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1.
J Pers Med ; 12(7)2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35887667

RESUMO

Background: To investigate the effects of the COVID-19 lockdowns on the vasculopathic population. Methods: The Divisions of Vascular Surgery of the southern Italian peninsula joined this multicenter retrospective study. Each received a 13-point questionnaire investigating the hospitalization rate of vascular patients in the first 11 months of the COVID-19 pandemic and in the preceding 11 months. Results: 27 out of 29 Centers were enrolled. April-December 2020 (7092 patients) vs. 2019 (9161 patients): post-EVAR surveillance, hospitalization for Rutherford category 3 peripheral arterial disease, and asymptomatic carotid stenosis revascularization significantly decreased (1484 (16.2%) vs. 1014 (14.3%), p = 0.0009; 1401 (15.29%) vs. 959 (13.52%), p = 0.0006; and 1558 (17.01%) vs. 934 (13.17%), p < 0.0001, respectively), while admissions for revascularization or major amputations for chronic limb-threatening ischemia and urgent revascularization for symptomatic carotid stenosis significantly increased (1204 (16.98%) vs. 1245 (13.59%), p < 0.0001; 355 (5.01%) vs. 358 (3.91%), p = 0.0007; and 153 (2.16%) vs. 140 (1.53%), p = 0.0009, respectively). Conclusions: The suspension of elective procedures during the COVID-19 pandemic caused a significant reduction in post-EVAR surveillance, and in the hospitalization of asymptomatic carotid stenosis revascularization and Rutherford 3 peripheral arterial disease. Consequentially, we observed a significant increase in admissions for urgent revascularization for symptomatic carotid stenosis, as well as for revascularization or major amputations for chronic limb-threatening ischemia.

2.
BJS Open ; 5(3)2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-34021325

RESUMO

BACKGROUND: Increased use of endovascular aneurysm repair (EVAR) and reduced open surgical repair (OSR), has decreased postoperative mortality after elective repair of abdominal aortic aneurysms (AAAs). The choice between EVAR or OSR depends on aneurysm anatomy, and the experience and preference of the vascular surgeon, and therefore differs between hospitals. The aim of this study was to investigate the current mortality risk difference (RD) between EVAR and OSR, and the effect of hospital preference for EVAR on overall mortality. METHODS: Primary elective infrarenal or juxtarenal aneurysm repairs registered in the Dutch Surgical Aneurysm Audit (2013-2017) were analysed. First, mortality in hospitals with a higher preference for EVAR (high-EVAR group) was compared with that in hospitals with a lower EVAR preference (low-EVAR group), divided by the median percentage of EVAR. Second, the mortality RD between EVAR and OSR was determined by unadjusted and adjusted linear regression and propensity-score (PS) analysis and then by instrumental-variable (IV) analysis, adjusting for unobserved confounders; percentage EVAR by hospital was used as the IV. RESULTS: A total of 11 997 patients were included. The median hospital rate of EVAR was 76.6 per cent. The overall mortality RD between high- and low-EVAR hospitals was 0.1 (95 per cent -0.5 to 0.4) per cent. The OSR mortality rate was significantly higher among high-EVAR hospitals than low-EVAR hospitals: 7.3 versus 4.0 per cent (RD 3.3 (1.4 to 5.3) per cent). The EVAR mortality rate was also higher in high-EVAR hospitals: 0.9 versus 0.7 per cent (RD 0.2 (-0.0 to 0.6) per cent). The RD following unadjusted, adjusted, and PS analysis was 4.2 (3.7 to 4.8), 4.4 (3.8 to 5.0), and 4.7 (4.1 to 5.3) per cent in favour of EVAR over OSR. However, the RD after IV analysis was not significant: 1.3 (-0.9 to 3.6) per cent. CONCLUSION: Even though EVAR has a lower mortality rate than OSR, the overall effect is offset by the high mortality rate after OSR in hospitals with a strong focus on EVAR.


