The piglet model, on the other hand, is more appropriate for preclinical evaluation of formulations and for validation, including determination of the likely effective therapeutic dose for humans
The piglet model, on the other hand, is more appropriate for preclinical evaluation of formulations and for validation, including determination of the likely effective therapeutic dose for humans. We conclude that 5C12, which is reactive against the Stx2 A subunit, is an excellent candidate for immunotherapy against HUS and that antibodies directed against the A subunit of Stx2, as opposed to those directed against the B subunit, have broad-spectrum activity that includes Stx2 variants. Acknowledgments This study was supported by NIH Public Health Service Difluprednate grants RO1-AI41326 and P30-DK-34928. We thank Jennifer Martineau for technical assistance. Notes J. the Stx2 A subunit, is an excellent candidate for immunotherapy against HUS and that antibodies directed against the A subunit of Stx2 have broad-spectrum activity that includes Stx2 variants, compared with those directed Difluprednate against the B subunit. Infection with Shiga toxin-producing (STEC) strains is associated with hemolytic-uremic syndrome (HUS) (2, 24, 27), the leading cause of acute renal failure in young children (11). Shiga toxins (Stx) are cytotoxins and are major virulence factors of STEC. There are two immunologically distinct Stx types, known as Stx1 and Stx2, of which Stx1 is largely homogeneous, whereas the Stx2 group is highly heterogeneous and consists of at least 10 Stx2 gene variants (10, 14, 23, 31, 32, 37, 41, 42, 50). Stx2 is the most prevalent genotype identified in STEC isolated from patients with HUS (9, 40), and Stx2c is the most common Stx2 variant associated with HUS (9). Stx2 variants other than Stx2c are found frequently in asymptomatic STEC carriers but can often cause uncomplicated diarrhea (9) and rarely cause HUS (14, 33, 38, 45). Stx2f, identified in from pigeons, has been identified only once in humans, in a patient with diarrhea in Canada (10). In addition to pathogenicity to humans, Stx2 is definitely more harmful to mice and piglets than Stx1. Stx2 is about 400 times more lethal to mice than Stx1 when given systemically (44). STEC strains generating Stx2 alone cause more-severe neurologic symptoms in gnotobiotic piglets than STEC strains generating both Stx1 and Stx2, or Stx1 only (8). The nomenclature of Stx2 is definitely confusing; Stx2vha and Stx2vhb (18), which are closely related to Stx2c (42), were originally identified as vtx2ha and vtx2hb (14). They were later shown to be triggered by intestinal mucus (21) and named Stx2d (22). However, the Stx2d we refer to in the present study is the Stx2d cluster defined by Pierard et al. (37), which comprises Stx2d-OX3a (32), Stx2d-Ount (37), and Stx2d-O111 (33). An Stx molecule consists of a monomeric A subunit and a pentameric B subunit. Among STEC strains with different Stx2 variants, genetic variations in either the A or the B subunit or in both often confer antigenic and practical variations. The amino acid sequence identities of the A subunits of variants Stx2c (42), Stx2vha (14), Stx2vhb (14), Stx2d-OX3a (32), Stx2d-Ount (37), Stx2d-O111 (33), Stx2e (39), and Stx2f (47) with the A subunit of Stx2 are 100, 99, 99, 95, 93, 95, 94, and 71%, respectively. For the B subunit the amino acid sequence homologies are 96, 96, 96, 87, 88, 88, 87, and 82%, FANCG respectively. The two current therapeutic methods for HUS involve neutralization of Stx either in the gut or in the bloodstream. The two methods attempted for Stx inactivation in the gut are (i) utilization of glycoconjugate polymers transporting Pk-trisaccharide sequences that serve as a receptor of Stx (1, 4, 5, 17) and (ii) use of recombinant bacteria showing a Stx-specific glycolipid (globotriose or globotetraose) receptor (29, 30). We believe that systemic administration of Stx-specific Difluprednate neutralizing antibodies is currently the most encouraging approach for prevention or treatment of Stx-mediated systemic complications, including HUS (7) and edema disease in pigs (15). Murine Stx1- and Stx2-specific monoclonal antibodies (MAbs) have been shown to neutralize both toxins in vitro and in vivo (13, 28, 43). However, murine MAbs are not considered appropriate for human use. Reshaping of a murine antibody against Stx2 into a humanized form has recently been shown to completely guard mice against a lethal challenge with STEC when the antibody is definitely given within 24 h after illness (51). The disadvantage of a humanized antibody is definitely that it still offers mouse parts and reduced affinity (12). Mukherjee et al. have recently generated a panel of 50 human being MAbs (HuMAbs) against Stx1 and Stx2 in transgenic mice (25, 26), from which we have selected a panel of 5 Stx2-specific HuMAbs that were shown to be highly protecting for piglets, even when given 12 h after an oral challenge with Stx2-generating STEC. In the present study, we used the mouse toxicity model (13, 25, 26, 28, 43) and the streptomycin-treated mouse model of STEC illness (22, 48, 49) to investigate the abilities of these five HuMAbs to protect against Stx2 variants. MATERIALS AND METHODS Bacteria Enterohemorrhagic (EHEC) O91:H21 strain B2F1, which generates Stx2 variants Stx2vha and Stx2vhb (14), was from the American Type Tradition Collection (ATCC 51435). A streptomycin-resistant clone of.