Protective cytotoxic T lymphocyte responses against paramyxoviruses induced by epitope-based DNA vaccines: involvement of IFN-gamma

Plasmid DNA vectors have been constructed with minigenes encoding a single cytotoxic T lymphocyte (CTL) epitope from either the M2 protein of respiratory syncytial virus (RSV) or from the nucleoprotein of measles virus (MV) with or without a signal sequence (also called secretory or leader sequence). Following intradermal immunization, plasmids in which the CTL epitopes were expressed in-frame with the signal sequence were more effective at inducing peptide- and virus- specific CTL responses than plasmids expressing CTL epitopes without the signal sequence. This immunization resulted in protection against MV-induced encephalitis and a significant reduction in viral load following RSV challenge. The reduction of viral load following RSV challenge was abrogated by prior injection with anti-IFN-gamma antibodies. These results highlight the ability of epitope-based DNA immunization to induce protective immune responses to well-defined epitopes and indicate the potential of this approach for the development of vaccines against infectious diseases.      

Effect of spark plasma sintering on the microstructure and in vitro behavior of plasma sprayed HA coatings

The crystalline phases and degree of crystallinity in plasma sprayed calcium phosphate coatings on Ti substrates are crucial factors that influence the biological interactions of the materials in vivo. In this study, plasma sprayed hydroxyapatite (HA) coatings underwent post-spray treatment by the spark plasma sintering (SPS) technique at 500 degrees C, 600 degrees C, and 700 degrees C for duration of 5 and 30 min. The activity of the HA coatings before and after SPS are evaluated in vitro in a simulated body fluid. The surface microstructure, crystallinity, and phase composition of each coating is characterized by scanning electron microscopy and X-ray diffractometry before, and after in vitro incubation. Results show that the plasma sprayed coatings treated for 5 min in SPS demonstrated increased proportion of beta-TCP phase with a preferred-orientation in the (214) plane, and the content of beta-TCP phase corresponded to SPS temperature, up to 700 degrees C. SPS treatment at 700 degrees C for 30 min enhanced the HA content in the plasma spray coating as well. The HA coatings treated in SPS for 5 min revealed rapid surface morphological changes during in vitro incubation (up to 12 days), indicating that the surface activity is enhanced by the SPS treatment. The thickest apatite layer was found in the coating treated by SPS at 700 degrees C for 5 min.     

In vitro studies of plasma-sprayed hydroxyapatite/Ti-6Al-4V composite coatings in simulated body fluid (SBF)

The bioactivity of plasma-sprayed hydroxyapatite (HA)/Ti-6Al-4V composite coatings was studied by soaking the coatings in simulated body fluid (SBF) for up to 8 weeks. This investigation was aimed at elucidating the biological behaviour of plasma-sprayed HA/Ti-6Al-4V composite coatings by analyzing the changes in chemistry, and crystallinity of the composite coating in a body-analogous solution. Phase composition, microstructure and calcium ion concentration were analyzed before, and after immersion. The mechanical properties, such as tensile bond strength, microhardness and Young's modulus were appropriately measured. Results demonstrated that the tensile bond strength of the composite coating was significantly higher than that of pure HA coatings even after soaking in the SBF solution over an 8-weeks period. Dissolution of Ca-P phases in SBF was evident after 24h of soaking, and, a layer of carbonate-apatite covered the coating surface after 2 weeks of immersion. The mechanical properties were found to diminish with soaking duration. However, slight variation in mechanical properties was found after supersaturation of the calcium ions was attained with the precipitation of the calcium phosphate layers.     

Paramagnetic macrocyclic complexes as contrast agents for MR imaging: proton nuclear relaxation rate enhancement in aqueous solution and in rat tissues

Paramagnetic macrocyclic chelates show promise as magnetic resonance (MR) imaging contrast agents due to stability and relaxivity comparable to those of DTPA-type chelates. For the three copper and manganese macrocyclic complexes studied in aqueous solution, T1 and T2 relaxivities ranged from 0.14 to 5.88 mM-1sec-1 at 6.25 MHz. In rats, the intravenous administration of 16 mumol/kg of Mn(cyclam) caused the liver T1 relaxation rate to double at 15 minutes after injection. T1 measurements by pulsed MR imaging and manganese analyses on excised tissue showed that both relaxation rate (1/T1) and manganese content of liver and kidney increase linearly with the dosage of Mn(cyclam). The linear relationship between 1/T1 and manganese content can be considered an "in tissue" relaxivity plot for the agent. The resulting relaxivity is 54 mM-1sec-1 in liver, compared with 3.1 mM-1sec-1 in aqueous solution. Although this work is preliminary, the implication for medical MR imaging applications is that macrocyclic contrast agents can be effective at approximately one-tenth the current typical dose used for gadolinium DTPA.     

