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1.
Anticancer Drugs ; 12(3): 235-45, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11290871

RESUMO

Prolonging tumor exposure to topoisomerase I inhibitors has been correlated to enhance the efficacy of those agents. Lurtotecan, a water-soluble camptothecin analog, was formulated as a liposomal drug, NX211, to enhance the delivery of drug to tumors. Tumor-bearing mice were treated with either [14C]NX211 containing [14C]lurtotecan, [3H]NX211 containing [3H]phosphatidylcholine or [14C]lurtotecan, euthanized at selected times post-injection, and tissues, plasma, urine and feces were collected. These studies demonstrated that KB tumors of [14C]NX211-treated mice had approximately 70-fold greater concentrations of [14C]lurtotecan at 24 h, respectively, compared to concentrations of [14C]lurtotecan of the KB tumors of [14C]lurtotecan-treated mice. The area under curve (AUC) from 0 to 48 h of [14C]lurtotecan for the KB tumors of [14C]NX211-treated animals was over 17-fold greater than the AUC of [14C]lurtotecan for the tumors of [14C]lurtotecan-treated animals. Treatment with [3H]NX211 demonstrated that the lipid component continually accumulated over 24 h in the tissues. HPLC analysis of extracted material from tumors of [14C]NX211-treated mice showed that more than 95% of the radioactive material was intact [14C]lurtotecan. These findings are one of the keys justifying the development of a liposomal formulation of lurtotecan, which has the intent to increase tumor exposure and increase antitumor efficacy.


Assuntos
Antineoplásicos/farmacocinética , Camptotecina/farmacocinética , Neoplasias/metabolismo , Animais , Antineoplásicos/administração & dosagem , Área Sob a Curva , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Lipossomos , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Distribuição Tecidual
2.
Clin Cancer Res ; 6(7): 2903-12, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10914740

RESUMO

Lurtotecan is a clinically active water-soluble camptothecin analogue that has been formulated into a low-clearance unilamellar liposome, NX 211. Comparative studies between free drug and NX 211 have been performed assessing pharmacokinetics in nude mice, tissue distribution in tumor-bearing mice, and antitumor efficacy in xenografts. Compared with lurtotecan, NX 211 demonstrated a significant increase in plasma residence time and a subsequent 1500-fold increase in the plasma area under the drug concentration curve. The volume of distribution was also greatly restricted, suggesting altered tissue distribution. Evaluation of tissues 24 h after administration of either [14C]NX 211 or [14C]lurtotecan to ES-2 tumor-bearing mice demonstrated a 40-fold increase in radiolabeled compound in the tumors of NX 211-treated mice compared with mice treated with lurtotecan. In single-dose efficacy studies, NX 211 produced a consistent 3-fold or greater increase in therapeutic index compared with lurtotecan in both the KB and ES-2 xenograft models. When compared at equitoxic levels in repeat-dose efficacy studies, NX 211 generated durable cures lasting >60 days and a 2-8-fold increase in log10 cell kill, compared with lurtotecan and topotecan, respectively. Together, these data demonstrate that NX 211 has significant therapeutic advantage over lurtotecan and that the improved antitumor activity is consistent with increased exposure and enhanced drug delivery to tumor sites.


Assuntos
Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Camptotecina/análogos & derivados , Sarcoma/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Área Sob a Curva , Camptotecina/administração & dosagem , Camptotecina/farmacocinética , Camptotecina/uso terapêutico , Radioisótopos de Carbono , Portadores de Fármacos , Feminino , Humanos , Células KB , Lipossomos , Camundongos , Camundongos Nus , Distribuição Tecidual , Topotecan/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Clin Exp Metastasis ; 16(1): 9-20, 1998 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9502073