Assuntos
Aneurisma da Aorta Abdominal , Implante de Prótese Vascular , Procedimentos Endovasculares , Aneurisma da Aorta Abdominal/cirurgia , Implante de Prótese Vascular/efeitos adversos , Hospitais com Alto Volume de Atendimentos , Humanos , Complicações Pós-Operatórias , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento
3.
Eur J Surg Oncol ; 44(4): 532-538, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29439836

RESUMO

BACKGROUND: Dutch national guidelines on the diagnosis and treatment of gastric cancer recommend the use of perioperative chemotherapy in patients with resectable gastric cancer. However, adjuvant chemotherapy is often not administered. The aim of this study was to evaluate hospital variation on the probability to receive adjuvant chemotherapy and to identify associated factors with special attention to postoperative complications. METHODS: All patients who received neoadjuvant chemotherapy and underwent an elective surgical resection for stage IB-IVa (M0) gastric adenocarcinoma between 2011 and 2015 were identified from a national database (Dutch Upper GI Cancer Audit). A multivariable linear mixed model was used to evaluate case-mix adjusted hospital variation and to identify factors associated with adjuvant therapy. RESULTS: Of all surgically treated gastric cancer patients who received neoadjuvant chemotherapy (n = 882), 68% received adjuvant chemo(radio)therapy. After adjusting for case-mix and random variation, a large hospital variation in the administration rates for adjuvant was observed (OR range 0.31-7.1). In multivariable analysis, weight loss, a poor health status and failure of neoadjuvant chemotherapy completion were strongly associated with an increased likelihood of adjuvant therapy omission. Patients with severe postoperative complications had a threefold increased likelihood of adjuvant therapy omission (OR 3.07 95% CI 2.04-4.65). CONCLUSION: Despite national guidelines, considerable hospital variation was observed in the probability of receiving adjuvant chemo(radio)therapy. Postoperative complications were strongly associated with adjuvant chemo(radio)therapy omission, underlining the need to further reduce perioperative morbidity in gastric cancer surgery.


Assuntos
Adenocarcinoma/terapia , Quimiorradioterapia Adjuvante , Gastrectomia , Complicações Pós-Operatórias/epidemiologia , Padrões de Prática Médica/estatística & dados numéricos , Neoplasias Gástricas/terapia , Adenocarcinoma/epidemiologia , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Países Baixos/epidemiologia , Assistência Perioperatória , Guias de Prática Clínica como Assunto , Sistema de Registros , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/patologia , Resultado do Tratamento
4.
Eur J Vasc Endovasc Surg ; 53(4): 520-532, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28256396

RESUMO

OBJECTIVE/BACKGROUND: The Dutch Surgical Aneurysm Audit (DSAA) is mandatory for all patients with primary abdominal aortic aneurysms (AAAs) in the Netherlands. The aims are to present the observed outcomes of AAA surgery against the predicted outcomes by means of V-POSSUM (Vascular-Physiological and Operative Severity Score for the enUmeration of Mortality and Morbidity). Adjusted mortality was calculated by the original and re-estimated V(physiology)-POSSUM for hospital comparisons. METHODS: All patients operated on from January 2013 to December 2014 were included for analysis. Calibration and discrimination of V-POSSUM and V(p)-POSSUM was analysed. Mortality was benchmarked by means of the original V(p)-POSSUM formula and risk-adjusted by the re-estimated V(p)-POSSUM on the DSAA. RESULTS: In total, 5898 patients were included for analysis: 4579 with elective AAA (EAAA) and 1319 with acute abdominal aortic aneurysm (AAAA), acute symptomatic (SAAA; n = 371) or ruptured (RAAA; n = 948). The percentage of endovascular aneurysm repair (EVAR) varied between hospitals but showed no relation to hospital volume (EAAA: p = .12; AAAA: p = .07). EAAA, SAAA, and RAAA mortality was, respectively, 1.9%, 7.5%, and 28.7%. Elective mortality was 0.9% after EVAR and 5.0% after open surgical repair versus 15.6% and 27.4%, respectively, after AAAA. V-POSSUM overestimated mortality in most EAAA risk groups (p < .01). The discriminative ability of V-POSSUM in EAAA was moderate (C-statistic: .719) and poor for V(p)-POSSUM (C-statistic: .665). V-POSSUM in AAAA repair overestimated in high risk groups, and underestimated in low risk groups (p < .01). The discriminative ability in AAAA of V-POSSUM was moderate (.713) and of V(p)-POSSUM poor (.688). Risk adjustment by the re-estimated V(p)-POSSUM did not have any effect on hospital variation in EAAA but did in AAAA. CONCLUSION: Mortality in the DSAA was in line with the literature but is not discriminative for hospital comparisons in EAAA. Adjusting for V(p)-POSSUM, revealed no association between hospital volume and treatment or outcome. Risk adjustment for case mix by V(p)-POSSUM in patients with AAAA has been shown to be important.


