• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    HIV-directed inhibitors of the natural 2′-5′A antiviral defense pathway, diagnostic procedures and materials, and modalities for treating viral disorders are described. HIV-induced inhibitors of RNase L in patients with lymphadenopathy (LAS), AIDS-related complex (ARC) and acquired immune deficiency syndrome (AIDS) were isolated and identified. Functional 2-5A is not able to activate RNase L in the peripheral blood mononuclear cells (PBMC) in these patients; endogenous dsRNA, especially mismatched dsRNA, activates 2-5A synthetase which results in an increased concentration by endogenous 2-5A.
    公开号:SU1687035A3
    申请号:SU884355467
    申请日:1988-03-03
    公开日:1991-10-23
    发明作者:А.Картер Вилльям
    申请人:Хем Рисерч Инк (Фирма)；
    IPC主号:
    专利说明:

    with
    with
    This invention relates to the field of virology, in particular, to a method for detecting a human immunodeficiency virus or another virus that produces a similar clinical picture.
    An inhibitory substance released or induced to be released by cells infected with human immunodeficiency viruses has been discovered. This inhibitor appears to be physically associated with the enzyme PH -ase L, the final product in the 2, 5-oligoadenyl t-PH-ase L system, and causes the entire immune system to fail, which allows viruses to multiply uncontrollably. Methods for identifying such inhibitors are described either directly or indirectly by the absence of the pH-base L or the inactivity of the pH-base L, or by the presence of intermediate biochemical substances in the 2-5 A-PH-ase system.
    L, wherein the inhibitor serves as a marker for detecting the presence of HIV or related viruses, giving essentially the same clinical picture.
    It also discusses the predictive value of such a marker in terms of the development of AIDS in full bloom in patients, in particular in patients who have antibodies to HIV in peripheral blood and tissues, including blood, blood fractions, organ transplanted cells and cell extracts. Describes how to activate the PH-basics L or deactivate the inhibitory PH-azu L substances. Part of the present invention are therapeutic methods for combating AIDS and related viruses and controlling the development of such viruses by activating the R-L basics and / or removing or deactivating the R-az inhibiting L substance (s) or
    about
    00
    ate

    Ca
    gibitiory virus. The invention includes methods for the production of antibodies to these viruses, both in vivo and in vitro, using a specific pH-L inhibitor or viral-directed inhibitors of biochemical intermediates in the 2-5A-PHH-a system of L. as antigen.
    In extracts of mononuclear cells of peripheral blood (PBMC) of people with lymph node syndrome (NWL), AIDS-related complex (CSC) and Acquired Immune Deficiency Syndrome (AIDS), the components of the 2-5A synthetase-PH-aza L. system were measured. Level 2 -5A synthetase is extremely overestimated in AIDS patients and is found at the basal level in patients with RSK or NWTC. However, regardless of fluctuations in the concentration of 2-5A synthetase (2-5A), high endogenous concentrations of 2-5A (up to 35 nM) were found in patients with NWT, RSK and AIDS. Extracts 2-5A of the plot were functional, as evidenced by the binding test, the cleavage of ribosomal RNA, and the test with pulp. Despite elevated concentrations of endogenous functional 2-5A, none of the patients with NWT, RSK or AIDS could not be shown activated PH-aza L. However, in all healthy people, pH-aza L in MCPM was activated, even though Endogenous 2-5A concentrations were less than 5 nM. Experiments with mixing indicate the presence of a RNase inhibitor L in five NWL patients. RSK and AIDS. Such an inhibitor can be transferred and can interfere with pH-based L-induced 2-5A-induced destruction of rRNA in extracts of L929 cells. One of these inhibitors of PH-basics, L, is precipitated with trichloroacetic acid (TCC) and ethanol. The inhibition of the PH-L action is removed by treatment with the PH-aza A or protease. Research suggests that in the MCPC of patients with NWT, CSC or AIDS, endogenous dsRNA activates 2-5A synthetase, which leads to an increase in the concentration of endogenous 2-5A. The ability of functional 2-5A to activate the PH-basics of L in the MCPC of patients with NWT, RSK and AIDS.
    Determining the concentration of 2-5A and the functioning of the PH-ase L in extracts of MCPC using specific and sensitive determinations shows that the defect of the antiviral system 2-5A synthetase-PH-ase L in MCPC of patients with NWPL, RSK and AIDS lies in
    the presence of an inhibitor of pH-basics L, but not in the synthesis of non-functional 2-5A. The data presented here emphasizes that seemingly paradoxical interactions,
    what is happening in a virus infected cell can now be explained by rational coordinated biochemical concepts.
