RESUMO
Background Pulsed cyclophosphamide or mycophenolate mofetil for lupus nephritis has limited efficacy. We previously reported a case of mixed-class IV + V lupus nephritis successfully treated with cyclophosphamide and tacrolimus. This study assessed the efficacy and safety of multitarget therapy with cyclophosphamide and tacrolimus for the treatment of lupus nephritis. Methods In a prospective, single-arm, open label pilot study, we recruited 15 patients aged 18-64 years with active lupus nephritis who met the American College of Rheumatology criteria for a diagnosis of systemic lupus erythematosus (1997). The treatment protocol was a starting dose of prednisolone of 0.6-1.0 mg/kg/day for 2 weeks and then tapered to a maintenance dose, intravenous cyclophosphamide (500 mg biweekly for 3 months) and tacrolimus (3.0 mg/day). Tacrolimus was continued as maintenance therapy. Complete remission was defined as a spot urine protein/creatinine ratio of < 0.5 g/gCr with no active urine casts and a serum creatinine level that was either normal or within 30% of a previously abnormal baseline level. We retrospectively compared results for the study patients with those of 18 historical controls conventionally treated with cyclophosphamide and prednisolone. Results At baseline, the mean patient age was 41.5 ± 14.6 years (male:female ratio 2:13), urine protein/creatinine ratio 3.9 ± 2.3 g/gCr and serum creatinine 84.6 ± 34.6 µmol/L. Lupus nephritis classifications included classes IV ( n = 8), III + V ( n = 1), IV + V ( n = 5) and unclassified ( n = 1). Eleven patients completed the treatment protocol and four withdrew. At 6 months, 12 of 15 (80.0%) had achieved complete remission using intention-to-treat analysis, significantly more than historical controls (seven of 18 patients, 38.9%). A transient increase in serum creatinine and gastric symptoms occurred in three cases. One patient withdrew due to cytomegalovirus antigenemia and severe diabetes, and one patient died of thrombotic microangiopathy. Conclusions Multitarget therapy with cyclophosphamide and tacrolimus can be a therapeutic option for lupus nephritis. Clinical trials registration Combination therapy of tacrolimus and intravenous cyclophosphamide for remission induction of lupus nephritis, UMIN: 000004893, URL: https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&type=summary&recptno=R000005830&language=E . Date of registration: 18 January 2011.
Assuntos
Ciclofosfamida/farmacologia , Nefrite Lúpica/tratamento farmacológico , Ácido Micofenólico/farmacologia , Tacrolimo/farmacologia , Administração Intravenosa , Adulto , Creatinina/sangue , Ciclofosfamida/administração & dosagem , Quimioterapia Combinada/métodos , Inibidores Enzimáticos/farmacologia , Feminino , Glucocorticoides/uso terapêutico , Humanos , Imunossupressores/farmacologia , Japão/epidemiologia , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/administração & dosagem , Projetos Piloto , Prednisolona/administração & dosagem , Prednisolona/uso terapêutico , Estudos Prospectivos , Indução de Remissão , Estudos Retrospectivos , Tacrolimo/administração & dosagem , Resultado do TratamentoRESUMO
INTRODUCTION: To achieve a high graft survival rate, patient adherence to immunosuppressive therapy is critical. It is extremely difficult to establish the actual adherence status of transplant recipients; only a few surveys on the issue have been performed in Japan. METHODS: We conducted a questionnaire survey mainly on treatment adherence to calcineurin inhibitors among renal transplant recipients. RESULTS: The survey demonstrated some degree of nonadherence in a relatively high percentage of the patients. The adherence rate was significantly lower for the evening than the morning dose (McNemar test, P < .001). It significantly decreased with time following transplantation for both the morning and the evening doses (logistic regression analysis, P = .025 and <.001, respectively). CONCLUSIONS: Immunosuppressive treatment places a substantial burden on patients, some of whom cannot continue regular treatment at specified time points due to daily life restrictions after they have returned to work.
