Modulation of mammary tumour progression using murine model by ethanol root extract of Saussurea costus (falc.) lipsch.

ETHNOPHARMACOLOGICAL RELEVANCE: Breast cancer is a major cause of death among human females across the globe. The anti-neoplastic agents or therapies used for the treatment of cancers can enhance longevity but are subsequently observed to deteriorate the quality of life due to the extensive side effects produced. Saussurea costus is a potential medicinal plant of the Himalayas with noticeable ethnopharmacological properties. The phytochemicals present in Saussurea costus are responsible for anti-carcinogenic potential and warranted nil or minimal side effects of Saussurea costus and directed to use this plant as a preventive or therapeutic drug candidate against cancers. AIM OF THE STUDY: The present study was planned to evaluate the anti-neoplastic activity of Saussurea costus root extract (SL) in rat mammary tumour model. MATERIALS AND METHODS: The anti-neoplastic activity of SL root extract at 3 different doses (100, 250 and 500 mg/kg BW) for 18 weeks against 12-dimethylbenz (a) anthracene (DMBA)-induced mammary tumours in Sprague Dawley (SD) female rats was analyzed through serum biochemistry (ALT, AST, ALP, Total protein, Creatinine and BUN), oxidative stress parameters (Lipid peroxidation, Catalase and Reduced glutathione), pro-inflammatory cytokines (TNF-α and NF-κB), immunohistochemical markers (Ki-67, MMP-9 and VEGF), real-time PCR (PCNA, p53, bax, bcl-2 and caspase-3, genes) and molecular docking. RESULTS: Inhibition of tumour parameters, minimal alteration in the liver (ALT, AST and ALP) and kidney enzymes (Creatinine and BUN), decreased activity of MDA, elevated levels of GSH and catalase, reduction in the levels of pro-inflammatory cytokines i.e. TNF-α and NF-κB, reduced gross and histomorphological changes, declined expression of Ki-67, MMP-9 and VEGF in vivo rat model, mRNA expression of cancer-related genes and docking of dehydrocostus lactone and costunolide with NF-κB and TNF-α demonstrated the chemopreventive action of SL root extract. CONCLUSIONS: The in-vivo trial elucidates anti-neoplastic activity of Saussurea costus root extract as demonstrated through the reduction of biochemical indices, oxidative stress parameters, histological changes, pro-inflammatory cytokines (NF-κB and TNF-α), cellular proliferation (Ki-67), metastases (MMP-9) and neovascularization (VEGF) markers with highest anti-neoplastic effect of SL extract at the dose of 500 mg/kg body weight. Therefore, the present study signifies the need to use the active principles present in the root extract of Saussurea costus against breast cancer as a therapeutic regimen.

Microwave assisted fast fabrication of zinc/iron oxides based polymeric nanocomposites and evaluation on equine fibroblasts.

We aimed to provide a tissue repair material, which can be synthesized rapidly, using polymers mimicking the natural environment in the extra-cellular matrix and metals/minerals. The components should have the potential to be used in tissue repair and simultaneously, reducing the side-effects of the incorporated molecules. It is challenging to manage the dispersibility of ZnO NPs in common solutions like water. Here, we report a novel method for preparing highly dispersible suspensions of ZnO NPs. In contrast to those synthesized by conventional methods, microwave assisted method allowed synthesis of dispersible ZnO NPs and the incorporation of zinc/Iron oxides NPs within alginate and gum matrix (AG) in a short span of time providing high yield of the product. The nanoformulations were characterized for size, morphology, interaction of various chemicals used during their synthesis by transmissible electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and energy dispersive X ray Spectrum. It was also evaluated for cytotoxicity and their effect on equine fibroblast cells. Microwave-assisted fabrication of zinc/iron oxides nanoparticles provided flowerlike morphology with good dispersibility and high yield in a short span of time. Our results revealed that ZnO NPs were more cytotoxic than AG ZnO NPs and doped AG Fe3O4 doped ZnO NPs at higher concentrations. Further metal nanoparticles capped with alginate/acacia with size range less than 100 nm demonstrated high stability, good biocompatibility, re-epithelization and enhanced mineralization in horse fibroblast cells.

Drug-induced reactive oxygen species-mediated inhibitory effect on growth of Trypanosoma evansi in axenic culture system.

