Introduction Neonatal inflammation, mediated in part through Toll-like receptor (TLR) and inflammasome signaling, contributes to adverse outcomes including organ injury. Pentoxifylline (PTX), a phosphodiesterase inhibitor which potently suppresses cytokine production in newborn cord blood, is a candidate neonatal anti-inflammatory agent. We hypothesized that combinations of PTX with other anti-inflammatory agents, the steroid dexamethasone (DEX) or the macrolide azithromycin (AZI), may exert broader, more profound and/or synergistic anti-inflammatory activity towards neonatal TLR- and inflammasome-mediated cytokine production. Methods Whole newborn and adult blood was treated with PTX (50–200 μM), DEX (10−10–10−7 M), or AZI (2.5–20 μM), alone or combined, and cultured with lipopolysaccharide (LPS) (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/adenosine triphosphate (ATP) (inflammasome induction). Supernatant and intracellular cytokines, signaling molecules and mRNA were measured by multiplex assay, flow cytometry and real-time PCR. Drug interactions were assessed based on Loewe's additivity. Results PTX, DEX and AZI inhibited TLR- and/or inflammasome-mediated cytokine production in newborn and adult blood, whether added before, simultaneously or after TLR stimulation. PTX preferentially inhibited pro-inflammatory cytokines especially TNF. DEX inhibited IL-10 in newborn, and TNF, IL-1β, IL-6 and interferon-α in newborn and adult blood. AZI inhibited R848-induced TNF, IL-1β, IL-6 and IL-10, and LPS-induced IL-1β and IL-10. (PTX+DEX) synergistically decreased LPS- and LPS/ATP-induced TNF, IL-1β, and IL-6, and R848-induced IL-1β and interferon-α, while (PTX+AZI) synergistically decreased induction of TNF, IL-1β, and IL-6. Synergistic inhibition of TNF production by (PTX+DEX) was especially pronounced in newborn vs. adult blood and was accompanied by reduction of TNF mRNA and enhancement of IL10 mRNA. Conclusions Age, agent, and specific drug-drug combinations exert distinct anti-inflammatory effects towards TLR- and/or inflammasome-mediated cytokine production in human newborn blood in vitro. Synergistic combinations of PTX, DEX and AZI may offer benefit for prevention and/or treatment of neonatal inflammatory conditions while potentially limiting drug exposure and toxicity.

Launch vehicle explicit guidance mechanism depends on the estimation of the desired burnout conditions and driving the vehicle to achieve these conditions. The accuracy of the vehicle at the target point depends on how tightly these conditions are achieved and what is the strategy used to define the trajectory. It has been observed in the literature that most of the guidance mechanisms during reentry use vacuum guidance equations that is during reentry the atmospheric effects are not considered. In order to achieve minimum miss distance at the target point the atmospheric effect'S are to be considered during the guided phase and appropriate corrections should be executed, otherwise depending on the reentry flight path angle and ballistic coefficient the errors can be as high as tens of nautical miles. In this paper, the authors develop a novel approach to these vacuum guided launch vehicle problems. The paper elaborates how to calculate a prior the reentry dispersion during the ascent phase guidance and provide guidance corrections such that the terminal conditions are achieved with higher accuracy.

The aim of any mission is to accomplish the final objective with desired accuracy and the same is valid for a generic launch vehicle. In many missions it is necessary to execute mid-course maneuvers with an intentional diversion trajectory to create a counter measure or to avoid certain specific known geographical locations. The current work elaborates a novel and practically implementable mid-course maneuver and an ascent phase guidance of a reentry vehicle executing an in-flight determined mid-course maneuver (trajectory reshaping) without compromising the accuracy of the final achieved target position. The robustness of the algorithm is validated with 6DoF simulation results by considering the dispersion of the burnout state vector conditions which arises due to variations in thrust profile, aerodynamics characteristics of the vehicle, atmosphere, etc.

