Diminished impact of cytomegalovirus infection on graft vasculopathy development in the antiviral prophylaxis era - a retrospective study.

Evidence concerning an association between cytomegalovirus (CMV) infection and accelerated cardiac allograft vasculopathy (CAV) is inconclusive. Data were analyzed retrospectively from 297 consecutive heart transplants between 1.1.2002 and 31.12.2012. Patients ≤18 years of age, survival, and follow-up ≤1-year post-transplant and patients with early CAV were excluded. CMV-infection was diagnosed and monitored closely in the first year. CAV was diagnosed by coronary angiography via left heart catheterization, and results were categorized according to the International Society of Heart and Lung Transplantation (ISHLT) scoring system. Risk factors for CAV were tested in a multivariable model. Median follow-up was 7.5 years (IQR: 5.6-10.3). CMV infection in the first year after transplantation occurred in 26% of patients (n = 78), CMV disease in 5% (n = 15). CAV ≥1 ISHLT was detected in 36% (n = 108). Incidence of CAV >1 ISHLT and severity of CAV increased over time. No statistically significant association between CMV infection and disease within the first year and risk of CAV after 1-year post-HTx was detected in the univariate (P = 0.16) and multivariable [hazard ratio (HR), 1.36; confidence interval (CI), 0.89-2.07; P = 0.16] Cox regression. In the multivariable Cox regression, donor age (HR, 1.04; 95% CI, 1.02-1.06; P < 0.01) and acute cellular rejection (ACR) ≥2R in the first year after HTx (HR, 1.77; 95% CI, 1.06-2.95; P = 0.03) were independent risk factors for CAV development. In our cohort, CMV infection and disease in the first year after transplantation did not significantly influence the risk of CAV in the long-term follow-up.

The protective effects of the lentivirus-mediated neuroglobin gene transfer on spinal cord injury in rabbits.

STUDY DESIGN: We introduced a lentiviral vector containing the neuroglobin (Ngb) gene into the injured spinal cords to evaluate the therapeutic potential of Ngb in a rabbit model of spinal cord injury (SCI). OBJECTIVES: It is not clear whether Ngb has the neuroprotective role to SCI. The purpose of this study was to investigate the possible protective effects of the Ngb overexpression on traumatic SCI in rabbits. SETTING: Department of Orthopedic Surgery, The First Affiliated Hospital, Fujian Medical University, Fuzhou, People's Republic of China. METHODS: A lentiviral vector containing Ngb gene was constructed and injected at the SCI sites 24 h after SCI. The rabbits' motor functions were evaluated by the Basso-Beattie-Bresnahan rating scale. Quantitative real-time PCRs, western blots, malondialdehyde (MDA) tests and terminal deoxynucleotidyl transferase-mediated UTP end labeling assays were also performed. RESULTS: The Ngb expression in the LV-Ngb group increased significantly at days 7, 14 and 21. A more significant functional improvement was observed in the LV-Ngb group compared with the improvements in all other groups at days 14 and 21. The traumatic SCI seemed to lead to an increase in the levels of MDA and in the number of the apoptotic cells, which could be prevented by the LV-Ngb treatment. CONCLUSION: This study demonstrated that the Ngb overexpression may have potential therapeutic benefits for both reducing secondary damages and improving the outcomes after traumatic SCI.

[Protective effects of PEG modified recombinant cytoglobin on acute liver injury in mice].

To investigate the protective effect of polyethylene glycol (PEG) modified recombinant cytoglobin (PEG-rCygb) on acute liver damage in mice. The acute liver injury model of KM mice was induced by CCl4 and then treated with PEG-rCygb, The liver and blood samples were collected for biochemical and histopathological analysis. The results showed that PEG-rCygb reduced the liver mass index and decreased significantly the levels of alanine amiotransferase (AST) and aspartate transaminase (ALT) in mouse serum. In liver tissues, the content of malondialdehyde (MDA) was decreased, whereas the content of glutathione (GSH) was increased in PEG-rCygb treated group. PEG-rCygb also elevated the activities of total super oxidedismutase (T-SOD) and catalase (CAT) in liver tissues. HE staining of liver tissue slices revealed that PEG-rCygb relieved fatty degeneration of liver, decreased inflammatory factors and reduced liver cell injury. Further in vitro experiments indicated that the protective effects of PEG-rCygb on hepatic stellate cell (HSC) against H2O2 were enhanced compared with that of rCygb. All results indicated that the PEG-rCygb promoted oxygen free radical scavenging ability and prevented acute liver injury in KM mice induced by CCl4.

