Detrimental effect of expression of Bt endotoxin Cry1Ac on in vitro regeneration, in vivo growth and development of tobacco and cotton transgenics.

High levels of expression of the cry1Ac gene from Bacillus thuringiensis cannot be routinely achieved in transgenic plants despite modifications made in the gene to improve its expression. This has been attributed to the instability of the transcript in a few reports. In the present study, based on the genetic transformation of cotton and tobacco, we show that the expression of the Cry1Ac endotoxin has detrimental effects on both the in vitro and in vivo growth and development of transgenic plants. A number of experiments on developing transgenics in cotton with different versions of cry1Ac gene showed that the majority of the plants did not express any Cry1Ac protein. Based on Southern blot analysis, it was also observed that a substantial number of lines did not contain the cry1Ac gene cassette although they contained the marker gene nptII. More significantly, all the lines that showed appreciable levels of expression were found to be phenotypically abnormal. Experiments on transformation of tobacco with different constructs expressing the cry1Ac gene showed that in vitro regeneration was inhibited by the encoded protein. Further, out of a total of 145 independent events generated with the different cry1Ac gene constructs in tobacco, only 21 showed expression of the Cry1Ac protein, confirming observations made in cotton that regenerants that express high levels of the Cry1Ac protein are selected against during regeneration of transformed events. This problem was circumvented by targeting the Cry1Ac protein to the chloroplast, which also significantly improved the expression of the protein.

Changing trends in sexually transmitted infections at a Regional STD Centre in north India.

BACKGROUND & OBJECTIVES: Sexually transmitted infections (STIs) a major public health problem in India show various trends in different parts of the country. However, there are limited data on the changing profile of laboratory proven STIs in the same clinic over the years. The present study was thus aimed to determine the changing trends of the profile of STIs and HIV seropositivity in STD clinic attendees over a 15 yr period, and also to detect change, if any, in the antimicrobial resistance pattern of Neisseria gonorrhoeae. METHODS: The STIs were diagnosed clinically and confirmed by standard laboratory techniques. Socio-demographic data were collected through pre-designed proformae. The STI profile and HIV seropositivity were compared between 1990-1993 (A), 1994-1997 (B), 1998-2001 (C) and 2002-2004 (D). Antimicrobial resistance pattern of N. gonorrhoeae was determined by standard techniques and compared between the last three periods. RESULTS: Of the 78,617 STD attendees, 12,709 (16.2%) had STIs. During period A, genital discharges and during B, C and D, genital ulcerative diseases were predominant. Syphilis was the commonest STI. There was significant rise in the cases of syphilis, herpes progenitalis and genital warts and reduction in that of chancroid, lymphogranulomavenereum (LGV), donovanosis, candidiasis, trichomoniasis and bacterial vaginosis cases. The number of cases with primary syphilis diminished significantly (P<0.001), with a concomitant rise in secondary and early latent syphilis. A rising trend was observed in the HIV seropositivity during the different periods. The association of HIV seropositivity was consistently more in patients presenting with genital ulcers specially syphilis, and rose significantly from A (0.6%) to C (8.8%), but became stationary during D. A drastic change in the antimicrobial resistance of N. gonorrhoeae from B to C and C to D and the emergence of less sensitive isolates to ceftriaxone during the later part of the study were observed. INTERPRETATION & CONCLUSION: Our study showed a definite changing trend in the profile of STIs in the clinic attendees of a major STD centre during a 15 yr period. However, the significant rise in the cases of viral STIs and syphilis contrasted with reduction in the rest.

CD4/CD8 lymphocyte counts in healthy, HIV-positive individuals & AIDS patients.