    Bound with acrylic balls (1
    0 mg) PH-ase A or bound with carboxymethylcellulose (1 mg) (Sigma chemical Co) is soaked in NP40 lysis buffer (16) (10 μl) for 2 hours at 30 ° C, then mixed with PBMC extract (100 mcg for testing)
    5 to a final volume of 15 μl and additionally incubated for 2 h at 30 ° C. During the reincubation, the contents of the tubes are gently mixed several times.
    0 is incubated without the full addition of the enzyme. Immobilized enzymes are precipitated by centrifugation (lOOOOx g, 5 min., Room temperature). Five microliters of centrifuged fractions are added to
    5 extracts of L929 cells (150 µg of protein per test) and rRNA digestion tests were performed to determine the pH-L-specific activity.
    Endogenous 2-5A data isolated from MCPC extracts are characterized by carrying out trials of rRNA disassociation. The products formed during the cleavage of the pH-isola L with the products isolated from PBMC 2-5A differ significantly in cell extracts.
    5 L929 from products derived from authentic 2-5A. For example, endogenous 2-5A, isolated from PBMC, activate the L-2929 RN-ase from L929 cells with cleavage of 28S and 18S rRNA and the formation of three specific
    0 splitting products. An explanation of the observed differences in the cleavage products may be that the endogenous 2-5A synthesized in the PBMC differs from the authentic 2-5A.
    5 To detect the level of free (non-2-5A-activated) PH-ase L, which is the target enzyme for 2-5A, was used more than radio-binding. Other target enzymes may also be used. In extracts of MCPC of patients with SEL, CSC and AIDS, the level of free pH-based L is 5-7 times higher than in healthy people. However, the radio binding test is of limited value for samples in which endogenous 2-5A competes with P / pCp for binding to the PH-ase L. Accordingly, the results of the radio-binding test do not accurately reflect the PH level of the L Thus, Essentially determining the level of 2-5A-activated PH-basics L. To achieve this, a new sensitive method was used to determine the activated PH-azu 1.0 extracts of PBMC, isolated from only one milliliter of peripheral blood, and this method is based on specific Spines of cleavage of 2-5A-activated RNase on rRNA.
    Due to the fact that rRNA is present in MCPC below uroon detection, extracts of HL 929 cells (subclone of L 929 cell strain with a pH deficiency L) are used as a source of rRNA. Using this method, it was possible to show that the PH-ase L is inactive in extracts of the MCPC of patients with NWPL, RSK and AIDS, where there is a high intracellular accumulation of functional 2-5A. MCPC extracts from all healthy people who were tested showed the presence of active pH-L, giving specific cleavage products despite the fact that 2-5A is present at a concentration of L 5nM.
    Biologically active 2-5A are present in extracts of PBMC infected with SZL, RSK and AIDS patients. Why is the inactive PH-ase L -To determine whether the absence of the activity of PH-ase L is due to the non-function of the PH-base L or the presence of an inhibitor of the activity of PH-ase L, the following mixing experiments were performed. Extracts of L929 cells containing functional PH-ase are mixed and incubated together with extracts of PBMC of patients with NWT, RSK and AIDS. No specific cleavage products for PH-ase L were detected.
    Endogenous 2-5A, which are responsible for the activation of the PH-ase L, give at least four-fold dilution relative to the cellular extracts from which these samples originate. These data indicate the presence of the PH-ase inhibitor (inhibitors) insoluble in TXK L in the MCPC of patients with NWHL, RSK and AIDS. Such an inhibitor is not found in healthy people. since their MCPC extracts generate RH-specific L cleavage products after co-incubation with extracts of L929 cells. Similarly, the soluble fraction of TCA PBMC from a healthy person generates specific degradation products. The presence of functional 2-5A and the fact that the putative pH-ase inhibitor L is precipitated by TCK and ethanol suggests that the PH-ase inhibitor L is not an analogue of 2-5A. as recently identified in cell cultures infected with HSV-1 and retrovirus.
    Experiments with the use of destructive enzymes immobilized on solid carriers give a completely new idea of the nature of the inhibitor (inhibitors) PH-ase L found in extracts of PBMC of patients with NWD, RSK and AIDS. If the MCPC extracts of AIDS patients are preincubated with immobilized protease or pH-ase A and then co-incubated with extracts of L929 cells, the pH-Lase L activity is restored, as evidenced by the appearance of specific cleavage products.