Enfermedad de Kawasaki: reporte de 3 casos / Kawasaki's disease: report on 3 cases

Se presentan 3 pacientes que acudieron al Hospital Pediátrico Juan M. Márquez con manifestaciones de exantema y fiebre. Se realizaron estudios para concluir el diagnóstico y en todos los casos los resultados fueron normales. Para arribar al diagnóstico de enfermedad de Kawasaki se aplicaron los criterios de la American Heart Association. Ninguno de los pacientes presentó complicaciones y se excluyeron otras enfermedades según el cuadro clínico y los exámenes complementarios realizados. Los 3 mejoraron con tratamiento de intacglobin en dosis de 400 mg/(kg∙día), durante 5 días

These three patients came to Juan Manuel Márquez with manifestations of exanthema and fever. We conducted studies to conclude the diagnosis and in all the cases results were normal. To arrive to diagnosis of Kawasaki's disease criteria from the American Heart Association were applied. No patient had complications excluding other diseases according to clinical picture and complementary examinations performed. The three patients improved with the 400 mg (kg/day) Intacglobin treatment for 5 days

Enfermedad de Kawasaki: reporte de 3 casos / Kawasaki's disease: report on 3 cases

Se presentan 3 pacientes que acudieron al Hospital Pediátrico Juan M. Márquez con manifestaciones de exantema y fiebre. Se realizaron estudios para concluir el diagnóstico y en todos los casos los resultados fueron normales. Para arribar al diagnóstico de enfermedad de Kawasaki se aplicaron los criterios de la American Heart Association. Ninguno de los pacientes presentó complicaciones y se excluyeron otras enfermedades según el cuadro clínico y los exámenes complementarios realizados. Los 3 mejoraron con tratamiento de intacglobin en dosis de 400 mg/(kg∙día), durante 5 días (AU)

These three patients came to Juan Manuel Márquez with manifestations of exanthema and fever. We conducted studies to conclude the diagnosis and in all the cases results were normal. To arrive to diagnosis of Kawasaki's disease criteria from the American Heart Association were applied. No patient had complications excluding other diseases according to clinical picture and complementary examinations performed. The three patients improved with the 400 mg (kg/day) Intacglobin treatment for 5 days (AU)

Neuroprotective roles and mechanisms of neuroglobin.

OBJECTIVE: The objectives of this work were to update and summarize recent experimental works on neuroglobin, mainly focus on its neuroprotective effects and the mechanisms. METHODS: The literature was reviewed using PubMed database, and some of the recent findings from our laboratory were included. RESULTS: Neuroglobin is a recently discovered tissue globin with a high affinity for oxygen and is widely and specifically expressed in neurons of vertebrate's central and peripheral nervous systems. Investigations in the past several years have advanced our knowledge on the functions and mechanisms of neuroglobin, but many issues remain unclear. Emerging reports have shown that overexpression of neuroglobin confers neuroprotection against neuronal hypoxia or ischemia-induced damage in cultured neurons and in cerebral ischemic animal models. Accumulating findings suggest several possible neuroprotective roles and mechanisms including ligand binding and oxygen sensing, modulation of cell signaling pathways and maintenance of mitochondria function. CONCLUSION: Emerging experimental works suggest that neuroglobin is neuroprotective against hypoxic/ischemic insults, probably via ligand binding and oxygen sensing, modulation of cell signaling pathways and maintenance of mitochondria function.

Stem cell engineering for the treatment of severe hemoglobinopathies.

