RESUMO
It is already established that cholinesterases (ChEs) appear in every embryonic blastema at a very early stage of development, independently from innervation. Embryonic butyrylcholinesterase (BChE) is typically found in cells engaged in proliferation processes, while acetylcholinesterase (AChE) is expressed by cells undergoing morphogenetic processes. In order to better define the regulation of cholinesterases during development, we examined their expressions during in vitro differentiation of two murine embryonic stem cell lines by reverse transcription polymerase chain reaction, histochemistry and enzyme activity measurements. AChE and BChE activity and mRNA were present in the undifferentiated stem cells. To test whether the ChEs expression is regulated during differentiation, we employed the embryoid bodies (EBs) culture method, allowing the cells to differentiate, to then collect them at various stages in culture. Interestingly, phases of differentiation were accompanied by increased AChE transcripts; BChE expression was constant, decreasing at later differentiation stages. Cholinesterase activities showed corresponding patterns, with AChE activity increasing at later stages in culture and BChE slightly decreasing. Histochemistry revealed that AChE and BChE activities were mutually exclusive, being expressed by different cell subpopulations. Thus, we have demonstrated that mouse embryonic stem cells express cholinesterases, the enzymes are functional and their expression is regulated during differentiation. Therefore, it appears that their functions under these conditions are not related to synaptic transmission, but for the developmental processes.
Assuntos
Acetilcolinesterase/genética , Butirilcolinesterase/genética , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Animais , Linhagem Celular , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The numbers of potential neurotoxicants in the environment are raising and pose a great risk for humans and the environment. Currently neurotoxicity assessment is mostly performed to predict and prevent harm to human populations. Despite all the efforts invested in the last years in developing novel in vitro or in silico test systems, in vivo tests with rodents are still the only accepted test for neurotoxicity risk assessment in Europe. Despite an increasing number of reports of species showing altered behaviour, neurotoxicity assessment for species in the environment is not required and therefore mostly not performed. Considering the increasing numbers of environmental contaminants with potential neurotoxic potential, eco-neurotoxicity should be also considered in risk assessment. In order to do so novel test systems are needed that can cope with species differences within ecosystems. In the field, online-biomonitoring systems using behavioural information could be used to detect neurotoxic effects and effect-directed analyses could be applied to identify the neurotoxicants causing the effect. Additionally, toxic pressure calculations in combination with mixture modelling could use environmental chemical monitoring data to predict adverse effects and prioritize pollutants for laboratory testing. Cheminformatics based on computational toxicological data from in vitro and in vivo studies could help to identify potential neurotoxicants. An array of in vitro assays covering different modes of action could be applied to screen compounds for neurotoxicity. The selection of in vitro assays could be guided by AOPs relevant for eco-neurotoxicity. In order to be able to perform risk assessment for eco-neurotoxicity, methods need to focus on the most sensitive species in an ecosystem. A test battery using species from different trophic levels might be the best approach. To implement eco-neurotoxicity assessment into European risk assessment, cheminformatics and in vitro screening tests could be used as first approach to identify eco-neurotoxic pollutants. In a second step, a small species test battery could be applied to assess the risks of ecosystems.