Assuntos
Aneurisma da Aorta Abdominal/cirurgia , Ruptura Aórtica/cirurgia , Implante de Prótese Vascular , Procedimentos Endovasculares , Hospitais , Idoso , Idoso de 80 Anos ou mais , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/mortalidade , Ruptura Aórtica/diagnóstico por imagem , Ruptura Aórtica/mortalidade , Benchmarking , Implante de Prótese Vascular/efeitos adversos , Implante de Prótese Vascular/mortalidade , Procedimentos Cirúrgicos Eletivos , Procedimentos Endovasculares/efeitos adversos , Procedimentos Endovasculares/mortalidade , Feminino , Mortalidade Hospitalar , Humanos , Masculino , Auditoria Médica , Países Baixos , Indicadores de Qualidade em Assistência à Saúde , Sistema de Registros , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento
5.
Int J Oral Maxillofac Surg ; 42(8): 939-48, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23583647

RESUMO

The aim of this study was to evaluate the suitability of tissue-engineered mucosa (TEM) as a model for studying the acute effects of ionizing radiation (IR) on the oral mucosa. TEM and native non-keratinizing oral mucosa (NNOM) were exposed to a single dose of 16.5Gy and harvested at 1, 6, 24, 48, and 72h post-irradiation. DNA damage induced by IR was determined using p53 binding protein 1 (53BP1), and DNA repair was determined using Rad51. Various components of the epithelial layer, basement membrane, and underlying connective tissue were analyzed using immunohistochemistry. The expression of cytokines interleukin-1ß (IL-1ß) and transforming growth factor beta 1 (TGF-ß1) was analyzed using an enzyme-linked immunosorbent assay. The expression of DNA damage protein 53BP1 and repair protein Rad51 were increased post-irradiation. The expression of keratin 19, vimentin, collage type IV, desmoglein 3, and integrins α6 and ß4 was altered post-irradiation. Proliferation significantly decreased at 24, 48, and 72h post-irradiation in both NNOM and TEM. IR increased the secretion of IL-1ß, whereas TGF-ß1 secretion was not altered. All observed IR-induced alterations in TEM were also observed in NNOM. Based on the similar response of TEM and NNOM to IR we consider our TEM construct a suitable model to quantify the acute biological effects of IR.


Assuntos
Mucosa Bucal/efeitos da radiação , Engenharia Tecidual , Membrana Basal/efeitos da radiação , Adesão Celular/efeitos da radiação , Técnicas de Cultura de Células , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Colágeno Tipo IV/análise , Colágeno Tipo IV/efeitos da radiação , Tecido Conjuntivo/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Desmogleína 3/análise , Desmogleína 3/efeitos da radiação , Epitélio/efeitos da radiação , Feminino , Fibroblastos/efeitos da radiação , Raios gama , Humanos , Integrina alfa6/análise , Integrina alfa6/efeitos da radiação , Integrina beta4/análise , Integrina beta4/efeitos da radiação , Interleucina-1beta/análise , Interleucina-1beta/efeitos da radiação , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/efeitos da radiação , Queratina-19/análise , Queratina-19/efeitos da radiação , Queratinócitos/efeitos da radiação , Masculino , Pessoa de Meia-Idade , Mucosa Bucal/citologia , Rad51 Recombinase/análise , Rad51 Recombinase/efeitos da radiação , Doses de Radiação , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/efeitos da radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Vimentina/análise , Vimentina/efeitos da radiação
6.
Calcif Tissue Int ; 79(4): 245-54, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17033726

RESUMO

Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone.


