A bispecific diabody directed against prostate-specific membrane antigen and CD3 induces T-cell mediated lysis of prostate cancer cells

Background Although cancer of the prostate is one of the most commonly diagnosed cancers in men, no curative treatment currently exists after its progression beyond resectable boundaries. Therefore, new agents for targeted treatment strategies are needed. Cross-linking of tumor antigens with T-cell associated antigens by bispecific monoclonal antibodies have been shown to increase antigen-specific cytotoxicity in T-cells. Since the prostate-specific membrane antigen (PSMA) represents an excellent tumor target, immunotherapy with bispecific diabodies could be a promising novel treatment option for prostate cancer. Methods A heterodimeric diabody specific for human PSMA and the T-cell antigen CD3 was constructed from the DNA of anti-CD3 and anti-PSMA single chain Fv fragments (scFv). It was expressed in E. coli using a vector containing a bicistronic operon for co-secretion of the hybrid scFv V_HCD3-V_LPSMA and V_HPSMA-V_LCD3. The resulting PSMAxCD3 diabody was purified from the periplasmic extract by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST) was used. For in vivo evaluation the diabody was applied together with human peripheral blood lymphocytes (PBL) in a C4-2 xenograft-SCID mouse model. Results By Blue Native gel electrophoresis, it could be shown that the PSMAxCD3 diabody is mainly a tetramer. Specific binding both to CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the diabody proved to be a potent agent for retargeting PBL to lyze C4-2 prostate cancer cells. Treatment of SCID mice inoculated with C4-2 tumor xenografts with the diabody and PBL efficiently inhibited tumor growth. Conclusions The PSMAxCD3 diabody bears the potential for facilitating immunotherapy of prostate cancer and for the elimination of minimal residual disease. P. Bühler and P. Wolf equally contributed to the work.     

Decreased glutamine synthetase,increased citrulline–nitric oxide cycle activities,and oxidative stress in different regions of brain in epilepsy rat model

To understand their role in epilepsy, the nitric oxide synthetase (NOS), argininosuccinate synthetase (AS), argininosuccinate lyase (AL), glutamine synthetase (GS), and arginase activities, along with the concentration of nitrate/nitrite (NOx), thiobarbituric acid reactive substances (TBARS), and total antioxidant status (TAS), were estimated in different regions of brain in rats subjected to experimental epilepsy induced by subcutaneous administration of kainic acid (KA). The short-term (acute) group animals were killed after 2 h and the long term (chronic) group animals were killed after 5 days of single injection of KA (15 mg/kg body weight). After decapitation of rats, the brain regions were separated and in their homogenates, the concentration of NOx, TBARS and TAS and the activities of NOS, AS, AL, arginase and glutamine synthetase were assayed by colorimetric methods. The results of the study demonstrated the increased activity of NOS and formation of NO in acute and chronic groups epilepsy. The activities of AS and AL were increased and indicate the effective recycling of citrulline to arginine. The activity of glutamine synthetase was decreased in acute and chronic groups of epilepsy compared to control group and indicate the modulation of its activity by NO in epilepsy. The activity of arginase was not changed in acute group; however it was decreased in chronic group and may favor increased production of NO in this condition. The concentration TBARS were increased and TAS decreased in acute and chronic groups of epilepsy and supports the oxidative stress in epilepsy.     

Efficacy and deficiencies of rapid biomonitoring in biodiversity conservation: a case study in South Africa

Rapid biomonitoring protocols, using biotic indices based on macroinvertebrate diversity to assess river ecosystem health, are widely used globally. Such quick assessment techniques are lauded for the rapid results obtained and the relatively easy protocol used to achieve an answer. However, do such quick assessments of water quality give enough information about ecosystems? Are important details being overlooked? When should a full faunal survey be used in preference? Important research programmes, including environmental impact studies, often misuse biomonitoring techniques, making influential management decisions using superficial, low-level data obtained using biomonitoring tools, inappropriate to address those management objectives. The value of using biomonitoring as a quick tool, versus a more detailed faunal assessment, is considered here. The assessment of teloganodid mayfly fauna occurring in South African rivers provides an example of the value of detailed studies versus superficial family level investigations, showing that a rapid biomonitoring approach should not be used as a shortcut when a more detailed survey is needed. Each situation should be assessed for its own merit in a given set of project circumstances. A checklist of criteria is presented, giving guidance on when rapid biomonitoring alone is valuable and when more detailed assessments would give a more relevant result.     

