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OBJECTIVE: To examine the roles of specific genetic polymorphisms as predictors of response to treatment of early rheumatoid arthritis (RA). METHODS: Subjects included 457 patients with early RA (duration of < or =3 years) who participated in a randomized controlled trial comparing weekly methotrexate and 2 dosages of etanercept (10 mg twice weekly and 25 mg twice weekly). Our primary outcome measure was achievement of 50% improvement in disease activity according to the criteria of the American College of Rheumatology (ACR50 response) after 12 months of treatment. Subjects were genotyped for HLA-DRB1 alleles and polymorphisms in the following genes: TNF, LTA, TNFRSF1A, TNFRSF1B, FCGR2A, FCGR3A, and FCGR3B. Univariate and multivariate analyses were performed to define the impact of specific polymorphisms and haplotypes on response to treatment. Covariates for the multivariate analyses included sex, ethnicity, age, disease duration, and baseline values for rheumatoid factor and the tender and swollen joint counts. RESULTS: The presence of 2 HLA-DRB1 alleles encoding the shared epitope (SE) (compared with 1 or 0 copies) was associated with response to treatment with standard-dose etanercept (odds ratio [OR] 4.3, 95% confidence interval [95% CI] 1.8-10.3). Among Caucasian patients, 2 extended haplotypes that included the HLA-DRB1 alleles *0404 and *0101 (both of which encode the SE) and 6 single-nucleotide polymorphisms in the LTA-TNF region were associated with response to treatment. In a multivariate model that included treatment received and the aforementioned covariates, the ORs for the association of these haplotypes with achievement of an ACR50 response at 12 months were 2.5 (95% CI 0.8-7.3) and 4.9 (95% CI 1.5-16.1) for the *0404- and *0101-containing haplotypes, respectively. CONCLUSION: Genetic variation in the HLA-DRB1 and the LTA-TNF regions is significantly associated with response to treatment of early RA. These findings may have clinical application through the identification of patients who are most likely to benefit from treatment with methotrexate or etanercept. [ABSTRACT FROM AUTHOR]

The enteric nervous system arises predominantly from vagal level neural crest cells that migrate into and along the developing gut. As the neural crest-derived cells migrate within the gut, a subpopulation begins to differentiate into enteric neurons. Here, we show that the differentiation of neural crest-derived cells into enteric neurons is delayed in L1-deficient mice, compared with littermate controls. However, glial cell differentiation is not affected in L1-deficient mice. These mice also show a delay in the differentiation of a neurotransmitter-specific subtype of enteric neuron within the gastrointestinal tract. Together, these results suggest a role for the cell adhesion molecule, L1, in the differentiation of neural crest-derived cells into enteric neurons within the developing enteric nervous system., ((c) 2009 Wiley-Liss, Inc.)

The enteric nervous system arises predominantly from vagal level neural crest cells that migrate into the foregut and then colonize the entire length of the gastrointestinal tract. Previous studies have demonstrated that glial cell line-derived neurotrophic factor (GDNF) promotes the migration of enteric neural crest-derived cells (ENCs) in vitro, but a role for GDNF in the migration of ENCs in vivo has yet to be demonstrated. In this study, the effects of Gdnf haploinsufficiency on ENC rate of migration and number during mid embryonic development were examined. Although the entire gut of embryonic Gdnf(+/-) mice was colonized, a significant delay in the migration of ENCs along the embryonic hindgut was found. However, significant effects of Gdnf haploinsufficiency on ENC number were detected before the stage at which migration defects were first evident. As previous studies have shown a relationship between ENC number and migration, the effects of Gdnf haploinsufficiency on migration may be due to an indirect effect on cell number and/or a direct effect of GDNF on ENC migration. Gdnf haploinsufficiency did not cause any detectable change in the rate of neuronal differentiation of ENCs.

Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respective tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.

Background & Aims: During development, the enteric nervous system is derived from neural crest cells that emigrate from the hindbrain, enter the foregut, and colonize the gut. Defects in neural crest migration can result in intestinal aganglionosis. Hirschsprung's disease (congenital aganglionosis) is a human condition in which enteric neurons are absent from the distal bowel. A number of clinical studies have implicated the cell adhesion molecule L1 in Hirschsprung's disease. We examined the role of L1 in the migration of neural crest cells through the developing mouse gut., Methods: A variety of in vitro and in vivo assays were used to examine: (1) the effect of L1 blocking antibodies or exogenous soluble L1 protein known to compromise L1 function on the rate of crest cell migration, (2) the effect of blocking L1 activity on the dynamic behavior of crest cells using time-lapse microscopy, and (3) whether the colonization of the gut by crest cells in L1-deficient mice differs from control mice., Results: We show that L1 is expressed by neural crest cells as they colonize the gut. Perturbation studies show that disrupting L1 activity retards neural crest migration and increases the number of solitary neural crest cells. L1-deficient mice show a small but significant reduction in neural crest cell migration at early developmental stages, but the entire gastrointestinal tract is colonized., Conclusions: L1 is important for the migration of neural crest cells through the developing gut and is likely to be involved in the etiology of Hirschsprung's disease.

Neural crest cells that originate in the caudal hindbrain migrate into and along the developing gastrointestinal tract to form the enteric nervous system. While they are migrating, neural-crest-derived cells are also proliferating. Previous studies have shown that the expression of glial-derived neurotrophic factor (GDNF) and endothelin-3 is highest in the embryonic caecum, and that GDNF alone or in combination with endothelin-3 promotes the proliferation of enteric neural-crest-derived cells in vitro. However, whether neural proliferative zones, like those in the central nervous system, are found along the developing gut is unknown. We used a fluorescent nucleic acid stain to identify dividing cells or BrdU labelling (2 h after administration of BrdU to the mother), combined with antibodies specific to neural crest cells to determine the percentage of proliferating crest-derived cells in various gut regions of embryonic day 11.5 (E11.5) and E12.5 mice. The rate of proliferation of crest-derived cells did not vary significantly in different regions of the gut (including the caecum) or at different distances from the migratory wavefront of vagal crest-derived cells. The phenotype of mitotic enteric crest-derived cells was also examined. Cells expressing the pan-neuronal markers, neurofilament-M and Hu, or the glial marker, S100b, were observed undergoing mitosis. However, no evidence was found for proliferation of cells expressing neuron-type-specific markers, such as nitric oxide synthase (at E12.5) or calcitonin gene-related peptide (at E18.5). Thus, for enteric neurons, exit from the cell cycle appears to occur after the expression of pan-neuronal proteins but prior to the expression of markers of terminally differentiated neurons.