by
Raffray S. Khan;
Mario D. Martinez;
Jay C. Sy;
Karl D. Pendergrass;
Pao-lin Che;
Milton Brown;
E. Bernadette Cabigas;
Madhuri Dasari;
Niren Murthy;
Michael Davis
There is a great need for the development of therapeutic strategies that can target biomolecules to damaged myocardium. Necrosis of myocardium during a myocardial infarction (MI) is characterized by extracellular release of DNA, which can serve as a potential target for ischemic tissue. Hoechst, a histological stain that binds to double-stranded DNA can be conjugated to a variety of molecules. Insulin-like growth factor-1 (IGF-1), a small protein/polypeptide with a short circulating-half life is cardioprotective following MI but its clinical use is limited by poor delivery, as intra-myocardial injections have poor retention and chronic systemic presence has adverse side effects. Here, we present a novel delivery vehicle for IGF-1, via its conjugation to Hoechst for targeting infarcted tissue. Using a mouse model of ischemia-reperfusion, we demonstrate that intravenous delivery of Hoechst-IGF-1 results in activation of Akt, a downstream target of IGF-1 and protects from cardiac fibrosis and dysfunction following MI.
Indoleamine 2,3 dioxygenase-1 (IDO-1) is an enzyme in the kynurenine pathway which augments tumor-induced immune tolerance. Previous studies in childhood acute myeloid leukemia (AML) have shown a negative correlation of IDO-1 mRNA expression with outcomes. The aim of our study was to develop a practical and objective immunohistochemical technique to quantify IDO-1 expression on diagnostic bone marrow biopsies of AML patients in order to facilitate its use in routine clinical practice. IDO-1 mRNA was extracted from diagnostic bone marrow specimens from 29 AML patients. IDO-1 protein expression was assessed in 40 cases via immunohistochemistry and quantified by a novel 'composite IDO-1 score'. In a univariate analysis, higher age (p = 0.0018), male gender (p = 0.019), high risk cytogenetics (p = 0.002), higher IDO-1 mRNA (p = 0.005), higher composite IDO-1 score (p < 0.0001) and not undergoing allogeneic stem cell transplant (SCT, p = 0.0005) predicted poor overall survival. In a multivariate model that included the aforementioned variables, higher composite IDO-1 score (p = 0.007) and not undergoing allogeneic SCT (p = 0.007) was found to significantly predict poor outcomes. Further, patients who failed induction had higher composite IDO-1 score (p = 0.01). In conclusion, 'composite IDO-1 score' is a prognostic tool that can help identify a certain subset of AML patients with 'early mortality'. This unique subset of patients can potentially benefit from specific IDO-1 inhibitor therapy, currently in clinical trials.
Bone marrow derived mesenchymal stem cells (MSCs) are regularly utilized for translational therapeutic strategies including cell therapy, tissue engineering, and regenerative medicine and are frequently used in preclinical mouse models for both mechanistic studies and screening of new cell based therapies. Current methods to culture murine MSCs (mMSCs) select for rapidly dividing colonies and require long-term expansion. These methods thus require months of culture to generate sufficient cell numbers for feasibility studies in a lab setting and the cell populations often have reduced proliferation and differentiation potential, or have become immortalized cells. Here we describe a simple and reproducible method to generate mMSCs by utilizing hypoxia and basic fibroblast growth factor supplementation. Cells produced using these conditions were generated 2.8 times faster than under traditional methods and the mMSCs showed decreased senescence and maintained their multipotency and differentiation potential until passage 11 and beyond. Our method for mMSC isolation and expansion will significantly improve the utility of this critical cell source in pre-clinical studies for the investigation of MSC mechanisms, therapies, and cell manufacturing strategies.
Atherosclerosis occurs preferentially in arterial regions exposed to disturbed blood flow. Targeting these pro-atherogenic regions is a potential anti-atherogenic therapeutic approach, but it has been extremely challenging. Here, using in vivo phage display approach and the partial carotid ligation model of flow-induced atherosclerosis in mouse, we identified novel peptides that specifically bind to endothelial cells (ECs) exposed to disturbed flow condition in pro-atherogenic regions. Two peptides, CLIRRTSIC and CPRRSHPIC, selectively bound to arterial ECs exposed to disturbed flow not only in the partially ligated carotids but also in the lesser curvature and branching point of the aortic arch in mice as well as human pulmonary artery branches. Peptides were conjugated to branched polyethylenimine-polyethylene glycol polymer to generate polyplexes carrying siRNA targeting intercellular adhesion molecule-1 (siICAM-1). In mouse model, CLIRRTSIC polyplexes carrying si-ICAM-1 specifically bound to endothelium in disturbed flow regions, reducing endothelial ICAM-1 expression. Mass spectrometry analysis revealed that non-muscle myosin heavy chain II A (NMHC IIA) is a protein targeted by CLIRRTSIC peptide. Further studies showed that shear stress regulates NMHC IIA expression and localization in ECs. The CLIRRTSIC is a novel peptide that could be used for targeted delivery of therapeutics such as siRNAs to pro-atherogenic endothelium.
