NOX1 loss-of-function genetic variants in patients with inflammatory bowel disease.

Genetic defects that affect intestinal epithelial barrier function can present with very early-onset inflammatory bowel disease (VEOIBD). Using whole-genome sequencing, a novel hemizygous defect in NOX1 encoding NAPDH oxidase 1 was identified in a patient with ulcerative colitis-like VEOIBD. Exome screening of 1,878 pediatric patients identified further seven male inflammatory bowel disease (IBD) patients with rare NOX1 mutations. Loss-of-function was validated in p.N122H and p.T497A, and to a lesser degree in p.Y470H, p.R287Q, p.I67M, p.Q293R as well as the previously described p.P330S, and the common NOX1 SNP p.D360N (rs34688635) variant. The missense mutation p.N122H abrogated reactive oxygen species (ROS) production in cell lines, ex vivo colonic explants, and patient-derived colonic organoid cultures. Within colonic crypts, NOX1 constitutively generates a high level of ROS in the crypt lumen. Analysis of 9,513 controls and 11,140 IBD patients of non-Jewish European ancestry did not reveal an association between p.D360N and IBD. Our data suggest that loss-of-function variants in NOX1 do not cause a Mendelian disorder of high penetrance but are a context-specific modifier. Our results implicate that variants in NOX1 change brush border ROS within colonic crypts at the interface between the epithelium and luminal microbes.

Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.

Co-expression of Flt-3 ligand gene ablates tumor immunity elicited by HER-2/neu DNA vaccine in transgenic mice.

Fms-like tyrosine-kinase 3 ligand (Flt-3L), is a powerful hematopoyetic growth factor, known to modulate the immune response against delivered antigens by acting either as an adjuvant or tolerogenic stimulus. In this study we evaluated the use of murine Flt-3 ligand plasmid (pFl) in combination with a DNA vaccine encoding rat-p185 oncoprotein extra cellular domain (pECD) in the prevention of mammary carcinogenesis in rat-neu HER-2 mutated (neuT) transgenic mice. We demonstrate that intramuscular (i.m.) co-immunization of pFl inhibits the production of anti-HER-2 antibody elicited by pECD vaccine, resulting in the development of spontaneous carcinomas in all co-immunized mice. The inhibitory effect on antibody production by mFlt3 gene appeared to be: dose-dependent, linked to the injection site and timing, and transient in nature. Additionally, we show that co-administration of pFI and pECD plasmids was unable to trigger cytotoxic T-cell immune response in neuT mice. On the other hand, we found that the combination of pFl with pECD had no impact on the ability of pECD to reject HER-2+ transplantable tumors in parental mice. In summary our results demonstrate that, depending on tumor model, co-administration of pFl gene can produce untoward effects to immune response, and thus its application as a vaccine adjuvant should be carefully evaluated.