Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes

<p>Abstract</p> <p>Background</p> <p>Human immunodeficiency virus type 1 (HIV-1)-based gene delivery systems are popular due to their superior efficiency of transduction of primary cells. However, these systems cannot be readily used for delivery of anti-HIV-1 genes that target constituents of the packaging system itself due to inimical effects on vector titer. Here we describe HIV-1-based packaging systems containing the Rev-response element (RRE), of simian immunodeficiency virus (SIV) in place of the HIV-1 RRE. The SIV RRE-containing packaging systems were used to deliver the anti-Rev gene, Rev M10, into HIV-1 susceptible target cells.</p> <p>Results</p> <p>An HIV-1 based packaging system was created using either a 272- or 1045-nucleotide long RRE derived from the molecular clone SIVmac239. The 1045-nucleotide SIV RRE-containing HIV-1 packaging system provided titers comparable to that of the HIV-1 RRE-based one. Moreover, despite the use of HIV-1 Rev for production of vector stocks, this packaging system was found to be relatively refractory to the inhibitory effects of Rev M10. Correspondingly, the SIV RRE-based packaging system provided 34- to 130-fold higher titers than the HIV-1 RRE one when used for packaging a gene transfer vector encoding Rev-M10. Jurkat T-cells, gene modified with Rev M10 encoding HIV-1 vectors, upon challenge with replication defective HIV-1 in single-round infection experiments, showed diminished production of virus particles.</p> <p>Conclusion</p> <p>A simple modification of an HIV-1 gene delivery system, namely, replacement of HIV-1 RRE with that of SIV, allowed efficient delivery of Rev M10 transgene into T-cell lines for intracellular immunization against HIV-1 replication.</p

Rev-Free HIV-1 Gene Delivery System for Targeting Rev-RRE-Crm1 Nucleocytoplasmic RNA Transport Pathway

The use of RNA transport elements from different viruses can provide novel attributes to HIV-1-based gene delivery systems such as improved safety or Rev independence. We previously described an HIV-1 based gene delivery system that utilized the simian immunodeficiency virus Rev-response element (RRE) in place of the HIV-1 RRE. Despite the use of Rev for the production of vector stocks, we showed the utility of this system for delivery of Rev M10, a dominant-negative mutant of HIV-1 Rev, into T-cells. Here, we investigated the use of RNA transport elements from Mason-Pfizer monkey virus or MPMV for the creation of high-titered Rev-free HIV-1-based packaging systems. The HIV-1 gag/pol expression constructs containing one or more copies of MPMV constitutive RNA transport element (CTE) were used to package similarly modified gene-transfer vectors in the presence or absence of Rev. An inverse correlation between the number of CTE modules and Rev dependency was noted for vector stock production. While packaging systems containing multiple CTEs were resistant to exogenously expressed Rev M10, the titers of vectors encoding Rev M10 were nevertheless reduced in comparison to vectors encoding only green fluorescent protein (GFP). In contrast, a gene transfer vector encoding the Rev M10 transgene and containing both RNA transport elements exhibited almost no loss in titer in comparison to a corresponding vector encoding only GFP. The optimized Rev-independent gene delivery system was used for delivery of Rev M10 transgene into T-lymphocytes. Upon challenge in single round infection assays with HIV-1, the modified T-cells produced fewer virus particles than control cells expressing GFP. This Rev-free packaging system may prove useful for targeting the Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway for inhibiting HIV replication

Clinical profile and surgical outcome of patients with congenital diaphragamatic hernia - A Prospective study.

