant zeta globin chain, and the hybridomas were generated by fusion of mouse spleen cells and myeloma cells NS-1. After three fusions and successive cloning, 3 hybridoma cell lines secreting the mAb against zeta were obtained and the antibodies were purified from the ascites, followed by identification with indirect enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA in combination with rabbit anti-zeta serum. RESULTS AND CONCLUSION: Three hybridoma cell lines secreting anti-zeta mAb were established, designated as 1A12, 3H9 and 4D11, respectively. Both of 3H9 and 4D11 mAbs belonged to IgG1 isotype and mAb 1A12 to IgG2a. All the mAbs could bind specifically to recombinant zeta and natural zeta globin from hemolysate of --(SEA) gene carrier. In addition, 1A12 and 3H9 mAbs could recognize different epitopes on zeta globin, suggesting the possibility of developing a detection system for screening alpha-thalassemia with these mAbs.

Purification of placenta-eluted gamma globulins and their strong effect against graft-versus-host reactions in vitro and in vivo.

In the 1980s, attempts were made to use placenta-eluted gamma globulins (PEGG) in patients with graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). Because production of PEGG had been discontinued for many years, we aimed to reestablish a method of production and further explore the mechanisms of the effect of these globulins on GVHD. PEGG were prepared by elution at acid pH from extensively washed human placenta followed by precipitation with saturated ammonium sulfate and absorption on a protein A Sepharose column. In vitro study showed PEGG significantly inhibited both the proliferative response of T-cells to phytohemagglutinin (PHA) and the mixed lymphocyte reaction (MLR). Results of flow cytometric analysis indicated that PEGG down-regulated the expression of CD25 and CD69 on T-cells stimulated by PHA. Cytokine quantification in MLR supernatant showed that PEGG decreased secretion of interferon 3 (IFN-3) but increased production of interleukin 4. In a murine GVHD model, we investigated the preventive effect of PEGG on lethal GVHD in irradiated recipients of allogeneic bone marrow cells and spleen cell transplants by in vivo administration. Compared with controls, recipients treated with PEGG had a markedly increased survival rate with less histopathological evidence of GVHD. These results suggest that PEGG may be a potent therapeutic agent for GVHD.

Analysis of interaction of cloned human and/or sheep fetal hemoglobin gamma-chain and LPS in augmenting induction of inflammatory cytokine production in vivo and in vitro.

We have reported earlier that purified preparations of sheep fetal hemoglobin, but not adult hemoglobin, in concert with non-stimulatory doses of lipopolysaccharide (LPS) (lipid A), act cooperatively to regulate in vitro production of a number of cytokines, including TNFalpha, TGFbeta and IL-6 from murine and human leukocytes. Following in vivo treatment of mice with the same combination of hemoglobin and LPS, harvested spleen or peritoneal cells showed a similar augmented capacity to release these cytokines into culture supernatants. We report below that genetically cloned gamma-chain of human or sheep fetal hemoglobin, but not cloned alpha- or beta-chains, can produce this cooperative effect, as indeed can HPLC purified, heme-free, gamma-chains derived from cord blood fetal hemoglobin, and that purified haptoglobin completely abolishes the cooperative interaction.

Protection studies with a globin-enriched protein fraction of Ostertagia ostertagi.

The protective capacity of an adult stage Ostertagia ostertagi globin antigen was tested in four vaccination experiments in cattle. In a preliminary experiment, calves were vaccinated three times intraperitoneally with 250 microg globin in Freund's adjuvant and challenged with a trickled infection of 25,000 infective larvae. In three subsequent field studies, calves were vaccinated twice or three times intramuscularly with 80-100 microg globin in Quil A and challenged with a natural gastrointestinal nematode infection on pasture. Higher globin-specific antibody levels were detected in the vaccinated calves than in the control animals in all vaccine trials. In the preliminary experiment, geometric mean cumulative egg counts in the globin group were reduced by 52% and total worm burdens were reduced by 28%, compared to the controls. In the first field trial cumulative faecal egg counts were reduced by 63% in the vaccinated calves. However, the reduction in faecal egg output in these two experiments was not statistically significant and no reduction in faecal egg counts was observed in the vaccinated animals in the two last field trials. In conclusion, vaccination of calves with O. ostertagi globin resulted in highly variable protection levels after challenge infection.

Simple non-invasive prenatal detection of Hb Bart's disease by analysis of fetal erythrocytes in maternal blood.

OBJECTIVE: To investigate a simple non-invasive technique for early detection of Hemoglobin (Hb) Bart's disease. METHOD: Maternal blood smears from 8 known Hb Bart's pregnancies and 40 at-risk pregnancies were investigated. Maternal peripheral blood smears were stained with fluorescence-labeled monoclonal antibodies against alpha- and embryonic zeta-globin chains. RESULTS: Fetal nonnucleated red blood cells, stained with anti-zeta but not with anti-alpha globin antibodies were found in 15 out of 16 affected pregnancies but were not detected in 23 out of 24 unaffected pregnancies. CONCLUSION: Results showed that non-invasive immunofluorescence staining of maternal blood is a feasible approach for screening Hb Bart's disease before ultrasound manifestation in affected pregnancies.