A role for annexin A7 (A7) is postulated in the obligatory fusion between lamellar bodies and the plasma membrane during surfactant secretion in alveolar type II cells. This study investigated if surfactant secretagogues increase cell surface A7, which could support A7 insertion into plasma membrane as annexin proteins reportedly lack membrane penetration ability. In vivo trafficking of A7 to cell surface was determined by immuno-staining after non-permeabilizing fixation of alveolar type II cells. Stimulation with various secretagogues increased protein kinase-dependent staining for A7 and ABCA3 in comparison to control cells. Biotin-labeling of surface proteins showed ~ 4% of total A7 in control cells, which increased ~ 3–4 folds in stimulated type II cells. Increased cell surface A7 was also observed by protein cross-linking studies showing ~ 70 kDa A7-adduct in the membranes but not in the cytosol fraction of PMA- or A23187-stimulated cells. In vitro phosphorylation increased the Ca2 +-dependent binding of recombinant A7 to lung plasma membranes; and subsequent cross-linking showed increased levels of ~ 70 kDa A7-adduct. PMA-stimulation of type II cells increased A7 trafficking to lipid rafts suggesting that the latter are involved in A7 trafficking to the cell surface. However, in vitro membrane insertion of recombinant A7 and its tryptophan mutants as determined by fluorescence quenching with doxylPC suggested only shallow membrane insertion by A7. Together, our studies support in vivo association between surfactant secretion and cell surface A7 occurring by insertion into plasma membrane and by fusion of A7 containing lamellar bodies.

The concept of biological osteosynthesis refers basically to the conservation of vascularity of the bone during surgical intervention to ensure the continued vitality of the individual fragments and to achieve improved fracture healing.Intramedullary nailing has become the standard of care for most displaced tibial shaft fractures. However, proximal tibial fractures are difficult to control with an intramedullary device, increasing the frequency of malalignment. Also, Metaphyseal comminution is a challenge to conventional plate fixation. The present study was carried out for evaluation & analysis of the role of minimally invasive plate osteosynthesis in cases of Proximal tibial fractures. Total of 50 patients were taken up & after proper pre-operative assessment, plating was done and the results were evaluated. On the basis of the finding of this study it was concluded that: MIPO technique preserves most of the osseous vascularity thus providing for a more biological repair.

Intra-capsular femoral neck fractures in elderly people are associated with high rate of morbidity and mortality. Primary cemented hip arthroplasty is a very useful procedure for intra-capsular fracture with osteoporotic bone in elderly people. This procedure has markedly improved as well as increased socioclinical and functional status of patients. Prosthetic replacement avoids the problems of avascular necrosis and non-union. The present study was conducted on 100 cases of intra-capsular fracture neck of femur above the age of 60 years. Out of 100 cases, 50 were those in whom cemented bipolar prosthesis was used while in other 50 total hip replacements arthroplasty was done. Arthroplasty is associated with more independent living and was cost effective than any kind of internal fixation for fracture neck of femur and it provides more satisfactory long term result.There was no significant difference between these two forms of arthroplasty (bipolar hip replacement and total hip replacement) in terms of hospital stay, time interval for partial or complete weight bearing and functional status. Bipolar arthroplasty has shown slightly better results than total hip arthroplasty in terms of pain and morbidity, but THR has shown high dislocation rate in these people due to cognitive impairments, impaired reflexes and weak musculature.So, we are of opinion that, cemented bipolar hip arthroplasty is little better than total hip arthroplasty in fracture neck of femur in elderly active and mobile patients.

Various treatment modalities have been described for treatment of isolated tears of anterior cruciate ligament injuries of knee.In midsubstance tear it is secondary repair with augumentation with grafts. In avulsion tears of ACL from tibial attachment, treatment is reattachmentof avulsed fragment in its bed with screws with washer or arthroscopic repair with biodegradable screws. In present casewe used primary Bunnell suture method to reattach the avulsed ACL.

