Weekly vs. tri-weekly cisplatin based chemoradiation in carcinoma cervix: a prospective randomized study of toxicity and compliance

BackgroundAddition of chemotherapy to radiation has improved 5-year survival by 6%. However, the optimal dose and schedule of concurrent cisplatin is not well defined, though widely accepted practice is the weekly schedule of 40 mg/m² for 5 weeks. Repeated admissions for weekly cisplatin drain the limited resources in high volume centres. We intended to study the compliance and toxicity of two cisplatin schedules in our patients diagnosed with carcinoma cervix.Materials and methodsBetween 2007–2011, 212 patients, histologically proven squamous cell carcinoma with stages IIB to IIIB were randomized into two arms. All patients were planned for external beam radiotherapy 45 Gy/25 frs over 5 weeks followed by Intracavitary or Interstitial brachytherapy to a total BED dose of 75–85 Gy. Single agent cisplatin given concomitantly, was scheduled weekly (40 mg/m²/cycle, 5 cycles) in an arm A and three weekly (100 mg/m²/cycle, 2 cycles) in an arm B. Toxicity and compliance were evaluated weekly according to the RTOG guidelines. Analysis of the compiled data was done using SSPS version 20.ResultsOf the evaluable 212, 109 patients received weekly cisplatin chemotherapy and 103 patients received three weekly cisplatin. The most common acute toxicity observed was grade I–II leucopoenia. The upper and lower gastrointestinal reactions were high in three weekly arms, which was statistically significant (57% and 42.7%, p < 0.05). Proctitis was observed in 10% of patients in both of the arms and only two patients had Gr1 Cystitis after 6 months of treatment.ConclusionsTri-weekly cisplatin based concurrent chemoradiation can be adopted in high volume centres with manageable haematological and gastrointestinal acute toxicities.     

ACTH-induced hydrolysis of cholesteryl esters in rat adrenal cells.

Rat adrenal cortical cells have been prepared by collagenase dissociation of trypsin-treated adrenal tissue. The content and compositions of cholesteryl ester, phospholipid, and triglyceride fatty acids compare favorably with those of undissociated rat adrenal tissue. During 2-hour control incubations of adrenal cortical cells, steroidogenesis was not detected, and the levels of sterol ester, phospholipid, and triglyceride fatty acids were not significantly altered. Incubations with adrenocorticotropic hormone (ACTH) resulted in coricosterone production and significant depletions of sterol ester and triglyceride fatty acids, but not of phospholipid fatty acids. Although all fatty acid esters of cholesterol were hydrolyzed under these conditions, the greatest contributions to the net decrease in sterol esters were by oleate, arachidonate, and adrenate. Incubations with dibutyryl cyclic adenosine monophosphate (0.5 mM) resulted in significantly greater levels of corticosterone production than did ACTH (250 muunits), but the effects on cellular lipids were comparable to those seen with the tropic hormone. This study represents the first demonstration of hormone-induced hydrolysis of sterol esters in an in vitro cell suspension system. The results are discussed with respect to hormone-sensitive sterol ester hydrolase of adrenal cortex, and to the role of endogenous cholesteryl esters in the steroidogenic pathway.     

Immunological comparison of cholesterol esterases.

Rat pancreas cholesterol esterase has been immunologically compared with rat intestinal cholesterol esterase. Monospecific precipitating antisera against purified rat pancreas cholesterol esterase were produced in rabbits. Immune IgG, isolated from the antisera, crossreacted with the cholesterol esterase of intestine in the immunodiffusion assay with a pattern of complete identity. Titration of the pancreatic and intestinal enzyme with immune IgG revealed a maximum precipitation (99 and 98%) and maximum inhibition of enzyme activity (66 and 65%) when the ratio of enzyme activity (units) to immune IgG (mg) was 4.1 and 4.0, respectively. The immunological identity demonstrated in these studies lend support to the concept that intestinal cholesterol esterase is derived from the pancreatic enzyme. In additional studies, the immune IgG was employed in the immunodiffusion assay to test for cross-reaction with cholesterol esterases prepared from rat aorta, adrenal, and liver and with cholesterol esterases prepared from the pancreas of rabbit, dog, cow, and guinea pig. There was no evidence of cross-reaction in any case. Further, cholesterol esterase prepared from the pancreas of rabbit, dog, and cow retained full enzymatic activity when titrated with immune IgG.     

Sterol carrier protein2 (SCP2)-mediated transfer of cholesterol to mitochondrial inner membranes

The fluorescence parameters of demetallized alpha-lactalbumin in the range from pH 8 to 2 show an extreme around pH 5-4 (a minimum in quantum yield and wavelength and a maximum in polarization). This extreme is not due to a competition between Ca2+ and protons but rather to a stabilization of the conformation of the protein near the isoelectric pH by the ionic interactions between local positive and negative charges on the protein. The calcium-free protein has similar fluorescence characteristics at pH 2 and 8 but the thermal transition curve is different. The influence of 0.1 M NaCl is also considered.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Sterol carrier and lipid transfer proteins

