Quantitative characteristic of circulating immune complexes in patients with lupus erythematosus and central nervous system disorders

Molecular weight and concentration characteristics of immune complexes (IC) from 19 sera of patients with systemic lupus erythematosus (SLE) and CNS impairment have been obtained by the rapid nephelometry assay. Basing on cranial CT findings, the examinees were divided into 2 groups. Group I included patients with cerebral cysts and local dilation of subarachonid spaces, group II those with the above dilatation or that of ventricles of the brain. Small-size IC were registered in 14 sera, their relative molecular mass being under the values derived for donors and SLE patients without CNS affections whereas their level exceeded such in donor sera. Larger IC relative concentrations were seen in group I patients than in group II ones (34 +/- 13 and 18.7 +/- 12, respectively). Five patients failed to demonstrate IC. The presence of small-size IC in high concentrations may be considered a marker of CNS involvement in SLE, the highest concentrations suggesting local impairment of the brain.     

Validation of the PEG-IgG screening test for soluble immune complexes by longitudinal studies in experimental acute serum sickness

A wide variety of tests for the detection of circulating immune complexes (IC) has been proposed by different authors, but there is very little to no information concerning the performance of IC screening assays in samples known to contain in vivo-formed IC. The purpose of our investigation was to compare the behavior of a non-specific assay, the PEG-IgG screening test for IC, with an antigen-specific assay in serum samples sequentially obtained from rabbits to which we induced acute serum sickness. Five animals were used in the study; we were able to detect an increase of IC constituted by the heterologous antigen (human serum albumin) and corresponding antibodies in all, and in 4 animals the results of the PEG-IgG assay closely correlated with the results of the antigen-specific assay (rho values between 0.975 and 1.00). The 4 animals in which IC showed a definite peak by both assays developed proteinuria and IC deposits at the glomerular level, while the animal that failed to develop IC detectable by the PEG-IgG test remained normal throughout the study. These results demonstrate the ability of the PEG-IgG test to detect in vivo-formed IC and suggest that the IC detected by this test have pathogenic potential.     

Autologous serum deprivation restored IL-1 receptor antagonist production by peripheral blood mononuclear cells in patients with gastric cancer

It has been established that cancer patients have immunosuppressive substances in their sera that depress cellular immunity. Although plasma exchanges have been attempted to remove these substances and to improve immunity to cancer, little is known about its mechanism from the viewpoint of cytokine pattern. The levels of the cytokines, tumour necrosis factor-alpha, interleukin 1beta, interleukin 6, interferon-gamma and interleukin-1 receptor antagonist (IL-1ra) by peripheral blood mononuclear cells (PBMC) were determined simultaneously by the whole-blood assay and the PBMC assay in 20 patients with gastric cancer and in 10 healthy volunteers. In both assays the cytokine levels were lower in patients with cancer compared with healthy controls, with the exception of IL-1ra. In the PBMC assay, the IL-1ra level in cancer patients was significantly higher than that in controls. No statistical correlation between the cytokine levels determined by the two assays was found. We suggest that autologous serum deprivation restored and enhanced IL-1ra production, and normalized the cytokine cascade in immune response, in patients with gastric cancer.     

Inhibition of phosphorylation of the colony-stimulating factor-1 receptor (c-Fms) tyrosine kinase in transfected cells by ABT-869 and other tyrosine kinase inhibitors