RESUMO
In benign and malignant ovarian tumor patients, human placental alkaline phosphatase (HPLAP) was determined in serum and extracts from surgical tumor biopsies using a highly specific enzyme-antigen immunoassay based on a mouse monoclonal antibody (E6) to HPLAP. Serum HPLAP levels greater than or equal to 0.1 unit/liter were found in 58% of ovarian cancer patients. Serum carcinoembryonic antigen levels were positive (greater than 5.4 ng/ml) in 17% of these patients. HPLAP was detected in extracts from 13 of the 14 tumors investigated (range, 2.4 to 557 milliunits/g). Only the mixed heterologous Müllerian sarcoma was negative. The highest HPLAP content of normal ovarian tissue was 1.1 milliunits/g. The amount of heat-stable and L-p-bromotetramisole-insensitive alkaline phosphatase was in all cases much higher than the fraction recognized by E6. The neoplastic origin of HPLAP was confirmed immunohistochemically on paraffin sections by an indirect avidin-biotin-peroxidase staining procedure using E6. The staining pattern was compared to the histochemical distribution of total alkaline phosphatase on adjacent sections. A consistency was found between the amount of HPLAP in tissue extracts and its immunohistochemical distribution. In all the tumors, staining for HPLAP was observed mainly on the plasma membranes of carcinoma cells. In 9 of the 10 carcinomas, the histological distribution of HPLAP and also of total alkaline phosphatase was heterogeneous. HPLAP staining, present in one of five normal ovaries, was restricted to germinal inclusion cysts. The present results support the hypothesis that serous ovarian tumors originate from these cysts.
Assuntos
Fosfatase Alcalina/análise , Isoenzimas/análise , Neoplasias Ovarianas/enzimologia , Antígeno Carcinoembrionário/análise , Eletroforese , Feminino , Histocitoquímica , Humanos , Neoplasias Ovarianas/patologia , Placenta/enzimologia , GravidezRESUMO
Human placental alkaline phosphatase (HPLAP), carcinoembryonic antigen (CEA), and cancer antigen 125 (CA 125) were localized immunohistochemically in paraffin sections of normal lung tissue from 16 patients, using monoclonal antibodies and an indirect avidin-biotin-peroxidase staining procedure. HPLAP and CEA were present in epithelial cells of respiratory bronchioli and alveolar type I pneumocytes. CEA was also observed in the tracheal, bronchial, and bronchiolar epithelium. CA 125 was present in the tracheal, bronchial, bronchiolar, and terminal bronchiolar epithelium; in the tracheal and bronchial glands; and in the pleural mesothelium. Normal and hyperplastic type II pneumocytes were negative for HPLAP, CEA, and CA 125 but were histochemically positive for nonspecific alkaline phosphatase. Fetal lung tissue between 11 and 15 weeks of gestation was negative for HPLAP, CEA, and CA 125. The fetal tracheal and bronchial epithelium, tracheal glands, and pleural mesothelium were positive for CA 125. For ten malignant pulmonary tumors investigated, HPLAP staining was observed in five, CEA in nine, and CA 125 in seven. The localization of HPLAP, CEA, and CA 125 in apparently normal constituents of all pulmonary specimens is in disagreement with the concept that the expression of these substances in the lung is indicative of abnormal cellular activity.
Assuntos
Fosfatase Alcalina/metabolismo , Antígenos de Neoplasias/análise , Antígeno Carcinoembrionário/análise , Neoplasias Pulmonares/imunologia , Pulmão/imunologia , Adolescente , Adulto , Idoso , Fosfatase Alcalina/imunologia , Humanos , Técnicas Imunológicas , Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Pessoa de Meia-Idade , Placenta/enzimologiaRESUMO
The aim of this study was to characterize the genotoxic action of UVA and UVB in human keratinocytes by application of the single cell gel electrophoresis assay (SCGE assay). Dose dependence of DNA damage, the time course of its repair, and the influence of cellular antioxidant status were assessed. Irradiation with UVA or UVB both resulted in a dose-dependent increase in the level of DNA damage. A time course study to evaluate the repair kinetics in keratinocytes irradiated with 5 J/cm2 UVA revealed an immediate occurrence of DNA effects which subsequently disappeared within about 1 h, indicating removal of DNA lesions. This rapid repair of DNA damage is consistent with the observation that 5 J/cm2 UVA did not impair cellular viability. In contrast, exposure to 15 mJ/cm2 UVB resulted in a prolonged repair of DNA damage which lasted about 25 h. Thus, the repair kinetics of UVA- and UVB-induced DNA damage clearly differed from each other, implicating the induction of different types of DNA lesions by UVA and UVB. Neither a pretreatment with Mg-ascorbyl phosphate or D,L-alpha-tocopherol, nor depletion of endogenous glutathione altered cellular sensitivity to UVB. In contrast, the DNA damaging effects of UVA could be counteracted by a pretreatment with these antioxidants. These observations confirm that the UVA-induced effects on DNA are related to radical mediated strand breaks and DNA lesions forming alkali-labile sites. The UVB-induced effects mainly occur as a consequence of excision repair-related strand breaks. The observed repair kinetics of DNA lesions and the influence of cellular antioxidant status may help to elucidate protective mechanisms against the carcinogenic effects of UV radiation present in sunlight.