Trypanosoma evansi, an extracellular haemoflagellate, has a wide range of hosts receptive and susceptible to infection, in which it revealed highly inconsistent clinical effects. Drugs used for the treatment of trypanosomosis have been utilized for more than five decades and have several problems like local and systemic toxicity. In the present investigation, imatinib and sorafenib were selected as drugs as they are reported to have the potential to cause reactive oxygen species (ROS)-mediated effect in cancer cells. Both have also been reported to have potential against T. brucei, T. cruzi and Leishmania donovani. To date, imatinib and sorafenib have not evaluated for their growth inhibitory effect against T. evansi. Imatinib and sorafenib showed significant (p < 0.001) inhibition on parasite growth and multiplication with IC50 (50% inhibitory concentration) values 6.12 µM and 0.33 µM respectively against T. evansi. Both the drug molecules demonstrated for the generation of ROS in T. evansi and were found up to 65% increased level of ROS as compared with negative control in the axenic culture system. Furthermore, different concentrations of imatinib and sorafenib were found non-toxic on horse peripheral blood mononuclear cells and Vero cell lines. Also, in conclusion, our results demonstrated that imatinib- and sorafenib-induced generation of ROS contributed inhibitory effect on the growth of Trypanosoma evansi in an axenic culture system.

Recombinant horse interleukin-4 and interleukin-10 induced a mixed inflammatory cytokine response in horse peripheral blood mononuclear cells.

BACKGROUND AND AIM: Interleukin (IL)-4 and IL-10 activate plethora of immune cells and induce the humoral immune response. However, recombinant version of horse IL-4 and IL-10 has not been investigated to understand their immunomodulating activities. This study aimed to produce recombinant horse mature IL-4 and IL-10 in Escherichia coli. Immune-modulating activities of recombinant horse IL-4 and IL-10 were investigated in peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: Equine PBMCs were stimulated with recombinant IL-4 and IL-10. A proliferation of PBMCs was measured by XTT assay and cytokines induction was measured by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis displayed a molecular weight of 15 kDa for IL-4 and 19 kDa for IL-10. Recombinant IL-4 and IL-10 significantly induced cell proliferation at 250 ng/ml. The results demonstrated that IL-4 enhanced expression of interferon-gamma (IFN-γ), IL-6, tumor necrosis factor-alpha (TNF-α), and IL-10, while recombinant horse IL-10 induced expression of IL-6, IFN-γ, and TNF-α. CONCLUSION: The present study demonstrated that biologically active horse IL-4 and IL-10 could be produced in E. coli.

Studies on pathology, cytokine gene expression and molecular typing of Mycobacterium avium subsp. paratuberculosis of naturally occurring Johne's disease in bullocks.

Pathology of Johne's disease (JD) in bullocks (castrated, adult male cattle) is rarely studied. Here, we report the pathology and cytokine gene expression of naturally occurring JD in bullocks. The small intestine and mesenteric lymph nodes collected from 404 bullocks, aged between 5 and 10years, were examined for JD lesions and detection of Mycobacterium avium subsp. paratuberculosis (Map). A total of 8.7% bullocks exhibited JD lesions, which were classified into multibacillary-diffuse granulomatous (n=2), paucibacillary-focal granulomatous (n=18) and paucibacillary-diffuse lymphocytic (n=15) lesions. The tissue cytokine gene expression profiles in all three forms of lesions corroborated with different immuno-pathological processes of JD in bullocks. The molecular typing and gene sequencing identified Map isolates from bullocks as bison type.

Lymphoproliferative response and its relationship with histological lesions in experimental ovine paratuberculosis and its diagnostic implications.

Lymphoproliferative response (LPR) was studied in 19 lambs orally infected (Group I) with Mycobacterium avium subsp. paratuberculosis (MAP) with in vitro lymphocyte stimulation test using MTT dye reduction assay. The non-specific LPR against Con A and specific LPR against sonicated antigen and johnin PPD (purified protein derivatives) were estimated on preinfection (0 day) and various days postinfection period (15 to 330 dpi) in the animals, which were classified according to histological and bacteriological evidence of paratuberculosis infection. Of the two antigens used, johnin PPD was found to be superior in terms of consistency and uniformity of response over an observation period of about a year. Significantly (P<0.05) higher LPR were observed in the infected sheep during postinfection period, as compared with preinfection values and values from uninfected control sheep. It was evident from the present study that the LPR in histologically infected animals fluctuated during the long course of infection and had a definite relationship with the gut pathology and the mycobacterial load. The LPR were stronger but variable in sheep with grades 1, 2 and 3 lesions (paucibacillary) and increased progressively from 30 dpi onwards. The sheep with the advanced lesions (grade 4, multibacillary) showed progressive decline in LPR till 120 dpi after initial stronger response at 30 dpi. Most of the animals were detected by LPR before initiation of faecal shedding of MAP. The results suggested that repeated testing was required while screening an infected flock for detecting most of the positive animals.

