Role of Macrophages in Muscle Transfection with pDNA/PLURONIC Formulation

Non-ionic amphiphilic block copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), Pluronics, arranged in a tri-block structure PEO-PPO-PEO, have raised a considerable interest in skeletal muscle Gene Therapy. Previous studies have demonstrated that co-administration of Pluronics with naked plasmid DNA (pDNA) by direct i.m. injection enhanced transgene expression not only in muscle but also in distal lymphoid organs (spleen and lymph nodes) and this response was strain-dependent; not observed in athymic (BALB/c nu/nu) mouse; suggesting a role of immune cells in gene transfer to skeletal muscles. Therefore, we first evaluated the role of inflammation and inflammatory cells, on muscle transfection. We showed that local inflammation in murine hind limb ischemia model (MHLIM) drastically increased DNA, RNA and transgene expression levels in muscles injected with pDNA/Pluronic. Ischemic muscles showed high number of GFP+ muscle fibers with GFP expression co-localized with desmin+ muscle fibers and CD11b+ macrophages (MØs). This suggested that MØs assist muscle transfection with pDNA/Pluronic. Moreover, adoptively transferred MØs were shown to pass the transgene to inflamed muscle cells. Hence, we developed an in vitro model of inflammation by co-culturing transfected MØs with myotubes (MTs) and used constitutive (cmv-luciferase) or muscle specific (desmin-luciferase) reporter gene expression to show that Pluronic P85 enhances the horizontal pDNA transfer from MØ to MTs. Systemic inflammation (MHLIM and peritonitis) also increased the transgene expression with pDNA/Pluronic but not pDNA alone. Second, we carried out an immunological profiling of adjuvant (P85/LPS/Alum) induced cellular recruitment and showed that P85 helps modulate the response towards MØs predominated recruitment. Moreover, the contribution of MØ predominance in assisting gene transfer after i.m. injection of x pDNA/P85 was reinforced by in vivo MØ depletion that resulted in near complete attenuation in gene expression to the level of naked pDNA alone. Finally, MØ recruitment response was harnessed to further increase muscle transfection by preinjecting P85 at the subsequent sites of pDNA/P85 injection resulting in improved muscle transfection. This dissertation study provides a key evidence about the role of MØs in assisting gene transfer in pDNA/Pluronic delivery approach. Altogether, we introduce Pluronics as simple and commercially available excipients to enhance gene transfer using pDNA/Pluronic admix compositions; are recognized pharmaceutical excipients in US and British Pharmacopoeia and therefore have great potential in human gene therapy clinical trials in healthy and disease pathologies that often involves inflammation

Pharmacovigilance analysis in a rural tertiary care hospital in North India: a retrospective study

Background: The main motive of PvPI (Pharmacovigilance Programme of India) is to collect valuable data so that signals can be generated from reported adverse drug events (ADEs). It also tries to establish their causality so that ADEs can be labelled as adverse drug reactions (ADRs) beyond any doubt.Methods: This retrospective observational study done in rural set up tertiary care teaching hospital collected data through voluntary reporting in ADR form of PvPI for period of 6 month. Causality assessment was done using WHO causality assessment scale.Results: In 150 reported cases, majority ADRs were due to tuberculosis, cancer and HIV treatments. Gastrointestinal tract and central nervous system were the major organs involved. Most ADRs occurred within first day of drug intake. Around 15% required hospitalization. 55% ADRs were probable and 41% were possible in nature. Vertigo and depression was most frequent ADR in MDR therapy. Rashes, pruritis, fever and joint pain was frequent in antiretroviral therapy. Dysguesia, dizziness, nausea, vomiting and constipation was frequent in patients taking anticancer drugs. Platins and antibiotics used for cancer therapy cause most cancer treatment ADRs.Conclusions: ADRs add to hospitalization expenses, insurance costs and increase in work loss days besides addition to patient suffering. Prior knowledge can help in better prescriptions and prevent valuable resource loss. Reasons for under-reporting of ADRs can be complacency, ignorance, lack of financial incentives for reporting, fear of litigation, claims of compensation and lack of time in busy hospital schedules

Correlation of glycosylated hemoglobin with microalbuminuria to predict renal damage in diabetic patients

Background: Regular screening of levels of glycosylated hemoglobin and microalbuminuria, diabetic nephropathy can be prevented. The aim was to assess and compare the levels of glycosylated hemoglobin, microalbuminuria and serum creatinine in type 2 diabetic patients divided in groups of those on default antidiabetic treatment compared with those on regular antidiabetic treatment and to assess its correlation in type 2 of diabetic nephropathy. Methods:Two hundred diabetic patients above 40 years of age and 200 age matched control subjects with levels of glycosylated hemoglobin < 6.5% and on regular antiglycemic therapy were selected. Fasting plasma sugar was estimated by the glucose oxidase (GOD) - glucose peroxidase (POD). Glycosylated hemoglobin and microalbuminuria level was measured by the immunoturbidimetric method and serum creatinine estimation was done by the Jaffe’s kinetic method. p value was drawn using the student’s paired t-test. Results: There is a strong correlation between the increase in the levels of glycosylated hemoglobin with the corresponding rise in the levels of microalbuminuria and serum creatinine. Conclusion: Periodic surveillance of the levels of microalbuminuria should be carried out in the type 2 diabetic patients to prevent further damage by early detection of diabetic nephropathy.