Assuntos
Antioxidantes/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Ácido Ascórbico/farmacologia , Butionina Sulfoximina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , DNA/metabolismo , Reparo do DNA , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Ágar , Humanos , Queratinócitos/química , Cinética , Protetores contra Radiação/farmacologia , Fatores de Tempo , Raios Ultravioleta/efeitos adversos , Vitamina E/farmacologiaRESUMO
HCVs constitute a genus within the Flaviviridae, with closest homology to the hepatitis G and GB viruses, and Pestiviruses. The positive-stranded RNA genome encodes at least nine proteins. Core, El, and E2 constitute the structural proteins; NS2, NS3, NS4A, NS4B, NS5A, and NS5B are nonstructural (NS) proteins. HCV isolates display high levels of sequence heterogeneity allowing classification into at least 11 types and 90 subtypes (1). HCV infection of the human liver is often clinically benign, with mild icterus in the acute phase, the disease may even go unnoticed in some cases of acute resolving hepatitis C. In the majority (>70%) of cases, however, HCV infection leads to chronic persistent or active infection, often with complications of liver cirrhosis and auto-immune disorders. Hepatocellular carcinoma may occur after about 20-35 yr (2); sometimes even without the intermediate phase of cirrhosis. No prophylaxis is available today and treatment with interferon-alpha (IFN-α) only leads to long-term resolution in about 4-36% of treated cases, depending on the HCV genotype (1).
RESUMO
The use of in vitro methods is increasingly recommended for the assessment and evaluation of cytotoxic effects resulting in cell damage. In most cases, cellular response and reaction of cell populations is determined by endpoint measurements. Even if a battery of such test systems is applied, information about the dynamics of cell damage and recovery is obtained only in part. However, phenomena of damage and recovery can be followed by observing the fate of individual cells and their progeny in culture over several days, which means over several cell cycles, by using light microscopy combined with image analysis. We have developed a recording system for such continuous observation and registration of toxic effects, based on image analysis. Growth rate, generation time, delay or shifting of cell cycle, identification of the progeny (pedigrees) and cellular locomotion can be recorded simultaneously.
RESUMO
The heat-shock response is a cellular defence mechanism against environmental stresses that is evolutionarily conserved from bacteria to man. Numerous reports demonstrate the beneficial effects of heat-shock protein induction on cell survival under toxic or oxidative stress, e.g., in cardiac and cerebral ischemia or prior to organ transplantation. However, there is little data on the effects of heat treatment on damage caused by UV irradiation. Applying three independent techniques, we have tested the influence of thermal pretreatment of skin cells (1 h, 43 degrees C) on the initial extent of UV-B-induced DNA damage and its subsequent repair. For cultured human epidermal keratinocytes and dermal fibroblasts we can show reduced levels of nucleotide-excision-repair-associated DNA strand incision in the comet assay. Moreover, immunostaining and flow cytometric quantitation of thymidine dimers immediately and one day after irradiation, respectively, reveal that the initial DNA damage is not (keratinocytes) or only moderately (fibroblasts) lower in heat-shocked cells as compared to untreated controls. However, excision repair of dimers is significantly attenuated during the first 24 h in both cell types. Furthermore, using a modified host-cell reactivation assay, we are able to demonstrate that repair of UV-B-damaged plasmid DNA is lower if the transfected cells are previously heat shocked. In summary, heat treatment (1 h, 43 degrees C) inducing heat-shock proteins reduces nucleotide excision repair of UV-B-mediated DNA lesions in fibroblasts and keratinocytes during the following 24 h. This is not necessarily caused by elevated heat-shock protein levels themselves. Possibly the direct thermal damage of repair enzymes is more severe than the potential protective effects of heat-shock proteins.
