The different expressed serum proteins in rhCygb treated rat model of liver fibrosis by the optimized two-dimensional gel electrophoresis.

Liver fibrosis, a common pathological process of chronic liver diseases, is the final stage of liver dysfunction that has severely deleterious impact on human health. Cytoglobin was first discovered in 2001 by proteomic analysis in rat stellate cells and was reported to play an important role in controlling tissue fibrosis. However, the mechanism by which cytoglobin inhibits or reverses the progression of fibrosis remains unclear. The present study examines the effect of recombinant human cytoblobin (rhCygb) in a rat model of liver fibrosis. Proteomic approaches were employed to identify differentially expressed proteins in the fibrosis model. Optimized conditions for two-dimensional gel electrophoresis were developed to provide improved protein detection and separation. A total of 43 spots were obtained and, through the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry, 30 differentially expressed proteins were identified. Gene ontology term annotation and KEGG pathway analysis allowed us to explore the function of the represented proteins. Based on these results, we provide a theory of the molecular mechanism related to rhCygb reversion of fibrosis and which will assist in the identification of biomarkers in patient serum to improve early diagnosis of liver fibrosis.

The Effect of rhCygb on CCl4-Induced Hepatic Fibrogenesis in Rat.

This study aims to investigate whether the use of recombinant human cytoglobin (rhCygb) impact on hepatic fibrogenesis caused by CCl4. SD (n = 150) rats were randomly divided into three groups of normal, CCl4 model and rhCygb groups. After model establishment, rats in rhCygb groups were administered daily with rhCygb (2 mg/kg, s.c.). Histological lesions were staged according to metavir. Serum parameters including ALT, AST, HA, LN, Col III and Col IV were determined. The liver proteins were separated by 2-DE and identified. As a result, the stage of hepatic damage and liver fibrosis in rhCygb groups were significantly milder than that in CCl4 model groups. Meanwhile, rhCygb dramatically reversed serum levels of ALT and AST, and also markedly decreased the liver fibrosis markers levels of LN, HA, Col III and Col IV. In 2-DE, 33 proteins among three groups with the same changing tendency in normal and rhCygb treated groups compared with CCl4 model group were identified. GO analysis showed that several identified proteins involved in oxidative stress pathway. The study provides new insights and data for administration of rhCygb reversing CCl4-induced liver fibrosis suggesting that rhCygb might be used in the treatment of liver fibrosis.

Pichia pastoris Production of Tat-NGB and Its Neuroprotection on Rat Pheochromocytoma Cells.

Neuroglobin (NGB) is a newly discovered neuroprotector and mainly localized in the neurons and retinal cells of the central and peripheral nervous systems in vertebrates, and its prokaryotic expression protein of which fused with HIV-1 virus-encoded Tat peptide exhibited significant antioxidant and anti-hypoxia. However, no study has documented on the anti-hypoxia of yeast expressed Tat-NGB. To address it, the NGB cDNA fragment with and without Tat tag was designed and conjugated to pPIC9K followed by electroporation, and positive colonies were screened. Subsequently, Tat-NGB-His and His-NGB-His proteins were expressed by inducer methanol and identified by SDS-PAGE, and purified with HisTrap™ FF crude column. After desalting, the transmembrane transduction of Tat-NGB was examined and identified by Western blot, and the anti-hypoxia activity was also examined by CCK-8 kit. Unexpectedly, Tat-NGB-His and His-NGB-His proteins were high yield and secretory expressed in GS115 Pichia pastoris. After purification, the high purified protein was prepared and exhibited a significant transmembrane transduction of Tat-NGB-His (**p < 0.01, compare to control and His-NGB-His). Significantly, Tat-NGB-His could protect hypoxia induced injury of PC12 cells and had an obviously difference when comparing to control and His-NGB-His groups (*p < 0.05, **p < 0.01). The present study first reported the yeast expressed production of Tat-NGB-His and His-NGB-His, and then elucidated the transduction and neuroprotection of Tat-NGB-His on PC12 cell. It not only provided a significant reference for high-yield expression of NGB in yeast expression system, but also provided a significant prevention and treatment of hypoxic and ischemic brain injury.

TAT-mediated delivery of neuroglobin attenuates apoptosis induced by oxygen-glucose deprivation via the Jak2/Stat3 pathway in vitro.