by
Fernanda C. Lessa;
Jennifer Milucky;
Nadine Rouphael;
Nancy M. Bennett;
H. Keipp Talbot;
Lee H. Harrison;
Monica Farley;
Jeremy Walston;
Fabiana Pimenta;
Robert E. Gertz;
Gowrisankar Rajam;
Maria da Gloria Carvalho;
Bernard Beall;
Cynthia G. Whitney
Streptococcus pneumoniae’s polysaccharide capsule is an important virulence factor; vaccine-induced immunity to specific capsular polysaccharide effectively prevents disease. Serotype 1 S. pneumoniae is rarely found in healthy persons, but is highly invasive and a common cause of meningitis outbreaks and invasive disease outside of the United States. Here we show that genes for polysaccharide capsule similar to those expressed by pneumococci were commonly detected by polymerase chain reaction among upper respiratory tract samples from older US adults not carrying pneumococci. Serotype 1-specific genes were predominantly detected. In five oropharyngeal samples tested, serotype 1 gene belonging to S. mitis expressed capsules immunologically indistinct from pneumococcal capsules. Whole genome sequencing revealed three distinct S. mitis clones, each representing a cps1 operon highly similar to the pneumococcal cps1 reference operon. These findings raise important questions about the contribution of commensal streptococci to natural immunity against pneumococci, a leading cause of mortality worldwide.
Ectodomain shedding of glycoprotein (GP) Ibα is thought to mediate the clearance of activated, aged or damaged platelets. A monoclonal antibody, 5G6, has been developed recently to specifically bind to the GPIbα shedding cleavage site and to inhibit its shedding. However, the molecular mechanism underlying antigen recognition and inhibitory specificity is not clear. To elucidate the structural basis for 5G6 binding to GPIbα, we determined the crystal structure of 5G6 Fab fragment in complex with its epitope peptide KL10 (GPIbα residues 461-470, KLRGVLQGHL), to 2.4-Å resolution. Key residues in both 5G6 and KL10 were mutated to validate their effects in antibody binding by using isothermal titration calorimetry. The 5G6 Fab-KL10 peptide complex structure confirmed the direct association of 5G6 with its target GPIbα residues and elucidated the molecular basis underlying its binding specificity and high affinity. The similar binding properties of 5G6 Fab fragment to GPIbα on human platelets as those to KL10 suggests that such an interaction may not be affected by the plasma membrane or nearby GPIbβ. This structural information may facilitate further antibody optimization and humanization.
by
Robinson Ramirez-Velez;
Jhonatan Camilo Pena-Ibagon;
Javier Martinez-Torres;
Alejandra Tordecilla-Sanders;
Jorge Enrique Correa-Bautista;
Felipe Lobelo;
Antonio Garcia-Hermoso
Evidence shows an association between muscular strength (MS) and health among young people, however low muscular strength cut points for the detection of high metabolic risk in Latin-American populations are scarce. The aim of this study was twofold: to explore potential age- and sex-specific thresholds of MS, for optimal cardiometabolic risk categorization among Colombian children and adolescents; and to investigate whether cardiometabolic risk differed by MS group by applying the receiver operating characteristic curve (ROC) cut point. MS was estimated by using a handle dynamometer on 1,950 children and adolescents from Colombia, using MS relative to weight (handgrip strength/body mass). A metabolic risk score was computed from the following components: waist circumference, triglycerides, HDL-c, glucose, and systolic and diastolic blood pressure. ROC analysis showed a significant discriminatory accuracy of MS in identifying the low/high metabolic risk in children and adolescents and in both genders. In children, the handgrip strength/body mass levels for a low metabolic risk were 0.359 and 0.376 in girls and boys, respectively. In adolescents, these points were 0.440 and 0.447 in girls and boys, respectively. In conclusion, the results suggest an MS level relative to weight for having a low metabolic risk, which could be used to identify youths at risk.
by
Alexandre Archanjo Ferraro;
Marco Antônio Barbieri;
Antonio Augusto Moura da Silva;
Carlos Grandi;
Viviane Cunha Cardoso;
Aryeh Stein;
Heloisa Bettiol
While birth weight and weight gain have been associated with hypertension (HT), the association of linear growth, independently of weight gains, has been less well studied. We assessed the independent association of body mass index (BMI) and length at birth and changes in BMI and height during the first two decades of life with adult blood pressure (BP). A birth cohort (n = 1141) was assembled in 1978-79, and followed up at school-age and adulthood. We used conditional length and BMI measures. BMI at birth was inversely associated with HT; c-BMI from school age to adulthood and c-height from birth to school age were positively associated with hypertension. Early adiposity accretion from birth to 9 years and late linear growth from 9 to 24 years were not associated with increased HT. Regarding BP, systolic and diastolic BP presented similar partterns: The lower the BMI at birth the higher the adult BP; the higher the BMI gains in the first 2 decades of life the higher the adult BP; linear accretion only in the first decade of life was associated with adult BP. Linear growth in the first decade of life and fat accretion in the second decade are associated with adults HT.