Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-11-14
2004-02-24
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S029000, C435S256800, C435S851000
Reexamination Certificate
active
06696270
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a submucosal tissue composition and the use of those compositions to promote growth of fastidious cells. More particularly, the present invention is directed to the use of submucosal tissue cell culture substrates to enhance the in vitro growth, and thus the identification and diagnosis, of vertebrate infections agents.
BACKGROUND OF THE INVENTION
Bacteria are a diverse group of organisms that live in a broad variety of environments. In particular, many bacterial species live both on and in vertebrate hosts. Bacteria that colonize the interior spaces of vertebrates typically exhibit specific interactions with the tissues that comprise the bacteria's optimal habitat in the host organism. Many bacteria express adhesions having fine tuned specificities for interacting with eukaryotic cell-surface proteins or carbohydrate structures so that only a restricted range of hosts and tissue that carry the appropriate receptors are available for bacterial colonization.
For example if bacteria cannot adhere to the mucosal layer of the vertebrate digestive system they will be removed rapidly by the local non-specific host-defense mechanisms (peristalsis, ciliary action and turnover of the epithelial cell populations and mucus layer). In addition, competition between bacteria for space and nutrients, and bacteria tolerance of biochemical parameters such as pH and antimicrobial peptides, select for bacterial species/strains that can colonize specific niches. The result of this selective process is often referred to as tissue tropism.
New strains of bacteria are continually being discovered as techniques for their detection improve. However, many detected strains have proven to be difficult to culture outside their natural micronenvironment due to the unique culture conditions required by these organisms. Accordingly, researchers attempting to culture microorganisms try to provide an in vitro microenvironment which mimics the in vivo environment in which the microorganisms grow. The ability to propagate microorganisms in vitro is of particular importance for the identification of infectious and pathogenic organisms, and for diagnosis of diseases. In addition, an in vitro microenvironment which mimics the in vivo environment enables the study of such organisms in vitro.
Many infectious agents when placed on existing culture media often fail to grow and therefore are not detected, given the state of the present technology. One medically significant organism that has proven difficult to culture in vitro is
Helicobacter pylori
, a gram negative spiral shaped microaerophilic bacteria.
H. pylori
live in the mucous layer lining the stomach of vertebrate species and are partially protected from the stomach's acid by the mucosal layer. The organisms secrete proteins that interact with the stomach's epithelial cells and attract phagocytic cells, such as macrophages and leukocytes, and those phagocytic cells induce inflammation and gastritis. In addition, the bacteria produce urease, an enzyme that helps to break down urea into ammonia and carbon dioxide. Ammonia can neutralize stomach acid allowing further proliferation of
H. pylori. H. pylori
also secretes toxins that contribute to the formation of stomach ulcers.
H. pylori
has been suggested to be a causative agent of chronic active gastritis and gastric duodenal ulcers. More recently,
H. pylori
infections have also been associated with the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma of the stomach.
Many bacteria cannot survive in an acidic environment, however
H. pylori
are not the only bacteria capable of colonizing the surface of a primate's stomach. Since the discovery of
H. pylori
bacteria, scientists have isolated 11 other organisms from the stomachs of other primates such as dog, cats, rodents, ferrets and even cheetahs. These bacteria, for now are considered to be members of the Helocobacter family. All are spiral shaped and highly mobile, properties that enable them to resist muscle contractions that regularly empty the stomach. These organisms grow best at oxygen levels of 5%, matching the level found in the stomachs mucus layer (ambient air is 21% oxygen).
Surveys using an antibody-based blood test to reveal the presence
H. pylori
have indicated that one-third to one-half of the world's population carry
H. pylori
. In the United States and Western Europe children rarely become infected, but the bacteria's prevalence rises with age such that more than half of all sixty year olds in those countries have the bacteria. In contrast, sixty to seventy percent of the children in developing countries show positive test results by age 10, and the infection rate remains higher for adults.
H. pylori
infection is also common in institutionalized children.
H. pylori
is capable of long term persistence in untreated individuals, and in the absence of treatment
H. pylori
remains persistent in the gastric mucosa for the lifetime of the host.
Although blood tests are useful for an initial screen for detecting the existence of an
H. pylori
infection, The blood test is based on detecting antibodies to
H. pylori
and thus is not a direct test for the presence of viable
H. pylori
bacteria. Screening for antibodies only provides information on whether the individual has been exposed to
H. pylori
. Furthermore, the blood test is know to give false positives. The present invention describes a direct assay for the presence of
H. pylori
that utilizes a unique cell culture matrix to grow
H. pylori.
In 1983
H. pylori
were cultured in vitro for the first time by using a complex media (Walkers media) and extending the culture timeperiods (5 days instead of the normal 2 day culture). This remains the current method for growing
H. pylori
, and accordingly, the method suffers the disadvantage of requiring long incubation times and the use of expensive complex media formulations. Furthermore, the presently used culture media fail to mimic the natural in vivo environment of
H. pylori
. Cellular morphology and metabolic activity of cultured cells are affected by the composition of the substrate on which they are grown. Presumably cultured cells function best (i.e. proliferate and perform their natural in vivo functions) when cultured on substrates that closely mimic their natural environment. Therefore, current studies of cellular function that are based on the in vitro growth of
H. pylori
, are limited by the lack of cell growth substrates that present the appropriate physiological environment for proliferation and development of the cultured cells. The compositions of the present invention provide a more appropriate physiological growth environment than is currently available for growing cells that naturally occupy the stomachs of vertebrate species.
SUMMARY OF THE INVENTION
The present invention is directed to an extracellular matrix, comprising vertebrate submucosal tissues, that provides the necessary microenvironment to allow the in vitro culture of fastidious organisms. Naturally occurring extracellular matrices have the ability to serve as a substrate for the growth of prokaryotic organisms, fungal agents and less well defined infectious agents of different genus and species which are difficult to grow in standard culture media. The extracellular matrixes of the present invention enhance the growth, and thus the detection of, and diagnosis of disease states caused by, these organisms. Likewise, cell culture substrates comprising submucosal tissue isolated from the stomach, the urinary tract, or the intestines can be utilized to mimic the natural in vivo environments of those tissue source organs. Thus cells growing on such substrates in vitro will provide more physiologically relevant data regarding the susceptibility of these organisms to various potential therapeutic agents.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term “contacting” as used herein with reference to cell culture is intended to
Badylak Stephen F.
Boder George B.
Afremova Vera
Barnes & Thornburg
Purdue Research Foundation
Saucier Sandra E.
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