RESUMO

Tumor establishment and metastasis are dependent on extracellular matrix proteolysis, tumor cell migration, and angiogenesis. Urokinase plasminogen activator (uPA) and its receptor are essential mediators of these processes. The purpose of this study was to investigate the effect of a recombinant human uPAR antagonist on growth, establishment, and metastasis of tumors derived from human cancer cell lines. A noncatalytic recombinant protein, consisting of amino acids 1-137 of human uPA and the CH2 and CH3 regions of mouse IgG1 (uPA-IgG), was expressed, purified, and shown to bind specifically to human uPAR and to saturate the surface of human tumor cells which express uPAR. Daily i.p. administration of uPA-IgG to nude mice extended latencies of unstaged tumors derived from Lox melanoma and SW48 colon carcinoma cells by 7.7 and 5.5 days, respectively. uPA-IgG treatment did not affect the growth of Lox or KB tumors staged to 200 mg before antagonist treatment commenced. The effect of uPA-IgG on the establishment of micrometastases was assessed in SCID mice. KB head/neck tumor cells were injected in the tail vein and allowed to seed for 48 h before initiation of daily i.p. injections of uPA-IgG for 24 days. The number of lung colonies ranged between 5 and 30% of vehicle-treated mice in two separate experiments. Furthermore, a single 800 microg dose of uPA-IgG administered 1 h prior to tail vein injection of KB cells reduced lung colony formation to just 3.5% of vehicle-treated SCID mice. These data demonstrate that antagonism of uPAR arrested metastasis and inhibited the establishment of primary tumors and micrometastases. Thus, small molecule uPAR antagonists may serve as useful adjuvant agents in combination with existing cancer chemotherapy.


Assuntos
Imunoglobulina G/uso terapêutico , Metástase Neoplásica/prevenção & controle , Proteínas de Neoplasias/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Progressão da Doença , Humanos , Imunoglobulina G/farmacologia , Camundongos , Camundongos Nus , Camundongos SCID , Proteínas de Neoplasias/efeitos dos fármacos , Transplante de Neoplasias , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes de Fusão/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos
4.
Gene ; 190(1): 139-44, 1997 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-9185859

RESUMO

Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.


Assuntos
Baculoviridae/genética , Imunoglobulina G/genética , Ativadores de Plasminogênio/genética , Proteínas Recombinantes de Fusão/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Linhagem Celular , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Humanos , Proteínas Recombinantes de Fusão/isolamento & purificação , Spodoptera
5.
Clin Exp Metastasis ; 14(2): 115-24, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8605725

RESUMO

Four potent, synthetic inhibitors of matrix metalloproteinases (MMPs) were assessed as inhibitors of tumor growth and spontaneous metastasis to the lung. Mat Ly Lu rat prostate tumor, LOX human melanoma and M27 murine Lewis lung tumor were implanted subcutaneously (s.c.) in mice and allowed to grow for 3-12 days. The lungs of the tumor-bearing mice were then removed and implanted s.c. into untreated mice, and the outgrowth of secondary tumors from the implanted lungs measured. The incidence and rate of outgrowth of secondary tumors increased with the length of primary tumor growth, validating these measurements as indices of spontaneous metastasis to the lung. Compounds were tested by s.c. implantation of minipumps which delivered compound throughout the period of primary tumor growth and spontaneous metastasis to the lung at steady-state drug concentrations orders of magnitude greater than the concentrations needed to either inhibit collagenase, gelatinase or stromelysin in vitro. Inhibitor treatment slowed the growth of primary s.c. Mat Ly Lu and LOX tumors by 40-60% but had no significant effect on the growth of primary M27 tumors. Surprisingly, inhibitor treatment had no significant effect on the ability of the lung to generate secondary tumors when reimplanted s.c. in untreated mice. Because of the possible importance of cathepsins B, H and L in tumor growth and metastasis, the irreversible inhibitor E-64 was also infused by s.c. minipump. E-64 had no effect on the growth or spontaneous metastasis of Mat Ly Lu or M27 tumors.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/antagonistas & inibidores , Metástase Neoplásica , Inibidores de Proteases/farmacologia , Células Tumorais Cultivadas/enzimologia , Animais , Divisão Celular/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/secundário , Metaloproteinase 3 da Matriz , Camundongos , Transplante de Neoplasias , Ratos , Células Tumorais Cultivadas/patologia
6.
J Med Chem ; 39(3): 713-9, 1996 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-8576914