Lung surfactant secretion involves lamellar body docking and fusion with the plasma membrane in alveolar type II cells. Annexin A7 (A7) is postulated to play a role in membrane fusion during exocytosis. Our recent studies demonstrated increased co-localization of A7 with ABCA3 in lamellar bodies in type II cells stimulated with established secretagogues of lung surfactant. In this study, we investigated in vivo and in vitro interactions of A7 with the t-SNARE protein, SNAP23. Immuno-fluorescence studies showed time-dependent increases in co-localization of A7 with SNAP23 in PMA- and in A23187-stimulated cells. PMA and A23187 also caused a time-dependent increase in co-localization of ABCA3 with SNAP23. The relocation of A7 to SNAP23 domains was inhibited in the presence of PKC inhibitor, similar to that previously reported for co-localization of A7 with ABCA3. The interaction of A7 and SNAP23 was confirmed by affinity binding and by in vitro interaction of recombinant A7 and SNAP23 proteins. The in vitro binding of recombinant A7 (rA7) to GST-SNAP23 fusion protein was calcium-dependent. Phosphorylation of rA7 with PKC increased its in vitro binding to SNAP23 suggesting that a similar mechanism may operate during A7 relocation to t-SNARE domains. Thus, our studies demonstrate that annexin A7 may function in co-ordination with SNARE proteins and that protein kinase activation may be required for annexin A7 trafficking to the interacting membranes (lamellar bodies and plasma membrane) to facilitate membrane fusion during surfactant secretion.

We have previously postulated that annexin A7 (A7) facilitates the obligatory membrane fusion between lamellar bodies and plasma membrane during surfactant secretion in alveolar type II (T2) cells. Recently, we reported that several secretagogues of lung surfactant promote protein kinase‐dependent co‐localization of A7 with the lamellar body marker protein ABCA3 in T2 cells. We now report that secretagogues also promote association of A7 with SNAP23, one of the SNARE proteins. Confocal immuno‐fluorescence microscopy of T2 cells showed a time‐dependent increase in co‐localization of A7 and SNAP23 in T2 cells stimulated with PMA or calcium ionophore A23187. The increased co‐localization was prevented by pre‐treatment of T2 cells with PKC inhibitor, BisI. Supporting evidence for interactions of A7 and SNAP23 was derived from in vitro binding studies. The binding of bacterially expressed A7 with GST‐SNAP23 was Ca2+‐dependent. The binding was observed at 0.2μM Ca2+, which increased further in Ca2+ concentration‐dependent manner. Thus, physiologic conditions causing calcium elevation would increase interaction of A7 with SNAP23. In vitro phosphorylation of A7 with PKC also increased the Ca2+‐dependent binding to GST‐SNAP23. We conclude that calcium elevation and increased protein phosphorylation that occur in secretagogue‐stimulated T2 cells would facilitate A7 interactions with SNAP23 during surfactant secretion. We speculate that A7 phosphorylation and association with lamellar bodies and SNARE proteins is to facilitate the membrane fusion process.

Osteoarthritis is the most common form of arthritis that leads to pain, infirmity and deformity of the knee joint. This study was done with the aim to evaluate the efficacy of total knee replacement in relieving knee symptoms associated with osteoarthritis knee. This study was conducted on 30 patients suffering from osteoarthritis of knee, having pain (for some even at rest), contractures, varus deformity and decreased range of motion and had no relief with conservative modes of management. Patients were evaluated according to knee society score system after one month, three months, six months one year and two years from the day of operation. In our study all were having poor grade preoperatively according to knee scoring system and postoperatively there were 22 excellent, 5 good and 3 fair results with no poor result in our study. It can thus be concluded from our study that total knee replacement provides an unparalleled improvement in the disease symptomatology, deformity correction and life style in patients suffering from osteoarthritis.

Membrane fusion between the lamellar bodies and plasma membrane is an obligatory event in the secretion of lung surfactant. Previous studies have postulated a role for annexin A7 (A7) in membrane fusion during exocytosis in some cells including alveolar type II cells. However, the intracellular trafficking of A7 during such fusion is not described. In this study, we investigated association of endogenous A7 with lamellar bodies in alveolar type II cells following treatment with several secretagogues of lung surfactant. Biochemical studies with specific antibodies showed increased membrane-association of cell A7 in type II cells stimulated with agents that increase secretion through different signaling mechanisms. Immuno-fluorescence studies showed increased co-localization of A7 with ABCA3, the lamellar body marker protein. Because these agents increase surfactant secretion through activation of PKC and PKA, we also investigated the effects of PKC and PKA inhibitors, bisindolylmaleimideI (BisI) and H89, respectively, on A7 partitioning. Western blot analysis showed that these inhibitors prevented secretagogue-mediated A7 increase in the membrane fractions. These inhibitors also blocked increased co-localization of A7 with ABCA3 in secretagogue-treated cells, as revealed by immuno-fluorescence studies. In vitro studies with recombinant A7 showed phosphorylation with PKC and PKA. The cell A7 was also phosphorylated in cells treated with surfactant secretagogues. Thus, our studies demonstrate that annexin A7 relocates to lamellar bodies in a phosphorylation-dependent manner. We suggest that activation of protein kinase promotes phosphorylation and membrane-association of A7 presumably to facilitate membrane fusion during lung surfactant secretion.