Assuntos
Dano ao DNA , Reparo do DNA , Pele/efeitos da radiação , Raios Ultravioleta , Adulto , Animais , Células Cultivadas , Fibroblastos/efeitos da radiação , Citometria de Fluxo , Proteínas de Choque Térmico HSP72 , Proteínas de Choque Térmico/análise , Temperatura Alta , Humanos , Queratinócitos/efeitos da radiação , Masculino , CamundongosRESUMO
The processes of aging and photoaging are associated with an increase in cellular oxidation. This may be in part due to a decline in the levels of the endogenous cellular antioxidant coenzyme Q10 (ubiquinone, CoQ10). Therefore, we have investigated whether topical application of CoQ10 has the beneficial effect of preventing photoaging. We were able to demonstrate that CoQ10 penetrated into the viable layers of the epidermis and reduce the level of oxidation measured by weak photon emission. Furthermore, a reduction in wrinkle depth following CoQ10 application was also shown. CoQ10 was determined to be effective against UVA mediated oxidative stress in human keratinocytes in terms of thiol depletion, activation of specific phosphotyrosine kinases and prevention of oxidative DNA damage. CoQ10 was also able to significantly suppress the expression of collagenase in human dermal fibroblasts following UVA irradiation. These results indicate that CoQ10 has the efficacy to prevent many of the detrimental effects of photoaging.
Assuntos
Antioxidantes/metabolismo , Envelhecimento da Pele/efeitos dos fármacos , Pele/efeitos dos fármacos , Ubiquinona/análogos & derivados , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Células Cultivadas , Coenzimas , Cosméticos , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Luz , Pele/efeitos da radiação , Fenômenos Fisiológicos da Pele/efeitos dos fármacos , Fenômenos Fisiológicos da Pele/efeitos da radiação , Ubiquinona/farmacologia , Ubiquinona/fisiologia , Ubiquinona/uso terapêuticoRESUMO
The proportion of patients with ischaemic stroke treated by intravenous (i.v.) recombinant tissue plasminogen activator (rt-PA) is an indicator of quality of stroke care. The objective of the study is to evaluate the rate of i.v. thrombolysis in the North-of-France region and its evolution over time. We determined the proportion of inhabitants treated by i.v. rt-PA in 2009-2010 (period A; 8 stroke units, no telemedicine) and 2012 (period B; population campaigns, 12 stroke units with telemedicine in 5). We used hospital registries from the 12 stroke units, and population-based data were collected in a subpopulation of 226,827 inhabitants (5.6% of the whole population). 1,563 inhabitants received i.v. rt-PA for stroke (period A: 835 in 24 months; period B: 728 in 12 months). Hospital and population data were similar. Annual rates of thrombolysis increased from 103 per million inhabitants [95% confidence interval (CI) 85-125] to 181 (95% CI 157-209; relative increase 76%, 95% CI 67-83%). This rate increased in 12 districts (significantly in 6), but the increase was greater in districts where new stroke units, telemedicine, or both were implemented. In conclusion, although the proportion of patients treated was already high in period A, there was still place for improvement. Implementation of new stroke units, extension of the telemedicine network and new population campaigns are necessary to improve the rate of thrombolysis in several areas, to ensure an equal access to treatment over the whole territory. The next step is now to determine whether this high rate of i.v. rt-PA delivery at the population level translates into clinical results.