Assuntos
Imunossupressores/uso terapêutico , Transplante de Rim/imunologia , Cooperação do Paciente/estatística & dados numéricos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Inibidores de Calcineurina , Esquema de Medicação , Feminino , Humanos , Imunossupressores/administração & dosagem , Masculino , Pessoa de Meia-Idade , Pacientes Desistentes do Tratamento/estatística & dados numéricos , Análise de Regressão , Inquéritos e QuestionáriosRESUMO
BACKGROUND: Dexamethasone, a synthetic glucocorticoid, has clinical benefit in patients with hormone-refractory prostate cancer (HRPC), but the mechanisms responsible for its effects are unknown. The nuclear factor-kappaB (NF-kappaB)-dependent cytokine interleukin (IL) 6 (IL-6) is thought to stimulate growth of HRPC. Because dexamethasone interferes with NF-kappaB activation, we determined whether dexamethasone inhibits prostate cancer growth by working through the glucocorticoid receptor (GR) to interfere with NF-kappaB-IL-6 pathway. METHODS: Three human prostate cancer cell lines (DU145, PC-3, and LNCaP) were assessed for GR expression and responsiveness to dexamethasone. Levels of GR, NF-kappaB, and the cytoplasmic NF-kappB inhibitor IkappaBalpha were determined by western blotting and of IL-6 by enzyme immunoassay. The subcellular localization of NF-kappaB was analyzed by immunofluorescence. The effects of dexamethasone (thrice weekly injections of 1 microg/mouse) on DU145 xenografts in nude and severe combined immunodeficient (SCID) mice were evaluated. GR expression in human prostate cancers was assessed by immunohistochemistry. All statistical tests were two-sided. RESULTS: Dexamethasone dose dependently decreased GR levels and inhibited the growth of DU145 and PC-3 but not LNCaP cells (DU145 cells, P< .001; PC-3 cells, P = .009). Dexamethasone increased IkappaBalpha protein levels and the cytosolic accumulation of NF-kappaB in DU145 cells and decreased secreted IL-6 levels to 37 pg/mL (95% confidence interval [CI] = 33 pg/mL to 41 pg/mL), compared with 164 pg/mL (95% CI = 162 pg/mL to 166 pg/mL) secreted by ethanol-treated control cells. Dexamethasone inhibited the growth of DU145 xenografts in nude (P = .006) and SCID (P = .026) mice without affecting GR levels. Eight of 16 human prostate cancers expressed GR at high levels (>or=30% GR-positive cells). CONCLUSION: Dexamethasone inhibited the growth of GR-positive cancers, possibly through the disruption of the NF-kappaB-IL-6 pathway.
Assuntos
Androgênios/fisiologia , Dexametasona/farmacologia , Proteínas I-kappa B , Neoplasias da Próstata/patologia , Animais , Western Blotting , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Nus , Modelos Animais , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo , Receptores de Glucocorticoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Transplante Heterólogo/patologia , Células Tumorais CultivadasRESUMO
Loss of imprinting (LOI) has been implicated in the pathogenesis of embryonal malignancies as well as adult cancers. Insulin-like growth factor II (IGF2) gene is an imprinted gene, normally transcribed only from the paternal allele. We investigated allele-specific expression of the IGF2 gene in 22 cases of renal cell carcinoma (RCC), a common adult-onset renal tumor. Sixteen cases (72%) were informative for IGF2 gene expression, and 9 (56%) of these cases showed biallelic expression of the IGF2 gene. Additionally, in four cases with biallelic expression from which uninvolved kidney tissue was available, LOI of the IGF2 gene was also demonstrated in the normal tissue. All cases with LOI of IGF2 were low-grade and low-stage tumors. LOI of the IGF2 gene in RCC was not associated with overexpression of IGF2 mRNA, whereas IGF2 overexpression was frequently observed in high-stage tumors. These results suggest that LOI of IGF2 predisposes to low-grade and low-stage tumors, whereas IGF2 overexpression may have a role in RCC tumor progression.