Assuntos
Fosfatase Ácida/biossíntese , Ossos do Braço/enzimologia , Isoenzimas/biossíntese , Ossos da Perna/enzimologia , Osteoclastos/enzimologia , Crânio/metabolismo , Fosfatase Ácida/deficiência , Fosfatase Ácida/genética , Animais , Catepsina K , Catepsina L , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Ativação Enzimática , Isoenzimas/deficiência , Isoenzimas/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fosfatase Ácida Resistente a Tartarato
7.
Calcif Tissue Int ; 78(4): 248-54, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16604288

RESUMO

Tartrate-resistant acid phosphatase (TRACP) is generally used as a marker of osteoclasts. Yet, other bone-associated cells, such as osteoblasts and osteocytes, may also express activity of this enzyme. Osteoblasts containing TRACP activity are seen particularly in the vicinity of areas of bone resorption, suggesting that osteoclasts somehow induce TRACP activity in osteoblasts. In a recent study, we found that osteoblast-like cells appeared to have the capacity to endocytose TRACP released by osteoclast precursors. In the present study, we investigated the endocytosis of TRACP in more detail as well as the fate of the endocytosed enzyme. We found that incubation of osteoblast-like cells with TRACP-coated beads resulted in attachment of a high number of beads to the cells. After culturing osteoblast-like cells with medium conditioned by blood monocytes that contain TRACP, activity of the enzyme was found in the cells. Following replacement of the medium by normal medium that did not contain TRACP, a decrease in the level of TRACP activity in osteoblast-like cells occurred. Our data strongly suggest that osteoblast-like cells recognize TRACP released by osteoclast precursors and that upon endocytosis inactivation of the enzyme occurs. We propose that uptake of the enzyme is important for the control of enzyme activity, thereby preventing degradation of matrix constituents.


Assuntos
Fosfatase Ácida/metabolismo , Reabsorção Óssea/metabolismo , Endocitose/fisiologia , Isoenzimas/metabolismo , Osteoblastos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Monócitos/metabolismo , Osteoclastos/metabolismo , Fosfatase Ácida Resistente a Tartarato
8.
Bone ; 36(6): 1065-77, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15878315

RESUMO

Tartrate-resistant acid phosphatase (TRACP) is produced by macrophages and other cells of the monohistiocytic lineage. In particular, osteoclasts are characterized for a high expression of this enzyme. Yet, several data suggest that other bone cell types, such as osteocytes and osteoblasts, may also express activity of this enzyme. This is particularly obvious at sites were osteoclasts resorb bone, suggesting that osteoclasts (or their precursors) somehow induce TRACP activity in osteoblasts. In the present study, we investigated this by culturing human osteoblast-like cells with and without conditioned medium (MCM) from human blood monocytes (as a source of osteoclast precursors). High levels of TRACP activity were found in osteoblast-like cells cultured with MCM. Depletion of TRACP from this medium resulted in the absence of its activity in osteoblast-like cells, thus suggesting that the TRACP activity in these cells was the result of endocytosed TRACP that was released by the monocytes in the MCM. Osteoblast-like cells cultured in control (non-conditioned) medium contained very low levels of TRACP-like activity. However, the cells expressed TRACP mRNA and incubation of extracts of these cells with active cathepsin B did induce activity of a TRACP-like enzyme. Inhibition of the activity of cysteine proteinases in general and of cathepsin B in particular, completely blocked TRACP activity of the osteoblast-like cells. This TRACP-like enzyme but not the alleged endocytosed fraction of TRACP was inhibited by fluoride, suggesting that the fractions may be different isoenzymes. Our data seem to indicate that osteoblast-like cells may contain two different fractions of TRACP, one that is released by monocytes and subsequently endocytosed by osteoblast-like cells and a second endogenous fraction that is present in an inactive proform. We hypothesize that the capacity of osteoblast-like cells to endocytose TRACP is important for the removal of this enzyme during or following the bone resorptive activity of the osteoclast.