RESUMO

Eleven water soluble 7-substituted quaternary ammonium salt derivatives of 10,11-(methylenedioxy)- and 10,11-(ethylenedioxy)-(20S)-camptothecin were synthesized via the Friedlander reaction followed by nucleophilic displacement with an aromatic amine. All of these compounds were more potent than camptothecin in the in vitro cleavable complex assay. These inherently charged camptothecin derivatives were cytotoxic against three different human tumor cell lines (SKOV3, an ovarian adenocarcinoma; SKVLB a multidrug resistant ovarian adenocarcinoma; and HT-29, a colon carcinoma). A selected group of five compounds was evaluated in the nude mouse HT-29 xenograft model. Two of these quaternary salts (17 and 18) were more efficacious than Topotecan in delaying tumor growth. In an extended in vivo model, 18 demonstrated tumor regression.


Assuntos
Antineoplásicos/farmacologia , Camptotecina/farmacologia , Inibidores da Topoisomerase I , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Animais , Antineoplásicos/química , Camptotecina/análogos & derivados , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Camundongos Nus , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Solubilidade , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Água
7.
Clin Cancer Res ; 2(1): 7-12, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9816083

RESUMO

We sought to develop an assay for measuring the inhibition of P-glycoprotein (Pgp) function in whole blood as an indicator of in vivo drug activity. Since the CD56(+) subset of peripheral blood lymphocytes (PBLs) has been shown to express functional Pgp, the changes in rhodamine 123 (R123) uptake by CD56(+) PBLs from GG918-treated and untreated whole blood were used as the basis for these studies. In an ex vivo study, heparin-treated whole blood was obtained from normal volunteers, and GG918 and R123 were added at various concentrations for time course analyses of dye loading. GG918 concentrations from 2.5 to 800 nm were tested in incubations ranging from 15 min to 3 h prior to R123 addition. R123 loading times ranged from 0 to 80 min. Flow cytometric analyses of the CD56(+) PBLs indicated that the resolution of Pgp inhibition was dependent on inhibitor concentration and time of R123 loading and independent of the R123 concentrations tested. In this ex vivo assay model, a dose-dependent response was seen for GG918 with a 2-fold increase in cellular R123 intensity being produced at a drug concentration of 80 nm. When this assay method was applied to blood samples from volunteers dosed p.o. with GG918, similar shifts in R123 fluorescence of the CD56(+) PBLs were observed with significant increases in R123 intensity occurring at serum concentrations as low as 40 nm. In contrast to assays in which target cell populations are enriched prior to testing, the addition of the substrate (R123) directly to the blood sample combined with the segregation of the target cells by specific immunofluorescence provides the investigator an indication of in situ activity of circulating drug. Thus, CD56(+) PBLs may prove useful as a surrogate target for monitoring multidrug resistance inhibitor activity in situ.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Acridinas/farmacologia , Resistência a Múltiplos Medicamentos , Citometria de Fluxo , Isoquinolinas/farmacologia , Tetra-Hidroisoquinolinas , Acridinas/metabolismo , Antígeno CD56/análise , Humanos , Isoquinolinas/metabolismo , Rodamina 123/farmacocinética
8.
J Med Chem ; 38(7): 1106-18, 1995 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-7707314