The properties of several multitargeted receptor tyrosine kinase inhibitors have been studied for their inhibition of colony-stimulating factor-1 receptor (CSF-1R) signaling. A structurally novel, multitargeted tyrosine kinase inhibitor (ABT-869), imatinib (STI571), and four compounds currently in clinical development (AG013736, BAY 43-9006, CHIR258, and SU11248) were tested for inhibition of CSF-1R signaling in both the enzymatic and cellular assays. ABT-869 showed potent CSF-1R inhibition in both the enzyme and cell-based assays (IC50s < 20 nmol/L). In contrast to a previous report, we have found that imatinib has activity against human CSF-1R in both assays at submicromolar concentrations. In enzyme assays, we have found that the inhibition of CSF-1R by both ABT-869 and imatinib are competitive with ATP, with Ki values of 3 and 120 nmol/L, respectively. SU11248 is a potent inhibitor of CSF-1R in the enzyme assay (IC50 = 7 nmol/L) and inhibits receptor phosphorylation in the cellular assay (IC50 = 61 nmol/L). AG013736 was also a potent inhibitor of CSF-1R in both assays (enzyme, IC50 = 16 nmol/L; cellular, IC50 = 21 nmol/L), whereas BAY 43-9006 is less potent in the enzyme assay (IC50 = 107 nmol/L) than in the cellular system (IC50 = 20 nmol/L). In contrast, we found that CHIR258 had less activity in the cellular assay (IC50 = 535 nmol/L) relative to its enzymatic potency (IC50 = 26 nmol/L). These results show the use of a cell-based assay to confirm the inhibitory activity of lead compounds and drug candidates, such as ABT-869, against the CSF-1R protein in situ.     

Absorbance nephelometry of immune complexes by reaction with anti-IgG after treatment with polyethylene glycol

We describe absorbance nephelometry of immunoglobulin G (IgG) in immune complexes (IC) with use of anti-IgG after precipitating the IgG from serum by three treatments with polyethylene glycol, 25 g/L final concentration. The three-step procedure removed all of the monomeric IgG, eliminating interference with the IC assay. Analytical recovery of IC was nearly 100%. Both the standard curve and results for control sera showed good day-to-day reproducibility, with CVs of less than 8%. Eighteen of 20 patients with diseases involving IC showed distinctly increased concentrations of IC; no increases were seen in 15 normal persons. Measurement of IC in 44 samples by this assay and a C1q assay gave a Spearman rank correlation coefficient of 0.76. The assay appears suited to routine use with manual double-beam spectrophotometers and stable nephelometers. The procedure is inexpensive, straightforward, and easy to perform.     

Similar diagnostic performances of antigen detection and nucleic acid detection of Cryptosporidium spp. in a low-prevalence setting

The diagnostic value of a real-time PCR assay for the detection of Cryptosporidium spp. in fecal samples was assessed as compared to the combination of the immunocromatographic assay (IC) and immunofluorescence assay (IF) currently performed in our laboratory for the diagnosis of cryptosporidiosis. On a total of 1040 samples collected from 2006 to 2010 and belonging to 533 patients suspected of having an intestinal parasitosis, Cryptosporidium spp. was detected in 31 samples (belonging to 12 patients) by IC and IF; the real-time PCR assay revealed Cryptosporidium spp. DNA in 5 additional samples for a total of 36 samples (13 patients). The real-time PCR assay exhibited higher sensitivity than IC and IF; however, its application to the diagnosis of cryptosporidiosis should be evaluated by every single laboratory, depending on the availability of trained personnel, financial resources, and the cost/effectiveness related to the prevalence of cryptosporidiosis.     