Assuntos
Administração Intravenosa/métodos , Isquemia Encefálica/complicações , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/etiologia , Ativador de Plasminogênio Tecidual/uso terapêutico , Adulto , Idoso , França , Humanos , Estudos Longitudinais , Pessoa de Meia-Idade , Estudos Retrospectivos , TelemedicinaRESUMO
We present a novel type of operatory chair which directs an operation microscope by remote control using infrared beams: XV, zoom, focal and accessories. The advantage of this system is the absence of transmission cables, and consequently represents an important improvement in terms of flexibility. It is a vast improvement as well for maintaining sterile conditions in the operation room. The ergonomics of this chair was especially designed for microsurgery. The adjustment and stability of the forearm has been considerably improved.
Assuntos
Microcirurgia/instrumentação , Equipamentos Cirúrgicos , Desenho de Equipamento , Humanos , Raios InfravermelhosRESUMO
Using a solid-phase monoclonal antibody enzyme immuno-assay, we evaluated in a multicenter study (18 laboratories) the utility of evaluating catalytic activity of human placental alkaline phosphatase (hPLAP, EC 3.1.3.1) in serum as a potential tumor marker. We determined hPLAP in serum samples from 130 patients with ovarian cancer, 79 patients with testicular cancer (53 seminoma testis, 26 nonseminoma testis), 537 patients with various other malignant diseases (95 lung, 39 gastrointestinal, 195 breast, 208 others), 291 patients with benign diseases, and 213 healthy controls. To assess the influence of smoking on hPLAP activity in serum, we evaluated 79 serum samples from patients with noncancerous diseases for whom smoking habits had been recorded. Our main findings are: (a) hPLAP activity is frequently increased (greater than 100 mU/L) in pre-operative serum samples from ovarian cancer patients (49%) and from testicular cancer patients (59% overall; 72% seminoma, 35% nonseminoma); (b) heavy smoking may increase hPLAP activity; (c) excluding heavy smokers, a 96% specificity for cancerous lesions was observed; (d) in patients with ovarian malignancies, CA 125 and hPLAP may behave as independent markers; and (e) in patients with seminoma, hPLAP is clearly more frequently increased than is beta-choriogonadotropin.
Assuntos
Fosfatase Alcalina/sangue , Antígenos de Neoplasias/sangue , Placenta/enzimologia , Anticorpos Monoclonais , Antígenos Glicosídicos Associados a Tumores/análise , Feminino , Humanos , Masculino , Neoplasias Ovarianas/enzimologia , Neoplasias Testiculares/enzimologiaRESUMO
We evaluate a new commercial enzyme immunoassay (EIA) of plasminogen activator inhibitor-1 (PAI-1) in plasma, the Innotest PAI-1. Because we wanted to measure PAI-1 in blood samples, we developed a procedure for evaluating the specificity of the assay for different PAI-1 forms in their natural environment. All molecular forms were prepared from a plasma that contained only active PAI-1. The recovery of the different molecular forms of PAI-1, relative to active PAI-1 (100%), was 99% +/- 7% for PAI-1 complexed with recombinant tissue-type plasminogen activator (t-PA), 104% +/- 4% for PAI-1 complexed with melanoma t-PA, 94% +/- 11% for PAI-1 complexed with high-M(r) urokinase, and 113% +/- 3% for latent PAI-1. The parallelism between the calibration curve of the EIA and the serial dilutions of the different PAI-1 forms was considered acceptable for clinical purposes. In selected clinical plasma samples, the PAI-1 values obtained with the Innotest PAI-1 EIA correlated well with those of the TintElize PAI-1 EIA (r = 0.913, n = 106); the observed correlation of the Innotest measurements with PAI activity was r = 0.795 (n = 79). The Innotest PAI-1 antigen assay appears to detect all molecular forms of PAI-1 to a similar degree, and comes close to being the so-called grand total assay for detecting the total molecular concentration of PAI-1 in plasma.