Pathology of naturally occurring paratuberculosis in water buffaloes (Bubalus bubalis).

Gross and histologic lesions of paratuberculosis were studied in water buffaloes. Small intestines and associated mesenteric lymph nodes of 405 water buffaloes were examined. Of these, 20 animals having visible changes of intestinal thickening, mucosal corrugations, and enlargement of mesenteric lymph nodes exhibited histologic alteration characteristics of mild to moderate granulomatous inflammation. The histologic lesions observed in these animals were classified into 3 grades on the basis of type of cellular infiltration, granuloma formation, and presence of acid-fast bacilli. Grade-1 lesions observed in 8 animals were marked by the presence of scattered epithelioid macrophages amid large number of lymphocytes in the intestinal villi and in the paracortical regions of the associated mesenteric lymph nodes. Another 8 animals classified under grade-2 revealed microgranulomas, infiltration with a larger number of epithelioid macrophages besides lymphocytes in the intestinal villi, and granulomas in the mesenteric lymph nodes. Grade-3 lesions observed in 4 animals were characterized by the presence of epithelioid granulomas and giant cells in the intestines and the mesenteric lymph nodes. The Ziehl-Neelsen's stained tissue sections revealed acid-fast bacilli in grade-3 and -2 animals and acid-fast granular debris in grade-1 animals. Among these 20 buffaloes, 14 (70%) were positive in the IS900 specific polymerase chain reaction and 6 (30%) were positive in the bacterial culture.

Comparison of IS900 tissue PCR, bacterial culture, johnin and serological tests for diagnosis of naturally occurring paratuberculosis in goats.

Comparative efficacy of an IS900 tissue PCR, bacterial culture, johnin, agar-gel immunodiffusion (AGID) and absorbed-ELISA tests was investigated in 43 goats naturally infected with paratuberculosis. On histological examination, tissue sections from all animals showed typical granulomatous inflammatory changes. The lesions were classified as multibacillary (MB) (n=30), which had diffuse granulomatous lesions with abundant acid-fast bacilli (AFB), and paucibacillary (PB) (n=13), which had focal or multifocal granulomatous lesions with few AFB. The sensitivities of johnin test, tissue culture, faecal culture, tissue PCR, AGID and ELISA were 68% (17/25), 100% (30/30), 84.6% (22/26), 100% (30/30), 96.2% (25/26) and 100% (26/26) in MB goats, and 88.8 (8/9), 46.1% (8/13), 40% (4/10), 61.5% (8/13), 50% (5/10), and 70% (7/10) in PB goats, respectively. Except for the johnin test, which showed higher sensitivity in PB goats, all other tests displayed significantly higher sensitivities in MB goats. The results indicate the usefulness of tissue PCR, culture and serological tests in the diagnosis of clinically affected paratuberculous goats, especially with multibacillary pathology.

Progressive immunopathological changes during early stages of experimental infection of goats with Mycobacterium avium subspecies paratuberculosis.

A dose of 10(10) Mycobacterium avium subspecies paratuberculosis was administered orally on seven occasions to produce experimental paratuberculosis infection in 10 5-8-week-old goat kids. Bacteriological, immunological, and histopathological changes, their relationships, and the efficacy of the commonly used diagnostic methods were studied during the progressive disease up to 270 days postinfection (DPI). Significant lymphocyte proliferative responses in the peripheral blood of five goats were detected as early as 60 DPI. A lymphoproliferative test was also performed on lymphocytes purified from different compartments of the guts of five infected and five control goats. Significant proliferative responses were observed in lymphocytes of jejunal compartments of all five goats, of which four had also significant lymphocyte proliferation in the blood. The ileal lymphocytes from two goats, one each at 120 and 270 DPI, had significant proliferation. The histological lesions were mainly observed in the gut-associated lymphoid tissues of the ileocecal valve, the ileum, and the terminal jejunum. Acid-fast bacilli were demonstrated in the lesions of two goats at 60 and 210 DPI. Bacterial culture showed poor sensitivity, detecting positive results for only one goat in the fecal and tissue samples at 210 DPI, whereas polymerase chain reaction (PCR) detected one goat in fecal sample at 210 DPI and two goats in tissue samples at 60 and 210 DPIs, respectively. Enzyme-linked immunosorbent assay and agar gel immunodiffusion test were found to be 100% sensitive from 180 and 210 DPI onwards, respectively.

