G.L. Sharma

In vitro and in vivo antimicrobial activities of seeds of Caesalpinia bonduc (Lin.) Roxb.

AIM OF THE STUDY Caesalpinia bonduc (Lin.) Roxb. is a known drug in Ayurveda to treat various diseases specifically tumors, cysts and cystic fibrosis (CF). The aim of this study was to assess in vitro as well as in vivo antimicrobial activity of Caesalpinia bonduc seeds. MATERIALS AND METHODS The in vitro antimicrobial activities of seed coat and seed kernel extracts were investigated by microbroth dilution assay. In vivo activities of hydro-alcoholic extracts were investigated in rat models of chronic Pseudomonas aeruginosa pneumonia mimicking that in patients with cystic fibrosis. RESULTS Various extracts of plant seeds exhibited in vitro antimicrobial activities in a range of 22-350 microg/ml. The extracts also showed activity against methicillin resistant (MR) Staphylococcus aureus and ampicillin resistant (AR) Pseudomonas aeruginosa as in the sensitive strains. In rat model of chronic Pseudomonas aeruginosa pneumonia, hydro-alcoholic extracts of Caesalpinia bonduc seed kernel (CBSK) and Caesalpinia bonduc seed coat (CBSC) were injected subcutaneously in the test groups of animals. The control groups were treated with cortisone and saline. Two weeks after challenge with Pseudomonas aeruginosa, the CBSK treated animals showed a significant bacterial clearance from the lungs (P<0.04) and less severe incidence of lung abscess (P<0.05). CONCLUSION Results showed that Caesalpinia bonduc may have the potential to be promising natural medicine, with other forms of treatments, for CF patients with chronic Pseudomonas aeruginosa lung infections.

T lymphocyte subset profile and serum alpha-1-antitrypsin in pathogenesis of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder characterized by the presence of non‐fully reversible airflow limitation. The study was undertaken to investigate the involvement of alpha‐1‐antitrypsin (α1AT) and T lymphocyte subsets in the pathogenesis of COPD. Blood samples of 50 subjects, including 25 healthy volunteers and 25 patients with COPD, were analysed. Serum trypsin inhibitory capacity (STIC) was determined by enzymatic assay. CD4+ and CD8+ T lymphocytes were enumerated in heparinized blood using a fluorescence activated cell sorter counter. The STIC in COPD patients was found to be decreased significantly than in controls (P < 0·01). In COPD patients with lower expression levels of α1AT, a highly significant decrease in the number of CD4+ T lymphocytes (P < 0·0009) and CD4/CD8 ratio was observed compared with control subjects (P < 0·008). The mean ± standard error of CD8+ lymphocytes was found to be little different (only marginally decreased) in COPD patients compared to healthy controls; however, an alteration in the individual count of CD8+ lymphocytes cells was observed in COPD patients. Using linear regression analysis, a negative correlation was observed between STIC and CD4+ lymphocytes and CD8+ lymphocytes (r = −0·40, P < 0·04; r = −0·42, P < 0·03, respectively) in COPD patients. An alteration in α1AT and T lymphocyte subsets in COPD patients suggested that interplay of these factors may be responsible for the progression of COPD.

Differential expression of Aspergillus fumigatus protein in response to treatment with a novel antifungal compound, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate.

A dihydropyridine derivative, diethyl 4‐(4‐methoxyphenyl)‐2,6‐dimethyl‐1,4‐dihydropyridin‐3,5‐dicarboxylate (2e), having potent antifungal activity against pathogenic species of Aspergillus was investigated for its possible molecular mechanism of action. The SDS‐PAGE coupled with nano‐high‐performance liquid chromatography–tandem mass spectrometry was used directly to assess both absolute abundance and differential expression of proteins in the secretory phases of Aspergillus fumigatus under the influence of 2e. It was observed that the compound inhibited the expression of two proteins of 60.99 and 79.77 kDa. Both of these secretory proteins that were inhibited by 2e, were analysed further by matrix assisted laser desorption ionization time‐of‐flight mass spectrometry. The 60.99‐ and 79.77‐kDa proteins were identified as probable retroelement pol polyprotein and elongation factor G respectively. These targeted proteins could be the products of potentially virulence‐related genes of A. fumigatus which may unravel the mode of action of 2e and pathobiology of A. fumigatus.