OBJECTIVES: Neuroglobin (Ngb), an identified globin in vertebrate brain, is a potential novel protective protein against brain ischemia. In our previous study, the human immunodeficiency virus trans-activator of transcription (TAT) protein transduction domain successfully delivered exogenous Ngb into neurons in the mouse, and protected the brain from cerebral ischemia-induced apoptosis. The aim of this study is to investigate the role of TAT-Ngb in attenuating oxygen-glucose deprivation (OGD) induced apoptosis and to explore the possible mechanism. METHODS: Nerve growth factor (NGF)-induced PC12 cells were divided into (1) the control group, (2) the OGD group (just OGD), (3) the Ngb treatment group (OGD and Ngb treatment), and (4) the TAT-Ngb treatment group (OGD and TAT-Ngb treatment). Cell viability and apoptosis were assessed by the MTT assay and the AnnexinV/propidium iodide (PI) staining, respectively. The mitochondrial transmembrane potential was measured by JC-1 staining. Caspase-3, Bcl-2, Bax, Stat3, Jak2, and Akt were determined by western blot analysis. RESULTS: Trans-activator of transcription effectively delivered Ngb into NGF-induced PC12 cells. Neuroglobin-mediated neuroprotection rescued cultured cells from OGD. We also confirmed previous findings that TAT-Ngb inhibited mitochondrial apoptosis following OGD. Inhibition of apoptosis by Ac-DEVD-CHO showed that caspase-3 was a crucial factor in OGD-induced apoptosis cascades. AG490, a specific Jak2 inhibitor, attenuated the protective effects of TAT-Ngb. The TAT-Ngb promoted expression of the anti-apoptotic protein Bcl-2 through the Jak2/Stat3 signal pathway, and inhibited apoptosis by blocking caspase-3 activation, while the Jak-Akt-Stat3 signal network was not involved. CONCLUSION: Our results demonstrate that TAT-Ngb can protect neuron-like cells against OGD-induced apoptosis by activating the Jak2/Stat3 pathway.

Effects of globin digest and its active ingredient Trp-Thr-Gln-Arg on galactosamine/lipopolysaccharide-induced liver injury in ICR mice.

AIMS: We investigated the effects of globin digest (GD) and its active ingredient Trp-Thr-Gln-Arg (WTQR) on galactosamine/lipopolysaccharide (GalN/LPS)-induced liver injury in imprinting control region (ICR) mice. MAIN METHODS: The effects of WTQR and GD on the liver injury were examined by measuring the survival rate, serum aminotransferase activities, hepatic components, antioxidant enzyme activities, histopathological analysis, serum levels and hepatic gene expression of tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2), and nitric oxide (NO) or inducible nitric oxide synthase (iNOS), and nuclear factor-kappa B (NF-κB) p65 content in GalN/LPS-treated ICR mice. RAW264 mouse macrophages were used to confirm the anti-inflammatory effects of WTQR and GD on the macrophages. KEY FINDINGS: WTQR and GD increased the survival rate, suppressed the serum aminotransferase activities, serum levels and hepatic gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in GalN/LPS-treated mice; decreased the oxidized glutathione content, increased the superoxide dismutase activity, and decreased the histopathological grade values of the hepatocyte necrosis and lobular inflammation in GalN/LPS-injured liver; and suppressed the release levels and gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in LPS-stimulated RAW264 macrophages. WTQR and GD may improve the antioxidant defense system and inflammatory status in GalN/LPS-injured liver. SIGNIFICANCE: These findings indicate that WTQR and GD have hepatoprotective effects on GalN/LPS-induced liver injury in ICR mice.

Protection by neuroglobin and cell-penetrating peptide-mediated delivery in vivo: a decade of research. Comment on Cai et al: TAT-mediated delivery of neuroglobin protects against focal cerebral ischemia in mice. Exp Neurol. 2011; 227(1): 224-31.

Over the last decade, numerous studies have suggested that neuroglobin is able to protect against the effects of ischemia. However, such results have mostly been based on models using transgenic overexpression or viral delivery. As a therapy, new technology would need to be applied to enable delivery of high concentrations of neuroglobin shortly after the patient suffers the stroke. An approach to deliver proteins in ischemia in vivo in a timely manner is the use of cell-penetrating peptides (CPP). CPP have been used in animal models for brain diseases for about a decade as well. In a recent issue of Experimental Neurology, Cai and colleagues test the effect of CPP-coupled neuroglobin in an in vivo stroke model. They find that the fusion protein protects the brain against the effect of ischemia when applied before stroke onset. Here, a concise review of neuroglobin research and the application of CPP peptides in hypoxia and ischemia is provided.

TAT-mediated delivery of neuroglobin protects against focal cerebral ischemia in mice.

