In Vitro Screening of Indigenous Lactobacillus Isolates for Selecting Organisms with Better Health-Promoting Attributes.

Lactobacilli have several attributes that provide health benefits to the host. The aim of this study was to screen indigenous lactobacilli from human gut and fermented foods for such attributes as production of ß- and α-galactosidase and also their ability to reduce serum cholesterol. Lactobacilli were cultured on MRS broth and ß-galactosidase activity was determined using o-nitrophenyl-ß-D-galactopyranoside (ONPG) as a substrate. Three isolates Lactobacillus fermentum GPI-3 and L. fermentum GPI-6 and Lactobacillus salivarius GPI-1(S) showed better ß-galactosidase activity than the standard strains Lactobacillus rhamnosus GG (LGG) and Lactobacillus plantarum ATCC 8014. The isolates showed variability in assimilating cholesterol during growth. Several isolates showed excellent cholesterol-lowering ability compared to standard strains LGG and L. plantarum ATCC 8014. Isolate L. rhamnosus SCB being the highest acid producer (pH 4.38) also showed the highest cholesterol reduction compared to other strains including standard strains. The ability of these isolates to produce α-galactosidase was also studied and the maximum α-galactosidase activity was found in isolate L. salivarius GPI-1(S) followed by L. fermentum FA-5 and Lactobacillus helveticus FA-7. This study therefore reports Lactobacillus isolates that have superior probiotic properties when compared to the standard strains; hence, they could be considered as potential probiotic strains, which can provide health benefits to the Indian population.

Indigenous lactobacilli strains of food and human sources reverse enteropathogenic E. coli O26:H11-induced damage in intestinal epithelial cell lines: effect on redistribution of tight junction proteins.

The aim of the study was to investigate the neutralizing effect of lactobacilli isolated from indigenous food and human sources on enteropathogenic Escherichia coli (EPEC) O26 : H11-induced epithelial barrier dysfunction in vitro. This was assessed by transepithelial electrical resistance (TEER) and permeability assays using intestinal cell lines, HT-29 and Caco-2. Furthermore, the expression and distribution of tight junction (TJ) proteins were analysed by qRT-PCR and immunofluorescence assay, respectively. The nine strains used in the study were from different species viz. Lactobacillus fermentum, Lactobacillushelveticus, Lactobacillus salivarius and Lactobacillus plantarum. All strains were able to reverse the decrease in TEER and corresponding increase in permeability across E. coli-infected monolayers. Maximum reversal was observed after 18 h [up to 93.8±2.0 % by L. rhamnosus GG followed by L. fermentum IIs11.2 (92.6±2.2 %) and L. plantarum GRI-2 (91.9±0.9 %)] of lactobacilli exposure following EPEC O26 : H11 infection. All strains were able to redistribute the TJ proteins to the cell periphery either partially or completely. Moreover, L. helveticus FA-7 was also able to significantly increase the mRNA expression of ZO-1 and claudin-1 (2.5-fold and 3.0-fold, respectively; P<0.05). The rapid reversal observed by these strains could be mostly because of the redistribution rather than increased mRNA expression of TJ proteins. In conclusion, L. helveticus FA-7, L. fermentum FA-1 and L. plantarum GRI-2 were good in all the aspects studied, and the other strains were good in some aspects. L. helveticus FA-7, L. fermentum FA-1 and L. plantarum GRI-2 can therefore be used for potential therapeutic purpose against intestinal epithelial dysfunction.

Effect of recombinant malarial antigen on monocyte functionality.

BACKGROUND: Recombinant proteins and vaccine candidates of Plasmodium vivax have met with limited success. One of the reasons could be their effect on monocytes which are important in malaria pathogenesis. Our aim was therefore to investigate the effect of selected recombinant malarial proteins on monocytes functions. METHODS: Phagocytosis rate and respiratory burst of healthy individuals' monocytes treated with antigens were examined. The homing capacity of monocytes was studied by examining the mRNA level of chemokine receptors from patients and healthy individuals. RESULTS: Phagocytosis rate was reduced in antigen treated monocytes whereas nitroblue tetrazolium (NBT) reduction was more in apical membrane antigen-1 (AMA-1) and merozoite surface protein-7 (MSP7) treated than in untreated and von Willebrand factor A domain-related protein (WARP) treated monocytes. Patient monocytes showed higher mRNA expression for CCR2 and CX3CR1 and reduced levels for CCR7 and CXCR4. AMA-1 and WARP treated monocytes showed lower expression for CCR2, CX3CR1 and CXCR4, but unchanged for CCR7. However, with MSP7, all the receptor levels were reduced. CX3CR1 in monocytes from activated PBMCs was either unchanged (AMA-1) or increased (MSP7, WARP) while remaining receptors were reduced. CONCLUSIONS: These antigens may modulate the monocyte functionality and hence may not have desired therapeutic effect.

