Selective Cytotoxic effect of Probiotic, Paraprobiotic and Postbiotics of L. casei strains against Colorectal Cancer Cells: Invitro studies

Abstract This study highlights the cytotoxic effect of three L. casei strains on colorectal cell lines in invitro conditions. Different concentrations of live, heat killed (HK) and cell free supernatant (CFS) of three L.casei strains were subjected to CaCo2 and MRC5 cell lines. The viability of the treated and untreated cells was determined after 72 hrs by MTT assay, and IC50 estimated. Apoptosis was evaluated by Annexin V-propidium iodide method using flow cytometry. The live, HK and CFS of the L. casei strains showed cytotoxic effects on colorectal cell lines with significant differences. The cytotoxicity effects of live cells on CaCo2 cells were significantly higher (p˂0.01) than the HK cells. A dose dependent response was observed, as higher concentrations resulted in enhanced cytotoxicity effects. Live L.casei 1296-2cells inhibited 91% of CaCo2 cell growth, with IC50 of less than 108 cfu/ml. MRS medium and concentrations of CFS at above 20% v/v, were cytotoxic to the normal cell lines. Flow cytometry analyses of L. casei 1296-2 indicated that cytotoxicity effects on CaCo2 cells is related to apoptotic induction. Invitro studies indicate that Live and CFS of L. casei 1296-2 might be promising candidate for the control of colorectal cancers

Higher expression level and lower toxicity of genetically spliced rotavirus NSP4 in comparison to the full-length protein in E. coli

Background: Rotavirus group A [RVA] is recognized as a major cause of severe gastroenteritis in children and new-born animals. Nonstructural protein 4 [NSP4] is responsible for the enterotoxic activity of these viruses in the villus epithelial cells. Amino acids 114-135 of NSP4 are known to form the diarrhea-inducing region of this viral enterotoxin. Therefore, developing an NSP4 lacking the enterotoxin domain could result in the introduction of a new subunit vaccine against rotaviruses in both humans and animals

Objectives: The aim of this study is the evaluation of rotavirus ANSP4 expression in E. coli expression system before and after removal of the diarrhea-inducing domain, which is the first step towards further immunological studies of the resulting protein

Materials and Methods: Splicing by overlap extension [SOEing] PCR was used to remove the diarrhea-inducing sequence from the NSP4 cDNA. Both the full-length [FL-NSP4] and the spliced [S-NSP4] cDNA amplicons were cloned into pET-32c and pGEX-6P-2. Expression levels of the recombinant proteins were evaluated in E. coli BL21 [DE3] by Western blot analysis. In addition, the toxicity of pET plasmids bearing the S-NSP4 and FL-NSP4 fragments was investigated by plasmid stability test

Results: For FL-NSP4, protein expression was detected for the strain containing the pGEX:FL-NSP4 plasmid, but not for the strain carrying pET:FL-NSP4. Hourly sampling up to 3 h showed that the protein production decreased by time. In contrast, expression of S-NSP4 was detected for pET:S-NSP4 strain, but not for pGEX:S-NSP4. Plasmid stability test showed that pET:S-NSP4 recombinant plasmid was almost stable, while pET:FL-NSP4 was unstable

Conclusions: This is the first report of production of rotavirus NSP4 lacking the diarrhea-inducing domain [S-NSP4]. SNSP4 shows less toxicity in this expression system and potentially could be a promising goal for rotavirus immunological and vaccine studies in the future

Effect of invasive aspergillosis infection on the immune responses of Cancer mice

Using a cancer murine model of invasive aspergillosis [IA], we investigated the expression of TLR-2, Dectin-1 and the level of cytokine production by CD4+ T helper cells in different groups of mice [with or without cancer], also, the effect of invasive aspergillosis on the immune response pattern of cancer mice. Patterns of susceptibility and resistance to infection obtained with different groups of mice injected with Aspergillus fumigatus conidia. TLR-2 and Dectin-1 analyzed applying flowcytometry and cytokine production of cultured splenocytes by ELISA method. Cancer mice that challenged with A. fumigatus conidia showed significant increase in TLR-2 and Dectin-1 levels compared with the two other control groups [normal mice challenged with A. fumigatus and non-infected cancer mice]. Moreover, it showed insignificant decrease in IFN-gamma and IL-10 levels and insignificant increase in TNF-alpha level. The data demonstrated remarkable rise in IL-4 level and the mortality of cancer mice that intravenously infected with A. fumigatus. Probably IA causes stimulation in innate immunity and Th2 cells, also some disorganization in cytokine production in CD4+ T helper cells. We hypothesize that concomitance of IA and cancer may change the microenvironment for local or systemic immune responses. Other complementary studies could help supporting our hypothesis

Inhibition of IL-13 by antisense oligonucleotide changes immunoglobulin isotype profile in cultured B-lymphocytes

The link between IL-13 and bronchial hyper-responsiveness has brought this cytokine as a potential therapeutic target for asthma and allergic diseases. At the present study, we address the role of B cell derived IL-13 in the IgE and other immunoglobulin development. Antisense oligo for human IL-13 m-RNA was used to study IgE down regulation. Human B-lymphocytes were purified by positive selection using magnetic cell sorting and were cultured in the complete medium plus anti-CD40 monoclonal antibody and recombinant human IL-4. Immunoglobulin assay was performed by ELISA in the presence and absence of antisense oligonucleotide. We demonstrated that IL-13 antisense causes the decrease of IgE and increase of IgA significantly and no significant changes in IgM and IgG levels [p<0.01]. We also demonstrated that both IL-13 inhibition and IL-4 removal cause the complete blocking of IgE and significant decrease of IgM and IgG levels. Our IL-13 antisense oligo can block B-cell IL-13 productions and consequently inhibits IgE production followed by IgA class switching in vitro. We suggest that in contrast to the IL-4, IL-13 is apparently more potent in the IgE switching and has no significant role in IgG and IgM levels