Assuntos
Carcinoma de Células Renais/genética , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , Neoplasias Renais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Northern Blotting , Carcinoma de Células Renais/metabolismo , Progressão da Doença , Feminino , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismoRESUMO
We recently cloned a novel transcription factor gene, hPSE, which belongs to the Ets gene family. hPSE mRNA was expressed specifically in prostate glandular epithelial cells and also in the human prostate carcinoma cell lines PC-3 and LNCaP. On the other hand, on immunoblot analysis with anti-hPSE antiserum, hPSE protein was detected only in human prostate tissue samples and not in PC-3 or LNCaP culture cells. Immunohistochemistry and in situ hybridization analysis revealed that hPSE protein was translated in normal prostate glandular epithelial cells, but not in carcinoma cells with hPSE transcripts. These findings suggest that expression of hPSE is regulated translationally in prostate epithelial cells and that hPSE protein is a candidate for a marker distinguishing normal cells from cancer cells in the prostate. It appeared that the 5'- and 3'-untranslated regions of hPSE transcripts might be necessary for translational control of hPSE, on the basis of results of transfection analysis in non-prostate lineage cells (HEK-293) using some deletion mutants of hPSE cDNA.
Assuntos
Próstata/metabolismo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transformação Celular Neoplásica/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Masculino , Mutação , Proteínas Proto-Oncogênicas c-ets , TransfecçãoRESUMO
We examined the spectrum of p53 mutations found in 40 UV-induced skin tumors of xeroderma pigmentosum group A gene (XPA)-deficient mice. p53 mutations were detected in 48% of the tumors. Nearly all of the mutations were induced at dipyrimidine sites. Ninety-three % of the mutations were G.C-->A.T transitions at dipyrimidine sites, including tandem transitions (CC-->TT), which are the hallmark of the UVB-induced mutation. Seventy-two % of the mutations at dipyrimidine sites could be ascribed to damage on the transcribed strand. In addition, no evident mutational hot spots were detected. This is in contrast to the UVB-induced skin tumors of normal mice, in which 92% of p53 mutations occurred as a result of DNA damage on the nontranscribed strand, and clear hot spots were observed. Thus, XPA-deficient mice showed significant mutation features that might be characteristic of the absence of nucleotide excision repair and may provide a good animal model for the analysis of the high incidence of skin cancer in xeroderma pigmentosum group A patients.
Assuntos
Genes p53 , Mutação , Neoplasias Induzidas por Radiação/genética , Neoplasias Cutâneas/genética , Raios Ultravioleta , Xeroderma Pigmentoso/genética , Animais , Sequência de Bases , Humanos , Camundongos , Camundongos MutantesRESUMO
Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [3H]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression. tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.
Assuntos
4-Butirolactona/análogos & derivados , Inibidores de Proteínas Quinases , Proteína do Retinoblastoma/metabolismo , 4-Butirolactona/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Histonas/metabolismo , Humanos , Fosforilação , Timidina/metabolismoRESUMO
We have previously reported on cloning of the human gene encoding Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-like protein (Bnip3L) and its growth inhibitory effect on cancer cells. Here we show that Bnip3L contains a motif similar to the BH3 domain which is conserved in Bcl-2 family proteins as well as containing a membrane-anchoring domain, and that Bnip3L interacts with Bcl-2 and Bcl-xL. Immunofluorescence microscopy revealed that Bnip3L was localized in the mitochondria, when in the presence of the membrane-anchoring domain. Transient expression of Bnip3L induced apoptosis of Rat-1 and HeLa cells and mutational analysis revealed that the BH3 domain and the membrane-anchoring domain were required for Bnip3L to induce cell death. Addition of recombinant Bnip3L to isolated mitochondria induced membrane potential loss and cytochrome c release both of which have been suggested to be prerequisite for apoptotic cell death. These results suggest that Bnip3L is one of the BH3-containing pro-apoptotic proteins and that it targets the mitochondria when inducing apoptosis.
