In mouse embryonic fibroblasts, neither caspase-8 nor cellular FLICE-inhibitory protein (FLIP) is necessary for TNF to activate NF-κB, but caspase-8 is required for TNF to cause cell death, and induction of FLIP by NF-κB is required to prevent it.

Binding of TNF to TNF receptor-1 can give a pro-survival signal through activation of p65/RelA NF-κB, but also signals cell death. To determine the roles of FLICE-inhibitory protein (FLIP) and caspase-8 in TNF-induced activation of NF-κB and apoptosis, we used mouse embryonic fibroblasts derived from FLIP and caspase-8 gene-deleted mice, and treated them with TNF and a smac-mimetic compound that causes degradation of cellular inhibitor of apoptosis proteins (cIAPs). In cells treated with smac mimetic, TNF and Fas Ligand caused wild-type and FLIP(-/-) MEFs to die, whereas caspase-8(-/-) MEFs survived, indicating that caspase-8 is necessary for death of MEFs triggered by these ligands when IAPs are degraded. By contrast, neither caspase-8 nor FLIP was required for TNF to activate p65/RelA NF-κB, because IκB was degraded, p65 translocated to the nucleus, and an NF-κB reporter gene activated normally in caspase-8(-/-) or FLIP(-/-) MEFs. Reconstitution of FLIP(-/-) MEFs with the FLIP isoforms FLIP-L, FLIP-R, or FLIP-p43 protected these cells from dying when treated with TNF or FasL, whether or not cIAPs were depleted. These results show that in MEFs, caspase-8 is necessary for TNF- and FasL-induced death, and FLIP is needed to prevent it, but neither caspase-8 nor FLIP is required for TNF to activate NF-κB.

Formulation, and physical, in vitro and ex vivo evaluation of transdermal ibuprofen hydrogels containing turpentine oil as penetration enhancer.

The aim of the present study was to investigate the transdermal permeation enhancing capability of turpentine oil for ibuprofen from hydrogels. Ibuprofen 1% w/v hydrogels were developed with carboxypolymethylene with and without turpentine oil. Turpentine oil was incorporated in increasing concentrations, i.e. 0.5, 1, 1.5, 2, 2.5 and 3% of the total gel formulation, and its permeation enhancing effect was examined. Gels were examined physically for pH, viscosity, spreadability, extrudability, smoothness and appearance. To study the in vitro and ex vivo permeation potential of formulated gels, permeation studies were performed with a Franz diffusion cell using cellulose membrane and excised rabbit abdominal skin. Ibuprofen hydrogel with 3% turpentine oil showed a maximum flux of 10.87 mg/cm2/h across artificial skin and 17.26 mg/cm2/h across rabbit abdominal skin.

Formulation and evaluation of controlled release matrices of ketoprofen and influence of different co-excipients on the release mechanism.

The present work reports the study of different controlled release formulations of ketoprofen, which is a non-steroidal anti-inflammatory drug (NSAID) and like other NSAIDs requires large and frequent daily doses, resulting in severe side effects and non-compliance. To avoid these problems, controlled release matrices were developed using different grades of ethylcellulose polymer with a drug-polymer ratio of 10:3 by the direct compression method. The effect on drug release of partial replacement of lactose by different co-excipients, HPMC K100 M, starch and CMC, was also studied. The tablets were tested for their drug content, weight variation, friability, hardness, thickness and diameter, all these physical properties being within the USP range. The release profile of all formulations containing polymer and co-excipients was compared with a formulation developed without polymer and co-excipients. After a 24-hour release study, it was concluded that formulations containing different grades of ethylcellulose polymer showed prolonged release for 6-18 hours, but the formulation containing the polymer Ethocel standard FP 7 Premium without co-excipient showed controlled release for 24 hours. DSC and FT-IR studies were performed to investigate any incompatibility between drug, polymer and co-excipient but no interaction was found. Different kinetic models were used, such as first order equation, zero order equation, Higuachi equation, Hixon Crowel's equation and Korsmeyer-Peppas to study the release mechanism. The formulations containing co-excipients showed an enhanced release rate.

Screening for gestational diabetes.

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HBsAg ELISA: comparison of serum and uncentrifuged plasma samples dried on filter paper.

To compare the serum and uncentrifuged plasma for hepatitis B surface antigen (HBsAg) enzyme linked immuno sorbent assay (ELISA), ninety samples were analysed. The study was conducted so that in the field, where no amenities are available especially in countries like Pakistan, the separated but uncentrifuged plasma may be preserved by spotting on the filter paper for sero-epidemiological studies later. It was found that there was no statistically significant difference between the serum and uncentrifuged plasma values.

Outpatient transesophageal echocardiography with intravenous propofol anesthesia in children and adolescents.

