Does larch arabinogalactan enhance immune function? A review of mechanistic and clinical trials.

The common cold is a viral infection with important economic burdens in Western countries. The research and development of nutritional solutions to reduce the incidence and severity of colds today is a major focus of interest, and larch arabinogalactan seems to be a promising supportive agent. Arabinogalactan has been consumed by humans for thousands of years and is found in a variety of common vegetables as well as in medicinal herbs. The major commercial sources of this long, densely branched, high-molecular-weight polysaccharide are North American larch trees. The aim of this article is to review the immunomodulatory effects of larch arabinogalactan derived from Larix laricina and Larix occidentalis (North American Larix species) and more specifically its role in the resistance to common cold infections. In cell and animal models, larch arabinogalactan is capable of enhancing natural killer cells and macrophages as well as the secretion of pro-inflammatory cytokines. In humans a clinical study demonstrated that larch arabinogalactan increased the body's potential to defend against common cold infection. Larch arabinogalactan decreased the incidence of cold episodes by 23 %. Improvements of serum antigen-specific IgG and IgE response to Streptococcus pneumoniae and tetanus vaccination suggesting a B cell dependent mechanism have been reported in vaccination studies with larch arabinogalactan, while the absence of response following influenza vaccination suggests the involvement of a T cell dependent mechanism. These observations suggest a role for larch arabinogalactan in the improvement of cold infections, although the mode of action remains to be further explored. Different hypotheses can be envisaged as larch arabinogalactan can possibly act indirectly through microbiota-dependent mechanisms and/or have a direct effect on the immune system via the gut-associated lymphoid tissue (GALT).

Evaluation of the anti-inflammatory and antioxidative potential of four fern species from China intended for use as food supplements.

Inflammation plays a major role in many diseases, for instance in arteriosclerosis, rheumatoid arthritis, autoimmune disorders and cancer. Since many plants contain compounds with anti-inflammatory activity, their consumption may be able to prevent the development of inflammatory-based diseases. Edible ferns are some of the most important wild vegetables in China and have traditionally been used both for dietary and therapeutic purposes. In this study we investigated the anti-inflammatory and antioxidant potential of fern extracts from Matteuccia struthiopteris, Osmundajaponica, Matteuccia orientalis and Pteridium aquilinum intended for use as nutraceuticals. Two modes of action were investigated: the inhibition of the pro-inflammatory gene expression of interleukin-1 beta (IL1-ß) and interleukin-6 (IL6), and the gene expression of iNOS by LPS-elicited macrophages. The results showed a decrease of IL1-ß gene expression for the five fern extracts. This effect was more pronounced for the extracts prepared from the roots of O. japonica (IC50 of 17.8 µg/mL) and the young fronds of M orientalis (50.0 µg/mL). Regarding the indirect measurement of NO, via iNOS gene expression, an interesting decrease of 50% was obtained with the extract of M. orientalis fronds at a low concentration (20 µg/mL) compared with P. aquilinum fronds (160 µg/mL) and leaves of O. japonica. The latter showed a higher decrease but at a high concentration of extract (160 µg/mL). The five fern extracts were also evaluated for their ability to scavenge 2,2-diphenyl-l-picrylhydrazyl (DPPH) radicals and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). All fern extracts exhibited antioxidant effects but the roots of O. japonica and the fronds of M orientalis were most efficient. The HPLC-MS analysis of the constituents of the fern extracts confirmed the presence of chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, kaempferol and apigenin, molecules known to exhibit antiinflammatory and/or antioxidant properties.

Signaling via class IA Phosphoinositide 3-kinases (PI3K) in human, breast-derived cell lines.

We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, ß, δ and γ) and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110ß>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not ß- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not ß- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/-) cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110ß, but not α- or δ- activity; in PTEN(-/-) MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110ß function in the absence of PTEN.

A case-control study of colorectal cancer detection by quantification of DNA isolated from directly collected exfoliated colonocytes.