Assuntos
Imunoensaio , Inibidor 1 de Ativador de Plasminogênio/sangue , Kit de Reagentes para Diagnóstico , Humanos , Imunoensaio/estatística & dados numéricos , Kit de Reagentes para Diagnóstico/estatística & dados numéricos , Sensibilidade e Especificidade , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The epidemiology of hepatitis C (HCV), especially on the African continent, is not well known. In this study, we investigated the presence of antibodies to HCV in 685 out-patients, seen in several health care centers or hospitals in different regions in Burundi from January to February 1991. Serological tests of the second generation were used. The global prevalence varied from 3.2% to 14.1% according to the center. Urban seroprevalence tended to be higher than rural prevalence. Also, with increasing age, a higher prevalence was observed. Anti-HCV antibodies were absent in patients younger than 21, while specific antibodies were detected in 23.1% of patients older than 50. Although the prevalence in men (10.4%) was higher than in women (7.4%), this difference was not statistically significant. Taking into account the selection of subjects participating in this evaluation, the results can not be extrapolated to the general population. No association between HCV and human immunodeficiency virus (HIV) was seen in this study. In contrast to previously described results from studies using reagents of the first generation, no cross-reactions were observed with anti-malarial antibodies.
Assuntos
Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/isolamento & purificação , Estudos Soroepidemiológicos , Adolescente , Adulto , Burundi/epidemiologia , Criança , Pré-Escolar , Feminino , Humanos , Testes Imunológicos , Lactente , Masculino , Pessoa de Meia-Idade , Pacientes Ambulatoriais , População Rural , População UrbanaRESUMO
Human placental alkaline phosphatase (hPLAP; EC 3.1.3.1), cancer antigen 125 (CA 125), and carcinoembryonic antigen (CEA) were determined in sera of patients with malignant and nonmalignant disorders. For CA 125 we used two different commercial assay systems, based on the same monoclonal antibody. hPLAP had the same sensitivity (20%) as CA 125 for detecting non-ovarian neoplasia, whereas that of CEA was 45%. For detecting ovarian cancer CA 125 (Cis kit) was slightly more sensitive (50%) than hPLAP (45%), much more than CEA (10%). hPLAP was increased in sera of 2% of patients with nonmalignant disorders, CA 125 in 23%, and CEA in 18%. hPLAP was increased in only one of 10 diabetic patients and two of 50 patients on chronic renal dialysis. CA 125 and CEA were respectively increased in 45% and 23% of all liver pathologies studied and in 12% and 17% of patients with renal insufficiency. The sensitivity of hPLAP for detecting ovarian cancer is slightly inferior to that of CA 125, but its specificity is much higher. We found the Abbott system for CA 125 to be more sensitive than the Cis system.
Assuntos
Antígenos de Neoplasias/sangue , Isoenzimas/análise , Neoplasias/análise , Fosfatase Alcalina , Anticorpos Monoclonais , Antígenos Glicosídicos Associados a Tumores , Antígeno Carcinoembrionário/sangue , Feminino , Proteínas Ligadas por GPI , Humanos , Masculino , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/imunologia , Kit de Reagentes para DiagnósticoRESUMO
An efficient and reproducible procedure was developed for extracting aminoglycosides from renal cortical tissue. It involves a double homogenization and two rinsings with trichloroacetic acid. A higher recovery is obtained compared with that of other previously reported methods.
Assuntos
Antibacterianos/isolamento & purificação , Córtex Renal/análise , Amicacina/isolamento & purificação , Aminoglicosídeos/isolamento & purificação , Animais , Feminino , Gentamicinas/isolamento & purificação , Netilmicina/isolamento & purificação , Ratos , Ratos EndogâmicosRESUMO
In patients with essential hypertension, an additional plasma protein with an apparent molecular weight of 15,000 has been observed by high resolution sodium dodecyl sulphate polyacrylamide gel electrophoresis in the presence of beta-mercaptoethanol. Gel filtration on Sephacryl S-300 superfine revealed a native molecule with a molecular weight of 105,000. After reduction of the disulfide bridges with beta-mercaptoethanol the native molecule is dissociated into two subunits of 45,000 and one of 15,000. The native plasma protein has been purified by a combination of gel filtration on Sephacryl S-300, affinity chromatography on Concanavalin A Sepharose and anion exchange chromatography on DEAE-Sepharose. This protein has been referred to as "hypertension associated protein" (HAP).
