RESUMEN

AIMS/HYPOTHESIS: Several lines of evidence suggest that incretin-based therapies suppress the development of cardiovascular disease in type 2 diabetes. We investigated the possibility that glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) can prevent the development of atherosclerosis in Apoe (-/-) mice. METHODS: Apoe (-/-) mice (17 weeks old) were administered GLP-1(7-36)amide, GLP-1(9-36)amide, GIP(1-42) or GIP(3-42) for 4 weeks. Aortic atherosclerosis, oxidised LDL-induced foam cell formation and related gene expression in exudate peritoneal macrophages were determined. RESULTS: Administration of GLP-1(7-36)amide or GIP(1-42) significantly suppressed atherosclerotic lesions and macrophage infiltration in the aortic wall, compared with vehicle controls. These effects were cancelled by co-infusion with specific antagonists for GLP-1 and GIP receptors, namely exendin(9-39) or Pro(3)(GIP). The anti-atherosclerotic effects of GLP-1(7-36)amide and GIP(1-42) were associated with significant decreases in foam cell formation and downregulation of CD36 and acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) in macrophages. GLP-1 and GIP receptors were both detected in Apoe (-/-) mouse macrophages. Ex vivo incubation of macrophages with GLP-1(7-36)amide or GIP(1-42) for 48 h significantly suppressed foam cell formation. This effect was wholly abolished in macrophages pretreated with exendin(9-39) or (Pro(3))GIP, or with an adenylate cyclase inhibitor, MDL12,330A, and was mimicked by incubation with an adenylate cyclase activator, forskolin. The inactive forms, GLP-1(9-36)amide and GIP(3-42), had no effects on atherosclerosis and macrophage foam cell formation. CONCLUSIONS/INTERPRETATION: Our study is the first to demonstrate that active forms of GLP-1 and GIP exert anti-atherogenic effects by suppressing macrophage foam cell formation via their own receptors, followed by cAMP activation. Molecular mechanisms underlying these effects are associated with the downregulation of CD36 and ACAT-1 by incretins.

Asunto(s)

Apolipoproteínas E/metabolismo , Aterosclerosis/tratamiento farmacológico , Incretinas/farmacología , Acetil-CoA C-Acetiltransferasa/metabolismo , Animales , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Western Blotting , Antígenos CD36/metabolismo , Línea Celular , Células Cultivadas , Células Espumosas/citología , Células Espumosas/efectos de los fármacos , Polipéptido Inhibidor Gástrico/farmacología , Péptido 1 Similar al Glucagón/análogos & derivados , Péptido 1 Similar al Glucagón/farmacología , Humanos , Masculino , Ratones , Ratones Noqueados , Microscopía Confocal , Fragmentos de Péptidos/farmacología , Péptidos/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

AIM: The aim of this study was to investigate the effects of ezetimibe on high-density lipoprotein (HDL) subspecies and serum amyloid A (SAA), an apolipoprotein mainly bound and transported by HDL particles, in patients with end-stage renal disease (ERSD), a condition typically characterized by high SAA- and low HDL-cholesterol (C ) levels. METHODS: 26 ERSD patients receiving hemodialysis (HD) were given ezetimibe (10 mg/d) for 6 - 8 weeks. HDL3 was separated from serum by a single precipitation method established by our group. HDL2 was estimated by subtracting HDL3 from total HDL. Serum amyloid A (SAA) was measured by the ELISA method. RESULTS: Ezetimibe significantly reduced remnant-like particle (RLP)-C, low-density lipoprotein (LDL)-C, and apolipoprotein (apo) B without affecting triglyceride, HDL-C and LCAT activities. HDL2-C levels were lower and HDL3-C was substantially lower in the HD patients than in the controls. Ezetimibe increased HDL2-apoAI but decreased HDL3-apoAI without affecting serum apoAI or AII. HDL-SAA was 5-fold higher in the HD patients than in the controls (56 ± 49 vs. 12 ± 9 µg/ml). Ezetimibe decreased HDL-SAA by 43 % (to 32 ± 36 µg/ml), and this inhibitory effect was exclusively attributable to a 72% reduction in HDL3-SAA in response to the ezetimibe treatment. The reduction of HDL3-SAA was significantly associated with increased HDL2-apo AI and reduced HDL3-apo AI. CONCLUSIONS: Ezetimibe treatment decreased "inflammatory" (SAA-containing) HDL3, and may thus have restored the anti-atherogenic function of HDL particles in ESRD patients.

