NON-INVASIVE APPROACH TO EARLY DIAGNOSIS AND PROGNOSIS OF ACUTE SEVERE PANCREATITIS BASED ON HEAT SHOCK PROTEINS MECHANISM OF ACTION

Abstract

This invention describes a method for early diagnostics of severe pancreatitis using cell membrane damage detection mechanisms. The method involves a non-invasive testing of human urine or other body fluids samples of patients that are suspected to be affected by acute pancreatitis. The invention is based on heat shock protein HSP-70, 90, 60, and 27 role of action in disease progression.

Claims

1. A method for determining acute pancreatitis disease initiation, wherein the method comprises the steps of measuring the levels of heat shock proteins HSP-70, 90, 60, 27 in human urine or other body fluids samples of patients admitted to emergency department within 72 hours since the onset of the symptoms of acute pancreatitis, analysing the data according to protocol, combining the data with routine chemical tests to provide a clinical diagnosis.

2. A method for determining lifetime risk for acute pancreatitis, wherein genetic testing of HSP-70, 90, 60, and 27 is performed for a subject in risk of AP.

3. A method according to claim 1 additionally involving a non-invasive radiological test to determine the need for surgical intervention.

Description

DESCRIPTION OF DRAWINGS

[0018] FIG. 1. Interaction between HSP70 and HSP90 with tBLM. A—Phase angle plot of EIS spectra: black—the initial spectra of tBLM, green—10 min after HSP70 of 0.2 uM injected, red—after 24 hours after injection of HSP70. tBLM was composed from DOPC and Cholesterol (in ratio % 60:40). B.—Phase angle plot of EIS spectra: black—the initial spectra of tBLM, green—10 min after HSP90 of 2 uM injected, red—after 24 hours after injection of HSP90. tBLM was composed from DOPC and Cholesterol (in ratio % 75:25).

[0019] FIG. 2. SPR sensograms of the adsorptions of HSP70 and HSP90 proteins on lipid membranes (DOPC:cholesterol in ratios 60:40 and 75:25, respectively). HSP70 and HSP90 concentrations are x and y, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Our invention involves using the methodology shown in patent LT 6424 entitled “Method of preparing immobilized on the surface phospholipid bilayer membrane (TBLM)” and applying approved methods in acute pancreatitis early diagnosis settings.

[0021] In one embodiment of the invention a prognostic and diagnostic biomarker or set biomarkers (diagnostic kit) are assembled for detection of acute pancreatitis by measuring Heat Shock Proteins (HSP) as major players in the mechanism of the severity of acute pancreatitis. The samples for testing are taken patients' urine, sweat, or saliva and blood serum/plasma levels are used as controls.

[0022] In another embodiment, the invention can serve as a therapeutic target to determine early multiple organ dysfunction syndrome in the setting of acute pancreatitis.

Materials and Methods

HSP-70

[0023] Recombinant Protein HSP70 was purchased from Sigma Aldrich (Germany) with purity of ≥70%. Recombinant HSP90 full length was donated by prof. D. Matulis from VU Life Sciences Center, Institute of Biotechnology, HSP90 was cloned and purified.

[0024] Serum HSP70 was analysed using Human Heat Shock Protein 70 (HSP70), (Cat no: 20,959) purchased from Bio Medical Assay (BMASSAY, Beijing, China) following the manufacturer's instructions. Their levels were expressed as ng/ml. The detection and quantification limits were set at <0.05 ng/ml for HSP70.

[0025] Serum HSP70 protein levels were determined using commercially available ELISA kits (StressGen Biotechnologies Corp). The concentrations of HSP70 protein were determined by comparison with a standard curve according to manufacturer's direction. The standard curve has a range of 0.78 to 50 ng/mL, and the sensitivity of the assay is 0.2 ng/mL.

[0026] Total sHsp70 levels in serum samples of humans were measured using a modified Hsp70 immunoassay (Duoset, DYC1663, R&D Systems, Minneapolis, Minn., USA). The ELISA is designed to detect human Hsp70 in buffer.

HSP-90

[0027] HSP90 ELISA (SUNRED Biotechnology Company, Shangai, People Republic of China) uses a double-antibody sandwich enzyme-linked immunosorbent assay to determine the level of human HSP90 in samples.

[0028] Serum HSP90a levels were determined in duplicate by a solid-phase ELISA technique (Assay Designs, Stressgen, Mich., USA).

[0029] Sandwich ELISA assays were performed in 96-well plates, using 100 μL peripheral blood plasma per well. When required, plasma was diluted in blocking buffer, in which case the concentration read from the standard curve was multiplied by the dilution factor. The following ELISA assays were used according to manufacturer recommendations: Hsp90a (human) ELISA Kit (Enzo Life Sciences, Farmingdale, N.Y.), Human beta 2-Microglobulin Quantikine IVD ELISA Kit (R&D Systems Inc., Minneapolis, Minn.) and Human IGFBP-2 DuoSet (R&D Systems In).

[0030] Lipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, USA) and used as received. All other solvents (AR grade) were used without purification, and all reactions were carried out under nitrogen.

