Over-expression of gene encoding heat shock protein 70 from Mycobacterium tuberculosis and its evaluation as vaccine adjuvant.

BACKGROUND: Heat shock proteins (Hsps) are evolutionary ancient and highly conserved molecular chaperons found in prokaryotes as well as eukaryotes. Hsp70 is a predominant member of Hsp family. Microbial Hsp70s (mHsp70s) have acquired special significance in immunity since they have been shown to be potent activators of the innate immune system and generate specific immune responses against tumours and infectious agents. OBJECTIVES: The present study was aimed to clone express and purify recombinant Hsp70 from the Mycobacterium tuberculosis and characterise it immunologically. The study also aimed at determining the potential of recombinant M. tuberculosis heat shock protein (rMTB-Hsp70) as adjuvant or antigen carrier. MATERIALS AND METHODS: Cloning of M. tuberculosis heat shock protein (MTB-Hsp70) amplicon was carried out using the pGEMT-Easy vector although for expression, pProExHTb prokaryotic expression vector was used. Purification of recombinant Hsp70 was carried out by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. For immunological characterization and determining the adjuvant effect of MTB-Hsp70, BALB/c mice were used. The data obtained was statistically analysed. RESULTS: Hsp70 gene was cloned, sequenced and the sequence data were submitted to National Center for Biotechnology Information (NCBI). Recombinant MTB-Hsp70 was successfully over-expressed using the prokaryotic expression system and purified to homogeneity. The protein was found to be immunodominant. Significant adjuvant effect was produced by the rMTB-Hsp70 when inoculated with recombinant outer membrane protein 31; however, effect was less than the conventionally used the Freund's adjuvant. CONCLUSION: Protocol standardised can be followed for bulk production of rHsp70 in a cost-effective manner. Significant adjuvant effect was produced by rMTB-Hsp70; however, the effect was than Freund's adjuvant. Further, studies need to be carried out to explore its applicability as carrier of antigen.

Serum prostate-specific antigen: hourly change/24 hours compared with prostatic acid phosphatase.

Simultaneous hourly changes in serum PSA and PAP levels per twenty-four-hour period was studied in 19 urologic patients with no prostatic disease. Serum PSA was determined using Hybritech PSA-R Kits, and PAP was determined using EIA method. PSA and PAP serum levels varied independently from each other. PSA level showed no diurnal variations. The level remained stable with minor variations around a low mean of 0.907 ng/mL, SD 0.09, and CV 9.9% in 16 of 19 (84%) patients, while PAP showed changes consistent with diurnal variations in 5 of those patients. Random variations with no discernible pattern in 7 and remained more or less constant in 4 other patients. Marked random variations in PSA serum level occurred in only 3 patients in the older age group and were accompanied by diurnal variations in PAP level in 2 patients and a constant high level of PAP in 1 patient. The highest PSA level obtained was 6.9 ng/mL which is important when selecting the cut-off level. PSA did not increase appreciably after prostatic massage, and on follow-up PSA returned to premassage level after twenty-four hours (except in 1 patient).

Diurnal variation of prostatic acid phosphatase (PAP) in urologic patients with nonprostatic disease.

Variation in serum prostatic acid phosphatase (PAP) level over a twenty-four-hour period was studied in 18 urologic patients with no prostatic disease. Hourly, twenty-four-hour serum PAP level was determined using Elisa method. Seven of 18 patients (38.8%) showed appreciable change with definite diurnal pattern. PAP level was highest in early AM hours and lowest in the PM period. The difference between the mean AM and PM periods was statistically significant (P less than 0.01). Four of 18 patients showed no change over the twenty-four hours while 7 patients showed some variation without definite diurnal pattern. Such changes may lead to variation in serum PAP level and may lead to false positive results.

Management of vesicouterine fistula following cesarean section.

Contrary to belief, urinary incontinence in vesicouterine fistulas following cesarean section is as common as cyclic hematuria with apparent amenorrhea. Review of the English literature revealed 21 cases and herein is added a new case. The presentations and management of such cases are discussed.

Diuretic renography and urodynamic pressure studies in evaluating dilated bilharzial ureters. A preliminary report.

A prospective trial was done in 30 ureters in 21 patients using perfusion pressure flow studies (PPFS) and diuretic renography with washout technique (DRWT) in dilated bilharzial ureters in order to diagnose the presence or absence of obstruction and to compare the results. PPFS could not be done in 11 ureters due to technical problems. Three categories of results were obtained: 1) no obstruction could be demonstrated by either PPFS or diuretic renography (11 ureters), 2) obstruction was confirmed by both methods (three ureters), and 3) obstruction was shown by DRWT but could not be proven by PPFS (five ureters). DRWT, which is a noninvasive method, gave the correct diagnosis in the majority of cases and should be the first method used to evaluate dilated bilharzial ureters. In cases where DRWT shows no obstruction, further evaluation is unnecessary, as PPFS did not show obstruction in these cases either. But in cases where obstruction is shown by DRWT, PPFS should be tried to confirm the obstruction before surgery is performed.

Surgical intervention in dilated bilharzial ureters.

Surgery has often been performed for presumed obstruction in dilated bilharzial ureters but with unrewarding results. In this report 55 non-refluxing dilated bilharzial ureters were studied. Perfusion pressure flow studies and diuresis renography with washout technique were used to diagnose the presence or absence of obstruction. Surgery was performed only when obstruction was proven by PPFS and diuresis renography; thus patients were spared an operation when the obstruction could not be confirmed. Antegrade ureteropyelographic findings, evaluation of peristalsis and transport of urine were the basis for the selection of operation performed. The various surgical procedures are discussed.

Some gross observations of the epididymides following vasectomy: a clinical study.

In 114 vasectomized men, 220 epididymides were grossly examined during vasovasostomy. Fullness, distention, turgidity, thickening, induration, and other gross changes of the epididymides, including the formation of cystic spermatic granuloma, or spermatocele, indicated inadequate removal of spermatozoa and testicular fluid from the sequestrated proximal seminal ducts and the epididymis. Further studies are necessary to determine whether the epididymal changes following vasectomy have any effect on sperm morphology and sperm physiology. The information obtained from these studies possibly may explain the poor pregnancy rates in the spouses of successfully vasanastomosed patients.