Doxorubicin-formaldehyde conjugates targeting alphavbeta3 integrin

We have reported the synthesis and biological evaluation of a prodrug to a doxorubicin active metabolite. Under physiologic conditions, release of the active metabolite, a conjugate of doxorubicin with formaldehyde, occurs with a half-life of 1 hour. To direct this prodrug to tumor, we designed two conjugates of the prodrug, doxsaliform, with the alphavbeta3-targeting peptides, CDCRGDCFC (RGD-4C) and cyclic-(N-Me-VRGDf) (Cilengitide). We now report the synthesis of these doxsaliform-peptide conjugates and their evaluation using MDA-MB-435 cancer cells. A hydroxylamine ether tether was used to attach 5'-formyldoxsaliform to RGD-4C in its acyclic form via an oxime functional group. The construct acyclic-RGD-4C-doxsaliform showed good binding affinity for alphavbeta3 in the vitronection cell adhesion assay (IC50 = 10 nmol/L) and good growth inhibition of MDA-MB-435 breast cancer cells (IC50 = 50 nmol/L). In its bicyclic forms, RGD-4C showed less affinity for alphavbeta3 and significantly less water solubility. Cyclic-(N-Me-VRGDf) was modified by substitution of D-4-aminophenylalanine for D-phenylalanine to provide a novel attachment point for doxsaliform. The conjugate, cyclic-(N-Me-VRGDf-NH)-doxsaliform, maintained a high affinity for alphavbeta3 (IC50 = 5 nmol/L) in the vitronectin cell adhesion assay relative to the peptide bearing only the tether (0.5 nmol/L). The IC50 for growth inhibition of MDA-MB-435 cells was 90 nmol/L. Flow cytometry and growth inhibition experiments suggest that the complete drug construct does not penetrate through the plasma membrane, but the active metabolite does on release from the targeting group. These drug conjugates could have significantly reduced side effects and are promising candidates for in vivo evaluation in tumor-bearing mice.     

Identification of a novel inhibitor of urokinase-type plasminogen activator

Urokinase-type plasminogen activator (uPA), a highly restricted serine protease, plays an important role in the regulation of diverse physiologic and pathologic processes. Strong clinical and experimental evidence has shown that elevated uPA expression is associated with cancer progression, metastasis, and shortened survival in patients. uPA has been considered as a promising molecular target for development of anticancer drugs. Here, we report the identification of several new uPA inhibitors using a high-throughput screen from a chemical library. From these uPA inhibitors, molecular modeling and docking studies identified 4-oxazolidinone as a novel lead pharmacophore. Optimization of the 4-oxazolidinone pharmacophore resulted in a series of structurally modified compounds with improved potency and selectivity. One of the 4-oxazolidinone analogues, UK122, showed the highest inhibition of uPA activity. The IC(50) of UK122 in a cell-free indirect uPA assay is 0.2 micromol/L. This compound also showed no or little inhibition of other serine proteases such as thrombin, trypsin, plasmin, and the tissue-type plasminogen activator, indicating its high specificity against uPA. Moreover, UK122 showed little cytotoxicity against CFPAC-1 cells (IC(50) >100 micromol/L) but significantly inhibited the migration and invasion of this pancreatic cancer cell line. Our data show that UK122 could potentially be developed as a new anticancer agent that prevents the invasion and metastasis of pancreatic cancer.     

The immunoglobulin G subclass composition of immune complexes in cystic fibrosis. Implications for the pathogenesis of the Pseudomonas lung lesion

It has been shown that pulmonary macrophage (PM) phagocytosis of Pseudomonas aeruginosa (PA) is inhibited in the presence of serum from cystic fibrosis (CF) patients colonized by Pseudomonas, and that these sera contain high concentrations of IgG2 antibodies. The goal of these studies was to investigate the role that IgG2-containing immune complexes (IC) play in this inhibition of both PM and neutrophil phagocytosis. We found that serum IgG2 concentrations were elevated significantly in CF patients with chronic PA colonization and that in selected sera from CF patients with chronic PA colonization (CF + IC, n = 10), the mean IC level was significantly elevated (2.90 +/- 0.22 mg/dl [SEM]). IgG2 comprised 74.5% of IgG precipitated in IC from CF + IC sera. An invitro phagocytic assay of [14C]PA uptake using CF + IC whole-sera opsonins confirmed that endocytosis by normal PM and neutrophils was significantly depressed. Removal of IC from CF + IC sera resulted in significantly decreased serum IgG2 concentrations without a significant change in the other subclass concentrations, and enhanced [14C]PA uptake by PM (26.6% uptake increased to 47.3%) and neutrophils (16.9% increased to 52.6%). Return of the soluble IgG2 IC to the original CF sera supernatants and the positive control sera resulted in return of the inhibitory capacity of the CF + IC sera. We conclude that immune sera from patients with chronic Pseudomonas infections characterized by elevated IgG2 subclass level functions poorly as an opsonin. In these individuals, IgG2 contributes significantly to circulating IC and removal of IC, matched by a simultaneous fall in IgG2, improves bacterial uptake by neutrophil and mononuclear phagocytes. IgG2 antibodies exert antiphagocytic effects by both direct inhibition and the formation of IC.     