Lung surfactant secretion requires fusion of lamellar bodies (LB) with the plasma membrane in type II (T2) cells. Annexin A7 (A7) contributes to such membrane fusion since 1) it promotes surfactant secretion in permeabilized T2 cells and 2) secretagogues increase its association with the LB and plasma membrane in T2 cells. In this study, we hypothesized that secretagogues would increase membrane insertion of A7 to facilitate its fusion promoting activity. As a result, A7 may be expressed at the cell surface. Isolated rat T2 cells were cultured for 20–22h on glass cover slips and stimulated for 2h without or with 100nM phorbol myristate acetate (PMA). The cells were fixed in 2% paraformaldehyde, (PFA, non‐permeabilized) or in ice‐cold methanol (permeabilized) and immuno‐stained for A7 and LB specific protein, ABCA3 (P180). With methanol fixation, control cells showed P180 and A7 staining at the cell surface as well as in the cell interior without significant co‐localization of two proteins. The PMA‐stimulated cells showed increased co‐localization of two proteins. With PFA fixation, control cells showed that A7 and some P180 staining at the cell surface without significant co‐localization of the two proteins. Very little staining was seen inside the cells. In PMA‐stimulated cells, increased staining and co‐localization of the two proteins were observed at the cell surface. Our results suggest that increased secretion is associated with increased insertion of A7 and P180 into the plasma membrane leading to exposure of these two proteins at the cells surface, possibly at the secretion site.

Objective: Forces transmitted to the neonate as a consequence of accelerations during transport have been associated with adverse neonatal outcomes including bronchopulmonary dysplasia. In this study, we sought to determine the relationship between the duration of transport and respiratory performance in the rat model.Methods: Four groups of Sprague-Dawley rat pups (10-12 pups/groups) were exposed to simulated medical transport on postnatal day of life 11 or 12. Each group was exposed to an average impulse of 27.4 m/s(2)/min for 0, 30, 60 or 90 min. During the exposure periods, impulse was monitored by computerized sampling using a digital accelerometer. Post-exposure, animals were immediately prepared, placed on mechanical ventilation and analyzed for elastance, tissue damping, airway resistance, ratio of damping to elastance (eta), hysteresivity, and inertance at positive end expiratory pressures (PEEPs) of 0, 3 and 6 cm(3) of H2O. Total phospholipid content and surfactant proteins A, B, and C mRNA levels in broncho-alveolar lavage fluid and lung tissue were obtained.Results: Increased transport time resulted in a significant step-wise increase in airway resistance at all levels of PEEP (P<0.01). Static compliance decreased significantly after 60 min at PEEPs of 3 and 6 cm H2O (P<0.01). Eta significantly decreased with greater transport time at a PEEP of 6 cm H2O (P<0.05). Tissue damping increased with duration of transport time across all PEEP levels, but only exhibited statistical significance at a PEEP of 0 cm H2O (P<0.05). No differences were seen in hysteresivity or inertance. Compared with controls, transport was associated with significant reductions in total phospholipid content and mRNA levels of surfactant proteins B and C.Conclusion: Rat pups experienced significant deterioration of respiratory function with increasing duration of simulated transport.