Outpatient transesophageal echocardiography (TEE) was performed in 10 children and adolescents (aged 3 to 19.5 years, mean 13.5 years; weight 12 to 91 kg, mean 49 kg), including two with Down's syndrome and one with autism, for diagnostic evaluation of issues unresolved by transthoracic echo examination (TTE). Issues for TEE: evaluation for atrial septal defect (two patients); anatomy of left ventricular outflow tract obstruction (one patient); aortic valve anatomy before valvuloplasty for insufficiency (one patient); evaluation for cause of cyanosis after Fontan operation (one patient); determination of source of high-velocity intracardiac turbulence after atrioventricular septal defect repair (one patient); rule out cardiac embolic source in patient with stroke (one patient); evaluate prosthetic valve function and rule out thrombus (one patient); determination of anatomic relationship of mitral valve to a ventricular septal defect before surgery for complex cyanotic heart disease (one patient); and evaluation for aortic dissection in Marfan's syndrome (one patient). Intravenous propofol anesthesia administered without endotracheal intubation by an anesthesiologist allowed successful outpatient TEE in nine patients; midazolam-conscious sedation was used in one. Outpatient TEE resolved diagnostic issues in all patients without complication, thereby avoiding cardiac catheterization in six patients and supplementing catheterization for preoperative planning in four patients. TEE can be performed safely and effectively with propofol anesthesia in the outpatient setting in carefully selected children and adolescents to provide vital diagnostic information. However, given the invasive nature of the procedure and the use of anesthesia, outpatient pediatric TEE should be used judiciously.

Erythroid burst formation in cultures of bone marrow and peripheral blood from patients with refractory anemia.

We have studied the growth of erythroid bursts in methylcellulose cultures from bone marrow (7 cases) and peripheral blood cells (17 cases) of patients with primary acquired refractory anemias. In most cases, erythroid burst-forming units (BFU-E) were either absent or present in lower than normal numbers, and these levels remained low to absent in sequential studies. In 2 patients, circulating BFU-E were initially higher than normal, but subsequently declined to normal levels. 1 patient had low-normal numbers of circulating BFU-E initially, but these declined to zero in a later study. No clinical or routine hematological features permitted distinction between patients with initially high BFU-E and those with initially low or absent BFU-E. These findings parallel those reported for granulocyte-macrophage progenitors in refractory anemias, and support the concept that erythroid progenitor cells in these disorders are influenced by the disordered hematopoiesis.

A rapid technique for measuring calcium uptake in mitogen-induced T and B lymphocytes.

The procedures for lymphocyte activation and for removing the cells from the radioactive loading solution in incubation medium were modified to routinely obtain significant and reproducible 45Ca2+ uptakes in mitogen-induced mouse T and B lymphocytes. Factors such as mouse strain, lymphocyte origin, and media pH were not critical to the 45Ca2+ uptake measurements. In contrast, factors such as lymphocyte cell concentration during mitogenic activation, filtering the 45Ca2+:3H2O mixtures, and the nature and purity of the B-cell mitogens were critical for obtaining maximal and reproducible 45Ca2+ uptakes. Centrifugation through silicone oil into sucrose was an efficient and rapid procedure for separating the cells from the radioactive loading solution in the incubation medium. Using optimal conditions, an approximate twofold increase in 45Ca2+ uptake (representing an influx of approximately 97 amol per lymphocyte and an increase in average cellular Ca2+ of approximately 0.72 mM) was routinely obtained with purified mouse lymphocytes activated with a variety of T- and B-cell mitogens (using concentrations resulting in maximal [3H]thymidine incorporation). A larger 45Ca2+ uptake was routinely obtained with mitogenic concentrations of A23187, a divalent cation ionophore stimulating T cells. Experiments employing [14C]sucrose and [14C]inulin with control and mitogen-induced lymphocytes showed that the trapped extracellular fluid measurements in the cell pellets should be used to correct the magnitude of the 45Ca2+ uptake measurements.

The PO protein. The major glycoprotein of peripheral nerve myelin.

A glycoprotein, referred to as PO protein, was isolated from rabbit sciatic nerve myelin by gel filtration on Agarose 0.5 m in dodecyl sulfate. The purified myelin was first defatted and extracted at pH 2. The water-soluble proteins such as myelin basic protein and P2 protein were extracted leaving a glycoprotein-rich residue, from which the PO protein was isolated. The purified protein showed a single band on gel electrophoresis in dodecyl sulfate when stained with Coomassie Blue of periodic acid-Schiff reagent. The carbohydrate, comprising 6.3% by weight, appears to exist as a nonasaccharide unit having 3 mannose, 3 N-acetylglucosamine, 1 sialic acid, 1 galactose and 1 fucose residue. The polypeptide moiety has a high content of non-polar amino acids. A single amino acid, isoleucine, was found at the NH2-terminal end by dansyl and Edman procedures. The PO protein is the major protein of peripheral nerve myelin.