No abstract

Degenerate oligonucleotides based on the published Escherichia coli glutamate decarboxylase (GAD) protein sequence were used in a polymerase chain reaction to generate a DNA probe for the E. coli GAD structural gene. Southern blots showed that there were two cross-hybridizing GAD genes, and both of these were cloned and sequenced. The two GAD structural genes, designated gadA and gadB, were found to be 98% similar at the nucleotide level. Each gene encoded a 466-residue polypeptide, named, respectively, GAD a and GAD 13, and these differed by only five amino acids. Both GAD a and GAD 13 contain amino acid residues which are highly conserved among pyridoxal-dependent decarboxylases, but otherwise the protein sequences were not homologous to any other known proteins. By restriction mapping and hybridization to the Kohara miniset library, the two GAD genes were located on the E. coli chromosome. gadA maps at 4046 kb and gadB at 1588 kb. Neither of these positions is in agreement with the current map position for gadS as determined by genetic means. Analysis of Southern blots indicated that two GAD genes were present in all E. coli strains examined, including representatives from the ECOR collection. However, no significant cross-hybridizing gene was found in Salmonella species. Information about the DNA sequences and map positions of gad4 and gadB should facilitate a genetic approach to elucidate the role of GAD in E. coli metabolism.The enzyme glutamate decarboxylase (GAD; also known as glutamic acid decarboxylase; EC 4.1.1.15) catalyzes the a-decaboxylation of glutamic acid to produce -y-aminobutyric acid. Within bacteria, GAD activity seems to be relatively unique to Escherichia coli (37). Gale (14) proposed a general role for the inducible bacterial amino acid decarboxylases, including GAD, in the maintenance of physiological pH under acidic conditions. E. coli GAD has been extensively characterized with respect to its biophysical and biochemical properties (1,14,30,31,(39)(40)(41)(42)(43)(44)(45)(46), and a partial protein sequence has been available for some time (42, 43). Based on genetic linkage studies in E. coli, the structural gene for GAD (gadS) and a potential regulatory gene (gadR) have been mapped between mtl at approximately 80.7 minutes and gltS at approximately 82.4 minutes (3,23,26,27), but these early results have not been followed up. For the purposes of our immunological studies, we were interested in obtaining the complete DNA and protein sequences of E. coli GAD. At the time this work was initiated, an extensive search of the literature and all available sequence data bases suggested that neither the complete protein sequence nor the gene sequence for E. coli GAD had been determined. To obtain this information, we generated a DNA probe for the gene based on a recent more extensive partial protein sequence (24). By using this probe on E. coli genomic DNA, we discovered that there were not one but two separate cross-hybridizing GAD genes. We report here the complete DNA sequences and map pos...

The nonobese diabetic (NOD) mouse is an excellent model of insulin-dependent (type I) human diabetes mellitus. We report that a single injection of complete Freund's adjuvant (CFA) given at an early age (5 wk) prevented the appearance of diabetes and greatly increased the life span of NOD mice without additional therapy. No treated mouse developed hyperglycemia by the age of 12 mo (n = 13), whereas all untreated mice died of diabetes before 8 mo of age (n = 38). All CFA-treated mice were alive and healthy at 12 mo of age. Some CFA-treated NOD mice that were monitored for long-term survival are still alive with no sign of disease at 18 mo of age (n = 5). Administration of CFA resulted in decreased in vitro splenic lymphocyte proliferative responses to alloantigen and mitogen. Cell-mixing experiments indicated that antigen-nonspecific inhibitory cells were elicited in the spleen and increased in the bone marrow. These regulatory cells were Thy-1- and nonadherent to nylon wool, as has been described for natural suppressor (NS) cells. These data lend support to a relationship between the boosting of endogenous NS activity and the establishment of tolerance to self in the context of autoimmunity. Our results suggest that early nonspecific immunotherapy of genetically predisposed individuals could prevent the development of autoimmune diabetes.

Insulin-dependent diabetes mellitus (IDDM) involves the destruction of the insulin-producing cells in the islets of Langerhans. One possible cure is by transplanting the islet cells; however, transplanted islets, even between identical twins, are subject to autoimmune destruction by the disease process, resulting in diabetes recurrence. We recently reported that complete Freund's adjuvant (CFA), an immunomodulating agent, prevented development of autoimmune diabetes in the NOD mouse. In this study, we evaluated adjuvant therapy in prevention of autoimmune destruction and rejection of transplanted islets in diabetic NOD mice. After transplantation, untreated syngeneic islet recipients (n = 16) initially became normoglycemic and then hyperglycemic, with a median survival time (MST) of the graft of 17 days. When CFA was administered at the time of transplantation, 11 of 13 CFA-treated syngeneic islet recipients remained normoglycemic long term (>100 days) with an MST >107 days. Ten of 11 mice maintained indefinite normoglycemia until the conclusion of follow-up (101 to 172 days). When adjuvant therapy was used in conjunction with allogeneic islet transplantation, graft survival was not extended, with MST being similar to the untreated allogeneic islet recipients (12 [n = 5] and 13 [n = 5] days, respectively). The extended acceptance of second syngeneic islet grafts by CFA-treated mice indicates that the persistent autoimmunity against the transplanted islets can be reversed in the diabetic NOD mice after CFA treatment.

SUMMARY:Studies of murine severe combined immune-deficient (scid/scid) fetuses gestating in transgene-tagged immune competent dams have established high frequencies of transplacental trafficking of nucleated maternal cells. Maternal cells first appeared in thymus at gestation day (gd) 12.5 and were present in more than 90% of late gestation fetuses. Morphologically heterogeneous maternal cells were located predominantly in bone marrow and thymus and also occasionally in liver, spleen, and nonlymphoid organs. We have now evaluated maternal cell chimerism in offspring with normal lymphoid development. Genetically normal blastocysts from random-bred CD1 mice were transferred to C57BL/6J-lacZ transgene-tagged ROSA26 females. Serial sectioning of fetuses followed by histochemistry for lacZ-expressing cells was used to comprehensively define organs containing maternal cells. Fetuses, sectioned in their entirety, had no detectable maternal cells before gd 16.5. Morphologically homogenous, nucleated maternal cells were first present in fetal bone marrow cavities at gd 16.5 and were evident in all offspring in later gestation. Postnatally, maternal cells were also present in bone marrow cavities into adulthood, as determined by lacZ histochemistry and PCR amplification of the maternal transgene. The frequency of maternally derived cells in postnatal bone marrow was increased compared with late gestation, and occasionally, maternal cells were detected in postnatal spleen. The normalcy of maternal cell transfer to genetically immune competent progeny and their long-term engraftment is suggestive of a functional role for maternal cells in offspring. (Lab Invest 2003, 83:673-681).