Difference in mononuclear cell cytokine profile of tuberculosis patients before and after treatment and its influence on in vitro multinucleate giant cell formation.

The present study focuses on correlation of in vitro multinucleate giant cell (MGC) forming ability with cytokine production in case of tuberculosis patients before and after treatment. In vitro MGC formation was carried out by treating monocytes with culture supernatant of Con A or PPD stimulated peripheral mononuclear cells. MGC formation was significantly lower for monocytes of untreated patients compared to control monocytes when incubated with respective autologous culture supernatant. MGC forming ability increased significantly for monocytes of treated patients incubated with autologous culture supernatant at the end of two months of treatment. Analysis of the supernatants revealed higher IL-10 levels and lower IFN-γ and IL-2 levels in untreated patients compared to treated patients and controls. The findings suggest that changes in IL-10, IFN-γ and IL-2 levels produced by mononuclear cells of patients before and after treatment might influence the functionality of monocytes and thereby the pathology of tuberculosis.

Isolation and characterization of lactobacilli from human faeces and indigenous fermented foods for their potential application as probiotics.

This study was conducted to select Lactobacillus strains from various sources on the basis of their probiotic attributes, such as acid and bile tolerance, binding to intestinal cells, and antimicrobial activity. Twelve isolates were obtained from human and food sources and were evaluated against standard probiotic Lactobacillus rhamnosus GG (LGG). Isolates were also studied for their antibiotic susceptibility. Isolate Lactobacillus fermentum GPI-6 showed the best survival profile at 0.3% and 1% bile salt, as compared with LGG. Isolates Lactobacillus plantarum GRI-2 and Lactobacillus salivarius GPI-4 showed no reduction in survival rate at pH 2.5. As expected, isolates showed strain-specific differences when comparing various attributes. Isolates GPI-4, GPI-7, and FA-5 showed better adhesion to HT-29, while isolate GPI-4 adhered better to Caco-2 cells than did LGG. However, when studying their ability to compete with Escherichia coli O26:H11, isolates GPI-6 and GPI-7 significantly inhibited E. coli adhesion to both HT-29 and Caco-2 cells compared with LGG. In conclusion, isolates GPI-4, GPI-7, and FA-5 showed excellent binding ability and antagonistic activity and better tolerance to acidic pH (pH 2.5) and to different bile salt concentrations in comparison with LGG, and hence, they could be considered as potential probiotic candidates.

IL-10 modulates in vitro multinucleate giant cell formation in human tuberculosis.

BACKGROUND: Multinucleated giant cells (MGC) are the histologic hallmark of granuloma which is known to limit tuberculosis infection. Both Th1 and Th2 type of cytokines regulate the immune response occurring within the granulomas. The objective of the study was to determine whether tuberculosis patient monocytes differed in their MGC forming ability as compared to healthy controls. METHODS: In vitro MGC formation was carried out by treatment of monocytes with cytokine containing culture supernatant of ConA or PPD stimulated peripheral mononuclear cells. IL-2, TNF-α, IL-4, IL-10 and TGF-ß cytokine levels were analysed in culture supernatants using ELISA. IL-4 and IL-10 were added to culture supernatant separately and simultaneously along with their respective neutralizing antibodies and their consequent effect on MGC formation was evaluated. RESULTS: MGC formation was significantly low in patient monocytes incubated with autologous culture supernatant as compared to control culture supernatant. Cytokine analysis of the culture supernatants revealed that while IL-4 levels were similar in patients and controls, increased IL-10 levels were found in patients. Exogenous addition of IL-10 resulted in reduced MGC formation. Contrastingly, when IL-4 was added exogenously, it led to increased MGC formation. The effects of both IL-10 and IL-4 were reversed upon addition of their respective antibodies. CONCLUSION: The findings suggest that one of the factors contributing to the disease could be the effect of cytokines on the functionality of monocytes, which are crucial in the fight against the organism. Significantly reduced MGC formation was observed on addition of IL-10. The findings imply an overriding role of IL-10 in MGC formation. The suppressive effect of IL-10 on MGC formation was further confirmed by addition of IL-10 neutralizing antibody.