RESUMO

A series of analogs based on a novel template, 11-aza-(20S)-camptothecin, were obtained from total synthesis and tested as potential anticancer drugs in the topoisomerase I enzyme cleavable complex assay. The parent compound 11-aza-(20S)-camptothecin (8) was derived from a Friedlander condensation between the known aminopyridine derivative 3-(3-amino-4-picolylidene)-p-toluidine and optically active tricyclic ketone 7. Compound 8 had activity approximately twice that of (20S)-camptothecin in the calf thymus topoisomerase I cleavable complex assay. Compounds were prepared wherein the 11-aza nitrogen atom was quaternized as either the corresponding N-oxide or methyl iodide. Compounds with quaternized N-11 showed improved water solubility and were equipotent to the clinically investigated camptothecin analog topotecan in the cleavable complex assay. These compounds were evaluated in vivo in nude mice bearing HT-29 human colon carcinoma xenografts. The analog 11-aza-(20S)-camptothecin 11-N-oxide was found to significantly retard tumor growth when compared to untreated controls. Finally, 7,10-disubstituted 11-azacamptothecin analogs were synthesized using Pd(0) coupling reactions of 10-bromo-7-alkyl-11-aza-(20S)-camptothecins 19 and 20, which in turn were available from a Friedlander condensation of the novel bromopyridine derivatives 17a and 17b with 7. Among the 10-substituted series, a number of analogs displayed extremely high in vitro potency against topoisomerase I and improved aqueous solubility. A significant number of the compounds were found to be active in whole cell cytotoxicity assays and several were evaluated in nude mice bearing the HT-29 tumor xenografts. The most effective of these proved to be (S)-11-aza-7-ethyl-10-(aminohydroximinomethyl)camptothecin trifluoracetic acid salt (27), a potent topoisomerase I inhibitor which demonstrated excellent efficacy in both short term and in extended in vivo assays. A comparison between in vitro enzyme data and in vivo data from nude mouse studies in other compounds in this series revealed a poor overall correlation between topoisomerase inhibition in vitro and antitumor efficacy in vivo.


Assuntos
Antineoplásicos/síntese química , Camptotecina/análogos & derivados , Inibidores da Topoisomerase I , Animais , Antineoplásicos/química , Camptotecina/síntese química , Camptotecina/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Solubilidade , Relação Estrutura-Atividade
9.
J Med Chem ; 38(3): 395-401, 1995 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-7853331

RESUMO

The synthesis and antitumor activities of the novel water soluble camptothecin derivatives 7-[(4-methylpiperazino)methyl]-10,11-(methylenedioxy)-(20S)-campto thecin trifluoroacetate (6) and 7-[(4-methylpiperazino)methyl]-10,11-(ethylenedioxy)-(20S)-camptot hecin trifluoroacetate (7) are described. The solubilities of compounds 6 and 7 were measured to be 4.5 and 5.8 mg/mL, respectively, in pH 5 acetate buffer in contrast to < 0.003 mg/mL for camptothecin in the same buffer. In the purified topoisomerase I cleavable complex enzyme assay, compounds 6 and 7 demonstrated potent inhibition of topoisomerase I with IC50's of 300 and 416 nM, respectively, in comparison to 679 nM for camptothecin and 1028 nM for topotecan. In human tumor cell cytotoxicity assays, compounds 6 and 7 demonstrated potent antitumor activity against ovarian (SKOV3), ovarian with upregulated MDRp-glycoprotein (SKVLB), melanoma (LOX), breast (T47D), and colon (HT29) with IC50's ranging from 0.5 to 102 nM. Compounds 6 and 7 induced tumor regressions in the HT29 human colon tumor xenograft model and demonstrated similar rank order of potency compared to in vitro assay results.


Assuntos
Antineoplásicos/síntese química , Camptotecina/análogos & derivados , Inibidores da Topoisomerase I , Animais , Antineoplásicos/farmacologia , Camptotecina/síntese química , Camptotecina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos , Solubilidade , Células Tumorais Cultivadas , Água/química
10.
Cancer Res ; 55(3): 603-9, 1995 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-7834631

RESUMO

The development of camptothecin-like compounds as inhibitors of topoisomerase I for the treatment of resistant tumors has generated clinical excitement in this new class of drugs. We have developed two novel water-soluble camptothecin analogues which are specific inhibitors of topoisomerase I and are potent cytotoxins with significant antitumor activity. We added water-solubilizing groups off position 7 in the B ring of either 10,11-ethylenedioxy- or 10,11-methylenedioxy-20(S)-camptothecin. These water-soluble camptothecin analogues were demonstrated to be nanamolar inhibitors of the topoisomerase I enzyme in the cleavable complex assay. The compounds, GI147211 [7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camp tot hecin], and GI149893 [7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-cam pto thecin], were compared to topotecan, a known water-soluble inhibitor of topoisomerase I. Both GI compounds were found to be slightly more potent than topotecan as inhibitors of topoisomerase I in the cleavable complex assay and were 1.5-2 times more soluble. Tumor cell cytotoxicity assays using 5 separate cell lines demonstrated that both GI compounds were 5-10 times more potent than topotecan, although by comparison all three topoisomerase I inhibitors were unaffected by the multidrug resistance P-glycoprotein. The antitumor activity of all three topoisomerase I inhibitors was compared concomitantly in two human colon xenograft models. In both models, GI147211 and GI149893 were able to induce regression of established HT-29 and SW-48 colon tumors by as much as 60%. The antitumor activity of both compounds were also demonstrated in the MX-1 and PC-3 xenografts. Microscopic examination of selected tissues indicated that drug-induced toxicity was primarily limited to the gastrointestinal tract and was comparable among the three compounds. Further clinical development of this class of compounds is ongoing.