Neuroglobin (Ngb), a newly identified globin in vertebrate brain, has been suggested to be able to protect against brain hypoxic-ischemic injury. However, owing to its large size, the impermeability of the blood-brain barrier (BBB) to Ngb limits its application in brain injury. Recently, the 11-amino-acid human immunodeficiency virus trans-activator of transcription (TAT) protein transduction domain was shown to successfully deliver macromolecules into the brain. This study explored whether the TAT-Ngb fusion protein can cross the BBB and protect the brain from cerebral ischemia. The TAT-Ngb fusion protein generated from Escherichia coli BL21 (DE3) was efficiently delivered into mice brain tissues by intravenous injection as demonstrated by immunohistochemistry and Western blotting. Two groups of mice were treated with filamentous middle cerebral artery occlusion (MCAO) for 30min or 2h followed by reperfusion. Each group was then divided into sub-groups and was injected intravenously with TAT-Ngb, Ngb, or saline respectively before MCAO or immediately after reperfusion. Compared with the Ngb- and saline-treated group, the group with TAT-Ngb treated 2h before MCAO showed significantly less brain infarct volume and had better neurologic outcomes (p<0.05). Furthermore, a TAT-Ngb injection following a 30-min MCAO treatment significantly increased neuronal survival in the striatum (p<0.05). Our results demonstrated that the exogenous Ngb fusion protein containing the TAT protein transduction domain could be efficiently transduced into neurons in the mouse and protect the brain from mild or moderate ischemic injury.

[Treatment of skin ulcers in collagen vascular diseases].

The causes of skin ulcers in collagen vascular diseases are complicated, and involve peripheral vascular complications, vasculitis, and thrombosis. It is necessary to determine the treatment, such as oral medication, infusion, ointment and surgery after careful consideration of its causes. This review shows skin manifestations of various collagen vascular diseases and the causes of skin ulcers in these diseases.

Locus control region elements HS1 and HS4 enhance the therapeutic efficacy of globin gene transfer in beta-thalassemic mice.

Globin gene transfer in autologous hematopoietic stem cells is a promising therapeutic option for subjects with beta-thalassemia major. In this approach, high level, erythroid-specific globin transgene expression should correct ineffective erythropoiesis and hemolytic anemia following the delivery of only 1 to 2 vector copies per cell. The generation of vectors that provide high-level globin expression and require low vector copy (VC) integration is therefore essential for both safety and efficacy. We show here the major roles played by 2 lesser-known locus control region elements, termed HS1 and HS4. Partial deletions within HS4 markedly reduce in vivo globin expression requiring multiple VC per cell to correct the anemia. Most strikingly, addition of HS1 to HS2-3-4 increases globin expression by 52%, yielding 9 g Hb/VC in beta-thalassemic mice. Thus, while vectors encoding HS2-3-4 provide curative levels of hemoglobin at 1 to 2 copies per cell, adding HS1 is a promising alternative strategy if upcoming clinical trials prove higher levels of expression to be necessary.

[Case of toxic shock-like syndrome affecting the neck].

Toxic shock-like syndrome (TSLS) is a form of rapidly progressing septic shock that can lead to multiple organ failure and has a high mortality rate of 30%. We report a rare case of TSLS affecting the head and neck. A 40-year-old man complained of redness and swelling of the neck with vomiting and diarrhea. His blood pressure dropped, and multiple organ failure occurred. Streptcoccus pyogenes, Group A, was identified in a blood culture, and he was diagnosed as having TSLS. He was treated with high-dose carbapenem, clindamysin, and gamma globulin. Continuous hemodiafiltration (CHDF) and PMX-DHP was applied to prevent sepsis and multiple organ failure. Debridement of the neck was performed on day 16. He recovered gradually and was discharged from hospital on day 45. A total resection is required to treat TSLS, but such a procedure is difficult to perform in the head and neck region. Our case improved without resection but after debridement and general control. TSLS should be first treated by medication and then by surgery, consisting of either debridement or resection.

Partial correction of murine beta-thalassemia with a gammaretrovirus vector for human gamma-globin.

