Bypassing luminal barriers, delivery to a gut addressin by parenteral targeting elicits local IgA responses

Induction of mucosal immunity, particularly to subunit vaccines, has been problematic. The primary hurdle to successful mucosal vaccination is the effective delivery of vaccine antigen to the mucosal associated lymphoid tissue. Physical and chemical barriers restrict antigen access and, moreover, immune responses induced in the mucosa can be biased towards tolerance or non-reactivity. We proposed that these difficulties could be circumvented by targeting antigen to the gastrointestinal associated lymphoid tissue via systemic (parenteral) rather than alimentary routes, using antibodies specific for the mucosal addressin cellular adhesion molecule-1 (MAdCAM). After intravenous or intramuscular injection of such rat antibodies in mice, we found a greatly enhanced (up to 3 logs) anti-rat antibody response. MAdCAM targeting induces a rapid IgA antibody response in the gut and vastly improves the systemic antibody response. Targeting also enhanced T cell proliferation and cytokine responses. Parenteral targeting of mucosal addressins may represent a generic technique for bypassing mucosal barriers and eliminating the need for adjuvants in the induction of proximal and systemic immunity.

Novel agents that potentially inhibit irinotecan-induced diarrhea

Irinotecan (CPT-11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) has exhibited clinical activities against a broad spectrum of carcinomas by inhibiting DNA topoisomerase I (Topo I). However, severe and unpredictable dosing-limiting toxicities (mainly myelosuppression and severe diarrhea) hinder its clinical use. The latter consists of early and late-onset diarrhea, occurring within 24 hr or ≥ 24 hr after CPT-11 administration, respectively. This review highlights novel agents potentially inhibiting CPT-11-induced diarrhea, which are designed and tested under guidance of disposition pathways and potential toxicity mechanisms. Early-onset diarrhea is observed immediately after CPT-11 infusion and probably due to the inhibition of acetylcholinesterase activity, which can be eliminated by administration of atropine. Lateonset diarrhea appears to be associated with intestinal exposure to SN-38 (7-ethyl-10-hydroxycamptothecin), the major active metabolite of CPT-11, which may bind to Topo I and induce apoptosis of intestinal epithelia, leading to the disturbance in the absorptive and secretory functions of mucosa. CPT-11 and SN-38 may also stimulate the production of pro-inflammatory cytokines and prostaglandins (PGs), thus inducing the secretion of Na⁺ and Cl^-. Early treatment of severe late-onset diarrhea with oral high-dose loperamide has decreased patient morbidity. Extensive studies have been conducted to identify other potential agents to ameliorate diarrhea in preclinical and clinical models. These include intestinal alkalizing agents, oral antibiotics, enzyme inducers, P-glycoprotein (PgP) inhibitors, cyclooxygenase-2 (COX-2) inhibitors, tumor necrosis factor-agr (TNF-α) inhibitors, or blockers of biliary excretion of SN-38. Further studies are needed to identify the molecular targets associated with CPT-11 toxicity and safe and effective agents for alleviating CPT-11-induced diarrhea.

Oxidised mannan as a novel adjuvant inducing mucosal IgA production.

Mannan, oxidatively coupled to recombinant protein antigens, has here been tested as a possible adjuvant for the production of antibody on the mucosa. Given intranasally, but not intraperitoneally, mannan markedly enhanced the production of IgA, IgG1 and IgG2a in the serum, and IgA locally in the lung and at remote mucosal sites, including tears, vaginal and salivary secretions. Oxidative coupling was critical to its action, since neither mannan simply mixed with protein nor mannan–protein conjugates which had been reduced by treatment with sodium borohydride, acted as adjuvants. Oxidatively coupled mannan was compared with the widely studied mucosal adjuvant, cholera toxin (CT). The use of oxidised mannan as an adjuvant induced better responses than CT judged by the induction of IgA in serum, vaginal washings and saliva. Thus, oxidised mannan, which is non-toxic and can be administered without injection, is a suitable adjuvant coupled with protective antigens for vaccinating against a number of infections that occur via the mucous membranes.