Assuntos
Antineoplásicos/uso terapêutico , Camptotecina/análogos & derivados , Neoplasias do Colo/tratamento farmacológico , Inibidores da Topoisomerase I , Animais , Antineoplásicos/toxicidade , Peso Corporal/efeitos dos fármacos , Camptotecina/uso terapêutico , Camptotecina/toxicidade , Bovinos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Nus , Timo/enzimologia , Topotecan , Transplante Heterólogo , Células Tumorais Cultivadas
11.
Cell Growth Differ ; 1(7): 313-8, 1990 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2278883

RESUMO

Rat pheochromocytoma cells (PC12) respond to nerve growth factor (NGF) by elaborating neurites. We have studied the effect of extracellular matrix proteins on responsiveness of PC12 cells to NGF. NGF elicited neurite outgrowth in cells plated on collagen, fibronectin, or laminin within 48-72 h. In contrast, cells entrapped in reconstituted basement membrane prepared from the Engelbreth-Holm-Swarm tumor (EHS-BM) responded within 24 h. Cells plated on collagen or fibronectin required a minimal dose of 50 ng/ml NGF to achieve the same effect as cells entrapped in EHS-BM at 1 ng/ml NGF. Neurites displayed by cells plated in EHS-BM were more extensively branched and remained intact up to 21 days following a single administration of NGF.


Assuntos
Neoplasias das Glândulas Suprarrenais/patologia , Membrana Basal/fisiologia , Fatores de Crescimento Neural/farmacologia , Feocromocitoma/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Colágeno , Técnicas de Cultura/métodos , Fibronectinas , Laminina , Fenótipo , Ratos , Estimulação Química , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/ultraestrutura
12.
Clin Immunol Immunopathol ; 48(3): 317-24, 1988 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-3042212

RESUMO

Antibody reactivity to melanocyte-derived cells was investigated in patients with alopecia areata or totalis by use of Western blot analysis of detergent-solubilized membrane antigens of a human melanoma cell line, M14. Reactivity was detected in the sera of 9 of 27 alopecia areata or totalis patients, 8 of 13 vitiligo patients, and 6 of 24 normal control subjects. Significant differences between patient and control sera were found in the number and distribution of antibody specificities detected. In vitiligo sera, there was an increased prevalence of reactivity to a melanoma antigen of 52,000 mol wt. In contrast, the predominant specificities in alopecia areata sera were for antigens of 74,500 and 70,800 mol wt, and the majority of positive sera were from patients with total hair loss. These findings suggest that autoreactivity to pigmented cells occurs in certain patients with alopecia areata or totalis.


Assuntos
Alopecia/imunologia , Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/imunologia , Doenças Autoimunes/imunologia , Melanoma/imunologia , Vitiligo/imunologia , Especificidade de Anticorpos , Imunofluorescência , Humanos , Técnicas de Imunoadsorção , Peso Molecular , Células Tumorais Cultivadas
14.
Hepatology ; 7(5): 906-12, 1987.
Artigo em Inglês | MEDLINE | ID: mdl-2820857

