Defining the caprine γδ T cell WC1 multigenic array and evaluation of its expressed sequences and gene structure conservation among goat breeds and relative to cattle.

Workshop cluster 1 (WC1) molecules are part of the scavenger receptor cysteine-rich (SRCR) superfamily and act as hybrid co-receptors for the γδ T cell receptor and as pattern recognition receptors for binding pathogens. These members of the CD163 gene family are expressed on γδ T cells in the blood of ruminants. While the presence of WC1+ γδ T cells in the blood of goats has been demonstrated using monoclonal antibodies, there was no information available about the goat WC1 gene family. The caprine WC1 multigenic array was characterized here for number, structure and expression of genes, and similarity to WC1 genes of cattle and among goat breeds. We found sequence for 17 complete WC1 genes and evidence for up to 30 SRCR a1 or d1 domains which represent distinct signature domains for individual genes. This suggests substantially more WC1 genes than in cattle. Moreover, goats had seven different WC1 gene structures of which 4 are unique to goats. Caprine WC1 genes also had multiple transcript splice variants of their intracytoplasmic domains that eliminated tyrosines shown previously to be important for signal transduction. The most distal WC1 SRCR a1 domains were highly conserved among goat breeds, but fewer were conserved between goats and cattle. Since goats have a greater number of WC1 genes and unique WC1 gene structures relative to cattle, goat WC1 molecules may have expanded functions. This finding may impact research on next-generation vaccines designed to stimulate γδ T cells.

Cervical FISH Testing for Triage and Support of Challenging Diagnoses: A Case Study of 2 Patients.

PATIENTS: A 29-year-old Caucasian woman (patient 1) and a 23-year-old Caucasian woman (patient 2).Chief Complaints: Abnormal Pap tests with high risk HPV positivity. PATIENT 1: In June 2014, this patient was diagnosed with atypical squamous cells of uncertain significance (ASCUS) after a routine screening cervical Papanicolaou (Pap) test. Reflex high risk human papilloma virus (HPV) testing was performed; the results were positive (genotyping was not performed). Subsequent endocervical curettage showed a small focus of immature, atypical squamous cells with abnormally positive p16 staining and an abnormally increased proliferative index staining pattern (evaluated with Ki-67) (Images 1A-1C). These findings strongly suggested a high-grade lesion; nevertheless, due to the minute and focal nature of these findings, a diagnosis was rendered of squamous dysplasia, cannot exclude high grade dysplasia. Additional follow-up was recommended, and the option of a fluorescent in situ hybridization (FISH) assay (HPV-4C, using reagent manufactured by Cancer Genetics Italia S.r.l.) was also suggested as a method of triage to be performed using the same Thinprep collection media used to create the Pap test. The results of the FISH assay were positive, with 6.6% of cells showing gain of the 3q26 region (Image 1D). With this knowledge, the gynecologist performed a cervical loop electrosurgical excision procedure (LEEP), which revealed moderate squamous dysplasia (cervical intraepithelial neoplasia grade 2 [CIN 2]) supported by strong and abnormal p16/Ki-67 co-expression (Images 1E-1G). PATIENT 2: In June 2013, this patient was diagnosed with atypical glandular cells (AGUS) and was shown to have high-risk human papillomavirus (HPV) positivity. (Again, genotyping was not performed.) The subsequent biopsy showed mild reactive atypia of the glandular cells, which did not completely correlate with the atypical cells revealed by the Papanicolaou (Pap) test. Therefore, further follow-up was recommended. A fluorescent in situ hybridization (FISH) assay was performed on the specimen assayed via the AGUS Pap test; the FISH assay yielded positive results, showing many cells with a gain of 3q26 and 5p15 regions above the established cutoff values. A repeat Pap test was performed, which also was interpreted as indicating AGUS. Results of a second HPV-4C FISH assay showed numerous (14.6%) cells with a gain of 3q26 and 5p15 regions (Image 2A). Repeat cervical and endocervical biopsies showed scant atypical glands in otherwise-generous biopsies (Image 2B). Supported by abnormal p16 and Ki-67 immunohistochemical staining results (Image 2C and 2D) and the knowledge of the abnormal FISH assay results, the pathologist diagnosed the patient with endocervical adenocarcinoma in situ (AIS). The results of a subsequent LEEP confirmed the diagnosis of endocervical adenocarcinoma in situ with negative resection margins. LABORATORY FINDINGS: Abnormal Papanicolaou (Pap) test results with high risk of human papilloma virus (HPV) positivity and scant lesional tissue, as revealed by cervical/endocervical biopsies.