Differential expression of alpha II spectrin in monocytes of tuberculosis patients.

Monocytes play a crucial role in immune response to tuberculosis. The present study focuses on identifying differences in the monocyte proteome profile of tuberculosis patients, household contacts and healthy controls. Differential protein expression was studied by two-dimensional (2D) gel electrophoresis. One of the spots consistently showed either lower intensity or was absent in patients and was identified as alpha II-spectrin. The decreased expression of αII-spectrin was further validated by quantitative PCR (qPCR) and western blot analysis. This study suggests the possible role of decreased levels of αII-spectrin in the pathology of tuberculosis.

Lactobacillus plantarum CS24.2 prevents Escherichia coli adhesion to HT-29 cells and also down-regulates enteropathogen-induced tumor necrosis factor-α and interleukin-8 expression.

The aim of the present study was to evaluate the potential of Lactobacillus plantarum CS24.2 to antagonize Escherichia coli adhesion and modulate expression of the responses by HT-29 cells of inflammatory molecules to E. coli adhesion. Experiments were performed under different adhesion conditions and findings compared with the responses of Lactobacillus rhamnosus GG. Tests of competitive adhesion, adhesion inhibition and displacement assays were performed for lactobacilli (L. rhamnosus GG and L. plantarum CS24.2) and E. coli O26:H11 to HT-29 cells. Both the lactobacilli significantly reduced E. coli adhesion to HT-29 cells (P < 0.05). The ability of lactobacilli to modulate tumor necrosis factor-α and interleukin-8 expression was analyzed in HT-29 cells stimulated with E. coli using qRT-PCR. L. plantarum CS24.2 significantly down regulated expression of both the genes induced by E. coli in HT-29 cells at 6 hr as well as 24 hr, which was more significant than the corresponding findings for L. rhamnosus GG. The present findings suggest that L. plantarum CS24.2 inhibits pathogen adhesion to a similar extent as does the established probiotic strain L. rhamnosus GG. It may also attenuate tumor necrosis factor-α and interleukin-8 expression in HT-29 cells stimulated with E. coli.

Liposomal formulations of Etoposide and Docetaxel for p53 mediated enhanced cytotoxicity in lung cancer cell lines.

The objective of present investigation was to develop and assess comparative enhancement in cytotoxicity of liposomal Etoposide and Docetaxel in non-small cell lung cancer cell lines after pre-treatment and co-administration of p53 tumor suppressor gene and to assess direct lung targeting of optimized formulations by dry powder inhaler technology. Cationic liposomes with and without drug were prepared and allowed to form p53-lipoplex for undertaking cytotoxicity studies in H-1299 (p53 null) and A-549 (p53 wt) cell lines. The optimized lipoplexes showed average size of 200-350 nm, zeta potential of 25-32 mV and sustained drug release up to 16-24 h. The developed liposomes and lipoplexes showed significant intracellular uptake and demonstrated enhanced cytotoxicity of 13-28 % after p53-drug co-administration and 41-63 % after p53 pre-treatment. The p53 mediated enhanced cytotoxicity by increased apoptosis and necrosis was also confirmed using Annexin V - FITC assay. The increased apoptosis suggested restored p53 function and reduced anti-apoptotic drug resistance theirby causing cell sensitization and synergism towards cytotoxicity. The studies conducted above demonstrated significant cell chemo-sensitization after p53 pre-treatment followed by Etoposide/Docetaxel liposomes administration than p53-Etoposide or p53-Docetaxel lipoplex co-administration; more significantly in Docetaxel and in H 1299 cell line. All the formulations when developed as dry powder inhalers showed significant in vitro lung deposition pattern in cascade impactor with fine particle faction of 33-37%. The study opens up a new strategy to treat lung cancer especially in cases of drug resistance. Moreover direct delivery to lung may provide an important role in complete remission of the disease due to target specificity.