The secretion of lung surfactant in alveolar type II (T2) cells requires fusion of lamellar bodies (LB) with the plasma membrane. Our in vitro studies have shown a role for Annexin A7 (A7) in such membrane fusion, since purified A7 binds to LB and plasma membranes and promotes their fusion. Using antibodies against recombinant A7, we have shown that secretagogues of lung surfactant promote membrane‐association of endogenous A7 in isolated T2 cells. We now show that activation of protein kinase is required for such membrane‐association. Isolated rat T2 cells were treated for 30min with phorbol myristate acetate (PMA), A23187, ATP or isoproterenol, and the A7 levels in the cytosol and membrane fractions were analyzed by immuno‐blot analysis. All agents increased relative proportion of A7 in the membranes when compared with the control cells. Immuno‐fluorescence staining of T2 cells showed that secretagogues promoted relocation of A7 to membranes including LB, as verified by co‐staining for ABCA3. Because of the significance of protein kinase activation in regulation of surfactant secretion, we evaluated the effect of protein kinase inhibitors on A7 relocation in treated cells. The PMA‐induced relocation was inhibited by pretreatment with bis‐indolylmaleimide I, a specific inhibitor of PKC, and the isoproterenol‐induced relocation was blocked with H‐89, a specific inhibitor of PKA. Thus, our studies provide further correlative evidence for the role of A7 in lung surfactant secretion.

To understand the role of reactive oxygen species in mechanosensory control of lung development a new approach to interfere with protein–protein interactions by means of a short interacting peptide was developed. This technology was used in the developing rodent lung to examine the role of NADPH oxidase (NOX), casein kinase 2 (CK2), and the cystic fibrosis transmembrane conductance regulator (CFTR) in stretch‐induced differentiation. Interactions between these molecules was targeted in an in utero system with recombinant adeno‐associated virus (rAAV) containing inserted DNA sequences that express a control peptide or small interfering peptides ( siPs ) specific for subunit interaction or phosphorylation predicted to be necessary for multimeric enzyme formation. In all cases only siPs with sequences necessary for a predicted normal function were found to interfere with assembly of the multimeric enzyme. A noninterfering control siP to nonessential regions or reporter genes alone had no effect. Physiologically, it was shown that siPs that interfered with the NOX‐CFTR‐CK2 complex that we call an “interactonome” affected markers of stretch‐induced lung organogenesis including Wnt/β‐catenin signaling. Developmental Dynamics 238:386–393, 2009. © 2009 Wiley‐Liss, Inc.

Background: Mechanicosensory mechanisms regulate cell differentiation during lung organogenesis. We have previously demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR) was integral to stretch-induced growth and development and that transient expression of antisense-CFTR (ASCFTR) had negative effects on lung structure and function. In this study, we examined adult alveolar type II (ATII) cell phenotype after transient knock down of CFTR by adenovirus-directed in utero expression of ASCFTR in the fetal lung.Results: In comparison to (reporter gene-treated) Controls, ASCFTR-treated adult rat lungs showed elevated phosphatidylcholine ( PC) levels in the large but not in the small aggregates of alveolar surfactant. The lung mRNA levels for SP-A and SP-B were lower in the ASCFTR rats. The basal PC secretion in ATII cells was similar in the two groups. However, compared to Control ATII cells, the cells in ASCFTR group showed higher PC secretion with ATP or phorbol myristate acetate. The cell PC pool was also larger in the ASCFTR group. Thus, the increased surfactant secretion in ATII cells could cause higher PC levels in large aggregates of surfactant. In freshly isolated ATII cells, the expression of surfactant proteins was unchanged, suggesting that the lungs of ASCFTR rats contained fewer ATII cells. Gene array analysis of RNA of freshly isolated ATII cells from these lungs showed altered expression of several genes including elevated expression of two calcium-related genes, Ca2+-ATPase and calcium-calmodulin kinase kinase 1 (CaMkk1), which was confirmed by real-time PCR. Western blot analysis showed increased expression of calmodulin kinase I, which is activated following phosphorylation by CaMkk1. Although increased expression of calcium regulating genes would argue in favor of Ca2+-dependent mechanisms increasing surfactant secretion, we cannot exclude contribution of alternate mechanisms because of other phenotypic changes in ATII cells of the ASCFTR group.Conclusion: Developmental changes due to transient disruption of CFTR in fetal lung reflect in altered ATII cell phenotype in the adult life.