    The present studies show that a defect in the antiviral system 2-5A-synthetase-PH-ase L in MCPC of patients with NWTC, CSC and AIDS can be attributed to the PH-ase inhibitor L, and due to the absence of functional 2-5A. The PH-ase inhibitor L is present in the extracts of the MCPC of patients with NWTCh. RSK and AIDS, which indicates the possibility of the existence of an inhibitor at all stages of development of typical retroviral diseases and that this inhibitor is also related to various subacute (chronic viral) pathogenic infections. Considering that in all cases of PBMC of patients with NWLS, RSK or AIDS tested, contain HIV RNA (determined by molecular hybridization) and HIV infection centers (determined by the co-cultivation of lymphocytes), it is quite likely that a PH-asease inhibitor L is induced by a virus.
    Virus replication modifies the 2-5A-synthetase-PH-ase L system so that the natural antiviral state cannot be fully expressed or regulated. It is likely that the protein (s) and RNA (s) encoded by HIV or another retrovirus coexisting with it can inhibit the activation of the PH-basics of L.
    Example. Peripheral blood mononuclear cells (PBMCs) were isolated on Ficoll-Hypagua. L929 and HL929 cells (L929 PH-ases L-deficient subclone) are obtained in monolayer culture, cytoplasmic extracts of L929 and HL929 cells are obtained in glycerol buffer, PBMC extracts are obtained in NP40 lysis buffer according to known methods.
    Protein concentration in extracts 1-15 µg / µl.
    The 2-5A synthetase activity was determined in isolated PBMC extracts (50 µg per test) using poly (1) -poly (C) -agarose. 2-5A was isolated from soluble TCC fractions of MCPC extracts after neutralization with freon-tri-n-acylamine. Extracted TCA 2-5A is then lysophilized and re-dissolved in water in such a way that all samples are standardized in volume of water to an equivalent of 5 µg of protein per 1 µl. Concentrations of 2-5A present in the PBMC extracts are measured in a radio binding test with extracts of L929 cells as a source of PH base L. From 18,900 dpm pzA4 / 32 / P / pCp (specific activity 3000 C / mmol, Amersham), added per test, 19,400 dpm are bound and ph-ase in the absence of 2-5A. Approximately 9-14 pZA4 / / P / pCp is replaced in tests in which 5 µl of 2-5A isolated samples are added. Concentration of 2-5A in PBMC is determined by comparison with a calibration curve for authentic 2-5A, this definition is based on counting cytoplasmic protein concentrations of 25 µg / ml. The concentration of 2-5A, isolated from PBMC, is also measured in the test with adrocellulose with L929 cells as a source of pH-base L The activation of pH-base L in this test is based on the conversion of poly (U) / 32P / pCp into soluble acid fragments. Approximately 5–20% of the total amount of poly (U) / P / pCp (15,000 dpm) is cleaved in the presence of 2.5 µl of the 2-2A isolated samples. Concentrations are determined from calibration curves for authentic 2-5A. The level of free pH-Ase L is measured by a radio binding test after adding 20,000 dpm P3 / A 1 / 32P / pCp to PBMC extracts (50 µg of protein per test).
    The activity of 2-5A synthetase, the concentration of endogenous 2-5A, the level of free PH Lase, and the presence of PH Lase inhibitor in extracts of MCPM in patients with NWT, RSK and AIDS are shown in the table.
    The activity of an interferon-induced enzyme (2-5A synthetase) in extracts of MCPC obtained from patients with NWTC, CSC and AIDS is measured in vitro and compared with the activity of extracts from edoroids (table). Concentrations of 2-5A synthetase in AIDS patients (3 and 5) were 16 and 20 times higher than in normal patients (table). Conversely, the concentration of 2-5A synthetase in patients with NWL (1),
    RSK (Ya2) and AIDS with Kaposi's Sarcoma (IC) (4) are only 1.3-5 times higher, which is confirmed by other major observations.
    To determine whether such in vitro 2-5A synthetase measurements are indicative of the functionality of the enzyme in vivo, 2-5A are extracted from PBMC. Biological activity of 2-5A from soluble in
    TCC fractions of PBMC extracts are strongly characterized in radio-binding tests and dro-cellulose tests. Concentrations of 2-5A are similar when tested by two methods (table). Similarity
    The results obtained by methods A and B (table) indicate that 2-5A, isolated from extracts of human MCPCs, can bind and activate the RN-azu L from L929 cells to the same extent as authentic2-5A.
