Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
2001-05-03
2004-05-11
Nolan, Patrick J. (Department: 1644)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
Reexamination Certificate
active
06733762
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to antigen-specific down-regulation of an immune response using Zot or zonulin. Specifically, the present invention provides a method for inhibiting antigen presenting cell-mediated antigen-specific lymphocyte proliferation in a dose-dependent manner by administering an effective amount of Zot or zonulin.
BACKGROUND OF THE INVENTION
I. Tight Junctions and the Actin Cytoskeleton
The tight junctions (hereinafter “tj”) or zonula occludens (hereinafter “ZO”) are one of the hallmarks of absorptive and secretory epithelia (Madara,
J. Clin. Invest
., 83:1089-1094 (1989); and Madara,
Textbook of Secretory Diarrhea
Eds. Lebenthal et al, Chapter 11, pages 125-138 (1990). As a barrier between apical and basolateral compartments, they selectively regulate the passive diffusion of ions and water-soluble solutes through the paracellular pathway (Gumbiner,
Am. J. Physiol
., 253(Cell Physiol. 22):C749-C758 (1987)). This barrier maintains any gradient generated by the activity of pathways associated with the transcellular route (Diamond,
Physiologist
, 20:10-18 (1977)).
There is abundant evidence that ZO, once regarded as static structures, are in fact dynamic and readily adapt to a variety of developmental (Magnuson et al,
Dev. Biol
., 67:214-224 (1978); Revel et al,
Cold Spring Harbor Symp. Quant. Biol
., 40:443-455 (1976); and Schneeberger et al,
J. Cell Sci
., 32:307-324 (1978)), physiological (Gilula et al,
Dev. Biol
., 50:142-168 (1976); Madara et al,
J. Membr. Biol
., 100:149-164 (1987); Mazariegos et al,
J. Cell Biol
., 98:1865-1877 (1984); and Sardet et al,
J. Cell Biol
., 80:96-117 (1979)), and pathological (Milks et al,
J. Cell Biol
., 103:2729-2738 (1986); Nash et al,
Lab. Invest
., 59:531-537 (1988); and Shasby et al,
Am. J. Physiol
., 255(
Cell Physiol
., 24):C781-C788 (1988)) circumstances. The regulatory mechanisms that underlie this adaptation are still not completely understood. However, it is clear that, in the presence of Ca
2+
, assembly of the ZO is the result of cellular interactions that trigger a complex cascade of biochemical events that ultimately lead to the formation and modulation of an organized network of ZO elements, the composition of which has been only partially characterized (Diamond,
Physiologist
, 20:10-18 (1977)). A candidate for the transmembrane protein strands, occluding, has been identified (Furuse et al,
J. Membr. Biol
., 87:141-150 (1985)).
Six proteins have been identified in a cytoplasmic submembranous plaque underlying membrane contacts, but their function remains to be established (Diamond, supra). ZO-1 and ZO-2 exist as a heterodimer (Gumbiner et al,
Proc. Natl. Acad. Sci., USA
, 88:3460-3464 (1991)) in a detergent-stable complex with an uncharacterized 130 kD protein (ZO-3). Most immunoelectron microscopic studies have localized ZO-1 to precisely beneath membrane contacts (Stevenson et al,
Molec. Cell Biochem
., 83:129-145 (1988)). Two other proteins, cingulin (Citi et al,
Nature
(London), 333:272-275 (1988)) and the 7H6 antigen (zhong et al,
J. Cell Biol
., 120:477-483 (1993)) are localized further from the membrane and have not yet been cloned. Rab 13, a small GTP binding protein has also recently been localized to the junction region (Zahraoui et al,
J. Cell Biol
., 124:101-115 (1994)). Other small GTP-binding proteins are known to regulate the cortical cytoskeleton, i.e., rho regulates actin-membrane attachment in focal contacts (Ridley et al,
Cell
, 70:389-399 (1992)), and rac regulates growth factor-induced membrane ruffling (Ridley et al,
Cell
, 70:401-410 (1992)). Based on the analogy with the known functions of plaque proteins in the better characterized cell junctions, focal contacts (Guan et al,
Nature
, 3:690-692 (1992)), and adherens junctions (Tsukita et al,
J. Cell Biol
., 1:1049-1053 (1993)), it has been hypothesized that tj-associated plaque proteins are involved in transducing signals in both directions across the cell membrane, and in regulating links to the cortical actin cytoskeleton.