BACKGROUND & OBJECTIVES: The enumeration of CD4 and CD8 positive cells, surrogate markers for HIV disease progression, is helpful in management and follow up of immunocompromised HIV-positive patients. In assessing the degree of immune deficiency in HIV-positive patients of a particular region, knowledge of reference range of T-cell subset counts of healthy individuals of that particular region is essential. The present cross-sectional study was undertaken to determine the reference range of T-cell subsets in healthy north Indians and to compare the values with those in HIV-positives. METHODS: Blood samples from 125 HIV seronegative healthy volunteers comprising group I (88 males, 37 females) and 452 HIV- positive patients, divided into group II of asymptomatic (n=137; 93 males, 44 females) and group III of AIDS patients (n=315; 253 males, 62 females) in the age group of 17-60 yr, were analysed for enumeration of CD4+, CD8+ cells/microl by flow cytometry. RESULTS: In group I, the CD4 and CD8 levels were 687 +/- 219 and 611 +/- 288 cells/microl in males and 740 +/- 255 and 546 +/- 246 cells/microl in females. Overall, a significant depressed level of CD4 (525 +/- 207 cells/microl) and elevated level of CD8 (1174 +/- 484 cells/microl) in group II and (170 +/- 115 and 1051 +/- 586 cells/microl) respectively in group III were observed. Group II patients had highest level of CD8 cells. No asymptomatic women had CD4 count of <200 cells/microl. INTERPRETATION & CONCLUSION: Our findings on T-cell subset reference ranges of normal healthy north Indians validate the utility of determination of CD4 cell count as a useful predictor of AIDS in Indian conditions and confirm that a significant per cent of AIDS patients had CD4 cell count below 200/microl.

Comparison of disc diffusion results with minimum inhibitory concentration (MIC) values for antimicrobial susceptibility testing of Neisseria gonorrhoeae.

BACKGROUND & OBJECTIVE: objectives: As antimicrobial susceptibility testing of Neisseria gonorrhoeae provides guidance for appropriate treatment, there is a need for simple, reliable and cost-effective method for susceptibility testing. The present study was aimed to compare the results of two methods of susceptibility testing, minimum inhibitory concentration (MIC) values by E test with disc diffusion results by Australian Gonococcal Surveillance Programme (AGSP) method in N. gonorrhoeae isolates. METHODS: Susceptibility testing for ciprofloxacin, penicillin and ceftriaxone using AGSP method was carried out for 301 confirmed consecutive isolates of N. gonorrhoeae. MIC of ciprofloxacin, penicillin and ceftriaxone was determined by E test in 301, 198 and 128 isolates respectively. The results of the two methods were compared by using Kappa statistics. RESULTS: Moderate levels of agreement for ciprofloxacin (kappa=0.44) and penicillin (kappa=0.54) were observed between the two methods. For ceftriaxone, 96.1 and 0.8 per cent isolates were found to be susceptible and less sensitive respectively by both the methods and per cent agreement between the two methods was 96.9 per cent. INTERPRETATION & CONCLUSION: Both the methods were easy to perform and gave reproducible results. However, disc diffusion method was cost-effective and more feasible in routine diagnostic laboratories in developing countries like India.

Evaluation of beta2 microglobulin level as a marker to determine HIV/AIDS progression.

Levels of beta2 microglobulin (beta2M) were evaluated to monitor the progression of HIV disease, as an alternate economical marker to RNA viral load and CD4 cell count in resource poor situations. A cross sectional study of 32 HIV sero-negative controls (Group I), 43 asymptomatic HIV sero-positives (Group II-A), 44 HIV sero-positives with clinical and/or laboratory proven STDs (Group II-B) and 30 with AIDS indicator conditions (Group III) was carried out. beta2M levels were determined using an enzyme immuno assay. Mean +3 SD (3.04mg/l) of concentration of beta2M in sero-negative controls was chosen as threshold of abnormality. A significant rise (p<0.001) in mean beta2M levels (mg/l) from 1.87 +/- 0.39 (Group I) to 2.59 +/- 1.09 (Group IIA), 3.01 +/- 1.27 (Group IIB) to 5.16 +/- 2.48 (Group III) was observed. Higher values of beta2M in the symptomatic phase than those in the asymptomatic phase indicated that elevated levels of beta2M parallel progression of HIV disease and suggest its use as an alternate marker for determining HIV progression.