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Title:  Diagnostic screens for type 1 diabetes (IDDM)

United States Patent:  6,620,583

Issued:  September 16, 2003

Inventors:  Corkey; Barbara E. (Boston, MA); Husni; Nicholas R. (Boston, MA)

Assignee:  Boston Medical Center Corporation (Boston, MA)

Appl. No.:  191207

Filed:  July 9, 2002

Abstract

The present invention features a method for identifying genes or proteins important in insulin-dependent diabetes mellitus (IDDM). The genes or proteins are useful in identifying IDDM-susceptible individuals, and in identifying and testing potential therapeutic agents for the treatment of IDDM.

SUMMARY OF THE INVENTION

The present invention features novel methods of diagnosing persons or subjects having diabetes or at risk for developing diabetes. In particular, the present invention features methods of diagnosing Insulin-Dependent Diabetes Mellitus ("IDDM"), also known as Type 1 diabetes. The present invention is based, at least in part, on the discovery of a striking difference in Ca2+ mobilization of human skin fibroblasts from patients with IDDM. In ten out of ten cultured cell lines from unrelated subjects with IDDM, hyper-responsive Ca2+ mobilization was observed as compared to the response in seven out of seven unrelated control cell lines. Accordingly, in one embodiment the present invention features methods for diagnosing IDDM in a person or subject (e.g., a test subject) which include detecting hyper-responsive Ca2+ mobilization in a cell sample obtained from the subject. In a preferred embodiment, detecting hyper-responsive Ca2+ mobilization in cells obtained from the subject (e.g., the test subject) includes comparing Ca2+ mobilization in those cells to Ca2+ mobilization in cells derived from a control subject. Ca2+ mobilization, according to the present invention is preferably induced by contacting cells with a stimulatory agent (e.g., bradykinin).

Hyper-responsive Ca2+ mobilization was observed in cells particularly in response to treatments that are known to affect the expression of genes and proteins. Exemplary treatments that caused hyper-responsive Ca2 mobilization to become apparent were exposure to the inflammatory cytokines, TNF.alpha. and IL-1.beta., that are elevated in newly diagnosed diabetics, and fatty acids, which are also elevated in diabetes. Accordingly, the methods of the present invention further feature contacting cells with a potentiating agent in order to facilitate detection of hyper-responsive Ca2 + mobilization in cells. In one embodiment, the potentiating agent is an inflammatory cytokine. Preferably, the potentiating agent is TNF-.alpha. or IL-1.beta.. In another embodiment, the potentiating agent is a component of the diabetic milieu. Preferably, the potentiating agent is a free fatty acid ("FFA"), for example, oleate or oleic acid. It is also within the scope of the present invention to contact cells with at least two potentiating agents, for example, prior to determining Ca2+ mobilization. For example, cells (e.g., cells from a test subject and/or cells from a control subject) can be contacted or treated with an inflammatory cytokine (e.g., TNF-.alpha. or IL-1.beta.) and a free fatty acid (e.g., oleic acid). These treatments change the Ca2+ signaling pathway which plays a major role in cell growth and transmitting information from the bloodstream to the interior of the cell.

Yet another aspect of the present invention includes methods for identifying (e.g., diagnosing) subjects at risk for developing IDDM (or having IDDM, for example, subjects whose disease is in the preclinical latency period) based on the striking difference in Ca2+ mobilization in fibroblasts from these patients (e.g., as compared to control subjects or to other standards). In one embodiment, the invention features identifying a person or subject (e.g., a test subject) at risk of developing IDDM which includes comparing Ca2+ mobilization in cells (e.g., a test cell sample) obtained from the subject to, for example, Ca2+ mobilization in cells from a control subject. Preferably, the person or subject at risk is identified by detecting a difference in Ca2+ mobilization in the test cell sample as compared to the control cell sample. In a preferred embodiment, a difference is detected in peak Ca2+ response (e.g., to a stimulatory or inducing agent). In yet another preferred embodiment, a difference is detected in steady state Ca2+ following stimulation with an inducing or stimulatory agent. In a more preferred embodiment, cells (e.g., test cells and/or control cells or an aliquot thereof) are contacted with a potentiating agent (e.g., TNF-.alpha. or IL-1.beta.), for example, prior to contacting with an inducing agent. In yet another embodiment, a difference is detected in the Ca2+ response increment (e.g., the incremental increase in response between cells treated with potentiating agent and untreated cells). Preferred subjects which benefit from the methodology described here are human subjects. The present invention further features kits for the diagnosis of IDDM or Type 1 diabetes.