Simultaneous determination of retinol and α-tocopherol by high pressure liquid chromatography in micro-volumes of serum

The lack of biochemical data to indicate the prevalence of marginal vitamin A deficiency (VAD) in the country is largely because of the difficulty of obtaining enough serum for analysis, especially from malnourished children. Efforts were therefore made to establish a high-pressure liquid chromatography system (HPLC) for the determination of retinol in micro-volumes of serum. Since our previous studies showed that tocopherol could be simultaneously determined in the same system, studies have been carried out for the determination of both vitamins using retinol acetate and -tocopherol acetate as internal standards. Trials were carried out to determine the most suitable sample treatment procedures and chromatographic system including composition of the mobile phase for handling 20 ml of serum. The HPLC system proposed enables successful separation and quantitation of retinol and α-tocopherol and their respective internal standards, retinol acetate and -tocopherol in less than 14 minutes. Reproducibility studies carried out with pooled sera showed a within day and between day variation of less than 8% and 13% respectively for retinol, whilst variations for α-tocopherol were higher, ranging from 8-16%. The proposed method is currently being applied to the determination of retinol and α-tocopherol in a group of malnourished children under six years of age.     

Simultaneous determination of B-vitamins and ascorbic acid in multi-vitamin preparations by reversed-phase HPLC

The tedious and time consuming methods employed for the analysis of individual B-vitamins can now be replaced by ion-pair reversed-phase high-performance liquid chromatographic (HPLC) methods. This laboratory has previously reported the simultaneous determination of eight water-soluble vitamin standards that is, B1, B2, B6, B12, C, niacin, niacinamide and folic acid. The proposed isocratic HPLC method, employing 3 channels of detection, adequately separated all eight vitamins in less than 20 minutes. This study reports another phase of the project whereby the method was employed for the analysis of pharmaceutical preparations. Different extraction procedures were first evaluated, namely acid, acid plus enzyme and alkaline hydrolysis methods, using vitamin standards, individual vitamin tablets and multivitamin preparations. The amounts obtained from the analysis were compared with the declared values. Recovery studies were also carried out. The method of acid hydrolysis with 0.1N sulphuric acid was found suitable for use and was thus adopted as the extraction procedure for the analysis of 10 multivitamin preparations obtained from various pharmaceutical outlets. For most of these preparations, the amount obtained were close to the declared values, except for folic acid and cyanocobalamin. Further trials on folic acid showed that the problem could be resolved by omitting the filtration step in the final extract after acid hydrolysis and diluting with 0.01N sodium hydroxide before processing for chromatography. Vitamin B12 was not detectable using the present chromatography system probably because of its low concentration in the samples studied.     

Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC- E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.      

STRESS FRACTURES: EFFECT OF PRIOR PHYSICAL ACTIVITY,SPORTS PARTICIPATION AND MILITARY TRAINING

A study was carried out to find out the effects of prior physical activity, sports participation and prior military training on the incidence of stress fractures among Gentlemen Cadets (GC''s) undergoing military training at Indian Military Academy (IMA). One thousand and fourteen GC''s were followed up for a period of 12 weeks. Thirty-seven GC''s developed stress fractures during the study period. The incidence of stress fractures was significantly higher in GC''s without any prior military training (p=0.0009). They were compared with 100 healthy controls drawn from the study population to study the influence of the other mentioned factors. There was no significant association between prior physical activity and stress fractures (OR=0.74, 95% CL=0.26 to 2.05, p=0.688). There was also no significant relationship between sports participation and stress fractures (OR=0.79. 95% CCL=0.35 to 1.81, p=0.684).KEY WORDS: Risk factors, Stress fractures