The beta-thalassemias and sickle cell anemia are severe congenital anemias for which there is presently no curative therapy other than allogeneic bone marrow transplantation. This therapeutic option, however, is not available to most patients due to the lack of an HLA-matched bone marrow donor. Emerging modalities based on cell engineering offer new prospects for potentially curative approaches that are applicable to more patients. The first is based on the transfer of a regulated globin gene in autologous hematopoietic stem cells (HSCs). This strategy, simple in principle, raises major challenges in terms of controlling transgene expression, which ideally should be erythroid-specific, differentiation and stage-restricted, elevated, position-independent, and sustained over time. Following the original report by May et al., several groups have reported that lentiviral vectors encoding slightly different combinations of proximal and distal transcriptional control elements of the normal human beta-globin gene permit lineage-specific and elevated beta-globin expression in vivo, resulting in therapeutic hemoglobin production and correction of anemia in beta-thalassemic mice. Clinical studies utilizing the TNS.3 vector are likely to be initiated in the US in 2009. While the addition of the wild-type beta-globin gene is naturally suited for treating beta-thalassemia, several alternatives have been proposed for the treatment of sickle cell disease, using either gamma- or mutant beta-globin gene addition, trans-splicing or RNA interference. The recent discovery that adult somatic cells can be reprogrammed to become pluripotent stem cells from which HSCs can be derived, provides yet another venue for developing stem cell engineering using either lentiviral vectors or homologous recombination techniques. Altogether, these recent advances bode well for the advent of curative stem cell-based therapies.

Cytoglobin: a novel potential gene medicine for fibrosis and cancer therapy.

Attempts have been made by conventional gene therapy to suppress hepatic fibrogenesis, but potential oncogenic activity may prevent its clinical use. Recently, a novel major approach has been developed for resolution of liver fibrosis and cirrhosis: inactivation of hepatic stellate cells (HSC) using the endogenous expressing gene, which could mediate the homeostatic adaptation of liver. Cytoglobin (Cygb), originally identified in cultured rat HSC, is in a new class of heme containing proteins known as the hexacoordinate globin superfamily. These proteins exhibit peroxidase activity against hydrogen peroxides and lipid hydroperoxides. Considerable attention has been focused on the potential benefits of use of Cygb in fibrosis therapy, as up-regulation of Cygb expression could reduce oxidant stress, suppress HSC differentiation to a myofibroblast-like phenotype and eventually prevent the progress of liver fibrosis. Cygb has also been found to be a candidate tumor suppressor gene that might provide a new option for cancer gene therapy. In this review we systematically analyze the potential of Cygb as a prospective gene medicine for curing fibrosis, cancer, and other diseases such as diabetes. The molecular structure, regulation and subcellular location of Cygb are reviewed as well.

Octamers and nanoparticles as hemoglobin based blood substitutes.

Progress in developing a blood substitute is aided by new biotechnologies and a better understanding of the circulatory system. For Hb based solutions, there is still a debate over the best set of fundamental parameters concerning the oxygen affinity which is correlated with the oxidation rate, the cooperativity, the transporter size, and of course the final source of material. Genetic engineering methods have helped discover novel globins, but not yet the quantity necessary for the high demand of blood transfusions. The expanding database of globin properties has indicated that certain individual parameters are coupled, such as the oxygen affinity and the oxidation rate, indicating that one must accept a compromise of the best parameters. After a general introduction of these basic criteria, we will focus on two strategies concerning the size of the oxygen transporter: Hb octamers, and Hb integrated within a nanoparticle.

Intracellular delivery of Neuroglobin using HIV-1 TAT protein transduction domain fails to protect against oxygen and glucose deprivation.

Neuroglobin (Ngb) is a heme protein that is primarily localised in the retina and the brain. Its physiological role is largely unknown. It has been reported that its overexpression protects neurons from hypoxia in vitro and in vivo, suggesting that the rapid modulation of the Ngb level in the nerve cells may be a promising stroke treatment strategy. In this study, we used a novel approach to overexpress Ngb and evaluate its ability to promote neuronal survival under hypoxic conditions. We constructed a human recombinant Ngb fused to the cell penetrating peptide (CPP) derived from HIV-1 TAT. Purified recombinant TAT-Ngb was able to efficiently transduce CHO and SHSY5Y cells, when added to the culture media. The potential neuroprotective action of Ngb was then examined by using an in vitro model of ischemia. The two neuronal cell lines RGC-5 and SH-SY5Y were subjected to oxygen glucose deprivation (OGD) after pre-treatment with TAT-Ngb. In both cell types, however, the treatment with the TAT-Ngb fusion protein did not show any effect on cell viability. This discrepancy to earlier reports might be due to the experimental model for oxygen glucose deprivation we employed. Alternatively, intracellular delivery of Ngb by the TAT/CPP might not have beneficial effects in the treatment of ischemic pathology.