Probiotic attributes of Lactobacillus strains isolated from food and of human origin.

Lactobacilli isolated from various sources were identified on the basis of 16S-23S rRNA gene intergenic region amplification and subsequent sequencing of the smaller intergenic region. An in vitro analysis of probiotic properties including binding, ability to tolerate different concentrations of bile, survival in acidic buffer and antimicrobial activity of four different isolates and two standard strains (Lactobacillus plantarum American Type Culture Collection (ATCC) 8014 and L. rhamnosus GG (LGG)) was carried out. The ability of each isolate to stimulate Caco-2 cells, human peripheral blood mononuclear cells (PBMC) and THP-1 cells resulting in immunomodulation of these cells was analysed. Isolates L. rhamnosus CS25 and L. delbrueckii M and standard strain ATCC 8014 showed broad antimicrobial activity, and isolates CS25 (percentage of survival 6.9 % at pH 2.5, 5.1 % at pH 2.0) and L. plantarum CS23 (5.7 % at pH 2.5, 4.9 % at pH 2.0) have shown good tolerance to acidic pH. Isolate CS23 showed a good survival (14 %) after 2 h incubation in de Man, Rogosa and Sharpe (MRS) medium containing 3 % bile salts. Isolates CS23, CS25 and L. fermentum ASt1 could stimulate Caco-2 cells, human PBMC and THP-1 cells for a strong and varied immunomodulatory response in these cells. Though LGG showed poor antimicrobial activity as well as bile and acid tolerance, it was found to be the best binding strain tested. Child faecal isolate CS23 from the present study showed high binding ability (seventeen bacteria/Caco-2), high tolerance to acidic pH and bile salts and significant immunomodulation; therefore it is a good potential probiotic candidate.

Intracellular delivery of nanoparticles of an antiasthmatic drug.

The aim of the investigation was to prepare and characterize wheat germ agglutinin(WGA)-conjugated poly(D: ,L-lactic-co-glycolic) acid nanoparticles encapsulating mometasone furoate (MF) as a model drug and assess changes in its fate in terms of cellular interactions. MF loaded nanoparticles were prepared using emulsion-solvent evaporation technique. WGA-conjugation was done by carbodiimide coupling method. The nanoparticles were characterized for size, zeta potential, entrapment efficiency and in-vitro drug release. The intracellular uptake of nanoparticles, drug cellular levels, and anti-proliferative activity studies of wheat germ agglutinin-conjugated and unconjugated nanoparticles were assessed on alveolar epithelial (A549) cells to establish cellular interactions. Prepared nanoparticles were spherical with 10-15 microg/mg of WGA conjugated on nanoparticles. The size of nanoparticles increased after conjugation and drug entrapment and zeta potential reduced from 78 +/- 5.5% to 60 +/- 2.5% and -15.3 +/- 1.9 to -2.59 +/- 2.1 mV respectively after conjugation. From the cellular drug concentration-time plot, AUC was found to be 0.4745, 0.6791 and 1.24 for MF, MF-nanoparticles and wheat germ agglutinin-MF-nanoparticles respectively. The in-vitro antiproliferative activity was improved and prolonged significantly after wheat germ agglutinin-conjugation. The results conclusively demonstrate improved availability and efficacy of antiasthmatic drug in alveolar epithelial cell lines. Hence, a drug once formulated as mucoadhesive nanoparticles and incorporated in dry powder inhaler formulation may be used for targeting any segment of lungs for more improved therapeutic response in other lung disorders as well.

Partial sequence analysis of VP1 of Indian isolates of foot-and-mouth disease virus type Asia-1.