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Hipertensão/sangue , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Humanos , Peso Molecular , Dodecilsulfato de SódioRESUMO
An anti-HIV-1/HIV-2 line immunoassay (LIA), using peptides and recombinant antigens was evaluated against commercially available Western blot tests for HIV-1 and HIV-2 antibodies. Two thousand one hundred and ten sera of European, African, and South American origin were used in the evaluation. The panel included 1066 sera with antibodies to HIV-1, 192 sera with antibodies to HIV-2, and 64 sera with antibodies to both. Using Western blot results interpreted according to the WHO criteria as a reference standard, the overall specificity obtained by this LIA was 100% and the sensitivity was 99.77% (97.51-100% for 95% confidence limits) when sera dually reactive in Western blot were included. Of the three sera negative in the LIA but positive in HIV-1 WB, two could be retested in a radioimmunoprecipitation assay and were negative. When dually reactive sera in the Western blot (WHO) were included, the LIA yielded 9.9% indeterminate results as compared with 15.5% for both assays (chi 2 = 29.30; p less than 0.001). Although only one HIV-2 specific peptide antigen (gp36) was used, the LIA yielded a specificity of 100% and a sensitivity of 100% as compared with the HIV-2 Western blot assay. When indeterminate results were included, the overall agreement between the LIA and the HIV-1 and HIV-2 Western blot (WHO criteria) was 89.9% and 90.1% respectively. These results indicate that the LIA provides reliable simultaneous detection of antibodies to HIV-1 and HIV-2, and at a cost which is substantially lower than the cost of Western blot tests.
Assuntos
Anticorpos Anti-HIV/análise , HIV-1/imunologia , HIV-2/imunologia , Imunoensaio , Western Blotting , Reações Cruzadas , Produtos do Gene env/imunologia , Produtos do Gene pol/imunologia , Antígenos HIV/imunologia , Proteína do Núcleo p24 do HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Humanos , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
In this enzyme-antigen immunoassay for human placental alkaline phosphatase (hPLAP; EC 3.1.3.1.) in serum and tissue extracts, polyclonal rabbit antiserum to mouse IgG2b is adsorbed to the wells of a microtiter plate, its excess binding sites are blocked, then it is incubated with murine monoclonal anti-hPLAP and mixed with serially diluted standard or sample antigen. The amount of antigen bound is determined by measuring its enzymic activity. The standard curve is linear for hPLAP concentrations of 0.2 to 1 U/L. The mean within-assay CV was 3.8% (SD 0.9%) for a serum sample and 6.1% (SD 3.0%) for a tissue extract. The respective mean between-assay CVs were: 6.7% (SD 2.0%), and 7.0% (SD 2.0%). Serum hPLAP concentrations, determined in four different dilutions, had a CV of 5.5%. We evaluated the method by standard additions and by comparing dilution curves for purified hPLAP, hPLAP in serum, and hPLAP in tissue extracts. The upper limit of activity in normal subjects was 0.1 U/L for serum samples, and 1.0 mU/g wet weight of tissue for tissue extracts. hPLAP activity was increased in 9.8% of all cancer patients, and in 40% of ovarian cancer patients. Almost half of the tumor biopsies were positive for hPLAP activity, and 94% of the biopsies from ovarian neoplasia had an increased activity of this isoenzyme. Of the nonmalignant tissues examined, normal lung tissue had the highest hPLAP activity.