Cloning, large-scale production and characterization of fusion protein (P-TUFT-ALT-2) of Brugian abundant larval transcript-2 with tuftsin in Pichia pastoris.

Lymphatic filariasis is a "disease of poor people" due to a large section of affected people with economic backwardness. Therefore, successful elimination of this disease requires a cost-effective prophylactic agent such as vaccine along with conventional drugs. The Abundant Larval Transcript-2 (BmALT-2) protein of Brugia malayi has been recognized as the most potential vaccine candidate. Tuftsin, a tetra-peptide immunopotentiator has already shown the enhanced immunogenicity of various vaccine antigens in earlier studies. This study deals with the development of tuft-alt-2 fusion construct and a suitable culture condition for its large-scale production in Pichia pastoris. The recombinant P. pastoris/tuft-alt-2 with 9-11 copies of the gene construct exhibited the highest expression level. The molecular weight of P-TUFT-ALT-2 was determined as 28 kDa in SDS-PAGE including 3 kDa due to glycosylation. The dry cell biomass was 57.4 gL-1 in the bioreactor. The P-TUFT-ALT-2 expression was measured as about 35 mg L-1, which was 102% higher than flask culture. The P-TUFT-ALT-2 produced the highest 65,000 IgG peak titer in Balb/c mice. Moreover, P-TUFT-ALT-2 exhibited about 9.46% higher splenocyte proliferation than E. coli expressed E-ALT-2 alone. The enhanced secreted production of P-TUFT-ALT-2 in bioreactor would step up its commercialization as an inexpensive commercial vaccine for human lymphatic filariasis.

Avocado fruit (Persea americana Mill) exhibits chemo-protective potentiality against cyclophosphamide induced genotoxicity in human lymphocyte culture.

Diets rich in fruits and vegetables have been associated with reduced risks for many types of cancers. Avocado (Persea americana Mill.) is a widely consumed fruit containing many cancer preventing nutrients, vitamins and phytochemicals. Studies have shown that phytochemicals extracted from the avocado fruit selectively induce cell cycle arrest, inhibit growth, and induce apoptosis in precancerous and cancer cell lines. Our recent studies indicate that phytochemicals extracted with 50% Methanol from avocado fruits help in proliferation of human lymphocyte cells and decrease chromosomal aberrations induced by cyclophosphamide. Among three concentrations (100 mg, 150 mg and 200 mg per Kg Body Weight), the most effective conc. of extract was 200 mg/Kg Body Wt. It decreased significant level of numerical and structural aberrations (breaks, premature centromeric division etc. up to 88%, p < 0.0001)), and accrocentric associtation within D & G group (up to 78%, p = 0.0008). These studies suggest that phytochemicals from the avocado fruit can be utilized for making active chemoprotective ingredient for lowering the side effect of chemotherapy like cyclophosphamide in cancer therapy.

In vitro evaluation of genotoxicity of avocado (Persea americana) fruit and leaf extracts in human peripheral lymphocytes.

Persea americana is much sought after both for the nutritional value of its fruit and the medicinal values of its various plant parts. A chromosomal aberration assay was undertaken to evaluate the potential genotoxicity of crude extracts from avocado fruits and leaves. Chromosomal aberrations were observed in cultured human peripheral lymphocytes exposed to separately increasing concentrations of 50% methanolic extracts of Persea americana fruit and leaves. The groups exposed to leaf and fruit extracts, respectively, showed a concentration-dependent increase in chromosomal aberrations as compared to that in a control group. The mean percentage total aberrant metaphases at 100 mg/kg, 200 mg/kg, and 300 mg/kg concentrations of leaf extract were found respectively to be 58 ± 7.05, 72 ± 6.41, and 78 ± 5.98, which were significantly higher (p < 0.0001 each) than that in the control group (6 ± 3.39). The mean percentage total aberrant metaphases at 100 mg/kg, 200 mg/kg, and 300 mg/kg concentrations of fruit extract were found to be 18 ± 5.49, 40 ± 10.00, and 52 ± 10.20, respectively, which were significantly higher (p = 0.033, p < 0.0001, and p < 0.0001, respectively) than that for control (6 ± 3.39). Acrocentric associations and premature centromeric separation were the two most common abnormalities observed in both the exposed groups. The group exposed to leaf extracts also showed a significant number of a variety of other structural aberrations, including breaks, fragments, dicentrics, terminal deletion, minutes, and Robertsonian translocations. The group exposed to leaf extract showed higher frequency of all types of aberrations at equal concentrations as compared to the group exposed to fruit extract.