Assuntos
Proteínas E1B de Adenovirus/metabolismo , Apoptose , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Bovinos , Linhagem Celular , Células HeLa , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-bcl-2/genética , Coelhos , Ratos , Homologia de Sequência de Aminoácidos , Proteína bcl-XRESUMO
The growth-suppressive activity of the retinoblastoma (RB) protein is suggested to be regulated by phosphorylation. In studies on the kinase that phosphorylates the RB proteins, we have previously found that RB proteins can be phosphorylated by purified cdc2 kinase. In this study, we noted that RB proteins immunoprecipitated from human cell lysates are weakly phosphorylated in the absence of purified cdc2 kinase. Immunoblot analysis showed the presence of p34cdc2 in the immunoprecipitates with anti-RB monoclonal antibody. In addition, the coprecipitated kinase was found to have the same substrate specificity as cdc2 kinase. The associated kinase activity was particularly high in cells arrested in G1/S and S phase by aphidicolin. Furthermore, RB proteins were shown to be phosphorylated in nuclear extracts by some endogenous cdc2-like kinase(s). These results suggest that cdc2-like kinase is the main kinase for phosphorylation of RB proteins in vivo.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteína do Retinoblastoma/metabolismo , Sequência de Aminoácidos , Animais , Afidicolina/farmacologia , Ciclo Celular , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Neoplasias do Colo , Células HeLa , Humanos , Leucemia , Neoplasias Mamárias Experimentais , Camundongos , Dados de Sequência Molecular , Neuroblastoma , Peptídeos/síntese química , Fosforilação , Especificidade por SubstratoRESUMO
Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-E2F-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated pRB but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the G0 and early G1 phase. Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdk2 in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdk2 may modulate its activity.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Baculoviridae/genética , Sequência de Bases , Ciclo Celular , Quinase 2 Dependente de Ciclina , DNA Recombinante , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosforilação , Testes de Precipitina , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Células Tumorais CultivadasRESUMO
It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evident for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk. Therefore, we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays. We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as well as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Proteínas Nucleares/metabolismo , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA/química , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Dados de Sequência Molecular , Fosforilação , Proteína 1 de Ligação ao Retinoblastoma , Proteína p107 Retinoblastoma-Like , Fator de Transcrição DP1RESUMO
We have performed a combined biochemical and immunochemical study on the identity of peptidylarginine deiminases (EC 3.5.3.15) present in various mammalian tissues. First, we purified peptidylarginine deiminase from rat skeletal muscle. It gave a single band of molecular weight 83,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Next we immunized rabbits with the purified enzyme. The resulting antibodies reacted specifically with the antigen in Western blot assay. Most of the enzyme activities present in rat skeletal muscle, brain, spinal cord, submaxillary gland and spleen could be characterized as the same muscle-type enzyme by immunoprecipitation and Western blot assay. The antibodies did not react with enzyme samples obtained from rat hair follicles and bovine epidermis. The lack of immunoreactivity of the epidermal enzyme could not be accounted for by the species difference, since the antibodies reacted with a 83 kDa polypeptide of bovine brain, which was thought to represent a bovine counterpart of the muscle-type enzyme. The epidermal enzyme could be distinguished from the other enzyme samples by its high activity towards benzoylarginine. These data suggest the existence of at least three types of peptidylarginine deiminase in mammalian tissues, i.e., a muscle type, a hair follicle type, and an epidermal type.
Assuntos
Hidrolases/isolamento & purificação , Animais , Especificidade de Anticorpos , Bovinos , Eletroforese em Gel de Poliacrilamida , Epiderme/enzimologia , Feminino , Cabelo/enzimologia , Hidrolases/imunologia , Imunoensaio , Masculino , Músculos/enzimologia , Testes de Precipitina , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Coelhos , RatosRESUMO
The International Society for the Study of the Aging Male (ISSAM) recommends that a diagnosis be based on a patient's total testosterone (TT), calculated free testosterone (cFT), or calculated bioavailable testosterone (cBT) for partial androgen deficiency of the aging male (PADAM). The purpose of this study was to confirm whether hypogonadism of patients with PADAM is related to symptoms and clarify which criteria of testosterone recommended by ISSAM is suitable for Japanese patients. A total of 90 patients with PADAM symptoms were included in this study. Endocrinologic profiles were reviewed as appropriate, and PADAM symptoms were judged by means of several questionnaires. Laboratory values and symptoms were compared between patients with and without hypogonadism. Even when any criterion of testosterone was used for diagnosis of hypogonadism, AMS (total and subscales), IIEF-5, or SDS scores of PADAM symptoms did not differ significantly between patients classified as having and not having hypogonadism. No other endocrinologic variables than testosterone differed significantly between them, either. PADAM symptoms are not related to testosterone level and it is still obscure whether ISSAM's criterion can be adopted for Japanese patients with PADAM. Other pathology needs to be addressed for evaluation and diagnosis of PADAM in Japan.