Yolk sac-derived primitive erythroblasts enucleate during mammalian embryogenesis.

The enucleated definitive erythrocytes of mammals are unique in the animal kingdom. The observation that yolk sac-derived primitive erythroid cells in mammals circulate as nucleated cells has led to the conjecture that they are related to the red cells of fish, amphibians, and birds that remain nucleated throughout their life span. In mice, primitive red cells express both embryonic and adult hemoglobins, whereas definitive erythroblasts accumulate only adult hemoglobins. We investigated the terminal differentiation of murine primitive red cells with use of antibodies raised to embryonic beta H1-globin. Primitive erythroblasts progressively enucleate between embryonic days 12.5 and 16.5, generating mature primitive erythrocytes that are similar in size to their nucleated counterparts. These enucleated primitive erythrocytes circulate as late as 5 days after birth. The enucleation of primitive red cells in the mouse embryo has not previously been well recognized because it coincides with the emergence of exponentially expanding numbers of definitive erythrocytes from the fetal liver. Our studies establish a new paradigm in the understanding of primitive erythropoiesis and support the concept that primitive erythropoiesis in mice shares many similarities with definitive erythropoiesis of mammals.

Sensitivity and specificity of the identification of fetal cells in maternal blood by combined staining with antibodies against beta-, gamma- and epsilon-globin chains.

OBJECTIVES: Antibodies against fetal and embryonic hemoglobins may identify fetal cells in maternal blood. Both gamma- and epsilon-globins are used as fetal cell markers. Gamma-globin is not fetus specific. So far epsilon-globin has been claimed to be fetus specific. In this communication, we compare the specificity of anti-epsilon- and anti-gamma-globin staining when combined with staining for beta-globin. METHODS: We applied single and double color immunofluorescent staining techniques in combination with XY chromosome hybridization. The blood sample was taken after chorion villus biopsy at 11 weeks of gestation from a woman carrying a male fetus. RESULTS: By gamma-globin staining alone, 21 fetal and 2 maternal nucleated red blood cells (NRBCs) were identified. Only 1 of the 2 maternally derived NRBCs expressed beta-globin. By epsilon-globin staining, 92 additional fetal NRBCs were identified. CONCLUSIONS: Epsilon-globin antibody and combined epsilon- and gamma-globin antibody staining of a blood sample from a pregnant woman at 11 gestational weeks showed higher sensitivity but lower specificity for the fetal origin of erythroblasts with combined compared with separate staining. The final decision of the origin of cells was made by gender determination by FISH. Out of 2 gamma-positive maternal cells 1 was beta-globin antibody positive, 1 was beta-globin negative, indicating that 100% specificity for fetal origin could not be obtained by combining all 3 hemoglobin types. Although only 1 blood sample was tested and only 2 gamma-positive maternal NRBCs were identified, the result indicates that beta-hemoglobin does not discriminate completely between gamma-positive NRBCs of fetal and maternal origin.

A globin in the nucleus!

Cytoglobin and neuroglobin are recently discovered members of the globin family. In situ hybridization localized neuroglobin mainly in brain and retina, while cytoglobin was expressed ubiquitously in all analyzed tissues. In the present study, polyclonal antibodies were raised against both proteins and the distribution of them was studied by immunocytochemistry at tissue and subcellular level. Cytoglobin immunoreactivity was uniformly distributed and found in all tissues studied. At the subcellular level, cytoglobin immunoreactivity was exclusively detected in the cell nucleus. In contrast, neuroglobin immunoreactivity was detected in specific brain regions with varying intensities and in the islet of Langerhans in the pancreas. The immunoreactivity was restricted to the cytoplasm of neurons and endocrine beta cells. The nuclear localization of cytoglobin opens new perspectives for possible function(s) of globin-folded proteins as transcriptional regulators.

Serum antibodies to lipophilin B detected in late stage breast cancer patients.

Lipophilin B mRNA is overexpressed in approximately 70% of breast tumors and shows a high degree of correlation with the mRNA expression profile of mammaglobin. This is further supported by the recent finding that, like other members of the secretoglobulin-uteroglobin family, mammaglobin and lipophilin B form a heteroduplex. The studies described show that there are pre-existing antibodies to lipophilin B peptide in the sera of breast cancer patients with different stages and grade of tumor and that this response is different from that seen to recombinant mammaglobin and native mammaglobin-lipophilin B complex. The highest titers were observed in later stage tumors. In addition, low levels of antibody were also seen in some patients with prostate and ovarian cancers, consistent with lipophilin B mRNA expression in these tumors at lower abundance than in breast tumors. In contrast, lipophilin B antibodies were absent in 20 healthy donor sera and 30 lung cancer sera. A polymorphism identified in Lipophilin B did not appear to influence human sera reactivity. The data indicate that humoral immune responses to lipophilin B may serve as a diagnostic indicator, particularly for breast cancer.