Nucleotide sequence of 3' end of VP1 (1D region) was determined using RT-PCR amplified DNA of 31 foot and mouth disease virus (FMDV) type Asia-1 field isolates originating from 11 different geographically distinct states of India during the period 1987-2000. These field strains exhibited an average of 7.5% divergence among them and were found to be divergent from the Indian vaccine strains Asia-1 WBN 117/85, IND 8/79, and IND 63/72, by an average 5.9, 14.8, and 7.4% divergence, respectively. Phylogenetic analysis of these 31 field isolates including 3 of the vaccine strains of India and sequences of 22 Indian field isolates obtained from the GenBank revealed that all the Indian FMDV type Asia-1 isolates belonged to a single genotype comprising of two distinct lineages (Lineages A and B). All the field isolates under study belonged to the Lineage-B comprising 8 different clusters, which also includes the vaccine strains WBN-117/85 and IND 8/79. Surprisingly, another vaccine strain IND 63/72 formed Lineage-A. Phylogenetic analysis of sequences of another 23 exotic type Asia-1 isolates from 15 different countries obtained from the GenBank along with the 56 Indian isolates revealed the existence of three distinct genotypes. The prototype strain Asia-1 PAK 1/54 belongs to a separate genotype. Two strains from India along with one strain each from China and Russia belongs to another genotype. The third genotype is formed by the remaining isolates including all the 31 isolates from the present study and exotic viruses from 14 other different countries. Comparison of deduced amino acid (aa) sequence indicated that majority of the mutations were found within two distinct regions corresponding to amino acid positions 130-160 and 193-211. The motif at aa positions 138-141 in vaccine strains WBN 117/85, IND 8/79 and in all the field isolates was ETTS/P; however, the same motif in IND 63/72 was TQPT. The motif 153-156 in majority of Indian isolates including vaccine strains WBN 117/85 and IND 8/79 was LSGQ/R whereas the same motif seen in IND 63/72 was VSNR. The study revealed that the FMDV type Asia-1 isolates circulating in the country are not highly heterogeneous, but showed considerable genetic variations. Certain mutations were also observed in the residues, which have been proved to be contributing to the formation of neutralizing epitopes. In neutralization studies employing polyclonal antisera, type Asia-1 WBN 117/85 revealed broader serological spectrum than other vaccine strains of India used in this study.

In vitro assessment of acyclovir permeation across cell monolayers in the presence of absorption enhancers.

The aim of the investigation was to establish transepithelial permeation of acyclovir across Caco-2 and Madin-Darby canine kidney (MDCK) cell monolayers and attempt to improve its permeation by employing absorption enhancers (dimethyl beta cyclodextrin, chitosan hydrochloride and sodium lauryl sulfate) and combinations thereof. Caco-2 and MDCK cell monolayers have been widely employed in studying drug transport, mechanisms of drug transport, and screening of absorption enhancers and excipients. Transepithelial electrical resistance and permeation of 99mTc-mannitol were employed as control parameters to assess the tight junction and paracellular integrity. Permeation of acyclovir in the presence of absorption enhancers was found to be significantly higher compared with drug permeation in their absence when assessed as apparent permeability coefficients (Papp). Synergistic improvements in Papp values of acyclovir were obtained in case-selected combinations of absorption enhancers; dimethyl beta cyclodextrin-chitosan hydrochloride, chitosan hydrochloride-sodium lauryl sulfate, and dimethyl beta cyclodextrin-sodium lauryl sulfate, were used. Recovery and viability assessment studies of both cell monolayers suggested reestablishment of paracellular integrity and no damage to cell membranes. Significantly improved permeation of acyclovir in the presence of selected combinations of absorption enhancers may be used as a viable approach in overcoming the problem of limited oral bioavailability of acyclovir.

Development and characterization of monoclonal antibodies against FMD virus type Asia-1 and determination of antigenic variations in the field strains.

Twelve mouse monoclonal antibodies (MAbs) were developed against an Indian vaccine strain of foot and mouth disease virus (FMDV) type Asia-1 WBN 117/85. The MAbs were tested for their ability to bind to whole virus particle, trypsin-treated 146S (TT-146S) virus particle, sub-viral (12S and disrupted virus) antigens by ELISA and to neutralize virus infectivity in cell culture. Extensive characterization of MAbs revealed the existence of three different groups based on the binding of non-overlapping epitopes. Eight type Asia-1 specific MAbs (RF7, RF8, RD10, RE11, RC11, RC10/O, RB11 and RC10/M), which formed group 1 (G1), were found to bind a neutralizing, trypsin-sensitive (TS) and conformational epitope. Two MAbs (WB8 and WC3) in group 2 (G2) were found to bind a non-neutralizing, trypsin-resistant, conformational and 12S-specific epitope, which was intertypically conserved in all the four serotypes of FMDV (O, A, C and Asia-1) prevalent in India. Two MAbs (KG10 and KF10), which formed group 3 (G3), were found to be against a non-neutralizing, TS and conformational epitope, common to types Asia-1 and A. Members of G1 were IgG2a isotype, while those of G2 and G3 were IgG1 and IgG2b isotypes, respectively. Antigenic analysis of 31 FMDV type Asia-1 field isolates and two vaccine strains, using a panel of type Asia-1-specific MAbs, revealed antigenic similarity of the virus isolates tested and non-existence of neutralization escape mutants. The developed MAbs have practical utility, especially in the manufacture of FMD vaccine, diagnosis and FMDV characterization.