Immunological evaluation of fusion protein of Brugia malayi abundant larval protein transcript-2 (BmALT-2) and Tuftsin in experimental mice model.

INTRODUCTION: Filariasis, a neglected tropical helminth disease needs vaccine besides mass drug administration for its successful eradication. METHODS: An attempt was made to produce a fusion protein (P-TUFT-ALT-2) of abundant larval transcript protein-2 and Tuftsin to enhance its immunogenicity. The fusion construct was expressed in Pichia pastoris, a nonexpensive commercial expression system. This study focused on the evaluation of immunological response produced by P-TUFT-ALT-2 in Balb/c mice. RESULT AND DISCUSSION: P-TUFT-ALT-2 showed an enhanced IgG peak titre compared to E. coli expressed E-ALT-2 and P. pastoris expressed P-ALT-2. IgG2b, IgG2a and IgG1 production were predominant indicating a balanced Th1/Th2 response. P-TUFT-ALT-2 also induced about 28% and 9.5% higher splenocyte proliferation over control and E-ALT-2 respectively. Splenocytes produced predominant IFN-γ followed by IL-5, IL-2 and IL-10 specifying a balanced Th1/Th2 response. P-TUFT-ALT-2 showed 55% to 80% with an average of 65% cytotoxicity in B. malayi L3 larvae in in vitro ADCC assay. CONCLUSION: This experiment validates P-TUFT-ALT-2 as a potential vaccine candidate for human lymphatic filariasis.

Elucidation of immunological response and its regulatory network by P-TUFT-ALT-2: a promising fusion protein vaccine for human lymphatic filariasis.

Human lymphatic filariasis, a mosquito-borne neglected tropical parasitic disease, needs an early development of prophylactic agents such as a vaccine for its successful elimination. Our earlier study suggested the enhanced immunological response by fusion protein (P-TUFT-ALT-2) of Tuftsin and ALT-2 in a mice model. We cultured human peripheral blood mononuclear cells (PBMCs) and treated cells with Escherichia coli-expressed ALT-2 (E-ALT-2) and P-TUFT-ALT-2. Real-time polymerase chain reaction was performed to identify the mRNA copy number of various cytokine and transcription factor genes. The recombinant vaccine candidate was also validated for humans by immunoreactivity with human sera samples of natural infection. In this study, P-TUFT-ALT-2 stimulated 12% higher PBMC proliferation in endemic normal (EN) individuals than E-ALT-2 alone. There was enhanced production of IFN γ, IL-2, IL-5 and IL-12, indicating a balanced Th1/Th2 response. However, higher expression of IL-5 and lower IL-4 validate the humoral response through an IL-5-dependent manner. Also, high level of IL-17 indicates a strong Th/Treg regulation over T-cell activation. The upregulated T-bet might have enhanced IFN-γ production, whereas GATA-3 was supposed to enhance IL-5 expression. The fusion protein also exhibited 15-16% higher reactivity with EN clinical sera, exposing the upregulation of IgG1 and IgM in natural infection. The higher reactivity of P-TUFT-ALT-2 with sera of natural infection (EN) was validated indirectly by B-cell activation through various cytokines and regulatory genes produced from different T cells. Thus, these findings endorse P-TUFT-ALT-2 as a potential vaccine candidate for human lymphatic filariasis.

Anticancer biology of Azadirachta indica L (neem): a mini review.

Neem (Azadirachta indica), a member of the Meliaceae family, is a fast growing tropical evergreen tree with a highly branched and stout, solid stem. Because of its tremendous therapeutic, domestic, agricultural and ethnomedicinal significance, and its proximity with human culture and civilization, neem has been called "the wonder tree" and "nature's drug store." All parts of this tree, particularly the leaves, bark, seed-oil and their purified products are widely used for treatment of cancer. Over 60 different types of biochemicals including terpenoids and steroids have been purified from this plant. Pre-clinical research work done during the last decade has fine-tuned our understanding of the anticancer properties of the crude and purified products from this plant. The anticancer properties of the plant have been studied largely in terms of its preventive, protective, tumor-suppressive, immunomodulatory and apoptotic effects against various types of cancer and their molecular mechanisms. This review aims at scanning scattered literature on "the anticancer biology of A. indica," related toxicity problems and future perspectives. The cogent data on the anticancer biology of products from A. indica deserve multi-institutional clinical trials as early as possible. The prospects of relatively cheaper cancer drugs could then be brighter, particularly for the under-privileged cancer patients of the world.