Timoma e inmunodeficiencia en un niño / Thymoma and immunodeficiency in an infant

Las inmunodeficiencias primarias más frecuentes son las humorales, en las que predominan las deficiencias de anticuerpos, y entre éstas las inmunodeficiencias con timoma son raras. En este trabajo se presenta un paciente masculino de 3 años de edad, sin antecedentes familiares de inmunodeficiencia, que a los 21 días de nacido presentó varicela afebril. A partir de un año de edad comenzó a presentar episodios frecuentes de infecciones de vías respiratorias altas (faringoamigdalitis, rinitis purulenta), y bajas como neumonías, o reacciones alérgicas a las picadas de insectos que se infectaban secundariamente. Los estudios inmunológicos revelaron una disminución de las concentraciones séricas de IgG, IgA, (IgG 7.4 g/l, IgA 0.52 g/l), IgM normal (IgM 1g/l) y niveles elevados de IgE (>200u/ml). El estudio radiológico mostró en el mediastino anterior una masa relacionada con el timo. El paciente recibió tratamiento con factor de transferencia, gammaglobulina y se realizó la timectomia por videotoracoscopía. El estudio histológico evidenció un timoma linfocítico benigno. Los linfocitos T CD2+, CD3+, CD8+ estaban disminuidos en el preoperatorio, mientras que en el post operatorio los linfocitos CD2+, CD4+ estaban disminuidos y los CD8+ aumentados

The most frequent primary immunodeficiency is the humoral, where the deficiency of antibodies prevails; among these, the immunodeficiency with thymoma is rare. In this work, a 3- yearold male is presented, without family antecedents of immunodeficiency, which 21 days after birth presented Chicken pox without fever. In the first year of age, he began to present frequent episodes of infections of the upper and the lower respiratory tract: tonsillitis, purulent rhinitis, and pneumonias. He also presented an allergic reaction to insect bites that became infected secondarily. The immunologic studies revealed a decrease of the seric concentrations of IgG, IgA (IgG 7.4 g/l, IgA 0.52 g/l), normal IgM values (IgM 1 g/l) and high levels of IgE (> 200u/ ml). The amount of CD2+, CD3+ and CD8+ T lymphocytes were diminished pre surgery. After surgery, the levels of CD2+, and CD4+ lymphocytes were diminished and those of CD8+ lymphocytes increased. The radiological study showed an anterior, mediastinal mass related to the thymus. The patient received treatment with transfer factor, gammaglobulin and underwent resection of the thymoma for videothoracoscopy. The histology study evidenced a benign lymphocytic thymoma

A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference.

The application of RNA interference (RNAi) to stem cell-based therapies will require highly specific and lineage-restricted gene silencing. Here we show the feasibility and therapeutic potential of coregulating transgene expression and RNAi in hematopoietic stem cells. We encoded promoterless small-hairpin RNA (shRNA) within the intron of a recombinant gamma-globin gene. Expression of both gamma-globin and the lariat-embedded small interfering RNA (siRNA) was induced upon erythroid differentiation, specifically downregulating the targeted gene in tissue- and differentiation stage-specific fashion. The position of the shRNA within the intron was critical to concurrently achieve high-level transgene expression, effective siRNA generation and minimal interferon induction. Lentiviral transduction of CD34(+) cells from patients with sickle cell anemia led to erythroid-specific expression of the gamma-globin transgene and concomitant reduction of endogenous beta(S) transcripts, thus providing proof of principle for therapeutic strategies that require synergistic gene addition and gene silencing in stem cell progeny.

