Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-07-16
2001-04-17
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06218154
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to an isothermal transcription based assay for the detection and quantitation of the &bgr; chemokines regulated-upon-activation, normal T expressed and secreted (RANTES), macrophage inflammatory protein-1&agr; (MIP-1&agr;) and macrophage inflammatory protein-1&bgr; (MIP-1&bgr;). The present invention is also directed to oligonucleotides for amplifying chemokine RNA and probes for use in the detection and quantitation of the amplification product.
BACKGROUND OF THE INVENTION
The RANTES chemokine has been shown to be a potent inhibitor of HIV-1 infection (Cocchi et al,
Science
270:1811, 1996). In this study it was shown that RANTES is one of the major suppressor factors produced by CD8+ cells. These suppressor factors have been implicated in the long term nonprogression of many asymptomatic HIV-1+ patients.
The invention described herein is a transcription based amplification method for the detection of the RANTES mRNA transcript, which will be detected in the PBL of HIV-1+ patients. The purpose of applying such an assay would be to determine the presence and level of the transcripts and use these results to predict prognosis and/or direct therapeutic management of the infected patient. The invention would involve primers that were found to be optimal for the detection/quantification of the RANTES transcript in vivo using NASBA. Qualitative and quantitative detection would involve the use of an internal control RNA standard. The preliminary sample processing and nucleic acid isolation would be as described by Boom et al. The detection technology would involve the colorimetric ELGA based system and the ECL based system.
The &bgr; chemokines MIP-1&agr;, MIP-1 &bgr; and RANTES inhibit infection of CD4
+
T cells by primary, non-syncytium-inducing HIV-1 strains at the virus entry stage, and also block env-mediated cell-cell membrane fusion. CD4
+
T cells from some HIV-1-exposed uninfected individuals cannot fuse with NSI HIV-1 strains and secrete high levels of &bgr;-chemokines. Expression of the &bgr;-chemokine receptor CC-CKR-5 in CD4
+
, non-permissive human and non-human cells renders them susceptible to infection by NSI strains, and allows env-mediated membrane fusion. CC-CKR-5 is a co-receptor for NSI primary strains of HIV-1. (Dragic et al,
Nature
, 381:667 (1996)).
The super family of chemoattractant cytokines (chemokines) and their receptors are involved in inflammation and infection. The chemokines range in size from 68 to 120 amino acids (in the mature form) and can be divided into three classes based on variations in a shared cysteine motif. The largest group, the C—C, or &bgr; chemokines has nearly 20 members identified to date. The smallest group, the C class, has but one. The C-X-C, or &agr; chemokine branch can be further subdivided into two groups based on structure and function. The largest of these groups contains proteins containing the E-L-R-C-X-C motif and the smaller group is made up of proteins without the E-L-R amino terminal to C-X-C.
The structural classes parallel function to a large extent in that most C-X-C chemokines with E-L-R are chemoattractants for neutrophils but not monocytes, whereas C—C chemokines generally attract monocytes and lymphocytes, but not neutrophils. Basopiuils and eosinophils are also affected predominantly by C—C chemokines. The C chemokine appears thus far to be lymphocyte specific.
A number of chemokine receptors have also been identified. These proteins are structurally related, with amino acid homology high in the transmembrane regions and some intracellular loops. There seems to be less homology at the N- and C-termini, and extracellular loops, which are presumed to be involved in ligand binding (N-terminal) and receptor specific interactions with signaling components.
Engagement of the chemokine receptors results ultimately in the movement of the cell. The steps of this process are thought to be: relay of information from the receptor through G-proteins; mobilization of intracellular second messengers; coordinated reorganization of the cytoskeleton; formation of focal adhesions and attachment to and detachment from the substrate with pseudopodial extension, and retraction to effect directional migration.
The involvement of the chemokines and chemokine receptors in HIV infection is beginning to be understood. It has been known for some time that binding of the viral glycoprotein gp120 to CD4 is not sufficient for viral fusion and entry, suggesting the need for an additional coreceptor for HIV infection. Isolates of HIV generally have a tropism for either transformed T-cells (T cell line tropic or simply T-tropic strains) or for monocytes or cultured macrophages, and primary T cells, but not transformed T cells (macrophage tropic). The difference in tropism appears to reside in the V3 region of the gp120.
It was also known that there were endogenous HIV suppressor factors secreted by CD8
+
T cells that inhibited viral replication in CD4
+
cells in vitro. The &bgr; chemokines RANTES, MIP-1&agr; and MIP-1&bgr; have now been identified as soluble suppressors of macrophage-tropic, but not T cell line tropic, HIV infection in vitro. (reviewed in Premack,
Nature Medicine
, 2(11): 1174 (1996)).
It has been observed that the resistance of persons who remain uninfected despite multiple high-risk sexual exposures is associated with the activity of the C—C chemokines RANTES, MIP-1&agr; and MIP-1&bgr;. The relative resistance does not extend to T-cell line-adapted strains (Paxton et al.,
Nature Medicine
, 2(4): 412 (1996)). These results are consistent with a model in which CCR5 is a coreceptor (with CD4
+
) for macrophage tropic strains of HIV, and RANTES, MIP-1&agr; and MIP-1&bgr; are also ligands to CCR5. It has been shown that RANTES, MIP-1&agr; and MIP-1&bgr; block the entry of HIV-1 into cells (Cocchi et al,
Nature Medicine
, 2(11): 1244 (1996)).
Chemokine levels in infected patients are therefore very important in determining the course of the disease. Detecting and quantitating the RNA for the chemokines is therefore an important factor in the clinical evaluation of HIV positive patients.
Because chemokines are involved in the activation of inflammatory cells, previous workers have investigated the levels of chemokines or chemokine RNAs in different diseases or conditions related to inflammation. These diseases and conditions include graft rejection (Strehlau et al,
Proc. Nat Acad. Sci
. 94(2): 695-700 (1997)); cancer (Burket et al,
Cytokine
8(7): 578-585, (1996)); HIV-1 infection (Schmidtmayerova et al,
Proc. Nat. Acad. Sci
. 93(2): 700-704, (1996)); human cytomegalovirus infection (Michelson et al,
J. Virol
. 71(9): 6495-6500 (1997)); asthma (Berkman et al,
Am. J. Respir. Crit. Care Med
. 154(6/1): 1804-11 (1996); and others.
Lastly, it may be easier to establish correlations between &bgr;-chemokines transcript levels and disease state due to the increased sensitivity of the amplification based transcript NASBA assay, and the typically larger dynamic range of the quantitative NASBA system.
An isothermal amplification method for detection or quantification of chemokine mRNAs has not been described.
SUMMARY OF THE INVENTION
The present invention provides isothermal transcription based amplification assays for the detection and quantitation of &bgr; chemokine RNAs. The detection assay uses primer pairs and probes for each of the targets, RANTES, MIP-1&agr; and MIP-1&bgr;. Quantitative assays use an internal control RNA (Q) that differs from the wild type RNA by a small internal randomized segment. Q RNA is spiked into the sample at a known copy number at lysis and is coextracted and coamplified with wild type RNA. The resulting amplificate is then subjected to two independent hybridization reactions with probes specific for the wild type and Q amplificates. Quantitation of the wt RNA is achieved by calculating the ratio of resulting signal of wild type to Q.
An isothermal amplification method starts with an RNA temp
Romano Joseph W.
Shurtliff Roxanne
Williams Kimberly G.
Akzo Nobel N.V.
Horlick Kenneth R.
Siew Jeffrey
Sullivan Michael G.
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