DETAILED DESCRIPTION OF THE INVENTION

Highly significant differences in Ca2+ transients between cultured skin fibroblasts from ten unrelated Type 1 diabetic (IDDM) and seven control subjects were observed. The defect in Ca2+ signaling, referred to herein as "hyper-responsive Ca2+ mobilization", was found in cells from ten of ten patients with IDDM and none of seven controls. There was no overlap between the groups. Non-diabetic siblings exhibited intermediate responses, suggesting a genetic basis for the effect. The three-fold enhancement was observed following exposure for a period of hours to a cytokine such as TNF.alpha. or IL-1.beta. or to free fatty acids (FFA) in the growth media. The changes were prevented by concurrent treatment with cyclohexamide, indicating the involvement of newly synthesized proteins in the defect. Although bradykinin (BK) was used as an agonist to monitor Ca2+ transients, the altered responses were not specific to bradykinin since the endoplasmic reticulum (ER) Ca2+ stores and the rate and extent of Ca2+ uptake were greater in the treated fibroblasts from IDDM subjects than from controls.

Because the defect in Ca2+ mobilization (i.e., hyper-responsive Ca2+ mobilization) is manifested in a common signal transduction pathway, mobilization of intracellular Ca2+, it is possible to identify novel specific genes and/or proteins that are different between controls and diabetics. Identification of these novel genes and/or proteins allows the development of screens to identify susceptible individuals and of appropriate screens for testing potential therapeutic agents. Furthermore, the defect in the Ca2+ pathway is predicted to affect responses to many hormones and agonists and because it is a well-understood pathway, provides a good target for testing the effectiveness of putative drugs that might prevent the changes from occurring in susceptible individuals and potentially preventing the development of IDDM. Identification of these novel genes and/or proteins allows the development of appropriate screens for testing potential therapeutic agents.

A. Diagnostic Assays

In one embodiment, the present invention involves a method for diagnosing IDDM in a test subject which includes detecting hyper-responsive Ca2+ mobilization in cells obtained from the test subject. The term "hyper-responsive Ca2+ mobilization" includes Ca2+ responsiveness or Ca2+ mobilization in a cells, in particular, mobilization of Ca2+ from intracellular stores into the cytoplasm of a cell. Ca2+ mobilization can result from contacting a cell, for example, with a stimulatory or inducing agent. The term "stimulatory agent" or inducing agent" includes any compound or agent that causes a cell (e.g., induces, triggers, stimulates) a cell to mobilize Ca2+, for example, from intracellular stores into the cytoplasm of a cell. "Stimulatory agents" or "inducing agents" of the present invention include any agent that acts on a cell through PLC activation. More preferably, inducing agents include any agent that causes release of calcium from the ER of a cell. Exemplary preferred inducing agents include bradykinin, epinephrine, calcium ionophores and the like. Also preferred are, for example, agents that inhibit Ca2+ -ATPase responsible for sequestering calcium in the ER (e.g., thapsigargin). Cells useful according to the diagnostic methods of the present invention include any cell obtained or isolated form the subject (e.g., test subject or control subject) which is capable of mobilizing calcium. Preferred cells (e.g., test cells and/or control cells include fibroblasts, for example fibroblasts cultured from a skin biopsy, white blood cells, for example peripheral blood leukocytes, fat cells, and the like. Cells can exist in populations as cultures or as single cells (e.g., with Ca2+ mobilization detected via an imaging system).

