It is hoped that the knowledge gained from such studies will suggest novel therapeutic strategies for these diseases. Acknowledgments We thank Professor Muramatsu and Dr Kurosu for their kind guidance, Mr J. VH were compared with the structure of Ig VH of the lacrimal glands and the peripheral blood cells. AID was expressed to varying degrees in lacrimal glands of all MD patients. Most IgG4-positive cells infiltrating lacrimal glands and in peripheral blood were polyclonal, although several clonally related pairs were detected. In one patient, two of the circulating MK-0773 IgG4 VH4-59 clones shared identical CDR3 sequences with the clones within the lacrimal glands. In conclusion, while most tissue-infiltrating and circulating IgG4-positive cells in MD are polyclonal, some clonally related IgG4 positive cells exist between lacrimal gland and peripheral blood, accounting for the clinical features MK-0773 of MD as an IgG4-related disease involving multiple organs. (CDR Rf or FR Rf) (CDRrelor FRrel), where = total number of observed mutations, Rf = replacement frequency inherent to CDR or FR sequences and CDRrel or FRrel = relative size of the CDRs or FRs. The probability (= n!/[k!(n ? k)!] qk (1 ? q)nCk, where = total number of observed mutations, k = number of observed R mutations in the CDRs or FRs and q = probability that an R mutation localizes to CDRs or FRs (q = CDRrel CDR Rf or FRrel FR Rf). In analysing in FRs, we exclude FR1 because the sequence primer was located in the middle of FR1. Results Detection of AID in patients with MD By analysing the expression of AID in the lacrimal glands of the five MD patients using RTCPCR, we confirmed that AID was expressed to Mouse monoclonal to Tag100. Wellcharacterized antibodies against shortsequence epitope Tags are common in the study of protein expression in several different expression systems. Tag100 Tag is an epitope Tag composed of a 12residue peptide, EETARFQPGYRS, derived from the Ctermini of mammalian MAPK/ERK kinases. varying degrees in all of them (Fig. 1a), and speculated that both SHM and CSR occur in lacrimal glands. Open in a separate windows Fig. 1 (a) Expression of activation-induced cytidine deaminase (AID) in lacrimal glands in patients with Mikulicz’s disease (MD). Lanes 1C5: patients; lane 6: RAMOS cell (Burkitt’s lymphoma-derived cell line) as a positive control; lane 7: hybridoma producing immunoglobulin M (IgM) monoclonal rheumatoid factor as a negative control. AID was expressed to varying degrees in all MD patients. (b) IgG subclass of infiltrating cells in lacrimal glands in patients with MD. SS is usually minor salivary glands in a patient with Sj?gren’s syndrome. IgG4 was detected at the same level as IgG1 in the lacrimal glands of MD. (c) Analysis of VH family of IgG4-positive cells infiltrating lacrimal glands and peripheral blood in case 1. Analysis of VH gene We examined the SHM in VH of IgG4 in lacrimal glands and peripheral blood in two MD patients. First, we performed Ig subclass-specific PCR. IgG4 was detected at the same level as IgG1 in the lacrimal glands of MD, but not in SS (Fig. 1b). We then performed VH family-specific PCR of IgG4, and detected VH1, 3, 4, 5 and 6 gene products in the lacrimal glands and peripheral blood in both patients (Fig. MK-0773 1c). Among the VH4 family of IgG4-positive cells, VH4-59 was the most common gene in the lacrimal glands in both cases (491% and 719% respectively) (Table 3). Table 3 Sequence analysis of VH4 in lacrimal glands and peripheral blood. = 0015, PB-24; 0001). In another pair, the R/S ratio was higher than would be expected by chance alone, although not statistically significantly so (vision-55; = 0132, PB-57; = 0055). In the FRs, the R/S ratio was statistically significantly lower than would be expected by chance alone in peripheral blood (PB-24; = 0001, PB-96; = 0043). The R/S ratio in lacrimal glands was also lower, although not statistically significantly so (vision-55; = 0076, vision-96; = 0067) (Table 4). Open in a separate windows Fig. 3 (a) CDR3 of genetically related clones between lacrimal glands and peripheral blood in case 1. (b) Sequence analysis of CDR1, CDR2, FR2 and FR3. Somatic hypermutation (SHM) were seen in these clones but did not share identical sequences. (c) Intraclonal diversification of genetically related clones between lacrimal glands and peripheral blood. Numbers beside the arrows refer to the number of amino acid exchanges. Underlined numbers such as 80 and 92 indicated different nucleotide exchanges. These data indicated that this IgG4-positive cells.