Tethered Phospholipid Bilayer Membrane Formation

[0031] Gold substrates for electrochemical impedance spectroscopy (EIS) measurements were prepared on 25×75 mm glass slides (ThermoFischer Scientific, UK). Gold substrates for SPR were prepared on BK7 glass slides (25 mm diameter, 1 mm thickness) obtained from AutoLab (Methorm, The Netherlands). Gold layers were deposited by the magnetron sputtering using PVD75 (Kurt J. Lesker Co., U.S.) system. The details on the synthesis and properties of the self-assembled monolayers used in this work were described earlier.

[0032] Tethered lipid membranes (tBLM) were prepared by multilamellar vesicle fusion method described in previous papers. Vesicle solution was prepared from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol (Avanti Polar Lipids, USA) in molar % ratio 6:4 and 7.5:2.5 in phosphate buffer solution (PBS) containing 0.1 M NaCl (Roth, Denmark), 0.01 M NaH2PO4 (Roth, Denmark), pH 4.5.

Electrochemical Impedance Spectroscopy

[0033] EIS measurements were registered using Zennium electrochemical workstation (Zahner GmbH, Germany) between 0.1 Hz and 100 kHz, with 10 logarithmically distributed measurement points per decade. Data were fitted using ZView software (Scribner Associates, Southern Pines, N.C.). All measurements were carried out in sodium phosphate buffer solution (PBS) 0.1 M NaCl, 0.01 M NaH2PO4 pH 7.2. A three-electrode cell configuration was used. Reference electrode was Ag/AgCl, NaCl (sat.) electrode. Platinum wire (Aldrich, 99.99% purity) was used as auxiliary electrode. Working electrode was Au coated glass slide. In this work 12 vial electrochemical cell was used. Surface area of the working electrode exposed to the solution in the cell was 0.25 cm.sup.2. All measurements were performed at room temperature (21±2° C.).

Surface Plasmon Resonance (SPR)

[0034] The SPR measurements were conducted on an Autolab Twingle system (Eco Chemie B.V., The Netherlands) equipped with a flow-through cell (volume—175 μL). The unit performs SPR spectra recording at a fixed wavelength of 670 nm. It automatically follows with a millidegree resolution the position of an incidence angle (ranging from 62 to 78°), at which minimum of the reflection due to an excitation of the surface plasmon resonance is observed. Model F34 refrigerating/heating circulator (Julabo, DEU) was used to stabilize temperature at 21±0.1° C.

[0035] Before each experiment, the baseline in buffer solution was recorded. All measurements were carried out at stopped-flow conditions. According to manufacturer's manual minimum shift by 122 millidegrees (m°) corresponds to a surface coverage of 100 ng/cm2 of an adsorbed protein. So, in this work, we used the relation 1.22 m°=1 ng/cm2 to recalculate the amount of adsorbed protein per surface area from the SPR data.

Results

[0036] Interaction of HSPs with Phospholipid Model Membranes by EIS and SPR.

[0037] The rise of the HSPs in the serum and urea is an important step in the pathogenesis of Acute pancreatitis. However, the mechanism by which HSP70 and HSP90 acts during AP is largely unknown. Data in the previous section suggests strong interaction of HSP70 and HSP90 preparations with phospholipid membranes.

[0038] The effect of reconstitution of HSP70 and HSP90 into the phospholipid membrane is shown in FIG. 1, protein action is readily seen form the changes of EIS plots. In particular, upon incubation of tBLMs with HSP70 (FIG. 1A). In addition, the phase minimum appears, and it moves towards higher frequencies as the incubation time of HSP70 increases (FIG. 1A). The conductance (Y) of the tBLM, which is the HSP70 induced conductance at the phase minimum (estimated as Y=1/Zphase min). Phase shift after interaction with HSP70 shows the damage of the membrane and alters the ion movement through the membrane (FIG. 1A).

[0039] Interaction of Hsp90 with phospholipid model membranes is showed in FIG. 1B. The opposite result comparing with interaction of HSP70 was observed. The phase minimum appears, and it moves towards lower frequencies as the incubation time of HSP90 increases (FIG. 1B). Such phase shift after interaction with HSP90 shows that protein is attaching the membrane and isolating any ion movement through the membrane (FIG. 1B).

[0040] FIG. 2 displays representative SPR traces obtained on tBLM exposed to the solutions of HSP70 (red curve) and HSP90 (black curve) monomers. In HSP70 case, SPR signal after washing (at 900th second) dropped below the measurement start. These changes indicate a decrease in the lipids on the surface of the sensor. This is an unusually unexpected result, because it was not known that proteins would disrupt the lipid membrane and this behaviour can be associated only with the specific protein properties. While another HSP90 protein causes an increase in the SPR signal. These changes are related to the adsorptions on lipids membrane.

[0041] Interaction of Hsp90 with phospholipid model membranes is showed in FIG. 1B. The opposite result comparing with interaction of HSP70 was observed. The phase minimum appears, and it moves towards lower frequencies as the incubation time of HSP90 increases (FIG. 1B). Such phase shift after interaction with HSP90 shows, that protein is attaching the membrane and isolating any ion movement through the membrane (FIG. 1B).

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