The phrase "detecting hyper-responsive Ca2+ mobilization" includes detecting any indicator of the trait defined herein as hyper-responsive Ca2+ mobilization. For example, Ca2+ mobilization can be determined in a cell according to any methodology familiar to one of ordinary skill in the art and compared to any suitable control or standard to determine that Ca2+ mobilization (e.g., Ca2+ mobilization in the test cell) is hyper-responsive. Ca2+ mobilization can be determined, for example, by loading cells with a calcium-sensitive dye (e.g., a fluorescent or colorimetric calcium-sensitive dye). A preferred calcium-sensitive dye is fura-2 acetoxymethyl ester, also referred to herein as fura-2 AM or fura-2. An exemplary Ca2+ mobilization response or Ca2+ response includes an increase and peak in intracellular Ca2+ concentration followed by a decrease and plateau in intracellular calcium concentration. The initial intracellular Ca2+ concentration is referred to herein and in the art as the basal intracellular Ca2+ concentration. The ending intracellular Ca2+ concentration is referred to as steady state intracellular Ca2+ concentration. As described previously, "detecting hyper-responsive Ca2+ mobilization" can include determining Ca2+ mobilization in a cell according to any methodology familiar to one of ordinary skill in the art and comparing it to any suitable control or standard to determine that Ca2+ mobilization is hyper-responsive. In a preferred embodiment, "detecting hyper-responsive Ca2+ mobilization" involves comparing Ca2+ mobilization in cells obtained from the test subject to Ca2+ mobilization in cells obtained from a control subject. Accordingly, the suitable control can be a control cell. The phrase "control cell" includes any cell which exhibits normal traits, as compared to diabetic traits (e.g, hyper-responsive Ca2+ mobilization). In one embodiment, control cells are cells of the same cell type as the test cell (or cell obtained or isolated from the test subject) but are obtained or isolated from a control subject (e.g., a subject devoid of IDDM or traits thereof). In another embodiment, control cells are cells obtained or isolated from the test subject but which exhibit normal traits. It is also within the scope of the present invention to use control cells, for example cell lines or cultures, which have predefined characteristics (e.g., have been previously determined to exhibit a normal phenotype). As defined herein, a suitable control can also include a predefined indication of normal phenotype. For example, a normal intracellular Ca2+ concentration, for example, for a particular cell type, can be predetermined form analysis of normal cells and that indication used as a control according to the present methodology. In one embodiment, a normal peak intracellular Ca2+ concentration can be determined (e.g, following bradykinin stimulation of normal cells) and that number (taking into account reasonable variation) can be used as a suitable control.

The term "detecting hyper-responsive Ca2+ mobilization" further includes detecting any protein characteristic of the hyper-responsive Ca2+ mobilization trait. For example, the present inventors have demonstrated that hyper-responsive Ca2+ mobilization is inhibitable by protein synthesis inhibitors. Accordingly, one of ordinary skill in the art can characterize proteins involved in conferring the hyper-responsive Ca2+ mobilization trait on cells and detect the abundance or activity of such proteins as indicating of the hyper-responsive Ca2+ mobilization trait.

In another aspect, the method includes the step of contacting the cells with a potentiating agent prior to comparing Ca2+ mobilization. Preferred potentiating agent include inflammatory cytokine (e.g., TNF-.alpha., IL-1.beta., IFN-.gamma. or LIF) as well as certain components of the diabetic milieu (e.g., free fatty acid (FFA), for example, oleic acid). The phrase "diabetic milieu" includes the extracellular environment experienced by a cell, for example, a cell within a diabetic donor. Components of the "diabetic milieu" include any agent that elevates cytosolic free calcium from intracellular stores and preferably can include free fatty acids and/or high glucose. Preferred components of the diabetic milieu include, for example, oleic acid.

Preferred methods of the present invention can further include contacting the cells with two potentiating agents prior to comparing Ca2+ mobilization (e.g., TNF-.alpha. or IL-1.beta. and FFA). The use of a potentiating agent is particularly desirable due to the fact that differences in Ca2+ mobilization between normal and control cells are augmented by inclusion of the potentiating agent. For example, peak Ca2+ mobilization response in cells from diabetic donors differs from that of control cells (e.g., is enhanced) when both are treated with a potentiating agent. Moreover, steady state Ca2+ concentrations in bradykinin-induced cells from diabetic donors differ from that of control cells when both are treated with a potentiating agent. Furthermore, Ca2+ mobilization increments (e.g., the incremental increase detected when comparing cells in the presence versus absence of potentiating agent) are detectable only when cells (or at least an aliquot of cells) are treated with potentiating agent. As exemplified herein, potentiation is a time-dependent effect. Accordingly, when cells are treated with a potentiating agent, they are preferably treated for at least 1 hour, preferably 2 hours, more preferably 34 hours, even more preferably between 4 and 12 hours, even more preferably between 12 and 24 hours or greater than 24 hours.