Assuntos
Fosfatase Ácida/metabolismo , Endocitose/fisiologia , Expressão Gênica/genética , Isoenzimas/metabolismo , Osteoblastos/enzimologia , Fosfatase Ácida/efeitos dos fármacos , Fosfatase Ácida/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Reabsorção Óssea/metabolismo , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Catepsina B/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/farmacologia , Meios de Cultivo Condicionados/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Vesículas Citoplasmáticas/química , Dipeptídeos/farmacologia , Ativação Enzimática , Humanos , Isoenzimas/efeitos dos fármacos , Isoenzimas/genética , Modelos Biológicos , Monócitos/citologia , Monócitos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Fluoreto de Sódio/farmacologia , Fosfatase Ácida Resistente a Tartarato
9.
J Bone Miner Res ; 19(10): 1722-31, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15355568

RESUMO

UNLABELLED: Precursors of osteoclasts seeded on top of a confluent layer of osteoblasts/bone lining cells induced retraction of the latter cells. The (pre)osteoclasts then migrated in the formed cell-free areas and fused to form osteoclast-like cells. Retraction of the osteoblasts/bone lining cells proved to depend on activity of matrix metalloproteinases, and TGF-beta1 prevented the retraction. INTRODUCTION: It is well known that osteoblasts have a profound effect on (pre)osteoclasts in inducing the formation of bone-resorbing osteoclasts. Whether, on the other hand, (pre)osteoclasts also modulate osteoblast activity is largely unknown. Because osteoblasts/bone lining cells have to retract from the surface before resorption of bone by osteoclasts, we addressed the question of whether (pre)osteoclasts have the capacity to induce such an activity. MATERIALS AND METHODS: Rabbit calvarial osteoblasts/bone lining cells or periosteal fibroblasts were cultured until confluency, after which rabbit peripheral blood mononuclear cells (PBMCs) were seeded on top of them. The co-cultures were maintained for up to 15 days in the presence or absence of the cytokines transforming growth factor (TGF)-beta1 and TNF-alpha and selective inhibitors of matrix metalloproteinases and serine proteinases. The formation of cell-free areas and the number of TRACP+ multinucleated osteoclast-like cells were analyzed. In addition, formation of cell-free areas was analyzed in co-cultures of osteoblasts with mature osteoclasts. RESULTS: The seeding of PBMCs on a confluent layer of osteoblasts/bone lining cells resulted in the following sequence of events. (1) A low number of PBMCs strongly attached to osteoblasts. 2) At these sites of contact, the osteoblasts retracted, thus forming cell-free areas. (3) The PBMCs invaded these areas and attached to the surface of the well, after which they fused and formed multinucleated TRACP+ osteoclast-like cells. Retraction was only seen if the cells were in direct contact; conditioned media from cultured PBMCs added to osteoblasts had no effect. Mature osteoclasts seeded on osteoblasts similarly induced retraction, but this retraction occurred at a much faster rate (within 2 days) than the retraction effectuated by the osteoclast precursors (after 8 days in co-culture). Inhibition of matrix metalloproteinase activity, but not of serine proteinases, strongly reduced retraction of the osteoblasts, thus indicating that this type of cell movement depends on the activity of matrix metalloproteinases. A similar inhibitory effect was found with TGF-beta1. TNF-alpha had no effect on osteoblast retraction but enhanced the formation of multinucleated osteoclast-like cells. Addition of PBMCs to confluent layers of periosteal fibroblasts resulted in similar phenomena as observed in co-cultures with osteoblasts. However, the cell-free areas proved to be significantly smaller, and the number of multinucleated cells formed within cell-free areas was three to four times lower. CONCLUSION: Our results indicate that osteoclast precursors and mature osteoclasts have the capacity to modulate the activity of osteoblasts and that, yet unknown, membrane-bound signaling molecules are essential in inducing retraction of osteoblasts and the subsequent formation of cell-free areas.