Sequential development of histologic lesions and their relationship with bacterial isolation, fecal shedding, and immune responses during progressive stages of experimental infection of lambs with Mycobacterium avium subsp. paratuberculosis.

Understanding pathogenesis during progressive stages of infection by Mycobacterium avium subsp. paratuberculosis (MAP) and finding suitable methods for its diagnosis are key to the control of Johne's disease in animals. Paratuberculosis was experimentally produced in 20 crossbred lambs by oral administration of MAP to study the sequential development of lesions between 10 and 330 days postinfection and to assess commonly used diagnostic methods such as bacterial culture, lymphocyte stimulation test (LST), and enzyme-linked immunosorbent assay (ELISA) during progressive stages of infection. Histologic lesions were classified into four grades from grade 1 (least severe) to grade 4 (most severe) on the basis of location of granulomatous lesions in different regions and layers of intestines, their association with intestinal lymphoid tissues, pattern and distribution of lesions, types of cellular infiltration, and presence of acid-fast bacilli. It is evident that infection first establishes in lymphoid tissues of the small intestine, possibly at multiple sites, producing segmental lesions and from there spreads to lamina propria and local lymph nodes. Wide variability in the histologic lesions in relation to postinfection periods and initial tropism of MAP to the intestinal lymphoid tissues (Peyer's patches) suggests a differential susceptibility of young animals, possibly because of compositional phenotypic variation of Peyer's patches influencing subsequent course of infection. Histopathology was found to be a better indicator of paratuberculous infection than bacteriology in sheep. The LST (reflecting the cellular immune response) and ELISA (reflecting the humoral immune response) had overall sensitivities of 65% (11 of 17) and 42% (8 of 19), respectively, in sheep with different types of pathology but when employed together could detect about 88% of infected animals.

Breastfeeding and childhood cancer.

Total duration of breastfeeding and of exclusive breastfeeding was studied and compared in 99 childhood cancer cases and 90 controls. The difference between the average duration of breastfeeding in cases and controls was significant (p < 0.05), but when average duration of exclusive breastfeeding was compared in cases and controls the difference was highly significant (p < 0.001). In lymphoma cases and controls the difference between the average duration of breastfeeding was moderately significant (p < 0.01). However, when average duration of exclusive breastfeeding was compared in lymphoma cases and controls the difference was highly significant (p < 0.001). When other cancer groups and controls were compared with respect to their total duration of breastfeeding and duration of exclusive breastfeeding the differences when insignificant (p > 0.05). Cases and controls were not different with respect to their age, sex, birth year, birth order, age and educational status of mothers, smoking of fathers and socioeconomic status. However, a positive family history of cancer was obtained in 4 (4%) of cases whereas in controls it was obtained in only 1 (1.1%).

PIP: Between April 1991 and June 1992 in India, physicians compared data on 99 childhood cancer cases with data on 90 sex, age, and hospital matched controls to examine the relationship between duration of total breast feeding and exclusive breast feeding and childhood cancer. Most of the children were patients at G.S.V.M. Medical College in Kanpur or at K.G. Medical College in Lucknow. Cases and controls were essentially the same in respect to birth order, age and educational status of mothers, smoking of fathers, and socioeconomic status. 65.7% of cases had leukemia. The duration of total breast feeding was significantly longer for controls than cases (10 months vs. 8 months; p .05). The difference between mean duration of exclusive breast feeding between mean duration of exclusive breast feeding between cases and controls was significant (4.6 months vs. 3.2 months; p .001). Controls were more likely to have undergone a longer duration of total breast feeding and exclusive breast feeding than were lymphoma cases (10 months vs. 6.15 months; p .01 and 4.6 months vs. 3 months; p .001, respectively). 58% of lymphoma cases had non-Hodgkins lymphoma. When the researchers compared other cancer groups and controls, no significant difference between the 2 groups existed in respect to total breast feeding and exclusive breast feeding. These results suggest that breast feeding has a protective effect against childhood cancer. Further, they indicate that exclusive breast feeding provides more beneficial immunological effects than breast feeding supplemented by artificial feeding. The high rate of breast feeding in India may explain the low incidence of childhood cancer in India (e.g., around 6/100,000 vs. 18/100,000 in Israel).

Role of lithium carbonate in pancytopenia following cytotoxic therapy.

Twenty-four patients presenting with pancytopenia following cytotoxic therapy were studied. Seventeen of them were given 900 mg/day of lithium carbonate apart from other supportive measures. Seven patients were used as controls and were not given any lithium. After 2 weeks, granulocyte count was significantly higher (p less than .05) in lithium-treated group. Platelet count was also higher though not statistically significant.