The discovery of the sterol carrier and lipid transfer proteins was largely a result of the findings that cells contained cytosolic factors which were required either for the microsomal synthesis of cholesterol or which could accelerate the transfer or exchange of phospholipids between membrane preparations. There are two sterol carrier proteins present in rat liver cytosol. Sterol carrier protein 1 (SCP1) (Mr 47 000) participates in the microsomal conversion of squalene to lanosterol, and sterol carrier protein 2 (SCP2) (Mr 13 500) participates in the microsomal conversion of lanosterol to cholesterol. In addition SCP2 also markedly stimulates the esterification of cholesterol by rat liver microsomes, as well as the conversion of cholesterol to 7 alpha-hydroxycholesterol - the major regulatory step in bile acid formation. Also, SCP2 is required for the intracellular transfer of cholesterol from adrenal cytoplasmic lipid inclusion droplets to mitochondria for steroid hormone production, as well as cholesterol transfer from the outer to the inner mitochondrial membrane. SCP2 is identical to the non-specific phospholipid exchange protein. While SCP2 is capable of phospholipid exchange between artificial donors/acceptors, e.g. liposomes and microsomes, it does not enhance the release of lipids other than unesterified cholesterol from natural donors/acceptors, e.g. adrenal lipid inclusion droplets, and will not enhance exchange of labeled phosphatidylcholine between lipid droplets and mitochondria. Careful comparison of SCP2 and fatty acid binding protein (FABP) using six different assay procedures demonstrates separate and distinct physiological functions for each protein, with SCP2 participating in reactions involving sterols and FABP participating in reactions involving fatty acid binding and/or transport. Furthermore, there is no overlap in substrate specificities, i.e. FABP does not possess sterol carrier protein activity and SCP2 does not specifically bind or transport fatty acid. The results described in the present review support the concept that intracellular lipid transfer is a highly specific process, far more substrate-specific than suggested by the earlier studies conducted using liposomal techniques.     

Phospholipids, sterol carrier protein2 and adrenal steroidogenesis

Rat adrenocortical cells and preparations of plasma membrane and mitochondria have been employed to assess the effects of phospholipids and of sterol carrier protein2 (SCP2) on specific aspects of adrenal steroidogenesis. With intact cells, liposomal dispersions of cardiolipin caused significant stimulation of corticosterone output, while preparations of phosphatidylcholine, phosphatidylinositol, or the 4'-phosphate and the 4',5'-diphosphate derivatives of phosphatidylinositol were without effect. With the adrenal plasma membrane preparation, none of the added phospholipids affected either sodium fluoride or ACTH-responsive adenylate cyclase activity. With intact mitochondria, only cardiolipin, among the various phospholipids, tested, caused a concentration-dependent stimulation of pregnenolone production. However, even at the highest concentration of cardiolipin tested (500 microM), the stimulatory effect was only half that observed with 0.7 microM SCP2, and the two effectors were not synergistic. SCP2 caused a redistribution of cholesterol from mitochondrial outer to inner membranes, while cardiolipin, which is an activator of cytochrome P-450scc, had no effect on distribution of mitochondrial membrane cholesterol.     

Normal cholesterol absorption in rats deficient in intestinal acyl coenzyme A:cholesterol acyltransferase activity

Acyl coenzyme A:cholesterol acyl transferase and/or cholesterol esterase may regulate the esterification and absorption of exogenous cholesterol. To assess this, mucosal acyl coenzyme A:cholesterol acyl transferase activity was inhibited selectively with three different drugs [Sandoz #58-035, inhibitor 1; Lederle inhibitor 2 and inhibitor 3] and the effect upon the absorption of a [4-14C]cholesterol meal was studied in the lymph fistula rat. Compared to control rats, ACAT activity measured in mucosal homogenates from the drug-treated rats was reduced 80-90%, 40%, and 30%, respectively, during the predicted time-frame for maximum mucosal esterification of cholesterol (i.e., after cholesterol is fed and before it appears in lymph). In contrast, [14C]cholesterol absorption in the drug-treated animals was unchanged from controls [5.7 +/- 1.2 (inhibitor 1) vs. 5.4 +/- 1.6 mumol/6 hr (control); 6.1 +/- 2.1 (inhibitor 2) and 5.2 +/- 1.5 (inhibitor 3) vs. 4.1 +/- 1.3 mumol/6 hr (control)]. Of the absorbed [14C]cholesterol, approximately 75% was esterified in all groups. Cholesterol esterase activity measured in the drug-treated rats was unchanged compared to controls nor did the drugs inhibit this enzyme in vitro. Under the conditions of this study, drugs causing substantial inhibition of acyl coenzyme A:cholesterol acyl transferase activity had no effect on the absorption of exogenous cholesterol.     

Cholesterol arachidonate as a prostaglandin precursor in adrenocortical cells

Rat adrenocortical cells were incubated with labeled arachidonate, and the radioactivity in unesterified fatty acids was reduced by washing with 2% albumin solutions. These cells were then incubated for two hours in the absence and presence of 7.1 × 10⁻¹⁰M ACTH. During subsequent incubation of prelabeled cells with ACTH, both the mass and radioactivity of arachidonate in adrenocortical cholesteryl esters was depleted to the same extent (30–32%). The released arachidonate was in part incorporated into phospholipids, and there was also a significant increase in unesterified arachidonic acid. During this period, there was also increased incorporation of arachidonate into labeled prostaglandins. Of this increase, 92% by isotope analysis, and 88% by radioimmunoassay techniques was attributable to prostaglandins of the E pathway. These data demonstrate that prostaglandin E synthesis is specifically increased during ACTH stimulation of rat adrenocortical cells and suggest that a major source of the arachidonate substrate for this synthesis is derived from hormone-dependent hydrolysis of cortical cholesteryl esters.