Platelet-derived VEGF, Flt-1, angiopoietin-1 and P-selectin in breast and prostate cancer: further evidence for a role of platelets in tumour angiogenesis

BACKGROUND: Cancer is a complex multi-factorial disorder that may commonly show abnormal angiogenesis in such patients. Recently, platelets have been postulated to have a major role in both these processes, suggesting that antiplatelet strategies may be useful in cancer treatment. MATERIALS AND METHODS: To further investigate the role of platelets in angiogenesis, we used a novel platelet lysate assay to analyse platelet contents in breast cancer (n = 30) and prostate cancer (n = 30) patients and age- and sex-matched controls (n = 60). Markers of angiogenesis (vascular endothelial growth factor (VEGF), angiopoietin-1 and-2 (Ang-1, -2), and their respective receptors (Flt-1 and Tie-2) plus a marker of platelet activation (P-selectin (P-sel)), were all measured in platelet lysate by enzyme-linked immunsorbent assay. RESULTS: Platelet lysate from breast cancer patients contained higher levels of VEGF (P < 0.0001). Ang-1 (P = 0.0186) and P-sel (P = 0.0002), compared to healthy controls. Platelet lysate from prostate cancer patients had elevated VEGF (P = 0.008) but not Ang-1 or P-sel. There were no significant differences between levels of Fit-1 between patients and controls, and both Ang-2 and Tie-2 were undetectable in both patient groups and control platelet lysate. CONCLUSION: We have shown that our previously developed platelet lysate technique could be used to measure indices of angiogenesis, and their respective receptors, and that this assay can be applied to patients with cancer. Our study also provides further evidence that platelets may influence angiogenic abnormalities in human cancer. The platelet may be a useful target in anti-cancer strategies.     

Monoclonal antibody-based ELISA to detect glycoprotein tumor-associated-antigen-specific immune complexes in cancer patients.

This report describes the development and applicability of a tumor-associated-antigen-specific immune complex (IC) detection assay to denote the presence of tumor cells in a cancer host. This assay utilizes a murine monoclonal antibody, AD1-40F4, which was produced to a glycoprotein tumor-associated antigen (TAA). In Western blot, the murine monoclonal antibody recognized a 90-100 kD subunit of the antigen. This antigen is immunogenic in cancer patients and induces formation of endogenous antigen-antibody complexes. Analysis of 250 sera from normal individuals and 419 sera from cancer patients revealed that a significantly (P less than .0005) greater proportion (234/419; 55.9%) of cancer sera was positive for the marker than the normal sera (8/250; 3.2%). The incidence of the 90 kD-TAA-specific-IC was consistently and significantly higher in melanoma (58.5%; 38/65), sarcoma (52.9%; 83/157), and carcinoma of breast (50.9%; 58/114), lung (68.2%; 30/44), and colon (64.1%, 25/39) than in the normal group. The age of serum donors did not affect the incidence of the reactivity. Also, at least in the cancer group the gender of the serum donor did not affect the incidence of the 90 kD-TAA-specific-IC positivity. In a retrospective study where sequential serum samples obtained postoperatively from 105 patients with melanoma were analyzed, the 90 kD-TAA-specific-IC could be detected in 72 (69%) of patients several years before the appearance of clinically detectable disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