RESUMO

Since antibodies to polyalbumin have been noted to occur in patients with hepatitis or cirrhosis, we investigated sera from 219 patients with a variety of acute and chronic liver diseases with and without HBsAg using an ELISA. The prevalence of circulating polyalbumin antibodies was shown to be significantly increased over that of healthy controls (6.7%) in patients with autoimmune chronic active hepatitis (67%), acute viral hepatitis (41%) and fulminant hepatic necrosis (38%), but such antibodies were absent in chronic hepatitis B patients and other liver diseases. Serial studies in acute hepatitis showed evidence of antibodies early in the course of illness with disappearance prior to full recovery. In acute hepatitis B, the presence of polyalbumin antibodies was significantly associated with female sex (p less than 0.01), 3-fold higher transaminase levels and shorter duration of clinical illness (less than 4 weeks in all cases). Polyalbumin antibodies appear to be associated with diseases characterized by active hepatocyte necrosis. Since they are evident early in acute hepatitis B when complexes of polyalbumin and virus are likely, these antibodies may play an adjunctive role to other hepatitis B-related antibodies in the clearance of hepatitis B virus infection.


Assuntos
Anticorpos/análise , Hepatopatias/imunologia , Albumina Sérica/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hepatite B/imunologia , Anticorpos Anti-Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/análise , Hepatite Crônica/imunologia , Hepatite Viral Humana/imunologia , Humanos , Imunoglobulinas/análise , Masculino , Receptores de Albumina , Receptores de Superfície Celular/metabolismo , Albumina Sérica Humana , Fatores Sexuais , Transaminases/sangue
15.
Hepatology ; 7(5): 825-30, 1987.
Artigo em Inglês | MEDLINE | ID: mdl-3115882

RESUMO

Evidence for increased plasma levels of complexes containing Gc (vitamin D-binding protein) and cellular actin has been previously reported during fulminant hepatic necrosis in man. In order to study this process in more detail, we produced liver injury in hamsters using increasing doses of acetaminophen, with serial collection of sera for up to 168 hr after acetaminophen injection. Hamster Gc was purified using a three-step procedure and was shown to resemble closely human Gc. Polyclonal antihamster Gc was prepared and used in rocket immunoelectrophoresis and radial immunodiffusion studies for quantitation of total serum Gc and the percentage of Gc complexed with actin. Serum Gc levels were depressed in animals having liver damage, and the extent of depression 42 hr after acetaminophen correlated with the extent of elevation of AST. The proportion of the total Gc that was present in the complexed form increased in relation to the severity of the liver disease. In serial studies, diminution in Gc level preceded the rise in AST and increase in the percent complexed. These changes closely resemble observations in man and suggest that the hamster-acetaminophen hepatotoxicity model may be of value in further study of interactions of Gc with intracellular actin components and its role in actin homeostasis in conditions of massive tissue necrosis.


Assuntos
Actinas/sangue , Hepatopatias/sangue , Fígado/patologia , Proteína de Ligação a Vitamina D/sangue , Acetaminofen , Actinas/fisiologia , Animais , Aspartato Aminotransferases/sangue , Doença Hepática Induzida por Substâncias e Drogas , Cricetinae , Homeostase , Imunodifusão , Imunoeletroforese , Masculino , Mesocricetus , Necrose , Proteína de Ligação a Vitamina D/fisiologia
16.
J Lab Clin Med ; 110(1): 83-90, 1987 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3598340

RESUMO

Increased complexing of circulating group-specific component (Gc, vitamin D-binding protein) has been found in humans under conditions of presumed increased actin release, and particularly fulminant hepatic necrosis (FHN). We therefore used a hamster model of acetaminophen-induced FHN to further investigate this phenomenon. Liver damage was monitored by aspartate aminotransferase (AST) activity and by histologic examination, and serum Gc was analyzed by polyacrylamide gel electrophoresis (PAGE) and transblotting with anti-Gc. In controls and treated animals displaying slight liver damage, greater than 90% of Gc was of mobility corresponding to native purified hamster Gc, whereas with more severe liver damage up to 100% of Gc was found in one of two cathodal configurations that appear to correspond to complexes with actin. Densitometric quantitation of complexed Gc demonstrated a strong correlation with the severity of liver damage. These results show PAGE to be a useful method of estimating the percentage of Gc complexed in serum, and suggest that cell damage may be followed by the appearance in the circulation of cellular actin that complexes with Gc.