INTRODUCTION : The migration of abdominal viscera through a defect in the diaphragm into the chest results in a diaphragmatic hernia. The congenital form exists as three distinct anatomical types: (i) Herniation through the posterolateral foramen of Bochdalek (ii) Herniation through the substernal foramen of Morgagni ; and (iii) Herniation through the esophageal hiatus. The term 'congenital diaphragmatic hernia' (CDH) refers to the herniation of abdominal viscera through the posterolateral foramen of Bochdalek. Congenital diaphragmatic hernia remains one of the most difficult challenges in the paediatric surgery. The surgical aspects are relatively straight forward but the medical management of the associated pulmonary hypoplasia and pulmonary hypertension still eludes us. With each passing decade, new approaches to the medical and surgical management of this entity have been advocated but have not stood the test of time. The multitude of treatment options for CDH reflects our limited understanding of the pathophysiology and explains the relatively fixed mortality rate seen. AIMS AND OBJECTIVES : To find out the incidence of congenital Diaphragmatic Hernia in Government Rajaji Hospital, Madurai. To evaluate the clinical presentation. To find out the incidence of Congenital Diaphragmatic Hernia (CDH) diagnosed antenatally. Total numbers treated and follow up for 1 year. MATERIAL AND METHODS : All cases of congenital diaphragmatic hernia admitted in Govt. Rajaji Hospital, Madurai were studied. All cases were given adequate preoperative preparation and those surgically treated were followed up for a period of 1 year and their outcome was noted. Those cases which were not eligible for surgical treatment were analyzed and their outcome was noted. CONCLUSION : In our institution when newborn were admitted with a prenatal diagnosis of Congenital Diaphragmatic Hernia (CDH)or when CDH babies became symptomatic after birth a careful protocol of respiratory assistance was followed. Immediate postnatal intubation was done; mask ventilation was avoided; whenever ventilators were available smallvolume, high frequency and reduced peak pressure mechanical ventilation was started and continued till stabilization of the baby for surgical repair. Pre-ductal and post-ductal percutaneous oxygen saturation measurements helped in the assessment of fitness of the child for surgical intervention. Inotropic drugs were used as necessary and cardiac function and heart anomalies were carefully evaluated. The goal of the preoperative treatments was “stabilization” of the child - PaO2 > 40 mmHg and PaCO2 < 60 mmHg, good myocardial function and adequate renal clearance with reduced or withdrawn inotropic drugs. Surgical repair of CDH was undertaken only after cardiorespiratory functions were stabilized. A policy of “delayed” surgery coupled with gentle ventilation and was followed. Congenital Diaphragmatic Hernia (CDH) still continues to show a significant mortality in our institution. This condition continues to remain a significant challenge for obstetricians, pediatric surgeons, neonatologists, and pediatricintensivists

PHARMACEUTICAL PRICING POLICY AND CONTROL– INDIAN PERSPECTIVE

ABSTRACTPharmaceutical sector is playing a vital role towards the wellness of the people and economic development of India. The innovation, development, production and marketing of medicines are accountable to pharmaceutical industry. Drugs and pharmaceuticals, healthcare delivery, medical devices among others come under the purview of healthcare sector. It is the duty of the Government to ensure the availability of the lifesaving drugs at reasonable prices by means of considering the interest of both the producers and the buyers. In order to safeguard the public health, National Pharmaceutical Pricing Authority (NPPA) is the watchdog in India, which controls the prices of drugs.Keywords: Pharmaceutical sector, Price, Drugs, Regulator,NPPA,DPC

Benign Prostatic Hyperplasia (BPH): A Comprehensive Analysis of the Malaise and Summarizing Possible Management Options through Phytotherapeutic Agents

Benign prostatic hyperplasia (BPH) is a severe illness affecting middle-aged and geriatric male patients. This disease normally occurs at the age of 40 or above and is also associated with sexual dysfunction. Alpha-blockers and 5α-reductase inhibitors are the preferred drugs practiced to treat BPH. However, invasive surgical therapy remains the gold standard for managing the condition in the case of refractory and intricate BPH conditions. Due to the fear of sexual dysfunction and the detrimental influence on their quality of life, most patients seek to avoid synthetic drugs and surgery. For this reason, several patients turn to phytotherapy and other alternative therapies. The authors looked at the existing perceptions of epidemiology, etiology, and pathophysiology associated with BPH in this review article. In addition, this article contained basic information on the pathological roles of inflammation in BPH and various diagnoses and treatment options. It was well reported that the administration of medicinal herbs played a vital role in managing BPH. In recent years, many researchers worldwide have reported the efficiency and safety of phytochemicals in managing numerous pathological disorders in-vivo and in-vitro conditions and the prevention of illness