Several studies have demonstrated that recombinant lentivirus vectors containing extended globin gene expression cassettes and regulatory elements can ameliorate the pathogenic sequela in murine models of beta-thalassemia and sickle cell disease. Similarly promising results have not yet been obtained with recombinant gammaretrovirus vectors. Of these two vector classes, only gammaretroviruses have been tested extensively in clinical trials, with a proven ability to transduce long-term reconstituting hematopoietic stem cells with an exceedingly low incidence of serious side effects. Toward the continuing goal of developing retrovirus vectors for the treatment of the beta-chain hemoglobinopathies, we report here the assessment of a recombinant gammaretrovirus vector for human gamma-globin in murine models of beta-thalassemia. In the beta-thalassemia intermedia Hbbth-3/+ model, we observed a dose-dependent but transient increase in total hemoglobin and red blood cells, with a 2.5 +/- 0.2 g/dL increase in hemoglobin for transduction rates > or = 33%. In the severe beta-thalassemia major Hbbth-3/Hbbth-3 model, we observed a modest but statistically significant increase in survival, from a median of 15 days to 30 days (P = 0.001). These studies provide the first evidence that globin gene transfer vectors based on recombinant gammaretroviruses may provide a viable option for the treatment of the beta-chain hemoglobinopathies.

Globin digest: no evidence for a weight loss mechanism.

This study was designed to document the mechanism through which globin digest, a dietary herbal supplement, might cause weight loss by exploring possible fat malabsorption, calorie malabsorption, energy expenditure, and fat oxidation. Six healthy subjects were placed on an outpatient diet for 14 days and given a meal containing 40.9 g of fat on days 5 and 11, and stools were collected for 72 hours after each meal for analysis of fecal fat content. Four grams of globin digest was given with one meal and placebo with the other. In another separate study, six subjects were placed on a 100-g fat, weight-maintaining diet for 14 days. All food was prepared by the Pennington Center (Baton Rouge, LA) metabolic kitchen. Globin digest (2 g) or placebo was given with each of three meals per day, and stool was collected for calorie determinations during the last 72 hours of each week. Subjects received globin digest during one of the 2 weeks and placebo during the other. Resting metabolic rate and respiratory quotient were measured on the last day of each 1-week period. There was no increase in 72-hour fecal fat or fecal calories by bomb calorimetry during either of the studies. There was no difference in the respiratory quotient. Globin digest did result in an increase in resting metabolic rate. However, this increase was not statistically significant. Globin digest, if effective, does not cause weight loss or fat loss through fat malabsorption or a relative increase in fat oxidation. Future studies are needed to document the efficacy of globin digest for weight loss in humans before further mechanistic investigation is attempted.

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.

Successful correction of the human beta-thalassemia major phenotype using a lentiviral vector.

beta-thalassemias are the most common single gene disorders and are potentially amenable to gene therapy. However, retroviral vectors carrying the human beta-globin cassette have been notoriously unstable. Recently, considerable progress has been made using lentiviral vectors, which stably transmit the beta-globin expression cassette. Thus far, mouse studies have shown correction of the beta-thalassemia intermedia phenotype and a partial, variable correction of beta-thalassemia major phenotype. We tested a lentiviral vector carrying the human beta-globin expression cassette flanked by a chromatin insulator in transfusion-dependent human thalassemia major, where it would be ultimately relevant. We demonstrated that the vector expressed normal amounts of human beta-globin in erythroid cells produced in in vitro cultures for unilineage erythroid differentiation. There was restoration of effective erythropoiesis and reversal of the abnormally elevated apoptosis that characterizes beta-thalassemia. The gene-corrected human beta-thalassemia progenitor cells were transplanted into immune-deficient mice, where they underwent normal erythroid differentiation, expressed normal levels of human beta-globin, and displayed normal effective erythropoiesis 3 to 4 months after xenotransplantation. Variability of beta-globin expression in erythroid colonies derived in vitro or from xenograft bone marrow was similar to that seen in normal controls. Our results show genetic modification of primitive progenitor cells with correction of the human thalassemia major phenotype.

Expression of an anti-sickling beta-globin in human erythroblasts derived from retrovirally transduced primitive normal and sickle cell disease hematopoietic cells.

