Botulism, a disease of humans characterized by prolonged paralysis, is caused by botulinum neurotoxins (BoNTs), the most poisonous substances known. There are seven serotypes of BoNT (A-G) which differ from each other by 34-64% at the amino acid level. Each serotype is uniquely recognized by polyclonal antibodies, which originally were used to classify serotypes. To determine if there existed monoclonal antibodies (mAbs) capable of binding two or more serotypes, we evaluated the ability of 35 yeast-displayed single-chain variable fragment antibodies generated from vaccinated humans or mice for their ability to bind multiple BoNT serotypes. Two such clonally related human mAbs (1B18 and 4E17) were identified that bound BoNT serotype A (BoNT/A) and B or BoNT/A, B, E and F, respectively, with high affinity. Using molecular evolution techniques, it proved possible to both increase affinity and maintain cross-serotype reactivity for the 4E17 mAb. Both 1B18 and 4E17 bound to a relatively conserved epitope at the tip of the BoNT translocation domain. Immunoglobulin G constructed from affinity matured variants of 1B18 and 4E17 were evaluated for their ability to neutralize BoNT/B and E, respectively, in vivo. Both antibodies potently neutralized BoNT in vivo demonstrating that this epitope is functionally important in the intoxication pathway. Such cross-serotype binding and neutralizing mAbs should simplify the development of antibody-based BoNT diagnostics and therapeutics.

Many patients with diabetes mellitus (both type 1 and type 2) require therapy to maintain normal fasting glucose levels. To develop a novel treatment for these individuals, we used phage display technology to target the insulin receptor (INSR) complexed with insulin and identified a high affinity, allosteric, human monoclonal antibody, XMetA, which mimicked the glucoregulatory, but not the mitogenic, actions of insulin. Biophysical studies with cultured cells expressing human INSR demonstrated that XMetA acted allosterically and did not compete with insulin for binding to its receptor. XMetA was found to function as a specific partial agonist of INSR, eliciting tyrosine phosphorylation of INSR but not the IGF-IR. Although this antibody activated metabolic signaling, leading to enhanced glucose uptake, it neither activated Erk nor induced proliferation of cancer cells. In an insulin resistant, insulinopenic model of diabetes, XMetA markedly reduced elevated fasting blood glucose and normalized glucose tolerance. After 6 weeks, significant improvements in HbA1c, dyslipidemia, and other manifestations of diabetes were observed. It is noteworthy that hypoglycemia and weight gain were not observed during these studies. These studies indicate, therefore, that allosteric monoclonal antibodies have the potential to be novel, ultra-long acting, agents for the regulation of hyperglycemia in diabetes.

The CYP21 gene, which encodes P450c21, the adrenal steroid 21-hydroxylase needed for glucocorticoid synthesis, lies in the major histocompatibility locus only 2.3 kilobase pairs (kb) downstream from the C4 gene. A 300-base pair (bp) proximal promoter and two upstream regions within C4 are needed for expression of mouse CYP21; the human gene also has a proximal promoter, but upstream elements have not been studied. To search for upstream regulatory elements in human CYP21B, we examined up to 9 kb of 5-flanking DNA by transient transfection into human adrenal NCI-H295A cells. The 300-bp proximal promoter had substantial activity, but constructs retaining the DNA between ؊4.6 and ؊5.6 kb had increased activity, indicating the presence of distal elements. This region does not correspond to the mouse upstream regions, lying further upstream within intron 35 of C4B, which encompasses the previously described "Z promoter." DNase I footprinting located two elements, F1 and F2, lying ؊186 to ؊195 bp and ؊142 to ؊151 bp upstream from the Z cap site (؊4862 to ؊4871 and ؊4818 to ؊4827 bp upstream of the CYP21B cap site). Each element formed a specific DNA-protein complex and conferred orientation-independent expression to a heterologous promoter. Mutations abolished formation of the DNA-protein complexes but only partially decreased expression. We identified a third site, F3, lying at ؊33 to ؊42 bp from Z. Competitive gel mobility supershift assays and co-transfection studies with SF-1 produced in vitro indicate F2 and F3 bind SF-1; BLAST searches and Southwestern blotting suggest that NF-W2 may bind F1. These results indicate that the Z promoter is a component of the CYP21 promoter needed to drive its adrenal-specific expression and that CYP21 transcription elements within C4 have kept these two genes linked during evolution.

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Degeneration in the macula region of the retina is a feature of a heterogeneous group of inherited, progressive disorders, causing blinding visual impairment. Autosomal dominant Doyne's honeycomb retinal dystrophy (DHRD) is characterised by the presence of drusen deposits at the level of Bruch's membrane in the macula and around the edge of the optic nerve head. We have studied 63 members of a large, nine-generation British pedigree by linkage analysis. Two-point analysis showed significant linkage to nine markers on the short arm of chromosome 2, a region overlapping that recently reported to be linked to Malattia leventinese. A maximum lod score (Zmax) of 7.29 (theta = 0.0) was obtained at marker locus D2S2251. Haplotype analysis of recombination events localised the disease to a 5 cM region between marker loci D2S2316 and D2S378. Striking clinical similarities between DHRD and the more common condition age-related macular degeneration (ARMD) suggest that the disease gene at this locus could be considered as the most likely candidate in future studies on ARMD.