Heterogeneous microbial oceanographic environments: Application of GIS technology in deciphering of microenvironment scenarios off the central west coast of India

In the vast oceanic microbial environment of 2468.83km 2, GIS modeling techniques involving sixty query steps, enabled the deciphering of Microenvironments as low as 1.19km 2 to 38.6 km 2 for the summer of 2004 and in case of summer 2005 where 84 query steps were involved to decipher Microenvironments of 10.55km 2 to 25.94km 2. Thirtythree sampling stations were established between Betul to Ankola off the central west coast of India accounting for a spatial coverage of 2468.83km 2. GIS query-modeling investigation was carried out using spatial layers of depth, optical parameters (k-Irradiance attenuation Coefficient, c-Beam attenuation coefficient), sediment size parameters (Sediment Mean Size and Sediment Sorting) and Benthic Foraminifera Suborders (Rotaliina, Textulariina, Miliolina, Lagenina). Foraminifera have been used as a surrogate parameter. However, any microbial parameter could proxy for foraminifers providing for the numerical deciphering of microenvironments. This is suggestive of the assimilation of GIS technology for a better appreciation of microbial oceanography

Effect of Proteolytic Processing at Two Distinct Sites on Shape and Aggregation of an Anchorless Fusion Protein of Human Respiratory Syncytial Virus and Fate of the Intervening Segment

AbstractWe have examined the consequences of cleaving the fusion glycoprotein (F) of human respiratory syncytial virus (HRSV) at two distinct furin-recognition sites. Purified anchorless F is a mixture of unaggregated cone-shaped molecules and rosettes of lollipop-shaped spikes. The unaggregated molecules contain a proportion of uncleaved F0 and an intermediate, FΔ1–109, cleaved only at site I, residues 106–109. Inhibition of cleavage at site I, by two amino acid changes (R108N/R109N), reduces the proportion of aggregated molecules with a concomitant increase in the amount of unprocessed F0. Inhibition of cleavage at site II, residues 131–136, by deletion of four amino acids (Δ131–134), abrogates aggregation of anchorless F and all molecules are seen as individual cone-shaped rods. In vitro cleavage of anchorless F, or mutant Δ131–134, with trypsin at 4, 20, or 37°C, under conditions in which cleavage at site II is complete in all molecules, leads to their aggregation in rosettes of lollipop-shaped spikes. Thus, cleavage at site II is required for the structural changes in anchorless F that lead to changes in shape and to aggregation. The segment between sites I and II, residues 110–136, is not associated with anchorless F in the supernatant of infected cell cultures, indicating that it is released from the processed protein when cleavage at sites I and II is completed

Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency &gt;3%, and were also present in the mutant library, had fitness levels that were &gt;40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

Cathelicidins have direct antiviral activity against respiratory syncytial virus in vitro and protective function in vivo in mice and humans.

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract infection in infants, causing significant morbidity and mortality. No vaccine or specific, effective treatment is currently available. A more complete understanding of the key components of effective host response to RSV and novel preventative and therapeutic interventions are urgently required. Cathelicidins are host defense peptides, expressed in the inflamed lung, with key microbicidal and modulatory roles in innate host defense against infection. In this article, we demonstrate that the human cathelicidin LL-37 mediates an antiviral effect on RSV by inducing direct damage to the viral envelope, disrupting viral particles and decreasing virus binding to, and infection of, human epithelial cells in vitro. In addition, exogenously applied LL-37 is protective against RSV-mediated disease in vivo, in a murine model of pulmonary RSV infection, demonstrating maximal efficacy when applied concomitantly with virus. Furthermore, endogenous murine cathelicidin, induced by infection, has a fundamental role in protection against disease in vivo postinfection with RSV. Finally, higher nasal levels of LL-37 are associated with protection in a healthy human adult RSV infection model. These data lead us to propose that cathelicidins are a key, nonredundant component of host defense against pulmonary infection with RSV, functioning as a first point of contact antiviral shield and having additional later-phase roles in minimizing the severity of disease outcome. Consequently, cathelicidins represent an inducible target for preventative strategies against RSV infection and may inform the design of novel therapeutic analogs for use in established infection