Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-03-01
2001-09-18
Geist, Gary (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S053000, C514S867000, C536S055100, C536S055200, C536S017200
Reexamination Certificate
active
06291435
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to treatment of diarrhea, particularly diarrhea caused by enteropathogenic
Escherichia coli
(EPEC). More specifically, the invention concerns compositions and methods which may be used to prevent EPEC infection or ameliorate symptoms caused by EPEC infection.
BACKGROUND OF THE INVENTION
Enteropathogenic
Escherichia coli
(EPEC) is a significant cause of diarrhea world-wide, with disease occuring most frequently in developing countries [1-3]. In these countries, disease occurs regularly in hospitals and clinics, as well as in the general community. EPEC outbreaks in developed countries, on the other hand, usually consist of sporadic, isolated incidents which are localized to neonatal nurseries of hospitals or day-care centers. Infants less than 6 months of age are most often affected, although EPEC is also capable of causing disease in children and adults. The transmission of EPEC infections is thought to occur primarily by the fecal-oral route as a result of contact with infected individuals or with contaminated surfaces or food. The isolation of EPEC from asymptomatic individuals has led to speculation that some individuals may be carriers who can also spread infection.
Clinical symptoms of EPEC infection in children consist of diarrhea which varies in duration (days to months) and severity [3,4]. In addition to profuse watery stool, symptoms include dehydration, fever, vomiting and weight loss. In protracted or severe cases, disease is often associated with the delayed growth of children, metabolic acidosis (decrease in blood pH resulting from a loss of bicarbonate [5,6]) and, in extreme cases, death. Adults participating in volunteer studies of EPEC infection displayed symptoms similar to those observed in children, but of shorter duration.
Results of volunteer studies indicate that, at least in adults, a relatively large infectious dose of organisms is required to produce symptoms which typically occur 7 to 16 h after infection [1,7]. For ethical reasons, similar information for EPEC infection in children is not available. It is speculated however, that a much lower number of organisms is required to infect children since transmission frequently occurs in hospitals or day-care facilities [1]. Treatment for EPEC infection usually consists of rehydration therapy and, if necessary, nutritional supplementation. Antibiotics are often used to treat EPEC infection though their overall effectiveness is uncertain [3].
Biopsies from children infected with EPEC reveal that the bacteria predominantly colonize the small intestine, although the large intestine can also be involved, presumably due to bacterial overgrowth [2,4,8,9]. The primary histopathological consequence of colonization is the atrophy or degeneration of microvilli at sites of bacterial attachment and the intimate association of bacteria with pedestal-like structures formed by host epithelial cells (e.g., enterocytes). This characteristic effect is referred to as an attaching and effacing (A/E) lesion [4,9-11]. Other features include the deterioration of the terminal web (apical region beneath microvilli consisting of cytoskeletal proteins which are physically associated with the central actin filaments of microvilli) of enterocytes, a reduction in mucosal thickness, and a general disordered arrangement of enterocytes. Bacteria are rarely found within intestinal epithelial cells or in the lamina propria, suggesting that EPEC are not invasive. An infiltration of inflammatory cells into the lamina propria of the intestine is also frequently observed during EPEC infection.
Clinical symptoms caused by EPEC result from bacterial attachment to the intestinal epithelium. Donnenberg and Kaper proposed a model in which EPEC attachment involves a three-step process [12]. The initial step consists of initial, non-intimate attachment of bacteria as microcolonies to epithelial cells. Next, the bacteria secrete several proteins which induce signal transduction pathways in epithelial cells. These signals initiate cytoskeletal rearrangement followed by the effacement of microvilli of host cells. In the final stage of attachment, cytoskeletal components are organized to form cup-like pedestal structures which partially surround adherent organisms. The latter steps of effacement and intimate attachment result in the characteristic A/E lesions associated with EPEC [11]. A modified version of this model has recently been suggested by Hicks, et al. In their model, three-dimensional microcolonies of EPEC are thought to develop after, not before, intimate attachment has occurred [13].
Since adherence is an important factor in EPEC pathogenesis, considerable research has been performed to identify bacterial and eukaryotic cell structures involved in attachment. So far, two bacterial structures have been relatively well characterized. The first, bundle-forming pili (BFP), are associated with the initial, non-intimate attachment of EPEC as microcolonies to discrete sites on epithelial cells, a pattern which is referred to as localized adherence (LA) [14,23,27]. Scanning electron micrographs of LA EPEC revealed that BFP are involved in mediating inter-bacterial linkages within microcolonies. Whether the BFP also function as adhesins for EPEC binding to epithelial cells remains to be resolved, however, since these structures appear to mediate bacterial binding to HEp-2 cells but not to human intestinal tissue in organ culture [13,27,47]. A second bacterial protein involved in attachment is intimin [36,48]. This protein is necessary for a later stage of EPEC attachment in that it focuses host cell cytoskeletal components beneath adherent bacteria to form A/E lesions [33].
Cravioto, et al. initially demonstrated that EPEC adhered to HEp-2 cells in greater numbers than other groups of
E. coli
studied, and that this adherence was not due to type I fimbriae [14]. Type I pili are structures which are expressed with similar frequency by pathogenic and non-pathogenic
Escherichia coli
(
E. coli
) strains, and whose binding is inhibited by mannose [15]. Subsequent investigations resulted in several different structures being proposed as EPEC adhesins. These structures included unidentified non-fimbrial [16] and fimbrial adhesins [17-19], fimbriae with N-terminal sequence homology to the fimbriae of uropathogenic and diffusely-adhering
E. coli [
20], and a 32 kDa outer membrane protein [21] (later reported to be OmpF [22]). However, the observation that EPEC grown in tissue culture medium attached to epithelial cells in a LA pattern [23,24], and that this phenotype was encoded by a large EPEC adherence factor (EAF) plasmid [25], led to the identification of a structure required for this pattern of binding.
In 1991, Giron, et al. described unqiue rope-like structures, termed BFP, [26] which appeared by scanning electron microscopy, to intercourse between bacteria to form microcolonies, and to attach the microcolonies to HEp-2 cells. Their role in attachment was supported by observations that antibodies raised against purified BFP partially inhibited EPEC attachment, and mutants lacking the EAF plasmid did not express BFP. The structural subunit of BFP is BfpA [52].
Following the effacement of microvilli, the formation of actin pedestals characteristic of A/E lesions requires the bacterial protein intimin [33,35,36]. Intimin is a 94 kDa outer membrane protein encoded by the eae gene of the locus of enterocyte effacement (LEE). The expression of this protein is necessary to focus host cytoskeletal proteins which accumulate beneath the organisms into pedestals, and for bacteria to become intimately associated (less than 10 mn separation) with this structure [11]. Based on serological and genetic techniques, EPEC intimins have been cl
Armstrong Glen D.
Yanmaele Rosa P.
Burns Doane , Swecker, Mathis LLP
Geist Gary
Khare Devesh
The Governs of the University of Alberta
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