Assuntos
Envelhecimento , Androgênios/deficiência , Andropausa/fisiologia , Testosterona/sangue , Humanos , Hipogonadismo/sangue , Hipogonadismo/diagnóstico , Hormônio Luteinizante/sangue , Masculino , Pessoa de Meia-Idade , Valores de Referência , Inquéritos e QuestionáriosRESUMO
MMP inhibitors are used clinically for the stabilization of tumor growth, thus prolonging survival in cancer patients. However, their role in the treatment of hematopoietic malignancies remains unclear. In the present study, we investigated the effects of a new MMP inhibitor, SI-27, in hematopoietic malignancies. SI-27 alone induces apoptosis in several human myeloid leukemia cell lines such as U937, NB4, and HL60 cells by activating caspase 8, 9, and 3. Apoptosis was measured with annexin V positive staining, a drop in mitochondrial transmembrane potential (deltapsim), presence of hypodiploid DNA, and cleavage of PARP and IkappaBalpha. Furthermore, at lowered concentrations, which did not directly induce apoptosis, SI-27 acted to sensitize U937 cells and other cells to tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. The accumulation of membrane Fas, the Fas ligand, and TNFR1 were not apparent due to exposure to SI-27, and antagonistic anti-Fas or anti-Fas ligand antibodies did not block SI-27-induced apoptosis. Thus, SI-27-induced apoptosis is not mediated by the Fas pathway. These results suggest that MMP inhibitors, alone or in combination with other cytotoxic agents, can provide a unique method for treating acute myeloid leukemia, refractory to classical anti-cancer drugs, and may thus suppress recurrence.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologia , Metaloendopeptidases/antagonistas & inibidores , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Humanos , Oligopeptídeos/uso terapêutico , Inibidores de Proteases/uso terapêutico , Células Tumorais CultivadasRESUMO
BACKGROUND: Chronic allograft nephropathy (CAN) is the main cause of renal transplant failure in the first decade posttransplant. The precise pathogenetic mechanism for CAN is not completely understood. A possible role of renin-angiotensin system for CAN has been suggested through clinical observations that angiotensin-converting enzyme inhibition and angiotensin II receptor blockers prevent CAN. METHODS: Distribution of renin-positive cells in allograft biopsy specimens was examined immunohistochemically in 23 renal transplant recipients diagnosed with CAN Biopsy specimens obtained from seven recipients with stable renal function were examined as controls. Histologic evaluation was performed based on the Banff 97 classification. RESULTS: Renin-positive cells were found in the juxtaglomerular apparatus (JGA) adjoining the afferent arterioles in both groups. When the number of renin-positive cells in JGA was defined as a renin index, it was significantly higher in the CAN than the control group (P = .007). There was no significant difference in age, interval between transplantation and biopsy, and blood pressure between groups. Only a significantly higher serum creatinine was found in the CAN group. CONCLUSIONS: The increased renin-positive cells in JGA suggest a significant role of the intrarenal renin-angiotensin system activation in the development of CAN.
Assuntos
Transplante de Rim/patologia , Renina/metabolismo , Adulto , Biomarcadores/análise , Doença Crônica , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Imunossupressores/classificação , Imunossupressores/uso terapêutico , Transplante de Rim/fisiologia , Masculino , Proteinúria , Estudos Retrospectivos , Fatores de Tempo , Transplante HomólogoRESUMO
We tested, in cynomolgus monkeys, the safety and effectiveness of a hybrid liposome vector, hemagglutinating virus of Japan (HVJ)--artificial viral envelope (AVE) liposomes, for human therapeutic gene transfer in a series of experiments. In a repetitive intramuscular administration study, vehicle control macaques (n = 2), which were treated with HVJ--AVE liposome suspension, received repetitive intramuscular injections of 2 ml of test substance. Human hepatocyte growth factor (HGF) cDNA-inserted expression vector (pUC-SR alpha/HGF) injection animals (n = 2), which were treated with HVJ--AVE liposome suspension containing pUC-SR alpha/HGF, received repetitive intramuscular injection of 2 ml of test substance. General body condition, hematology, blood chemistry, and serum HGF were determined sequentially before treatment and 7, 21, 28, and 29 days after treatment. Elevations in HGF were detected in monkeys injected with pUC-SR alpha/HGF. After this observation period, macaques were killed for autopsy and histological examination. pUC-SR