Uses of mammalian genes and related reagents

Methods for treating, diagnosing, or evaluating mucosal surface medical conditions. Correlations of chemokine or receptor expression with gastrointestinal status are provided.

EXAMPLES I. General Methods Some of the standard methods are described or referenced, e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York. Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g., Ausubel, et al. (1987 and periodic supplements); Deutscher (1990) “Guide to Protein Purification” in Meth. Enzymol., vol. 182, and other volumes in this series; and manufacturer's literature on use of protein purification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combination with recombinant techniques allow fusion to appropriate segments, e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence. See, e.g., Hochuli (1990) “Purification of Recombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.) Genetic Engineering. Principle and Methods 12:87-98, Plenum Press, N.Y.; and Crowe, et al. (1992) QIAexpress: The High Level Expression & Protein Purification System QIAGEN, Inc., Chatsworth, Calif. Computer sequence analysis is performed, e.g., using available software programs, including those from the GCG (U. Wisconsin) and GenBank sources. Public sequence databases were also used, e.g., from GenBank and others. II. RNA Preparation RNA was extracted from intestinal tissue either using guanidinium thiocyanate followed by centrifugation in cesium chloride for surgical samples or RNA STAT-60 (Tel-Test) for biopsies. RNA quality was assessed by agarose gel electrophoresis. Five &mgr;g of total RNA was treated with RNAse-free DNAse I (Boehringer Mannheim) in First Strand Synthesis Buffer in the presence of RNAsin (Promega). Samples were incubated for 20 minutes at 37° C., heated for 10 minutes at 70° C., and then immediately chilled on ice. A mixture of 2.5 &mgr;g of oligo d(T) 12-15 (Boehringer Mannheim) and 250 ng of random hexamers (Promega) was added to each sample. Samples were heated to 70° C. for 10 minutes, rapidly chilled on ice, and then briefly spun in a microfuge. cDNA was generated from the RNA using Superscript II reverse transcriptase (GIBCO-BRL) according to manufacturer's instructions in a final volume of 100 &mgr;L. III. Patient Evaluation Patient intestinal samples were grouped two different ways. In the first grouping patient samples were into one of five groups, based on the clinical and pathological diagnoses of the patients. Non-lesional samples from the same patients were also studied, however, none of these samples was statistically significant from the control group. A. Controls: There were twenty-eight samples in the control group, including samples from both colon and small intestine. Samples in this group were surgical samples from patients with normal adjacent intestinal tissue removed during excision of a tumor (n&equals;12), biopsies from patients with chronic colitis (n&equals;4), surgical specimens from patients diagnosed with diverticulosis (n&equals;5), and biopsies from patients undergoing routine endoscopy (n&equals;2). In addition, five intestinal samples were collected during autopsy. B. Crohn's Diseas, lesional, no steroids: There were six samples from patients, diagnosed with Crohn's Disease who had no history of recent steroid use. Four of these were biopsies and two surgical samples. C. Crohn's Disease, lesional, steroids: There were four samples from patients, diagnosed with Crohn's Disease who had recently been treated with steroids. All four were surgical samples. D. Ulcerative colitis, lesional, no steroids: There were eight samples from patients, diagnosed with ulcerative colitis who had no history of recent steroid use. Seven of these were biopsies and one a surgical sample. E. Ulcartive colitis, lesional, steroids: There are five samples from patients, diagnosed with ulcerative colitis and who had recently been treated with steroids. All five were surgical samples. In addition to the above groups the data collected from the above samples were all grouped as described below and analyzed as described in the next section. A. Controls: This group was the same as described above. B. Crohn's Disease, lesional, all: This group had 15 samples and included all samples in both Crohn's Disease lesional groups described above as well as five additional samples for which treatment history was not available. C. Ulcartive colitis, lesional, all: This group had 22 samples and included all samples in both Ulcerative colitis lesional groups described. IV. Gene Expression Analysis by TaqMan Ten ng of cDNA/reaction was analyzed for expression of selected chemokines, chemokine receptors, and ubiquitin on a GeneAmp 5700 Sequence Detector (PE Applied Biosystems) in a 25 &mgr;L reaction. Expression of chemokines and chemokine receptors was detected using primers and probe (PE Applied Biosystems) with TaqMan Universal Master Mix (PE Applied Biosystems) or primers alone and SYBR Green PCR Master Mix (PE Applied Biosystems). TaqMan reagents to detect expression of given chemokines or their receptors were extensively tested to verify that they only recognized the appropriate target sequence and not closely related family members. Ubiquitin was detected using 200 nM primers (F: CACTTGGTCCTGCGCTTGA; R: CAATTGGGAATGCAACAACTTTAT) with SYBR Green PCR Mater Mix. The data was analyzed to calculate a cycle threshold value (Ct) for each sample with GeneAmp 5700 SDS Software (PE Applied Biosystems). The relative level of CCL18 mRNA in the tissue using the following formula: 1.8(Ct of ubiquitin −Ct of gene of interest)10,000 for each sample. Mean and standard error were calculated for each group. V. Statistical analysis Statistical analysis was performed with JMP 3.2.2 (SAS Institute, Inc.). The TaqMan data for the relative level of chemokine and chemokine recpetors was log transformed and a one-way ANOVA performed. The log transformed of chemokine and chemokine recpetor level was used as the dependent variable and the disease group was used as the independent variable. All groups were compared to the control group using a modified Student t-test, Dunnett's method. Increased expression of several chemokine and their receptors in lesional tissue samples from patients with inflammatory bowel diseases, Crohn's Disease and ulcerative colitis was detected (see Table 1). 1 TABLE 1 Gene expression levels of chemokines and receptors. Bold type indicates a statistically significant fold increase in gene expression over that of the control group. Fold Increase in Gene Expression over Controls n TARC CCR4 CCR8 MIP-3&agr; CCR6 CCR7 TNF-&agr; Controls 28 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Crohn's Lesional, 6 3.8 7.9 8.3 7.4 6.2 3.9 4.4 no steriods Crohn's Lesional, 4 1.6 1.8 0.9 2.5 0.9 2.3 0.5 steriods Crohn's Lesional, 15 2.9 4.4 3.5 2.4 2.5 4.2 1.6 all Ulcerative colitis, 8 2.7 10.0 7.5 5.0 2.5 3.1 2.4 no steriods Ulcerative colitis, 5 2.4 3.3 1.2 1.6 0.8 2.0 0.9 steriods Ulcerative colitis, 22 2.4 5.2 2.2 2.4 1.2 2.7 1.4 all The chemokine receptors studied here are expressed on lymphocytes and antigen expressing cells. Recruitment of these cell types, which promote immune responses, into intestinal lesions may result in the maintenance and magnification of the chronic underlying these debilitating diseases. While the expression of several of the chemokines and chemokine receptors studied here is decreased by steroid treatment, it is not abolished in all cases. The results above indicate that these molecules may represent new targets for dampening the immune response, without the broad systemic effects induced by chronic steroid use. All citations herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled; and the invention is not to be limited by the specific embodiments that have been presented herein by way of example.