Control samples for use as standards for evaluating apoptosis in a selected tissue

a technology of control samples and tissue, applied in the field of medicine and pharmacological research, can solve problems such as limitations of each of these methodologies, tissue atrophy or hypertrophy, autoimmune disease or degenerative disorder,

Inactive Publication Date: 2005-03-17
HEWITT CHARLES W
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Abnormalities in either of these processes may cause tissue atrophy or hypertrophy, either of which can lead to cancer, autoimmune disease or degenerative disorders.
Each of these methodologies has certain limitations.
The information gathered from the use of cultured cell lines is of limited value, however, because the physiological profile of cells in culture often does not accurately reflect the physiological profile of corresponding primary cells contained in a tissue or organ.
One disadvantage of this practice is the lack of appropriate standardized controls.
As such, it is of little value in diagnosis of early or intermediate stages of the disease.
Biopsied tissue also is often not readily available when needed, and moreover can present a health hazard in that it may contain infectious agents.
From a practical standpoint, it is expensive and time-consuming to use animals in research.
Moreover, results may not be reproducible from one animal study to another.
Furthermore, there are numerous diseases and pathological conditions for which no relevant animal model exists.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Thymic Apoptosis in Microgravity Culture

[0055] Materials and Methods:

[0056] The following media and solutions were prepared: [0057] 1. Phosphate buffered saline (PBS) for rinsing tissue. [0058] 2. Control media, consisting of RPMi 1640 containing 15% heat-inactivated fetal bovine serum, 100 μg / mL penicillin, 100 μg / mL streptomycin, 100 μg / mL L-glutamine, and 2.5 μg / mL amphotericin B. [0059] 3. Experimental media, consisting of control media containing 1 μM dexamethasone.

[0060] Two microgravity bioreactors (Synthecon, Inc., Houston Tex.) were utilized; one containing control media and one containing experimental media.

[0061] The thymus was removed from two Lewis rats, washed in cold PBS to remove blood, and stripped of excess tissue. Thymus tissue was cut into sections of about 3 mm3, while in cold media.

[0062] The bioreactors were filled with control (CON) or experimental (DEX) media. Approximately 24 thymus pieces were placed in each bioreactor, for incubation at 37° C. at a r...

example 2

Apoptosis of Several Tissue Types in Microgravity Culture

[0068] The experiment described in Example 1 was repeated with each of the following tissue types: heart, kidney, liver, spleen, lymph node and skin. Results paralleled those observed and set forth above for thymic tissue.

example 3

Detection of Apoptosis Markers in Microgravity Cultured Tissues

[0069] The experiments described in Examples 1 and 2 were repeated with one or more of the following tissue types: thymus, heart, kidney, liver, spleen, lymph node and skin. Markers of apoptosis were detected immunohistochemically in the treated tissue, and / or by Western blot in culture fluid. Specific apoptosis marker proteins assayed for included annexin, one or more caspases, and Fas / FasL.

[0070] Results showed that markers of apoptosis increased in a graded fashion, similar to the results observed from TUNEL assays of the tissues. In addition, the marker proteins accumulated in the culture fluid in a similar graded fashion over time.

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Abstract

A method for producing standard control samples to be used to evaluate disease states or trauma that involve apoptosis or suppression of apoptosis is disclosed. Also disclosed are standard control samples produced by the method. The control samples comprise natural or artificial tissues treated in vitro to display reproducible, predetermined indicators of apoptosis that are equivalent to indicators of apoptotic status of corresponding tissues and organs of a living subject.

Description

FIELD OF THE INVENTION [0001] The present invention relates generally to the fields of medicine and pharmacological research. More specifically, the invention provides standard control samples to be used to evaluate disease states or trauma that involve apoptosis or suppression of apoptosis. The control samples comprise natural or artificial tissues treated in vitro to display reproducible, predetermined indicators of apoptosis that are equivalent to indicators of apoptotic status of corresponding tissues and organs of a living subject. BACKGROUND OF THE INVENTION [0002] Several scientific or patent publications are referenced in this patent application to describe the state of the art to which the invention pertains. Each of these publications is incorporated by reference herein, in its entirety. [0003] A coordination and balance between cell proliferation and cell death is critical for normal development and homeostasis of tissues and organs. Abnormalities in either of these proce...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N1/30
CPCG01N1/30
Inventor HEWITT, CHARLES W.
Owner HEWITT CHARLES W
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