Horizontal gene transfer from macrophages to ischemic muscles upon delivery of naked DNA with Pluronic block copolymers

Intramuscular administration of plasmid DNA (pDNA) with non-ionic Pluronic block copolymers increases gene expression in injected muscles and lymphoid organs. We studied the role of immune cells in muscle transfection upon inflammation. Local inflammation in murine hind limb ischemia model (MHLIM) drastically increased DNA, RNA and expressed protein levels in ischemic muscles injected with pDNA/Pluronic. The systemic inflammation (MHLIM or peritonitis) also increased expression of pDNA/Pluronic in the muscles. When pDNA/Pluronic was injected in ischemic muscles the reporter gene, Green Fluorescent Protein (GFP) co-localized with desmin+ muscle fibers and CD11b+ macrophages (MØs), suggesting transfection of MØs along with the muscle cells. P85 enhanced (~4 orders) transfection of MØs with pDNA in vitro. Moreover, adoptively transferred MØs were shown to pass the transgene to inflamed muscle cells in MHLIM. Using a co-culture of myotubes (MTs) and transfected MØs expressing a reporter gene under constitutive (cmv-luciferase) or muscle specific (desmin-luciferase) promoter we demonstrated that P85 enhances horizontal gene transfer from MØ to MTs. Therefore, MØs can play an important role in muscle transfection with pDNA/Pluronic during inflammation, with both inflammation and Pluronic contributing to the increased gene expression. pDNA/Pluronic has potential for therapeutic gene delivery in muscle pathologies that involve inflammation

ECMO: a lifesaving modality in ARDS during puerperium

Acute respiratory distress syndrome (ARDS) is an uncommon condition encountered in pregnancy. The incidence of ARDS in pregnancy has been reported to be 1 in 6229 deliveries with mortality rates to range from 24% to 39% in pregnant patients. An essential component in management of ARDS involves good communication between the obstetrics team and critical care specialist and a fundamental understanding of mechanical ventilatory support. In critically ill patients where both cardiorespiratory support is required, Extracorporeal Membrane Oxygenation (ECMO) can be used to help maintain the vital functions. ECMO is a temporary cardio respiratory or respiratory support in critically ill patients who are unresponsive to conventional management.  In present case a young female with post-partum ARDS was successfully managed with extra corporeal membrane oxygenation (ECMO)

Sex differences in metabolic and adipose tissue responses to juvenile-onset obesity in sheep

Sex is a major factor determining adipose tissue distribution and the subsequent adverse effects of obesity-related disease including type 2 diabetes. The role of gender on juvenile obesity and the accompanying metabolic and inflammatory responses is not well established. Using an ovine model of juvenile onset obesity induced by reduced physical activity, we examined the effect of gender on metabolic, circulatory, and related inflammatory and energy-sensing profiles of the major adipose tissue depots. Despite a similar increase in fat mass with obesity between genders, males demonstrated a higher storage capacity of lipids within perirenal-abdominal adipocytes and exhibited raised insulin. In contrast, obese females became hypercortisolemic, a response that was positively correlated with central fat mass. Analysis of gene expression in perirenal-abdominal adipose tissue demonstrated the stimulation of inflammatory markers in males, but not females, with obesity. Obese females displayed increased expression of genes involved in the glucocorticoid axis and energy sensing in perirenal-abdominal, but not omental, adipose tissue, indicating a depot-specific mechanism that may be protective from the adverse effects of metabolic dysfunction and inflammation. In conclusion, young males are at a greater risk than females to the onset of comorbidities associated with juvenile-onset obesity. These sex-specific differences in cortisol and adipose tissue could explain the earlier onset of the metabolic-related diseases in males compared with females after obesity

Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells

AbstractExosomes have recently come into focus as "natural nanoparticles" for use as drug delivery vehicles. Our objective was to assess the feasibility of an exosome-based drug delivery platform for a potent chemotherapeutic agent, paclitaxel (PTX), to treat MDR cancer. Herein, we developed different methods of loading exosomes released by macrophages with PTX (exoPTX), and characterized their size, stability, drug release, and in vitro antitumor efficacy. Reformation of the exosomal membrane upon sonication resulted in high loading efficiency and sustained drug release. Importantly, incorporation of PTX into exosomes increased cytotoxicity more than 50 times in drug resistant MDCKMDR1 (Pgp+) cells. Next, our studies demonstrated a nearly complete co-localization of airway-delivered exosomes with cancer cells in a model of murine Lewis lung carcinoma pulmonary metastases, and a potent anticancer effect in this mouse model. We conclude that exoPTX holds significant potential for the delivery of various chemotherapeutics to treat drug resistant cancers.From the Clinical EditorExosomes are membrane-derived natural vesicles of ~40 - 200 nm size. They have been under extensive research as novel drug delivery vehicles. In this article, the authors developed exosome-based system to carry formulation of PTX and showed efficacy in the treatment of multi-drug resistant cancer cells. This novel system may be further developed to carry other chemotherapeutic agents in the future