Modulation of ganciclovir intestinal absorption in presence of absorption enhancers.

The purpose of this investigation was to study the influences of absorption enhancers in increasing oral bioavailability of Ganciclovir (GAN) by assessing the transepithelial permeation across cell monolayers in vitro and bioavailability in rats in vivo. The permeation of GAN across Caco-2 and MDCK cell monolayers in the absence/presence of dimethyl-beta-cyclodextrin (DMbetaCD), chitosan hydrochloride (CH), sodium lauryl sulphate (SLS), and their combinations was studied for a 2-h period. GAN was administered to rats in absence/presence of absorption enhancers and drug contents in plasma were estimated. We found that the apparent permeability coefficient (Papp) of GAN in absence of absorption enhancers (control) were 0.261 +/- 0.072 x 10(-6) and 0.486 +/- 0.063 x 10(-6) cm/s in Caco-2 and MDCK cell monolayers, respectively, whereas in the presence of DMbetaCD, CH, SLS, and their combinations, Papp of GAN increased by 5- to 25-fold and 7- to 33-fold as compared to control in Caco-2 and MDCK cell monolayers, respectively. However, in rats, the maximum enhancement in bioavailability of GAN during coadministration of these absorption enhancers was only fivefold compared to GAN control. To conclude, the absorption enhancers-DMbetaCD, CH, SLS, and their combinations demonstrated significant improvement in transepithelial permeation and bioavailability of GAN.

Role of 99mTc-mannitol and 99mTc-PEG in the assessment of paracellular integrity of cell monolayers.

AIM: To assess the role of 99mTc-mannitol and 99mTc-polyethylene glycol 4000 in the evaluation of paracellular integrity of Caco-2 and Madine-Darby canine kidney (MDCK) cell monolayers, and confirm it in the presence of absorption promoters. METHODS: Radiolabelling of mannitol and polyethylene glycol was performed by a simple reduction method. Transepithelial electrical resistance values were measured to gain information regarding the integrity of tight junctions of Caco-2 and MDCK cell monolayers. Permeabilities of 99mTc-mannitol/99mTc-polyethylene glycol across cell monolayers were studied in the absence and presence of absorption promoters, namely dimethyl-beta-cyclodextrin, chitosan hydrochloride and sodium lauryl sulfate, and during recovery studies to assess paracellular integrity. RESULTS: Values for the apparent permeability coefficient (Papp) of Tc-mannitol were found to be 0.286 x 10 cm x s(-1) and 0.507 x 10 cm x s(-1) in Caco-2 and MDCK cell monolayers, respectively, whereas corresponding values for 99mTc-polyethylene glycol were 0.046 x 10 cm x s(-1) and 0.065 x 10 cm x s(-1). The insignificant Papp values of the marker molecules demonstrated the paracellular integrity of the cell monolayers. Significant increases in the Papp values in the presence of absorption promoters and their combinations due to opening of paracellular pathways and a return of Papp values to almost baseline values during recovery studies confirm the role of these marker molecules in the assessment of paracellular integrity of cell monolayers. CONCLUSION: 99mTc-labelled marker molecules can be attractive, useful and viable alternatives to the conventionally used markers in the assessment of paracellular integrity because of the absence of tissue-damaging corpuscular radiation and the ease of production of radiochemically pure and stable molecules at a reasonable cost.

Role of Caco-2 cell monolayers in prediction of intestinal drug absorption.

The usefulness of Caco-2 cell monolayers in determining the intestinal drug absorption of potential drug candidates as such and from delivery systems, elucidating the underlying mechanisms thereof, presystemic metabolism, cellular uptake and cytotoxicological assessment has been exemplified in this review. The role of Caco-2 cell monolayers in studying the effectiveness, involved mechanism and toxicity of various excipients for drug absorption promotion has also been discussed.