Interaction of N-myristoyldimyristoylphosphatidylethanolamine with dimyristoylphosphatidylcholine investigated by differential scanning calorimetry: binary phase diagram

The temperature-composition phase diagram was derived for hydrated, binary mixtures of N-myristoyldimyristoylphosphatidylethanolamine (N-14 DMPE) and dimyristoylphosphatidylcholine by high sensitivity differential scanning calorimetry. Gel phase immiscibility was detected in mixtures containing up to 20 mol% N-14 DMPE and there was no evidence for compound formation between the two components. In the fluid phase nearly complete miscibility is indicated by the calorimetric data. These results are relevant to understanding the role of N-acylphosphatidylethanolamines in the stress combating responses of organisms and in their application to developing liposome-based drug delivery systems.     

Computational identification of putative miRNAs and their target genes in pathogenic amoeba Naegleria fowleri

Naegleria fowleri is a parasitic unicellular free living eukaryotic amoeba. The parasite spreads through contaminated water andcauses primary amoebic meningoencephalitis (PAM). Therefore, it is of interest to understand its molecular pathogenesis. Hence,we analyzed the parasite genome for miRNAs (microRNAs) that are non-coding, single stranded RNA molecules. We identified245 miRNAs using computational methods in N. fowleri, of which five miRNAs are conserved. The predicted miRNA targets wereanalyzed by using miRanda (software) and further studied the functions by subsequently annotating using AmiGo (a geneontology web tool).     

The Impact of Dyspepsia on Symptom Severity and Quality of Life in Adults with Headache

Background

Dyspepsia and headache frequently co-exist, but the clinical implication of this association is uncertain. We planned to examine the prevalence and impact of dyspepsia in adults with headache.

Methods

A cross-sectional study was conducted in a secondary care setting. Clinical, psychological and health-related quality of life (HRQOL) data were compared between subjects with headache and controls (non-headache subjects). The impact of dyspepsia was analysed further in subjects with headache alone.

Results

280 subjects (93 cases with headache and 187 matched controls) were recruited. The following baseline characteristics of subjects were as follows: mean age 45.0±17.3 years, 57.0% females and ethnic distribution—Malaysian = 45 (48.4%), Chinese n = 24 (25.8%) and Indians n = 24 (25.8%). Headache sub-types among cases with headache were as follows: tension-type headache (TTH) n = 53 (57.0%) and migraine n = 40 (43.0%). Dyspepsia was more prevalent in cases with headache compared to controls (25.8% vs 12.8%, p = 0.011), and headache was independently associated with dyspepsia (OR 2.75, 95% CI 1.39–5.43). Among cases with headache, there was a trend towards a higher prevalence of dyspepsia in those with migraine (27.5%) compared to TTH (24.5%). Subjects with headache and dyspepsia, compared to those with headache alone, had a greater severity of headache symptoms (63.67±22.85 mm vs 51.20 ±24.0 mm VAS, p = 0.029). Overall HRQOL scores were lower in headache subjects with dyspepsia (EQ-5D summary score 0.82±0.18 vs 0.90 ±0.16, p = 0.037 and EQ-5D VAS 62.08±17.50 mm vs 72.62 ±18.85 mm, p = 0.018), compared to those without dyspepsia.

Conclusion

Dyspepsia is associated with more severe headache symptoms and results in a lower HRQOL in patients with headache.     

Discovery of matrix metalloproteases selective and activated peptide–doxorubicin prodrugs as anti-tumor agents

To selectively target doxorubicin (Dox) to tumor tissue and thereby improve the therapeutic index and/or efficacy of Dox, matrix metalloproteinases (MMP) activated peptide–Dox prodrugs were designed and synthesized by coupling MMP-cleavable peptides to Dox. Preferred conjugates were good substrates for MMPs, poor substrates for neprilysin, an off-target proteinase, and stable in blood ex vivo. When administered to mice with HT1080 xenografts, conjugates, such as 19, preferentially released Dox in tumor relative to heart tissue and prevented tumor growth with less marrow toxicity than Dox.     

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization

Two carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CM-cellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS–PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS–PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K_m of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K_m was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45 °C (ME-III) and 37 °C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35 °C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB).