M-cell targeted biodegradable PLGA nanoparticles for oral immunization against hepatitis B

The transcytotic capability and expression of distinct carbohydrate receptors on the intestinal M-cells render it a potential portal for the targeted oral vaccine delivery. PLGA nanoparticles loaded with HBsAg were developed and antigen was stabilized by co-encapsulation of trehalose and Mg(OH)(2). Additionally, Ulex europaeus 1 (UEA-1) lectin was anchored to the nanoparticles to target them to M-cells of the peye's patches. The developed systems was characterized for shape, size, polydispersity index and loading efficiency. Bovine submaxillary mucin (BSM) was used as a biological model for the in vitro determination of lectin activity and specificity. The targeting potential of the lectinized nanoparticles were determined by Confocal Laser Scanning Microscopy (CLSM) using dual staining technique. The immune stimulating potential was determined by measuring the anti-HBsAg titre in the serum of Balb/c mice orally immunized with various lectinized formulations and immune response was compared with the alum-HBsAg given intramuscularly. Induction of the mucosal immunity was assessed by estimating secretary IgA (sIgA) level in the salivary, intestinal and vaginal secretion. Additionally, cytokines (interleukin-2; IL-2 and interferon-gamma; IFN-gamma) level in the spleen homogenates was also determined. The results suggest that HBsAg can be successfully stabilized by co-encapsulation of protein stabilizers. The lectinized nanoparticles have demonstrated approximately 4-fold increase in the degree of interaction with the BSM as compared to plain nanoparticles and sugar specificity of the lectinized nanoparticles was also maintained. CLSM showed that lectinized nanoparticles were predominantly associated to M-cells. The serum anti-HBsAg titre obtained after oral immunization with HBsAg loaded stabilized lectinized nanoparticles was comparable with the titre recorded after alum-HBsAg given intramuscularly. The stabilized UEA-1 coupled nanopartilces exhibited enhanced immune response as compared to stabilized non-lectinized nanoparticles. Furthermore, the stabilized lectinized nanoparticles elicited sIgA in the mucosal secretion and IL-2 and IFN-gamma in the spleen homogenates. These stabilized lectinized nanoparticles could be a promising carrier-adjuvant for the targeted oral-mucosal immunization.     

The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity

NK cell cytotoxicity is controlled by numerous NK inhibitory and activating receptors. Most of the inhibitory receptors bind MHC class I proteins and are expressed in a variegated fashion. It was recently shown that TIGIT, a new protein expressed by T and NK cells binds to PVR and PVR-like receptors and inhibits T cell activity indirectly through the manipulation of DC activity. Here, we show that TIGIT is expressed by all human NK cells, that it binds PVR and PVRL2 but not PVRL3 and that it inhibits NK cytotoxicity directly through its ITIM. Finally, we show that TIGIT counter inhibits the NK-mediated killing of tumor cells and protects normal cells from NK-mediated cytoxicity thus providing an “alternative self” mechanism for MHC class I inhibition.     

Structural imperatives impose diverse evolutionary constraints on helical membrane proteins

The amino acid sequences of transmembrane regions of helical membrane proteins are highly constrained, diverging at slower rates than their extramembrane regions and than water-soluble proteins. Moreover, helical membrane proteins seem to fall into fewer families than water-soluble proteins. The reason for the differential restrictions on sequence remains unexplained. Here, we show that the evolution of transmembrane regions is slowed by a previously unrecognized structural constraint: Transmembrane regions bury more residues than extramembrane regions and soluble proteins. This fundamental feature of membrane protein structure is an important contributor to the differences in evolutionary rate and to an increased susceptibility of the transmembrane regions to disease-causing single-nucleotide polymorphisms.     

Peptides modulating conformational changes in secreted chaperones: From in silico design to preclinical proof of concept

Blocking conformational changes in biologically active proteins holds therapeutic promise. Inspired by the susceptibility of viral entry to inhibition by synthetic peptides that block the formation of helix–helix interactions in viral envelope proteins, we developed a computational approach for predicting interacting helices. Using this approach, which combines correlated mutations analysis and Fourier transform, we designed peptides that target gp96 and clusterin, 2 secreted chaperones known to shift between inactive and active conformations. In human blood mononuclear cells, the gp96-derived peptide inhibited the production of TNFα, IL-1β, IL-6, and IL-8 induced by endotoxin by >80%. When injected into mice, the peptide reduced circulating levels of endotoxin-induced TNFα, IL-6, and IFNγ by >50%. The clusterin-derived peptide arrested proliferation of several neoplastic cell lines, and significantly enhanced the cytostatic activity of taxol in vitro and in a xenograft model of lung cancer. Also, the predicted mode of action of the active peptides was experimentally verified. Both peptides bound to their parent proteins, and their biological activity was abolished in the presence of the peptides corresponding to the counterpart helices. These data demonstrate a previously uncharacterized method for rational design of protein antagonists.     