    The apparent lack of quantitative correlation between the levels of 2-5A synthetase 11 endogenous 2-5A in NWT. RSK and AIDS (table) may be first summarized as follows. The determination of 2-5A synthetase in in vitro tests is based on partial purification through poly (g) -poly (hC) -agarose, respectively, in such a test only free
    2-5A synthetase, and not 2-5A synthetase activated in vivo using dsRNA. However, endogenous 2-5A synthetase plus degraded 2-5A enzymes (for example, 2-phosphodiesterase and
    phosphatase), Thus, the accumulation of 2-5A MCPC of patients with NWTC, CSC and AIDS indicates a low activity of degraded 2-5A-enzymes. The presence of 2-5A in the MCPC also indicates that
    权利要求:
    Claims (1)
    [1]
    There is an endogenous dsRNA, so far not detected in healthy people, and in patients with AIDS. DETAILED DESCRIPTION OF THE INVENTION A method for detecting the presence of a human immunodeficiency virus or another virus giving a similar clinical picture, including determining the presence of a biological marker in a sample of biological material, characterized in that RNAase inhibitor L is used as a marker, and monogonous cells are used as a biological material. peripheral blood.
    Note.
    a - standard deviation of 9%;
    b - determined in the test by radio communication
    to (duplicated); c - determined in test with cellulose
    (duplicated), standard deviation M "; d - determined in the test by radio linking
    (duplicated), standard deviation 10%; d - determined in the test for splitting RRIK; e - on average for 7 control samples (duplicate
     vano); BUT - not defined.
    类似技术:
    公开号 | 公开日 | 专利标题
    Ranki et al.1987|Long latency precedes overt seroconversion in sexually transmitted human-immunodeficiency-virus infection
    SU1687035A3|1991-10-23|Method for detection of human immunodeficiency virus | or of other virus producing similar clinical picture
    Johnston et al.2001|Monocyte activation and differentiation augment human endogenous retrovirus expression: implications for inflammatory brain diseases
    US8338180B2|2012-12-25|Methods for treating lentivirus infections
    Juffroy et al.2010|Dual mechanism of impairment of interleukin-7 | responses in human immunodeficiency virus infection: decreased IL-7 binding and abnormal activation of the JAK/STAT5 pathway
    Kleiman et al.2004|HERV‐K | GAG/ENV antibodies as indicator for therapy effect in patients with germ cell tumors
    Lazzi et al.1998|HIV-associated malignant lymphomas in Kenya |
    Kessing et al.2017|High number of activated CD8+ T cells targeting HIV antigens are present in cerebrospinal fluid in acute HIV infection
    Meddows-Taylor et al.1998|Reduced expression of interleukin-8 receptors A and B on polymorphonuclear neutrophils from persons with human immunodeficiency virus type 1 disease and pulmonary tuberculosis
    Roger et al.1999|Highly active anti-retroviral therapy | is associated with a lower level of CD4+ T cell apoptosis in HIV-infected patients
    US5776690A|1998-07-07|Detection of chronic fatigue syndrome by decreased levels of RNase L inhibitor mRNA
    Jabado et al.1997|gp160 of HIV or anti‐CD4 monoclonal antibody ligation of CD4 induces inhibition of JNK and ERK‐2 activities in human peripheral CD4+ T lymphocytes
    Narita et al.2000|Actinomycin D and staurosporine, potent apoptosis inducers in vitro, are potentially effective chemotherapeutic agents against glioblastoma multiforme
    Koizumi et al.1999|Apoptosis in favourable neuroblastomas is not dependent on Fas | expression but on activated caspase 3 |
    CA2222513C|2009-10-13|Agents binding specifically to biotin for detecting damaged nucleic acid bases
    Gozlan et al.1998|Human immunodeficiency virus type 1 induction mediated by genistein is linked to cell cycle arrest in G2
    Beuhler et al.2004|The Meixner test in the detection of α-amanitin and false-positive reactions caused by psilocin and 5-substituted tryptamines
    Giraldo et al.1998|Frequency of the Asn-108 and Thr-108 point mutations in the dihydrofolate reductase gene in Plasmodium falciparum from southwest Colombia.