To meet the many diverse physiological and pathological challenges to which epithelia are subjected, the ZO must be capable of rapid and coordinated responses that require the presence of a complex regulatory system. The precise characterization of the mechanisms involved in the assembly and regulation of the ZO is an area of current active investigation.
There is now a body of evidence that tj structural and functional linkages exist between the actin cytoskeleton and the tj complex of absorptive cells (Gumbiner et al, supra; Madara et al, supra; and Drenchahn et al,
J. Cell Biol
., 107:1037-1048 (1988)). The actin cytoskeleton is composed of a complicated meshwork of microfilaments whose precise geometry is regulated by a large cadre of actin-binding proteins. An example of how the state of phosphorylation of an actin-binding protein might regulate cytoskeletal linking to the cell plasma membrane is the myristoylated alanine-rich C kinase substrate (hereinafter “MARCKS”). MARCKS is a specific protein kinase C (hereinafter “PKC”) substrate that is associated with the cytoplasmic face of the plasma membrane (Aderem,
Elsevier Sci. Pub
. (
UK
), pages 438-443 (1992)). In its non-phosphorylated form, MARCKS crosslinks to the membrane actin. Thus, it is likely that the actin meshwork associated with the membrane via MARCKS is relatively rigid (Hartwig et al,
Nature
, 356:618-622 (1992)). Activated PKC phosphorylates MARCKS, which is released from the membrane (Rosen et al,
J. Exp. Med
., 172:1211-1215 (1990); and Thelen et al,
Nature
, 351:320-322 (1991)). The actin linked to MARCKS is likely to be spatially separated from the membrane and be more plastic. When MARCKS is dephosphorylated, it returns to the membrane where it once again crosslinks actin (Hartwig et al, supra; and Thelen et al, supra). These data suggest that the F-actin network may be rearranged by a PKC-dependent phosphorylation process that involves actin-binding proteins (MARCKS being one of them).
II. Zonula Occludens Toxin (“Zot”) and Zonulin
Most
Vibrio cholerae
vaccine candidates constructed by deleting the ctxa gene encoding cholera toxin (CT) are able to elicit high antibody responses, but more than one-half of the vaccinee still develop mild diarrhea (Levine et al,
Infect. Immun
., 56(1):161-167 (1988)). Given the magnitude of the diarrhea induced in the absence of CT, it was hypothesized that
V. cholerae
produce other enterotoxigenic factors, which are still present in strains deleted of the ctxa sequence (Levine et al, supra). As a result, a second toxin, zonula occludens toxin (hereinafter “Zot) elaborated by
V. cholerae
, and which contribute to the residual diarrhea, was discovered (Fasano et al,
Proc. Nat. Acad. Sci., USA
, 8:5242-5246 (1991)). The zot gene is located immediately adjacent to the ctx genes. The high percent concurrence of the zot gene with the ctx genes among
V. cholerae
strains (Johnson et al,
J. Clin. Microb
., 31/3:732-733 (1993); and Karasawa et al,
FEBS Microbiology Letters
, 106:143-146 (1993)) suggests a possible synergistic role of Zot in the causation of acute dehydrating diarrhea typical of cholera. Recently, the zot gene has also been identified in other enteric pathogens (Tschape, 2nd
Asian
-
Pacific Symposium on Typhoid fever and other Salmonellosis
, 47(Abstr.) (1994)).
It has been previously found that, when tested on rabbit ileal mucosa, Zot increases the intestinal permeability by modulating the structure of intercellular tight junctions (Fasano et al, supra). It has been found that as a consequence of modification of the pericellular pathway, the intestinal mucosa becomes more permeable. It also was found that Zot does not affect Na
+
-glucose coupled active transport, is not cytotoxic, and fails to completely abolish the transepithelial resistance (Fasano et al, supra).
More recently, it has been found that Zot is capable of reversibly opening tight junctions in the intestinal mucosa, and thus Zot, when co-administered with
Fasano Alessio
Lu Ruiliang
Sztein Marcelo B.
Tanner Michael K.
Nolan Patrick J.
University of Maryland Baltimore
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