Antifungal Treatments Delineate a Correlation between Cathepsins and Cytokines in Murine Model of Invasive Aspergillosis

In the pathogenesis of invasive pulmonary aspergillosis both fungal and host factors play roles. Though cytokines and phagocyte, as host factors, have been shown to participate in defence against Aspergillus species yet the role of cysteine proteases, that is cathepsins, a lysosomal enzymes of phagocytes, remains unknown in fungal infection. Studies are available which shows that cytokines regulate the cysteine proteases processed immune molecules for their further action but their relationship with each other under fungal infection is not clear. Therefore, in this study, we demonstrate the substantial role of cathepsins and cytokines in aspergillosis. In the present murine model of invasive pulmonary aspergillosis, on seventh day of Aspergillus fumigatus infection, both kidney and liver showed significant (P<0.05) fungal burdens, which was also confirmed by histological analyses. The activity profiles of four cathepsins in the kidney and liver tissue were analysed and correlated with blood cytokines level in the presence and absence of antifungal compounds (amphotericin B, a standard drug and 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrole-2-yl)-1-methylethyl pentanoate, isolated in our laboratory from natural source) treatment. The data illustrate that the reduction in fungal load in both organs probably results in a decreased local inflammatory response, as measured by decreased levels of interleukin-4 and interleukin-10 and increased level of interferon gamma in the antifungal compounds treated mice. Interestingly, this altered level of cytokines relates well with the activity level of cathepsins, that is decreased in interleukines (interleukinL-4/interleukin-10) and cathepsins (cathepsin B, cathepsin C and cathepsin L); and increase in interferon gamma and cathepsin H levels in the mice treated with antifungal compounds were observed. These observations support not only the negative (cathepsin B, cathepsin C and cathepsin L) and positive (cathepsin H) role of cathepsins in aspergillosis but also prove the role of cytokines in remodelling of immune response. Overall, the study reveals a correlation between cathepsins and cytokines and their regulatory role in fungal mediated infection.

Antifungal effects of 3-(4-Phenyl-thiazol-2-yl)-2-thioxo-2, 3-dihydro-1H-quinazolin-4-one against Aspergillus Species

Aspergillus infections have become an important health problem with the increasing number of patients. Available antifungal drugs are lack with their spectrum, toxic or immunosuppressive in nature, so that need to develop new compound with high efficacy. To evaluate antifungal efficacy of synthesized compound and to identify the protein profile of Aspergillus fumigatus treated with antifungal. Clinical isolates of A. fumigatus, A. flavus and A. niger were cultured and efficacy of compound were conducted by Disc Diffusion Assay (DDA), Microbroth Dilution Assay (MDA). Percent of spore germination inhibition assay (PSGI), Time kill analysis and toxicity assay. The culture filtrate containing secretory proteins was collected after 24 h growth and expression of downregulated proteins were identified. We developed a new and useful quinazoline derivatives expected to antifungal activity. The result of anti-Aspergillus evolution revealed that one of the 3-(4-Phenyl-thiazol-2-yl)-2-thioxo-2, 3-dihydro-1H-quinazolin-4-one (DDVKT4Q) exhibited appreciable activity. The potency of compound was found concentration of 3.125 µg/disc by disc diffusion assay (DDA) and 15.625 µg/ml. by Microbroth Dilution Assay (MDA). The compound was nontoxic up to concentration 625 µg/ml and its lysed only 35.9% of human erythrocytes, at the highest dose tested. It’s observed that the treatment of pathogen with DDVKT-4Q targeted the expression of four proteins having molecular weights 18 kDa 37 KDa and 43 KDa proteins was completely inhibited or down regulated by the compound the extra cellular. The novel compound DDVKT-4Q, having antifungal activity Can be exploited further to develop new ideal antimycotic drugs.