This pilot study aimed to assess an original test based on the analysis of exfoliated colonocytes as a new approach to colorectal cancer (CRC) detection. DNA was isolated from exfoliated cells collected from the surface of the rectal mucosa by a standardized minimally invasive procedure in a case-control trial involving 66 patients with CRC diagnosis and 110 healthy volunteers (age 50-70). PicoGreen staining and quantitative real-time PCR (QRTPCR) were used for DNA quantification. Mean DNA scores in microg/ml obtained for the control and cancer groups were 2.1 (95% CI 1.7-2.5) and 9.0 (CI 6.7-11.2) respectively (p < 0.001) for PicoGreen and 0.8 (CI 0.6-0.9) and 3.8 (CI 1.9-5.7) respectively (p = 0.003) for QRTPCR. The PicoGreen assay better detected CRC presence. At DNA score cut-off point of 2.5 microg/ml this assay gave sensitivities of 77.8% (CI 52.4-93.6) for proximal tumours, 91.4% (CI 76.9-98.2) for distal CRC and 86.8% (CI 74.7-94.5) for all CRC with specificity at 74.0% (CI 64.0-82.4). Increasing the cut-off point to 5.0 microg/ml resulted in sensitivities of 38.9% (CI 17.3-64.3) for proximal tumours, 71.4% (CI 53.7-85.4) for distal CRC and 60.4% (CI 46.0-73.5) for all CRC. Specificity for this cut-off point increased to 94.8% (CI 88.3-98.3). The new procedure of exfoliated cell collection from the surface of the rectal mucosa is a simple, safe and well-tolerated technique providing high quality cells. These early results suggest that exfoliated cell collection in combination with DNA quantification can potentially be employed as a tool for CRC early detection.

Expression of GIMAP1, a GTPase of the immunity-associated protein family, is not up-regulated in malaria.

BACKGROUND: GIMAP (GTPase of the immunity-associated protein family) proteins are a family of putative GTPases believed to be regulators of cell death in lymphomyeloid cells. GIMAP1 was the first reported member of this gene family, identified as a gene up-regulated at the RNA level in the spleens of mice infected with the malarial parasite, Plasmodium chabaudi. METHODS: A monoclonal antibody against mouse GIMAP1 was developed and was used to analyse the expression of the endogenous protein in tissues of normal mice and in defined sub-populations of cells prepared from lymphoid tissues using flow cytometry. It was also used to assess the expression of GIMAP1 protein after infection and/or immunization of mice with P. chabaudi. Real-time PCR analysis was employed to measure the expression of GIMAP1 for comparison with the protein level analysis. RESULTS: GIMAP1 protein expression was detected in all lineages of lymphocytes (T, B, NK), in F4/80+ splenic macrophages and in some lymphoid cell lines. Additional evidence is presented suggesting that the strong expression by mature B cells of GIMAP1 and other GIMAP genes and proteins seen in mice may be a species-dependent characteristic. Unexpectedly, no increase was found in the expression of GIMAP1 in P. chabaudi infected mice at either the mRNA or protein level, and this remained so despite applying a number of variations to the protocol. CONCLUSION: The model of up-regulation of GIMAP1 in response to infection/immunization with P. chabaudi is not a robustly reproducible experimental system. The GIMAP1 protein is widely expressed in lymphoid cells, with an interesting increase in expression in the later stages of B cell development. Alternative approaches will be required to define the functional role of this GTPase in immune cells.

Colorectal cancer detection by measuring DNA from exfoliated colonocytes obtained by direct contact with rectal mucosa.

The purpose of the study was to explore the potential of direct exfoliated colonocyte collection from human rectal mucosa for colorectal cancer screening. A special device was designed for standardized collection of exfoliated cells from the surface of human rectal mucosa. Material was collected from 120 outpatients selected for colonoscopy and 36 patients with confirmed diagnosis of colorectal cancer or large polyps. Determination of total DNA amounts in the collected samples (DNA scores) by PicoGreen assay and real-time PCR was employed alongside cytological assessment. Well preserved cells with cytological patterns characteristic for different colorectal conditions (cancer, inflammatory bowel disease) were detected in the collected material. In the outpatient group DNA scores were higher in patients with cancer and inflammatory bowel disease compared to those with no abnormalities detected, diverticular disease and small polyps (P<0.001 for PicoGreen assay; P=0.002 for real-time PCR). The sensitivity and specificity of the quantitative DNA test (PicoGreen assay; cut-off point 3.0 microg/ml) for detecting serious colorectal conditions were 1.00 and 0.74, respectively. In the group with confirmed tumours, the PicoGreen assay performed better for distal colorectal cancer (sensitivity 0.83; specificity 0.76) compared with proximal colon malignancies (sensitivity 0.57; specificity 0.76). It can be concluded that the proposed technique of direct collection of exfoliated cells from the surface of human rectal mucosa provides abundant cellular material suitable for diagnostic and research applications. Further refinement of the quantitative DNA test may lead to a new approach for colorectal cancer early detection and screening.