Assuntos
Osteoblastos/citologia , Osteoclastos/citologia , Fosfatase Ácida/metabolismo , Animais , Aprotinina/farmacologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Adesão Celular , Fusão Celular , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Isoenzimas/metabolismo , Leucócitos Mononucleares/citologia , Osteoblastos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Coelhos , Inibidores de Serina Proteinase/farmacologia , Fosfatase Ácida Resistente a Tartarato , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
10.
Anal Chem ; 73(14): 3426-31, 2001 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-11476244

RESUMO

The influence of a variety and combination of both ionic surfactants and different chain lengths of the polyelectrolyte poly(ethylene glycol) (PEG) on the performance characteristics (with particular reference to signal response) of a homogeneous, latex agglutination immunoassay was investigated. The test analyte was human serum C-reactive protein (CRP), and the antibody reagent consisted of a sheep polyclonal anti-CRP IgG fraction covalently coupled to 50-nm-sized latex including a glycine-capped chloromethylstyrene shell. The amount and rate of immunoagglutination was monitored turbidimetrically after sample addition. It was found that 2.5 mmol/L concentrations of the small cationic surfactant tetramethylammonium hydroxide (TMH), when present alone, substantially increased both reaction rates and sensitivity in the lower clinical ranges of CRP concentration when compared to normally used assay conditions containing PEG and the anionic detergent Gafac. The nonspecific binding (NSB) was also found to be unchanged. Evidence is presented that the TMH enhances the actual antibody-antigen interaction as opposed to the known effects of other surfactants in immunocomplex dissociation or in maintenance of colloidal stability. We suggest that the enhancement seen with TMH could be an alternative to PEG and may provide a new means of further extending detection limits. The utility of this type of immunoassay technology could therefore be increased whenever clinically required.


Assuntos
Proteína C-Reativa/análise , Testes de Fixação do Látex/métodos , Polietilenoglicóis/química , Compostos de Amônio Quaternário/química , Tensoativos/química , Ânions/química , Cátions/química , Humanos , Imunoensaio/métodos
11.
Anal Chem ; 73(14): 3417-25, 2001 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-11476243

RESUMO

The role of the solution environment for a light-scattering, latex-particle-enhanced, homogeneous immunoassay of C-reactive protein (CRP) has been investigated in order to assess and optimize the immunoagglutination response. Latex particles of 50-170-nm sizes were covalently coupled with an IgG polyclonal antibody and subjected to an extensive optimization regime. This consisted of conditions responsible, in different degrees, for the principal attractive/repulsive forces affecting both colloidal stability and the antibody/antigen interaction: particle size, antibody concentration, ionic strength and species, pH, and amino acid chemistry of the particle surface. Careful control of these parameters was found to be necessary to achieve the desired effects of balancing high colloidal stability in the absence of antigen but promoting a rapid, sensitive, and dose-dependent agglutination with pathological serum samples. In addition, the estimation of fundamental properties governing intermolecular interaction (i.e. the "Hamaker" constant and critical coagulation concentration) was attempted to order to investigate a simple, practical means of defining a colloidal/immunoassay system under "real conditions" as well as "real time". It is concluded that because each antibody system is unique, a similar optimization should be performed in diagnostic immunoassays of this type to maximize their clinical utility.


Assuntos
Proteína C-Reativa/análise , Testes de Fixação do Látex/métodos , Imunoensaio/métodos , Imunoglobulina G/química , Luz , Concentração Osmolar , Tamanho da Partícula , Espalhamento de Radiação , Soluções , Propriedades de Superfície
12.
Mutat Res ; 227(3): 167-72, 1989 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2509905

RESUMO

The induction of micronuclei (MN) by vincristine, mitomycin C and cyclophosphamide was compared in purified lymphocytes and in whole-blood cultures. With both assays, cytokinesis was blocked by cytochalasin B and MN were only scored in binucleate cells. The data suggest that whole-blood cultures may be considered a better experimental condition for the detection of MN induced by chemicals in vitro.


Assuntos
Linfócitos/efeitos dos fármacos , Testes para Micronúcleos , Mutagênicos , Células Sanguíneas , Separação Celular , Células Cultivadas , Ciclofosfamida/toxicidade , Feminino , Humanos , Linfócitos/citologia , Mitomicina , Mitomicinas/toxicidade , Vincristina/toxicidade
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