Fulminant pertussis: A multi‐center study with new insights into the clinico‐pathological mechanisms

Pertussis carries a high risk of mortality in very young infants. The mechanism of refractory cardio‐respiratory failure is complex and not clearly delineated. We aimed to examine the clinico‐pathological features and suggest how they may be related to outcome, by multi‐center review of clinical records and post‐mortem findings of 10 patients with fulminant pertussis (FP). All cases were less than 8 weeks of age, and required ventilation for worsening respiratory symptoms and inotropic support for severe hemodynamic compromise. All died or underwent extra corporeal membrane oxygenation (ECMO) within 1 week. All had increased leukocyte counts (from 54 to 132 × 10⁹/L) with prominent neutrophilia in 9/10. The post‐mortem demonstrated necrotizing bronchitis and bronchiolitis with extensive areas of necrosis of the alveolar epithelium. Hyaline membranes were present in those cases with viral co‐infection. Pulmonary blood vessels were filled with leukocytes without well‐organized thrombi. Immunodepletion of the thymus, spleen, and lymph nodes was a common feature. Other organisms were isolated as follows; 2/10 cases Para influenza type 3, 2/10 Moraxella catarrhalis, 1/10 each with respiratory syncytial virus (RSV), a coliform organism, methicillin‐resistant Staphylococcus aureus (MRSA), Haemophilus influenzae, Stenotrophomonas maltophilia, methicillin‐sensitive Staphylococcus aureus (MSSA), and candida tropicalis. We postulate that severe hypoxemia and intractable cardiac failure may be due to the effects of pertussis toxin, necrotizing bronchiolitis, extensive damage to the alveolar epithelium, tenacious airway secretions, and possibly leukostasis with activation of the immunological cascade, all contributing to increased pulmonary vascular resistance. Cellular apoptosis appeared to underlay much of these changes. The secondary immuno‐compromise may facilitate co‐infection. Pediatr Pulmonol. 2009; 44:970–980. ©2009 Wiley‐Liss, Inc.     

PTEN and SHIP2 regulates PI3K/Akt pathway through focal adhesion kinase

Our laboratory has established a novel role of focal adhesion kinase (FAK) in vitro and in vivo, as a positive regulator of insulin signaling pathway. In vitro studies reported tyrosine dephosphorylation of FAK under insulin resistance in C2C12 skeletal muscle cells. A decrease in FAK tyrosine phosphorylation was also observed in skeletal muscle of insulin resistant Sprague–Dawley rats fed on high-fat-diet. Present study was undertaken to explore the cellular mechanism of FAK dephosphorylation under insulin resistance in C2C12 skeletal muscle cells. Here we report that PTEN and SHIP2, the phosphatases widely implicated as negative regulators of insulin signaling, to be responsible for dephosphorylation of FAK. Data propose that under insulin resistance upregulation of PTEN and SHIP2 act through changes in FAK phosphorylation to impair insulin signaling suggesting FAK to be a key mediator of PTEN and SHIP2 in the regulation of insulin signaling. Thus data elucidates a part of molecular mechanism of insulin resistance in skeletal muscle cells.     

Acquired Left Atrial-to-Right Ventricular Shunt with Mitral Valve Incompetence: A Rare Sequela after Repair of Atrioventricular Septal Defect

Acquired left ventricular-to-right atrial communication is encountered periodically. This condition is chiefly attributable to surgical mishaps, trauma, endocarditis, or endomyocardial biopsy. In a few instances, a Gerbode-like defect develops after the repair of an atrioventricular septal defect. Our search of the worldwide medical literature revealed just 1 report of a “mirror” occurrence of a Gerbode-like defect: a shunt between the left atrium and the right ventricle.Herein, we present the case of a 22-year-old woman who had severe mitral valve incompetence accompanying an acquired shunt between the left atrium and the right ventricle—a late sequela of the earlier repair of an atrioventricular septal defect. After surgical correction of the shunt and the associated mitral incompetence, the patient experienced a good outcome.Echocardiographic and intraoperative findings are presented, along with a plausible explanation for the mechanism and presentation of the condition in our patient. To our knowledge, this is only the 2nd report of an acquired shunt between the left atrium and the right ventricle, and the 1st such case to be accompanied by severe mitral valve incompetence.Key words: Cardiac surgical procedures, cardiomyopathies/etiology/ultrasonography, heart septal defects, atrial/physiopathology/surgery/ultrasonography, heart septal defects, ventricular/complications, postoperative complications/etiology, mitral valve insufficiency/etiology/surgery, reoperation, time factors, treatment outcomeAcquired left ventricular-to-right atrial communications are encountered from time to time. These are chiefly attributable to surgical mishaps, trauma, endocarditis, or even endomyocardial biopsy.¹ A few cases are on record wherein endocarditis was thought to have led to a Gerbode-like defect after the repair of an atrioventricular septal defect (AVSD).² However, we could find just 1 report of an acquired shunt between the left atrium (LA) and the right ventricle (RV).³ Here, we report a 2nd such case, that of a 22-year-old woman who also had late-onset severe mitral valve regurgitation. A point of interest is not merely the rare occurrence of this condition, but also the quest for a plausible explanation for its mechanism and presentation.