双层探测器光谱CT定量参数与肺癌及其病理特征的关系

目的  分析双层探测器光谱CT定量参数与肺癌及其病理特征的关系。 方法  收集2021年3月~2022年1月共87例肺癌患者的临床资料纳入肺癌组,并选择同期入院治疗的36例肺炎性结节患者作为对照组,两组患者治疗前2周内均行双层探测器光谱CT扫描。比较两组常规CT参数及光谱CT定量参数差异,并分析肺癌组不同病理特征者光谱CT定量参数差异。 结果  两组棘突征、CT值及动脉期碘密度(IC)、标准化IC(NIC)、有效原子序列(Zeff)比较,差异无统计学意义(P > 0.05),肺癌组分叶征、毛刺征、胸膜凹陷征及血管集束征CT征象多于对照组(P < 0.05),肺癌组静脉期IC、NIC及Zeff均低于对照组(P < 0.05)。肺癌组不同病理特征者动脉期IC、NIC、Zeff的差异无统计学意义(P > 0.05),腺癌者静脉期IC、NIC及Zeff高于鳞癌及小细胞癌者(P < 0.05),鳞癌者则高于小细胞癌者(P < 0.05)。 结论  双层探测器光谱CT定量参数对鉴别肺癌与肺炎性结节有利,且能辅助判断肺癌病理特征,具有良好应用前景。     

Highly active anti-Pneumocystis carinii compounds in a library of novel piperazine-linked bisbenzamidines and related compounds

Trimethoprim-sulfamethoxazole and pentamidine isethionate have been used extensively for the prophylaxis and therapy of pneumonia caused by Pneumocystis jirovecii. Problems associated with toxicity and potential emerging resistance for both therapies necessitate the development of safe and effective analogs or new treatment strategies. In the present study, a library of 36 compounds was synthesized by using the pentamidine molecule as the parent compound modified by a 1,4-piperazinediyl moiety as the central linker to restrict conformation flexibility. The compounds were evaluated for anti-Pneumocystis carinii activity in a bioluminescent ATP-driven assay. Four of the compounds were highly active, with 50% inhibitory concentration (IC(50)) values of <0.01 microg/ml; four had very marked activity (IC(50) < 0.10 microg/ml); ten had marked activity (IC(50) < 1.0 microg/ml); nine had moderate activity (IC(50) < 10 microg/ml); one had slight activity (IC(50) = 34.1 microg/ml); and the remaining eight did not demonstrate activity in this assay system. The high level of activity was specifically associated with an alkyl chain length of five to six carbons attached to one of the nitrogens of the bisamidinium groups. None of the highly active compounds and only one of the very marked compounds exhibited any toxicity when evaluated in three mammalian cell lines. The strategy of substitution of 1,4-piperazine-linked bisbenzamidines produced compounds with the highest level of activity observed in the ATP assay and holds great promise for the development of efficacious anti-P. carinii therapy.     

CA 72-4 radioimmunoassay for the detection of the TAG-72 carcinoma-associated antigen in serum of patients

Monoclonal antibody (MAb)B72.3 has been used to detect the presence of TAG-72 in the serum of carcinoma patients. We have developed new anti-TAG-72 MAbs and have selected one of these, CC49, as the "catcher" MAb with 125I-B72.3 as the detecting antibody in a double-determinant immunoradiometric assay. This combination enabled the development of a sequential assay (designated CA 72-4) that showed optimal quantitative properties as demonstrated by such parameters as linear dose-response, high re-producibility, and lack of serum-matrix and "hook-back" effects. Only 3.5% of 744 normal sera and 6.7% of 134 sera from patients with benign gastrointestinal diseases had TAG-72 levels greater than 6 U/ml. Approximately 40% of 303 patients with gastrointestinal malignancies had serum TAG-72 levels of greater than 6 U/ml (55% of the patients with advanced disease). Thirty-six percent of patients with adenocarcinomas of the lung and 24% of patients with ovarian cancer (53% stage IV patients) also had elevated serum TAG-72 levels. A poor correlation was found between the carcinoembryonic antigen (CEA) and TAG-72 values of sera obtained from gastric cancer patients. Thirty-four percent of CEA negative cases were scored positive in the CA 72-4 assay, suggesting the complementarity of the CA 72-4 assay to CEA assays in the analysis of sera from patients with certain malignancies.     