alpha/HGF was detected by polymerase chain reaction (PCR) analysis in the liver, spleen, and at the injection site. In single intravenous administration study, control macaques (n = 4) received a single intravenous injection of 10 ml of physiological saline. Vehicle control animals (n = 5) received a single intravenous injection of 10 ml of HVJ--AVE liposome suspension. DNA-treated animals (n = 7) received a single intravenous injection of 10 ml of HVJ--AVE liposome suspension containing plasmid DNA [pcDNA 3.1(+)]. General body condition, body weight, hematology, blood chemistry, and urine composition were determined sequentially before treatment and 1, 14, 21, and 28 days after treatment. After this observation period, macaques were killed for autopsy and histological examination. pcDNA 3.1(+) was detected by PCR analysis on day 1 in lung, liver, and spleen of all monkeys, in kidney of one of two monkeys, and in heart of one of two monkeys. However, no DNA was detected in any of the tissues examined on days 14, 21, and 28. No virus genomic RNA was detected by reverse transcription (RT)-PCR analysis with HVJ-specific primers. In this series of safety evaluations, the animals tolerated the safety study with no change in body weight or general condition. No hematological changes or alterations in blood chemistry or urine composition was detected. Moreover, no histological changes were observed. This safety evaluation study demonstrates the safety, feasibility, and therapeutic potential of the novel transfection vehicle, HVJ--AVE liposomes, in humans.
Assuntos
Vetores Genéticos , Lipossomos/metabolismo , Respirovirus/genética , Animais , DNA/metabolismo , DNA Complementar/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Fator de Crescimento de Hepatócito/sangue , Fator de Crescimento de Hepatócito/genética , Humanos , Hipersensibilidade , Fígado/metabolismo , Macaca fascicularis , Masculino , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/metabolismo , Fatores de Tempo , Distribuição Tecidual , TransfecçãoRESUMO
Male juvenile spermatogonial depletion (jsd) mutant mice are sterile because of spermatogenic failure and so may provide a model for genetically caused human male infertility. To test the effects of testosterone suppression therapy on spermatogenesis in jsd/jsd mice, we treated them with Nal-Glu, a GnRH antagonist. Treatment with Nal-Glu at 2500 microg/kg/day was started at 5.5 or 8 weeks of age and continued for 4 or 8 weeks. Differentiation of spermatogonia was evaluated by the percentage of tubules containing two or more spermatocytes (% of differentiating tubules). Nal-Glu treatment caused a marked decrease in the weights of the testes and seminal vesicles and intratesticular testosterone concentrations. However, in contrast to a value of 1.3% in untreated jsd/jsd mice, the mean % of differentiating tubules was 59.9% and 25.1% in treatment groups started at 5.5 and 8 weeks of age, respectively. We propose that spermatogonial differentiation in jsd/jsd mutant mice is sensitive to the high intratesticular levels of testosterone and can only proceed when testosterone production is suppressed.
Assuntos
Dipeptídeos/farmacologia , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Antagonistas de Hormônios/farmacologia , Mutação/fisiologia , Espermatogônias/citologia , Espermatogônias/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Masculino , Camundongos , Camundongos Mutantes , Tamanho do Órgão/efeitos dos fármacos , Glândulas Seminais/anatomia & histologia , Túbulos Seminíferos/efeitos dos fármacos , Testículo/anatomia & histologia , Testículo/metabolismo , Testosterona/antagonistas & inibidoresRESUMO
The Fas system-based rejection mechanism has not been studied well in terms of cytotoxic T cell activity in graft rejection. We investigated the Fas and Fas ligand level in renal grafts with acute and chronic rejection in a rat model using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Fas ligand in renal allografts was detected as early as 1 day after transplantation in an acute rejection model. It was highly expressed at day 4 and began to decline at day 6 after transplantation. In contrast, Fas ligand in normal kidneys was almost undetectable. Fas ligand in isografts was increased, but the expression level was much lower than in allografts. Interestingly, when Fas ligand expression began to decline in renal allografts, it increased in the spleens of recipients. Fas ligand expression in chronically rejecting allografts was slightly increased, but it was stronger than in isografts. In contrast to Fas ligand gene expression, Fas was constitutively expressed in isografts, allografts, and normal kidneys. However, the Fas level in renal allografts was higher than in normal kidneys. Our data demonstrated that the Fas system might play an important role in acute and chronic rejection by causing apoptosis, and the spleen may eliminate the lymphocytes strongly expressing Fas ligand after completion of the acute rejection.