Assuntos
Hepatopatias/metabolismo , Proteína de Ligação a Vitamina D/metabolismo , Actinas/sangue , Animais , Cricetinae , Densitometria , Desoxirribonucleases/sangue , Eletroforese , Fígado/patologia , Masculino , Mesocricetus , Necrose/metabolismo , Proteína de Ligação a Vitamina D/sangue
18.
Biol Neonate ; 52(5): 250-5, 1987.
Artigo em Inglês | MEDLINE | ID: mdl-3676365

RESUMO

Because of the possible involvement of group-specific component (Gc) or vitamin-D-binding protein in the immunological functions of mononuclear cells and the increased risk of central nervous system infections in early infancy, we studied Gc levels in the cerebrospinal fluid (CSF) of children. CSFs were examined for the Gc concentration using ELISA and rocket immunoelectrophoresis, with purified Gc as standard. The results showed a significant inverse correlation (p less than 0.05) between the age of the patients and CSF Gc levels. Gc levels in the CSF were significantly increased in infants less than 2 months of age (12.5 micrograms/ml), as compared to infants greater than 2 months (1.7 micrograms/ml, p less than 0.0028). In children greater than 2 months of age a significant correlation was found between Gc levels and those of other CSF proteins (albumin, IgG and total protein, p less than 0.002). However, no significant correlation between Gc levels and those of other CSF proteins was apparent in infants less than 2 months of age, indicating the possibility that the concentration of Gc in the CSF may be selectively increased in this age group.


Assuntos
Envelhecimento/líquido cefalorraquidiano , Proteína de Ligação a Vitamina D/líquido cefalorraquidiano , Infecções Bacterianas/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/líquido cefalorraquidiano , Lactente , Recém-Nascido
19.
Biochemistry ; 25(21): 6467-72, 1986 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-3790535

RESUMO

Profilin purified from human platelets formed a 1:1 molar ratio complex with rabbit skeletal muscle G-actin but was displaced by purified serum Gc (vitamin D binding protein) in a dose-dependent fashion as assessed by chromatography and ultrafiltration. This suggested that Gc and profilin competed for the same binding area on G-actin, with Gc-G-actin complexes being more stable than profilin-G-actin complexes in vitro. The binding domain for Gc on G-actin was localized to a 16,000-Da C-terminal fragment of G-actin generated by Staphylococcus aureus V8 protease, as judged by comigration on two-dimensional electrophoresis and also by overlaying electrophoresis gels with 125I-Gc. Previous studies have reported that residues 374 and 375 of G-actin are essential for binding of profilin. In this study, experiments involving tryptic removal of Cys-374 labeled with the fluorescent probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)-ethylenediamine showed that these C-terminal amino acids were not necessary for interaction with Gc.


Assuntos
Actinas/metabolismo , Proteínas Contráteis , Proteínas dos Microfilamentos , Proteínas/metabolismo , Proteína de Ligação a Vitamina D/farmacologia , Animais , Plaquetas/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Cinética , Peso Molecular , Músculos/metabolismo , Profilinas , Proteínas/isolamento & purificação , Coelhos , Ultrafiltração
20.
Life Sci ; 38(8): 735-42, 1986 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-3951329

RESUMO

Limited proteolysis of G-actin was performed with trypsin and chymotrypsin to compare the binding sites for Gc and DNase. DNase I bound to the N-terminal area corresponding to the major cleavage site on G-actin (residues 62-68) and inhibited proteolysis, but did not bind the 33.5K C-terminal fragment (G-actin33.5) generated. In contrast, Gc did not exert any inhibitory effect upon proteolysis of the intact native G-actin42.0 molecule, although its presence protected G-actin33.5 from further proteolysis. This was shown by gel filtration to be due to the formation of complexes between Gc and G-actin33.5.


Assuntos
Actinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteína de Ligação a Vitamina D/metabolismo , Sítios de Ligação , Quimotripsina/metabolismo , Desoxirribonucleases/metabolismo , Eletroforese em Gel de Poliacrilamida , Tripsina/metabolismo
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