In another embodiment, the present invention involves a method for identifying a subject at risk of developing IDDM or a subject having IDDM which includes obtaining a test cell sample from a test subject, determining Ca2+ mobilization in the test cell sample, comparing the Ca2+ mobilization in the test cell sample to Ca2+ mobilization in a control cell sample from a normal subject, and identifying a subject at risk of developing IDDM or a subject having IDDM by detecting a difference in Ca2+ mobilization in the test cell sample as compared to the control cell sample. In a preferred embodiment, the test cell sample and the control cell sample are contacted with a stimulatory agent (e.g., bradykinin) to induce Ca2+ mobilization. In another embodiment, the test cell sample and the control cell sample are contacted with a potentiating agent prior to stimulation with the stimulatory agent. A preferred potentiating agent is, for example, an inflammatory cytokine (e.g., TNF-.alpha. or IL-1.beta.).

In another embodiment, the present invention involves a method for identifying a subject at risk of developing IDDM or a subject having IDDM which includes obtaining a test cell sample from a test subject, contacting the test cell sample with a stimulatory agent (e.g., bradykinin), determining steady state Ca2+ levels in the test cell sample following response of the cell to the stimulatory agent, comparing the steady state Ca2+ levels in the test cell sample following response of the cell to the stimulatory agent to steady state Ca2+ levels in a control cell sample from a normal subject following response of the control cell to the stimulatory agent, and identifying a subject at risk of developing IDDM or a subject having IDDM by detecting a difference in steady state Ca2+ levels in the test cell sample as compared to the control cell sample. In another embodiment, the test cell sample and the control cell sample are contacted with a potentiating agent prior to stimulation with the stimulatory agent. A preferred potentiating agent is, for example, an inflammatory cytokine (e.g., TNF-.alpha. or IL-1.beta.).

In yet another embodiment, the present invention involves a method for identifying a subject at risk of developing IDDM or a subject having IDDM which includes obtaining a test cell sample from a test subject, contacting the test cell sample with at least one component of the diabetic millieu (e.g., FFA and/or glucose), determining Ca2+ mobilization in the test cell sample, comparing the Ca2+ mobilization in the test cell sample to Ca2+ mobilization in a control cell sample from a normal subject following response of the control cell to at least one component of the diabetic millieu, and identifying a subject at risk of developing IDDM or a subject having IDDM by detecting a difference in Ca2+ mobilization in the test cell sample as compared to the control cell sample. In a preferred embodiment, the test cell sample and the control cell sample are contacted with a stimulatory agent (e.g., bradykinin) to induce Ca2+ mobilization. In another embodiment, the test cell sample and the control cell sample are contacted with a potentiating agent prior to stimulation with the stimulatory agent. A preferred potentiating agent is, for example, an inflammatory cytokine (e.g., TNF-.alpha. or IL-1.beta.).

In yet another embodiment, the present invention involves a method for identifying a subject at risk of developing IDDM or a subject having IDDM which includes obtaining a test cell sample from a test subject, contacting the test cell sample with at least one component of the diabetic millieu (e.g., FFA and/or glucose), contacting the test cell sample with a stimulatory agent (e.g., bradykinin), determining steady state Ca2+ levels in the test cell sample, comparing steady state Ca2+ levels in the test cell sample to steady state Ca2+ levels in a control cell sample from a normal subject following response of the control cell to at least one component of the diabetic millieu and the stimulatory agent, and identifying a subject at risk of developing IDDM or a subject having IDDM by detecting a difference in steady state Ca2+ levels in the test cell sample as compared to the control cell sample. In another embodiment, the test cell sample and the control cell sample are contacted with a potentiating agent prior to stimulation with the stimulatory agent. A preferred potentiating agent is, for example, an inflammatory cytokine (e.g., TNF-.alpha. or IL-1.beta.).

Examplary methods of determining mobilization of Ca2+ and steady state Ca2+ levels in cells are described in detail in the following examples. Furthermore, it is intended that such determinations can be made using alternative methods known in the art for determining mobilization of Ca2+ and steady state Ca2+ levels in cells.

Claim 1 of 4 Claims

We claim:

1. A method for identifying specific genes or proteins that differ between control and diabetic subjects, said method comprising comparing Ca2+ responsiveness in cells from said control and diabetic subjects and selecting a gene or protein based on a detectable difference in said Ca2+ responsiveness.




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