High-throughput screen identifies novel inhibitors of cancer biomarker α-methylacyl coenzyme A racemase (AMACR/P504S)

α-methylacyl coenzyme A racemase (AMACR) is a metabolic enzyme whose overexpression has been shown to be a diagnostic indicator of prostatic adenocarcinoma and other solid tumors. Here, we confirm that attenuation of AMACR expression diminishes the growth of prostate cancer cell lines by using stably expressed short-hairpin RNA constructs. This observation strongly suggests that the AMACR enzyme may be a target for therapeutic inhibition in prostate cancer. To this end, we report here a novel assay capable of screening libraries of diverse small molecules for inhibitors of AMACR activity. This assay facilitated the screening of approximately 5,000 unique compounds and the discovery of 7 distinct chemical entities capable of inhibiting AMACR at low micromolar concentrations. The most potent inhibitor discovered is the seleno-organic compound ebselen oxide [inhibitory concentration (IC(50)): 0.80 μmol/L]. The parent compound, ebselen (IC(50): 2.79 μmol/L), is a covalent inactivator of AMACR (K(I)((inact)): 24 μmol/L). Two of the AMACR inhibitors are selectively toxic to prostate cancer cell lines (LAPC4/LNCaP/PC3) that express AMACR compared to a normal prostate fibroblast cell line (WPMY1) that does not express the protein. This report shows the first high-throughput screen for the discovery of novel AMACR inhibitors, characterizes the first nonsubstrate-based inhibitors, and validates that AMACR is a viable chemotherapeutic target in vitro.     

Quantification of circulating immune complexes by chicken anti-C4 micro ELISA

A quantitative assay for C4-containing immune complexes (IC) by a solid phase anti-C4 micro ELISA is described. It is based upon the use of an affinity purified chicken anti-human C4 antibody to capture the immune complex, and protein A-alkaline phosphatase for detection. The chicken antibody was chosen as capture antibody because it does not react with rheumatoid factor, does not activate the human complement system and is not detected by anti-mammalian IgG antibodies or protein A. Increased levels of C4 containing circulating immune complexes (CIC) were detected in sera from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and lung cancer, when compared with normal sera. Normal levels of C4 containing immune complexes were found in sera from patients with Bell's palsy.     

实性肺癌能谱CT参数与Ki-67表达水平的相关性研究

目的探讨实性肺癌能谱CT定量参数与Ki-67表达水平之间的关系。方法回顾性分析了152例经手术或活检证实的实性肺癌患者的临床资料。采用免疫组织化学检测Ki-67阳性癌细胞百分率(Ki-67值),反映Ki-67表达水平。根据Ki-67值分为3组:高水平组(大于30%),中水平组(10%~30%),低水平组(小于等于10/%)。用GSI Viewer软件测量和计算增强胸部能谱CT(静脉期)的定量参数,包括碘浓度(IC)、40和70 keV的CT值(CT40keV、CT70keV)、能谱衰减曲线的斜率(K)和标准碘浓度(NIC)。用单因素方差分析比较3组间能谱参数的差异。计算定量参数与Ki-67表达水平之间的Spearman相关系数。应用受试者工作特征曲线(ROC)分析,计算曲线下面积(AUC)、敏感度、特异度来衡量各参数的诊断性能。结果单因素方差分析显示3组间各定量参数均有显著差异(P<0.05)。LSD-t两两比较分析显示,高水平组分别与低水平组、中表达组间的IC、NIC、CT40keV、CT70keV、K有显著差异(P<0.05)。各定量参数与Ki-67表达水平呈负相关(|r|均<0.45,P<0.001)。ROC曲线分析表明,IC和K在鉴别高水平组和非高水平组方面表现最好(AUC分别为0.745和0.746)。IC和K的敏感度均为69.4%,特异度74.4%。结论能谱CT定量参数(IC、NIC、CT40keV、CT70keV、K)可用于鉴别实性肺癌Ki-67高表达与非高表达水平,并与Ki-67表达水平呈负相关,为评估实性肺癌癌细胞增殖能力提供了参考。