Globin gene transfer for treatment of the beta-thalassemias and sickle cell disease.

The beta-thalassemias and sickle cell disease are severe congenital anemias that are caused by mutations that alter the production of the beta chain of hemoglobin. Allogeneic hematopoietic stem cell (HSC) transplantation is curative, but this therapeutic option is not available to the majority of patients. The transfer of a functional globin gene in autologous HCSs thus represents a highly attractive alternative treatment. This strategy, simple in principle, raises major challenges in terms of controlling the expression of the globin transgene, which ideally should be erythroid specific, differentiation-stage restricted, elevated, position independent, and sustained over time. Using lentiviral vectors, we have demonstrated that an optimised combination of proximal and distal transcriptional control elements permits lineage-specific, elevated expression of the beta-globin gene, resulting in therapeutic hemoglobin production and correction of anemia in beta-thalassemic mice. Several groups have now confirmed and extended these findings in various mouse models of severe hemoglobinopathies, thus generating enthusiasm for a genetic treatment based on globin gene transfer. Furthermore, globin vectors represent a general paradigm for the regulation of transgene function and the improvement of vector safety by restricting transgene expression to the differentiated progeny within a single lineage, thereby reducing the risk of activating oncogenes in hematopoietic progenitors. Here we review the principles underlying the genesis of regulated vectors for stem cell therapy.

Extended beta-globin locus control region elements promote consistent therapeutic expression of a gamma-globin lentiviral vector in murine beta-thalassemia.

Since increased fetal hemoglobin diminishes the severity of beta-thalassemia and sickle cell anemia, a strategy using autologous, stem cell-targeted gene transfer of a gamma-globin gene may be therapeutically useful. We previously found that a gamma-globin lentiviral vector utilizing the beta-globin promoter and elements from the beta-globin locus control region (LCR) totaling 1.7 kb could correct murine beta-thalassemia. However, therapeutic consistency was compromised by chromosomal position effects on vector expression. In contrast, we show here that the majority of animals that received transplants of beta-thalassemic stem cells transduced with a new vector containing 3.2 kb of LCR sequences expressed high levels of fetal hemoglobin (17%-33%), with an average vector copy number of 1.3. This led to a mean 26 g/L (2.6 g/dL) increase in hemoglobin concentration and enhanced amelioration of other hematologic parameters. Analysis of clonal erythroid cells of secondary spleen colonies from mice that underwent transplantation demonstrated an increased resistance of the larger LCR vector to stable and variegating position effects. This trend was also observed for vector insertion sites located inside genes, where vector expression was often compromised, in contrast to intergenic sites, where higher levels of expression were observed. These data emphasize the importance of overcoming detrimental position effects for consistent therapeutic globin vector expression.

Antisickling effects of an endogenous human alpha-like globin.

Gene replacement or gene reactivation therapies for sickle-cell disease (SCD) typically target the mutant beta(S)-globin subunits of hemoglobin-S (alpha(2)beta(S)(2)) for substitution by nonpathological beta-like globins. Here we show, in vitro and in vivo in a transgenic mouse model of SCD, that the adverse properties of hemoglobin-S can be reversed by exchanging its normal alpha-globin subunits for zeta-globin, an endogenous, developmentally silenced, non-beta-like globin.

Beneficial effect of steroid pulse therapy on acute viral encephalitis.

Corticosteroids are often used in the treatment of acute viral encephalitis, although the efficacy of corticosteroid therapy has not been proven. We examined the effects of high-dose corticosteroid therapy on acute viral encephalitis in 5 patients with progressive disturbances of consciousness. In 3 patients who were treated within 5 days after the onset of illness, pulse therapy dramatically reduced the degree of consciousness disturbance. They became alert within 24 h, and then neurological symptoms gradually improved. Corticosteroid therapy in the other 2 patients, in whom treatment was started more than 3 weeks after the onset of illness, was not as effective, but repeated therapy at 2-week intervals resulted in complete recovery. These findings suggest that high-dose corticosteroid therapy is effective, particularly for disturbances of consciousness, an important prognostic factor in acute viral encephalitis.