    CN112055715A|2020-12-08|Quantitative detection of CD4T cell injury and prediction of CD4T cell recovery following antiretroviral therapy using COFILIN phosphorylation
    Haraguchi et al.2011|Developmental regulation of P‐glycoprotein activity within thymocytes results in increased anti‐HIV protease inhibitor activity
    Komoto et al.2002|Ability to induce p53 and caspase-mediated apoptosis in primary CD4+ T cells is variable among primary isolates of human immunodeficiency virus type 1
    Guillerm et al.1998|An anti-CD4 | monoclonal antibody inhibits human immunodeficiency virus type 1 envelope glycoprotein-induced apoptosis
    Albuquerque et al.2007|Low CD4 T-cell counts despite low levels of circulating HIV: insights from the comparison of HIV-1 infected patients with a discordant response to antiretroviral therapy to patients with untreated advanced HIV-2 disease
    AU2010227529A1|2011-10-13|Methods for the detection and treatment of aberrant prion disease
    WALDERICH et al.1997|Sensitivity of Entamoeba histolytica and Entamoeba dispar patient isolates to human complement
    同族专利:
    公开号 | 公开日
    AU660973B2|1995-07-13|
    FI880960A|1989-06-24|
    EP0325018A2|1989-07-26|
    JP2690493B2|1997-12-10|
    AU1256488A|1989-06-29|
    PT86880B|1993-01-29|
    PT86880A|1988-04-01|
    CA1337277C|1995-10-10|
    AU1618792A|1992-09-10|
    HU205460B|1992-04-28|
    FI880960A0|1988-03-02|
    NO880935L|1989-06-26|
    JPH01171500A|1989-07-06|
    NO880935D0|1988-03-02|
    KR890010562A|1989-08-09|
    DK112988A|1989-06-24|
    RU1836101C|1993-08-23|
    DK112988D0|1988-03-02|
    EP0325018A3|1990-01-17|
    ZA881470B|1988-11-18|
    PH25365A|1991-05-13|
    HUT47745A|1989-03-28|
    OA08815A|1989-03-31|
    CN1043569A|1990-07-04|
    IL85577D0|1988-08-31|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    FR2505845A1|1981-05-13|1982-11-19|Choay Sa|Substrate system for oligo-adenylate synthetase assay - used to diagnose infection and measure interferon concn.|
    EP0113162B1|1982-09-16|1989-07-19|Hem Research, Inc.|Anti-proliferative action of dsnras on tumor cells|
    CA1326450C|1985-08-26|1994-01-25|William A. Carter|Modulation of aids virus-related events by double stranded rnas |US5278042A|1989-02-06|1994-01-11|The United States Of America As Represented By The Department Of Health & Human Services|Method for detecting inhibitors of tat protein|
    FR2725214A1|1994-09-30|1996-04-05|Inst Nat Sante Rech Med|NOVEL COMPOUNDS FOR PREPARING ANTI-INFECTIOUS AGENTS|
    US5985565A|1996-10-09|1999-11-16|Temple University-Of The Commonwealth System Of Higher Education|Chronic fatigue syndrome diagnosis|
    US6506559B1|1997-12-23|2003-01-14|Carnegie Institute Of Washington|Genetic inhibition by double-stranded RNA|
    WO1999049029A1|1998-03-20|1999-09-30|Benitec Australia Ltd|Control of gene expression|
    AUPP249298A0|1998-03-20|1998-04-23|Ag-Gene Australia Limited|Synthetic genes and genetic constructs comprising same I|
    AU776150B2|1999-01-28|2004-08-26|Medical College Of Georgia Research Institute, Inc.|Composition and method forandattenuation of gene expression using double stranded RNA|
    US6207366B1|1999-04-14|2001-03-27|Temple University- Of The Commonwealth System Of Higher Education|Chronic fatigue syndrome diagnosis|
    US6423885B1|1999-08-13|2002-07-23|Commonwealth Scientific And Industrial Research Organization |Methods for obtaining modified phenotypes in plant cells|
    US20020132257A1|2001-01-31|2002-09-19|Tony Giordano|Use of post-transcriptional gene silencing for identifying nucleic acid sequences that modulate the function of a cell|
    CN112088903A|2020-09-28|2020-12-18|武汉愔紫生物科技有限公司|Application of macromolecular protein in antibacterial and antiviral disinfectant|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US13697887A| true| 1987-12-23|1987-12-23|
    [返回顶部]