Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-23
2001-10-02
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S005000, C435S007800, C435S007210, C435S091200, C435S320100, C435S029000, C435S034000, C435S091500, C435S863000, C435S007320, C435S007240, C435S002000, C435S811000, C436S094000, C436S501000, C436S518000, C436S811000, C436S507000, C436S544000, C436S546000, C436S820000, C514S017400, C514S018700, C536S022100, C536S028500, C536S027400, C536S026110, C536S029200
Reexamination Certificate
active
06297015
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to compositions and methods for diagnosing and treating Crohn's disease, and, more particularly, to such compositions and methods for screening for a presence of a bacterium believed involved in causing Crohn's disease and for treating patients shown by the screening method to be infected with the bacterium.
2. Description of Related Art
Crohn's disease is an inflammatory bowel disease that affects 2-3 million Americans, with a typical onset between 15 and 25 years of age. Crohn's is a chronic disorder that causes inflammation or ulceration in the small and/or large intestine, extending into the deeper layers of the intestinal wall. Sometimes the inflammation may also affect the mouth, esophagus, stomach, duodenum, appendix, or anus. Although Crohn's is a chronic condition, periods of remission may occur, with recurrences unpredictable. Two forms of the disease, perforating and nonperforating, are believed to occur.
Common symptoms of Crohn's disease include abdominal pain and diarrhea. There may also be rectal bleeding, weight loss, and fever. The bleeding may be serious and persistent, leading to anemia. Children may suffer delayed development and stunted growth.
Current diagnoses are performed by blood test to detect anemia and elevated white blood cell count, colon biopsy, and lower gastrointestinal x-ray series.
There are drugs that can be helpful in controlling Crohn's disease, but at present there is no cure. Treatment is aimed at correcting nutritional deficiencies, controlling inflammation, relieving the symptoms of abdominal pain, diarrhea, and rectal bleeding. Drugs known to be used for this condition can help, but side effects can be deleterious. Surgeries that may be performed to alleviate symptoms include the removal of inflamed areas, draining of abscesses, and bowel resection.
The cause of Crohn's disease has been debated since its recognition in the early part of the twentieth century. There are those who believe that at least some of the cases are caused by a bacterium, specifically
Mycobacterium paratuberculosis
, which is endemic in foods derived from cattle and water supplies in the Western world. Crohn's patients have been reported to have been cured by an antibiotic or a multidrug antibiotic regime having activity against that organism.
Mycobacterium paratuberculosis
is an obligate pathogen; that is, it cannot multiply outside the cells of animals. It is known to be present in a wide variety of animals, including primates and humans. The best-studied animal paratuberculosis is bovine Johne's disease (BJD), a disease that causes chronic diarrhea, weight loss, and malnutrition in cattle and affects up to 25% of the dairy cattle in the United States. Cows infected with BJD are known to secrete
Mycobacterium paratuberculosis
in their milk, which is not destroyed by standard milk pasteurization methods, but only by ultrapasteurization. This bacterium has also been cultured from a municipal water supply in the United States.
One can be exposed to
Mycobacterium paratuberculosis
and not develop Crohn's disease, if the immune system is capable of fighting the bacterium, resulting only in a transient intestinal infection with no after-effects. However, those susceptible to inflammatory bowel disease, including those with a genetic predisposition or being immunosuppressed, can develop the disease.
Mycobacterium paratuberculosis
occurs in two forms, the bacillary form and the spheroplast form, in which no cell wall is present. The former, which may be required to cause disease, is easily detected in animals by a simple chemical test; the latter, however, has only been found to be detectable with genetic testing techniques, such as polymerase chain reaction (PCR), to detect the 1451-bp IS900 insertion sequence unique to
Mycobacterium paratuberculosis
. PCR methods, however, can fail under conditions in which the amount of spheroplast present in the tissue is low or when tissue preserved in wax-embedded paraffin blocks is used. Another method is direct culturing of the organism followed by IS900 detection.
Currently available serological tests for Johne's disease are believed to have poor sensitivity and specificity, and fecal smear microscopy and fecal culture in early stages of infection are of limited value. El-Zaatari et al. (
Current Microbiol
. 29, 177-184, 1994) have reported using the chromosomal DNA of a
Mycobacterium paratuberculosis
strain to construct an expression genomic library in
E. coli
. A recombinant clone, p35, that expresses a protein of approximately 35K was identified, and its gene product was used in the serodiagnosis of Johne's disease by immunoblotting. El-Zaatari et al. reported a diagnostic yield with this clone that was higher than those reported using commercially available diagnostic tests. They suggest that the high sensitivity and specificity of p35 indicates a potential utility for the diagnosis of animals at all stages of Johne's disease.
El-Zaatari et al. also tested the clone's potential as a probe, and found that it hybridized specifically to nine bacterial strains representing the
Mycobacterium avium
complex species and none of the other mycobacterial species or other related and unrelated bacteria.
Recently El-Zaatari et al. (U.S. Pat. No. 5,776,692) have reported a recombinant clone pMptb #48 that expresses a 36K
M. paratuberculosis
antigen and its use as a test, as well as a mycobacterial genus-specific DNA probe corresponding to a 1.4 kb BamH 1-DNA insert in pMptb #48. Serological tests are suggested for using the clone and/or the p36k protein or fragments thereof
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a composition and method for screening for an infection caused by
Mycobacteriurm avium
, such as Crohn's disease.
It is an additional object to provide such a composition and method that have improved specificity and sensitivity over prior known compositions and methods.
It is a further object to provide such a composition and method that do not require a surgical procedure to obtain a test sample.
It is another object to provide such a composition and method that are serological in nature.
An additional object is to provide a composition and method for predicting a predisposition to Crohn's disease.
A further object is to provide a composition and method for treating patients shown by the screening method to be infected with
Mycobacterium paratuberculosis.
These objects and others are attained by the present invention, a composition and associated methods for detecting and treating a
M. para
. infection such as Crohn's disease in a human and for predicting a genetic predisposition thereto.
The detecting composition in a first embodiment comprises a combination of the p35 and p36 clones.
In a second embodiment the detecting composition comprises a combination of the 35K and 36K proteins expressed by the p35 and p36 clones, respectively.
In a first embodiment of the detection method of the present invention, a combination of the 35K and 36K proteins is used as an antigen and reacted with serum samples to detect for the presence of a
M. para
. infection, and thus for the presence of Crohn's disease, at least some cases of which are believed to be caused by
M. paratuberculosis.
An embodiment of the treatment composition of the present invention comprises at least one antibiotic effective in the eradication of
M. paratuberculosis.
In an embodiment of the treatment method, the effective antibiotic is administered to a patient having been found positive for
M. paratuberculosis
by the serologic method of the invention.
The features that characterize the invention, both as to organization and method of operation, together with further objects and advantages thereof, will be better understood from the following description.
REFERENCES:
patent: 4918178 (1990-04-01), Hurley et al
Allen Dyer Doppelt Milbrath & Gilchrist, P.A.
Cook Lisa V
Le Long V.
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