A natural hypomorphic variant of the apoptosis regulator Gimap4/IAN1.

The Gimap/IAN family of GTPases has been implicated in the regulation of cell survival, particularly in lymphomyeloid cells. Prosurvival and prodeath properties have been described for different family members. We generated novel serological reagents to study the expression in rats of the prodeath family member Gimap4 (IAN1), which is sharply up-regulated at or soon after the stage of T cell-positive selection in the thymus. During these investigations we were surprised to discover a severe deficiency of Gimap4 expression in the inbred Brown Norway (BN) rat. Genetic analysis linked this trait to the Gimap gene cluster on rat chromosome 4, the probable cause being an AT dinucleotide insertion in the BN Gimap4 allele (AT(+)). This allele encodes a truncated form of Gimap4 that is missing 21 carboxyl-terminal residues relative to wild type. The low protein expression associated with this allele appears to have a posttranscriptional cause, because mRNA expression was apparently normal. Spontaneous and induced apoptosis of BN and wild-type T cells was analyzed in vitro and compared with the recently described mouse Gimap4 knockout. This revealed a "delayed" apoptosis phenotype similar to but less marked than that of the knockout. The Gimap4 AT(+) allele found in BN was shown to be rare in inbred rat strains. Nevertheless, when wild rat DNA samples were studied the AT(+) allele was found at a high overall frequency ( approximately 30%). This suggests an adaptive significance for this hypomorphic allele.

Expression of the Ian family of putative GTPases during T cell development and description of an Ian with three sets of GTP/GDP-binding motifs.

Reports suggest that two members of the novel immune-associated nucleotide (Ian) GTPase family, Ian1 and Ian5, play roles in T cell development. We performed real-time PCR analysis of the expression of Ian genes of the rat during T cell maturation, in macrophages and in cell lines. We found that all of the genes were expressed at relatively low levels at the early double-negative thymocyte stage but were expressed more strongly at later cell stages. Our study also revealed the fact that the previously reported Ian9, Ian10 and Ian11 genes are, instead, parts of a single gene for which we retain the name Ian9, potentially encoding a GTPase with a highly unusual triplicated structure. Antisera were developed against both Ian1 and Ian9. We established that Ian9 is produced as an approximately 75-kDa protein in both T cells and thymocytes. We observed that levels of both Ian1 and Ian9 proteins are profoundly reduced in T cells from lymphopenic rats as compared with wild-type rats. It was demonstrated that thymocytes and B cells from lymphopenic rats (Ian5 null) did not show enhanced sensitivity to gamma-irradiation-induced apoptosis.

Measurement of faecal immunoglobulin a levels in young children.

A nephelometric immunoassay was developed to quantify immunoglobulin A (IgA) in children's stools. This method enables IgA in faecal protein extracts to be measured over a large range of concentrations (1.61-51.50 mg/L) with good accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation (CVs) of 1-6%). An excellent recovery (105%) was obtained in stool samples overloaded by purified colostral IgA, demonstrating that the method used for faecal IgA extraction is adapted, not to induce significant IgA degradation, and probably allow a complete extraction of IgA. The amount of faecal IgA, as determined in stool samples from 125 children (6-24 months old), was an average of 14 mg per 100 g of stools (about 10% of the total protein stool content), with large individual variation (3-30 mg per 100 g of stools). No correlation was observed between faecal IgA amounts and the children's age or sex. Such an immunoassay could enable exhaustive noninvasive investigations of the maturation of the intestinal immune system, as well as accurate studies of the effect of oral dietary supplementation on IgA regulation.