Oral immunization of an animal is generally hard to achieve unless large quantities of antigen are administered. In this study a number of antigens were tested for their ability to elicit a systemic immune response upon oral administration. It was found that bacterial pili, LTB, lectins, and a viral hemagglutinin were all able to elicit significant antibody titers upon oral feeding. The immune response thus generated to LTB and K99 pili could be completely abolished by cofeeding a number of sugars that have close structural homology to the terminal sugars of the GM1 and GM2 gangliosides to which these molecules are known to bind. All of the proteins that were active in oral immunization are known to possess "lectin or lectin-like" binding activities. It is therefore proposed that these molecules are able to bind to glycolipids and glycoproteins on the intestinal mucosa and to stimulate these cells to transport the proteins into the systemic circulation, thereby eliciting a systemic immune response. Molecules that did not possess this binding activity were unable to elicit significant responses at the doses tested.

Insulin-dependent diabetes mellitus (IDDM) is associated with serum antibodies that precipitate a 64-kDa pancreatic islet cell protein reported to be glutamic acid decarboxylase (GAD; glutamate decarboxylase, EC 4.1.1.15). Previously, antibodies to GAD were found in the rare neurological disorder stiff man syndrome. To demonstrate directly antibodies to GAD, enzymatically active GAD was first purified from fresh human cerebellum. Brain GAD activity was precipitated by noninhibitory antibodies in the sera of 16/26 (62%) subjects defined as having preclinical IDDM (islet cell antibody-positive first-degree relatives of a person with IDDM), 3/13 (23%) with recent-onset IDDM, and 3/3 with the stiff man syndrome. In addition, sera of 5/26 (19%) preclinical and 2/13 (15%) recent-onset IDDM subjects contained antibodies that precipitated GAD but inhibited its activity. Thus, overall, 21/26 (81%) preclinical and 5/13 (38%) recent-onset IDDM subjects had antibodies that precipitated GAD activity.Antibodies to GAD were not detected in sera from subjects with other autoimmune diseases (n = 29) or healthy controls (n = 14). GAD affinity-purified to homogeneity (specific activity, 58 units/mg) was specifically immunoprecipitated as a single 60-kDa species by the IDDM sera. In an ELISA incorporating whole mouse brain GAD captured by the GAD-6 monoclonal antibody the frequencies of GAD antibodies for all subject groups were indistinguishable from those found by precipitation of human brain enzymatic activity. We conclude that (i) GAD is an (auto)antigen in a majority of subjects operationally dermed as having preclinical IDDM, (u) pancreatic islet and brain GAD are likely to be cross-reactive, and (Ui) the majority of GAD antibodies are directed away from the catalytic site of the brain enzyme. The lower frequency of GAD antibodies in recent-onset IDDM subjects indicates either that immunoreactivity is lost with near-total (3-cell destruction or that GAD antibodies denote a low risk of progression to clinical disease.

SUMMARY1. Rat phaeochromocytoma (PC12) cells were used to investigate the expression of Ca2" channel types during neuronal differentiation. Neuronal differentiation was induced by treatment with nerve growth factor (NGF) or by activation of a temperature-sensitive tyrosine kinase (pp60v"8Tc) in genetically modified PC12 (PC12/v-src) cells. PC12 cells differentiated morphologically in the presence of NGF. When grown at the permissive temperature of 37 TC which activates the kinase activity of pp60v-rc, PC12/v-src cells differentiated morphologically with the extension of neurites. In contrast, PC12/v-src cells grown at the non-permissive temperature of 40 'C continued to divide and were morphologically indistinguishable from control PC12 cells.2. Whole-cell Ca2+ currents were measured in PC12 cells using Ba2+ as the charge carrier. Ba2+ currents measured at the peak of the current-voltage curve from a holding potential of -80 mV were -0-28 + 004 nA (mean +s.E.M.) in control PC12 cells compared to -1-25+0-16 nA in NGF-differentiated cells. The current density increased from 9-4 + 0 7 pA/pF in control PC12 cells to 22-8 + 2-4 pA/pF in NGFdifferentiated PC12 cells. Ba2+ currents were -0-24 + 0-04 nA in undifferentiated PC12/v-src cells grown at the non-permissive temperature of 40 0C compared to -0-95 +016 nA in differentiated PC12/v-src cells grown at the permissive temperature of 37 'C. The current density increased from 4-5 + 0-5 pA/pF in PC12/vsrc cells grown at the non-permissive temperature of 40 'C to 13-3 + 24 pA/pF in PC12/v-src cells grown at the permissive temperature of 37 'C.3. The sensitivity of Ba2+ currents to w)-conotoxin GVIA (w)-CgTX) was determined for currents measured at the peak of the current-voltage curve (0 mV in 10 mm Ba2+) from a holding potential of -80 mV. In NGF-differentiated PC12 cells, 10 jtM CWCgTx inhibited 68-1+ 3-2 % of the total Ba2+ current compared to 35-9 +4-1% in control cells. The density of the w)-CgTX-sensitive current increased from 3-3 ± 04 pA/pF in control cells to 15-7 + 2-0 pA/pF in NGF-differentiated cells. In

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