The effect of folic acid supplementation on dna biomarkers of colorectal cancer risk (uracil misincorporation, global and gene-specific dna hypomethylation) : a randomised intervention study

Background - Alterations in folate status are associated with colorectal carcinogenesis. Folate’s role has been postulated to be either via prevention of changes in DNA methylation or uracil misincorporation.
Objective - To investigate the effect of folic acid supplementation on colonocyte folate status and DNA biomarkers.
Design - Twenty individuals harbouring colonic adenomas were randomised to receive folic acid (600 g daily) or placebo for 6 months post polypectomy. Systemic and colonocyte folate status was determined at baseline and following the intervention. Modified Comet assays were used to determine uracil misincorporation and global DNA hypomethylation at the site adjacent to the polyp and a site distal to the polyp.
Outcomes - Supplementation resulted in increased colonocyte folate, which approached significance, at the site adjacent to the polyp (P= 0.06) but not distal to the polyp (P= 0.36); correspondingly there was a reduction in uracil misincorporation at the site adjacent to the polyp (P = 0.02) and the distal site showed no such trend (P= 0.39). There were no significant changes in global DNA hypomethylation at either site post-intervention.
Conclusions - Folic acid supplementation resulted in increased colonocyte folate and decreased uracil misincorporation at the site of the adenoma but not distal to the adenoma. This supports the hypothesis that localised areas of folate deficiency may exist in human colonic mucosa which respond to folic acid supplementation through increasing colonocyte folate and improving folate-related DNA biomarkers of cancer risk.

Defining the interaction of HIV-1 with the mucosal barriers of the female reproductive tract

Worldwide, HIV-1 infects millions of people annually, the majority of whom are women. To establish infection in the female reproductive tract (FRT), HIV-1 in male ejaculate must overcome numerous innate and adaptive immune factors, traverse the genital epithelium, and establish infection in underlying CD4(+) target cells. How the virus achieves this remains poorly defined. By utilizing a new technique, we define how HIV-1 interacts with different tissues of the FRT using human cervical explants and in vivo exposure in the rhesus macaque vaginal transmission model. Despite previous claims of the squamous epithelium being an efficient barrier to virus entry, we reveal that HIV-1 can penetrate both intact columnar and squamous epithelial barriers to depths where the virus can encounter potential target cells. In the squamous epithelium, we identify virus entry occurring through diffusive percolation, penetrating areas where cell junctions are absent. In the columnar epithelium, we illustrate that virus does not transverse barriers as well as previously thought due to mucus impediment. We also show a statistically significant correlation between the viral load of inocula and the ability of HIV-1 to pervade the squamous barrier. Overall, our results suggest a diffusive percolation mechanism for the initial events of HIV-1 entry. With these data, we also mathematically extrapolate the number of HIV-1 particles that penetrate the mucosa per coital act, providing a biological description of the mechanism for HIV-1 transmission during the acute and chronic stages of infection.

The orexinergic system in rainbow trout Oncorhynchus mykiss and its regulation by dietary lipids

In this study, we report the distribution of orexin A (OXA), orexin B (OXB), and orexin receptor (OX2R) immunoreactive (ir) cells in the hypothalamus and gastrointestinal tract of Oncorhynchus mykiss fed diets with different dietary fatty acid compositions. Trout were fed five iso-energetic experimental diets containing fish oil, or one of four different vegetable oils (olive, sunflower, linseed, and palm oils) as the added dietary lipid source for 12 weeks. OXA, OXB, and OX2R immunoreactive neurons and nervous fibers were identified in the lateral and ventro-medial hypothalamus. OXA, OXB, and OX2R ir cells were found in the mucosa and glands of the stomach and in the mucosa of both the pyloric cecae and intestine. OX2R ir cells were localized in the mucosa layer of both the pyloric cecae and intestine. These immunohistochemical (IHC) results were confirmed via Western blotting. Antibodies against preproorexin (PPO) crossreacted with a band of ∼16 kDa in the hypothalamus, stomach, pyloric cecae, and intestine. Antibodies against OX2R crossreacted with a band of ∼38 kDa in the hypothalamus, pyloric cecae, and intestine. The presence and distribution of OXA, OXB, and OX2R ir cells in the hypothalamus and gastrointestinal tract did not appear to be affected by dietary oils. The presence of orexin system immunoreactive cells in the stomach, pyloric cecae, and intestine of rainbow trout, but not in the enteric nervous system, could suggest a possible role of these peptides as signaling of gastric emptying or endocrine modulation, implying a main local action played by orexins.