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Rim/imunologia , Glicoproteínas de Membrana/biossíntese , Receptor fas/biossíntese , Doença Aguda , Animais , Antígenos de Superfície , Doença Crônica , Proteína Ligante Fas , Rejeição de Enxerto/metabolismo , Rim/imunologia , Rim/metabolismo , Ligantes , Masculino , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , RNA Mensageiro/química , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Baço/imunologia , Baço/metabolismo , Receptor fas/imunologia , Receptor fas/metabolismoRESUMO
Hyperlipidemia is frequently developed following renal transplantation and results in worsening of the patient's prognosis. In study 1, the effects of immunosuppressants, cyclosporine (CsA) and tacrolimus on serum lipids were compared in-patients undergoing renal transplantation. The study included 32 cases of renal transplantation recipients who randomized to the CsA treatment group (15 patients) and the tacrolimus group (17 patients). Before and 1 month after the transplantation, we assessed the serum lipid levels, apolipoprotein levels, the concentrations of cholesterol in the respective lipoprotein fractions and the enzyme activities related to lipid-metabolism. The serum lipid levels in both groups were significantly increased at 1 month after renal transplantation. In the CsA group, there were significant increases in cholesterol contents in very-low-density lipoprotein (VLDL), LDL2 and HDL2 fractions, whereas, in the tacrolimus group, cholesterol content was increased in VLDL and HDL2 fractions. In study 2, 1 month after renal transplantation, 19 patients with hypercholesterolemia (total cholesterol (TC) >200 mg/dl) and hypertriglyceridemia (triglyceride (TG) >150 mg/dl) were treated with simvastatin 5-10 mg/day for 6 months. Simvastatin treatment significantly decreased serum TC (240+/-29-200+/-22 mg/dl, P<0.001), low-density lipoprotein cholesterol (LDL-C; 114+/-20-99+/-17 mg/dl, P<0.05) and TG levels (217+/-103-130+/-38 mg/dl, P<0.01). In addition, there were significant decreases in very-low-density lipoprotein cholesterol (VLDL-C; 53+/-20-34+/-15 mg/dl, P<0.001). The Cmax and AUC of simvastatin were increased about eight-fold, when simvastatin was given in combination with CsA. In contrast, no significant changes in simvastatin levels were observed when combination with tacrolimus. Although simvastatin levels were increased with CsA, there were no abnormal changes in renal and liver functions, creatinine phosphokinase (CPK) levels or in incidence of adverse effects.
Assuntos
Ciclosporina/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hiperlipidemias/tratamento farmacológico , Hipolipemiantes/uso terapêutico , Imunossupressores/uso terapêutico , Transplante de Rim/efeitos adversos , Lipídeos/sangue , Sinvastatina/uso terapêutico , Tacrolimo/uso terapêutico , Adulto , Apolipoproteínas/sangue , Colesterol/sangue , Feminino , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/etiologia , Lipoproteínas/sangue , Masculino , Estudos ProspectivosRESUMO
In the joint experimental and computational efforts reported here to obtain novel chemical entities as growth hormone secretagogues (GHSs), a small database of peptides and non-peptides known to have GHS activity was used to generate and assess a 3D pharmacophore for this activity. This pharmacophore was obtained using a systematic and efficient procedure, "DistComp", developed in our laboratory. The 3D pharmacophore identified was then used to search 3D databases to explore chemical structures that could be novel GHSs. A number of these were chosen for synthesis and assessment of their ability to release growth hormone (GH) from rat pituitary cells. Among the compounds tested, those with a benzothiazepin scaffold were discovered with micromolar activity. To facilitate lead optimization, a second program, a site-dependent fragment QSAR procedure was developed. This program calculates a library of chemical and physical properties of "fragments" or chemical components in a known pharmacophore and determines which, if any, of these properties are important for the observed activity. The combined use of the 3D pharmacophore and the results of the site-dependent fragment QSAR analysis led to the discovery and synthesis of a novel series of potent GHSs, a number of which had nanomolar in vitro activity.