OBJECTIVE: Recent improvements in human beta-globin vector design have fueled interest in gene therapy approaches to the treatment of human thalassemia and sickle cell disease (SCD). The present study was undertaken to determine whether human beta-globin mRNA and protein could be obtained in the erythroid progeny of more primitive human target cells transduced with a retrovirus containing murine stem cell virus long terminal repeats, a phosphoglycerate kinase promoter driving the expression of a green fluorescence protein (GFP) cDNA, and an anti-sickling beta-globin (beta87(+)) gene under the control of an HS2, HS3, HS4 enhancer cassette. MATERIALS AND METHODS: A two-step pseudotyping strategy was devised to obtain useful preparations of this virus. Primitive cells present in normal human cord blood (CB) and adult SCD patients' blood samples were infected and the level of gene transfer (% GFP(+) cells) and erythroid-specific beta87(+)-globin expression assessed. RESULTS: Analysis of the proportion of infected cells that became GFP(+) showed that this virus transduced approximately 50% of initial CD34(+) CB and SCD cells and up to 23% of cells able to regenerate both lymphoid and myeloid cells in sublethally irradiated primary and secondary NOD/SCID mice. beta87(+)-globin transcripts were readily detected in erythroblasts generated from primitive transduced CB cells and SCD progenitors. Evidence of beta87(+)-derived protein in transduced CB cell-derived erythroblasts also was obtained. CONCLUSION: These findings demonstrate that retroviral vector-based gene transfer approaches can be used to achieve human beta-globin protein expression in the erythroid progeny of transplantable human precursors.

Factors relating to the cardiac sequelae of Kawasaki disease one month after initial onset.

UNLABELLED: This study aimed to determine the risk factors related to the presence of cardiac sequelae 1 mo after initial onset and to examine the preventive effect of the early administration of high-dose gamma-globulin (GG) on cardiac sequelae in patients with Kawasaki disease. Patients treated with high-dose GG of 2000 +/- 100 mg kg(-1) were selected as subjects from the 15th nation-wide survey in Japan. Univariate and logistic multiple variable analyses were used to test the effects of background variables such as age and gender, variables relating to laboratory findings such as the percentage of neutrophil leucocytes, and variables relating to the GG treatment on the presence of cardiac sequelae. The odds ratios were significantly higher for males (1.48), those younger than 1 y of age (1.71), recurrent cases (2.42), and those with a low haematocrit (<32.5%) (1.45) and high percentage of neutrophil leucocytes (>68%) (1.63). The odds ratio was low for those who started GG administration in less than 6 d from onset between the patients with and without cardiac sequelae. The odds ratio for the duration of GG treatment was not significantly different between those with and without cardiac sequelae. CONCLUSION: Patients who received early administration of GG, less than 6 d from onset of the disease, had a lower risk than those received GG more than 6 d from the onset. The percentage of neutrophil leucocytes and the haematocrit level are useful indicators in predicting the development of cardiac sequelae.

Intravenous gamma-globulin therapy in bronchial asthma.

Two possible uses exist for intravenous gamma-globulin (IVIG) therapy in asthma. First, it has been suggested that high-dose IVIG can serve as an anti-inflammatory, immunomodulatory agent in steroid-dependent asthma patients. Second, IVIG can be used as a replacement treatment in those asthma patients with frank hypogammaglobulinemia or more subtle antibody deficiencies. The mechanisms by which IVIG functions are widely different in these two potential uses. Clear characterization of the patients' immune status is pivotal in choosing whether to use IVIG. The assessment should not be limited to simple determination of serum immunoglobulin A (IgA), IgG, IgM, and IgG subclass levels. When clinically warranted, the specific antibody response to active immunization with antigens such as those in Pneumovax may be invaluable in identifying patients with subtle antibody-deficiency disorders. Asthma in those patients may be improved markedly if infection is prevented by antibody-replacement therapy with IVIG.

Successful treatment of murine beta-thalassemia intermedia by transfer of the human beta-globin gene.

The beta-thalassemias are caused by more than 200 mutations that reduce or abolish beta-globin production. The severity of the resulting anemia can lead to lifelong transfusion dependency. A genetic treatment based on globin gene transfer would require that transgene expression be erythroid specific, elevated, and sustained over time. We report here that long-term synthesis of chimeric hemoglobin (mualpha(2):hubeta(A)(2)) could be achieved in mice with beta-thalassemia intermedia following engraftment with bone marrow cells transduced with a lentiviral vector encoding the human beta-globin gene. In the absence of any posttransduction selection, the treated chimeras exhibit durably increased hemoglobin levels without diminution over 40 weeks. Ineffective erythropoiesis and extramedullary hematopoiesis (EMH) regress, as reflected by normalization of spleen size, architecture, hematopoietic colony formation, and disappearance of liver EMH. These findings establish that a sustained increase of 3 to 4 g/dL hemoglobin is sufficient to correct ineffective erythropoiesis. Hepatic iron accumulation is markedly decreased in 1-year-old chimeras, indicating persistent protection from secondary organ damage. These results demonstrate for the first time that viral-mediated globin gene transfer in hematopoietic stem cells effectively treats a severe hemoglobin disorder.