Asunto(s)

Anticolesterolemiantes/farmacología , Azetidinas/farmacología , Lipoproteínas HDL3/análisis , Diálisis Renal , Proteína Amiloide A Sérica/análisis , Adulto , Anciano , Anciano de 80 o más Años , Apolipoproteína A-I/sangre , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Ezetimiba , Femenino , Humanos , Masculino , Persona de Mediana Edad

RESUMEN

OBJECTIVES: To test the hypothesis that CX3CL1 contributes to the pathogenesis of microscopic polyangiitis. METHODS: Serum samples from 18 patients with microscopic polyangiitis (MPA), who fulfilled the revised criteria of the American College of Rheumatology (ACR), were collected during both the newly diagnosed, untreated active disease states and inactive disease states. Also serum was from patients with large vessel vasculitis (LVV), including giant cell arteritis (n=4) and Takayasu arteritis (n=3), and from 52 healthy individuals. Soluble (s)CX3CL1 levels in serum were measured using an enzyme-linked immunosorbent assay. Disease activity was assessed using Birmingham vasculitis activity scores (BVAS). Expression of CX3CR1 was examined by flow cytometry. RESULTS: Serum sCX3CL1 levels were significantly higher in MPA patients than in either LVV group or healthy individuals. The elevated sCX3CL1 levels seen in MPA patients correlated positively with BVAS, as well as with CRP levels and ESR, and similarly increased expression of cell-surface CX3CR1 was seen on peripheral blood CD4 and CD8 T cells from patients with MPA. Notably, sCX3CL1 levels and CX3CR1 expression were diminished during clinical remission following treatment. CONCLUSION: Our findings suggest that CX3CL1 may be involved in the pathogenesis of MPA, and may serve as a useful serologic marker of disease activity in systemic vasculitis.

Asunto(s)

Quimiocina CX3CL1/sangre , Vasculitis/sangre , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Estudios de Casos y Controles , Quimiocina CX3CL1/metabolismo , Estudios de Cohortes , Citometría de Flujo , Arteritis de Células Gigantes/sangre , Humanos , Masculino , Microvasos/metabolismo , Persona de Mediana Edad , Arteritis de Takayasu/sangre , Vasculitis/inmunología

RESUMEN

To investigate the nature of recent HIV outbreaks among injecting drug users (IDUs) near the Vietnam-China border, we genetically analyzed 24 HIV-positive blood specimens from 2 northern provinces of Vietnam (Lang Son and quang Ninh) adjacent to the China border, where HIV outbreaks among IDUs were first detected in late 1996. Genetic subtyping based on gag (p17) and env (C2/V3) sequences revealed that CRF01_AE is a principal strain circulating throughout Vietnam, including the provinces near the China border. The majority of CRF01_AE sequences among IDUs in Quang Ninh and Lang Son showed significant clustering with those found in nearby Pingxiang City of China's Guangxi Province, sharing a unique valine substitution 12 amino acids downstream of the V3 loop. This particular subtype E variant, uniquely found among IDUs in northern Vietnam and southeastern China, is designated E(v). The genetic diversity of CRF01_AE distributed in Quang Ninh (1.5 +/- 0.6%) and Pingxiang City (1.9 +/- 1.2%) was remarkably low, indicating the emerging nature of HIV spread in these areas. It is also noted that the genetic diversity of CRF01_AE among IDUs was consistently lower than that in persons infected sexually, suggesting that fewer closely related CRF01_AE variants were introduced into IDUs and, conversely, that multiple strains of CRF01_AE had been introduced via the sexual route. The data in the present study provide additional evidence that HIV outbreaks among IDUs in northern Vietnam were caused by the recent introduction of a highly homogeneous CRF01_AE variant (E(v)) closely related to that prevailing in nearby southern China.

Asunto(s)

Brotes de Enfermedades , Genoma Viral , Infecciones por VIH/virología , VIH-1/genética , Abuso de Sustancias por Vía Intravenosa/virología , Adolescente , Adulto , Secuencia de Aminoácidos , Sustitución de Aminoácidos , China/epidemiología , Clonación Molecular , Femenino , Genes env/genética , Genes gag/genética , Infecciones por VIH/epidemiología , VIH-1/clasificación , Humanos , Masculino , Datos de Secuencia Molecular , Factores de Riesgo , Conducta Sexual , Valina/genética , Vietnam/epidemiología

RESUMEN

We have previously shown that HIV-1 env subtypes B' (a Thai-B cluster within subtype B) and E (CRF01_AE) are distributed in Yangon, the capital city of Myanmar. However, HIV strains from the rest of country have not yet been genetically characterized. In the present study, we determined env (C2/V3) and gag (p17) subtypes of 25 specimens from central Myanmar (Mandalay). Phylogenetic analyses identified 5 subtype C (20%), in addition to 10 CRF01_AE (40%) and 4 subtype B' (16%). Interestingly, the remaining six specimens (24%) showed discordance between gag and env subtypes; three gag subtype B'/env subtype C, one gag subtype B'/env subtype E, one gag subtype C/env subtype B', and one gag subtype C/env subtype E. These discordant specimens were found frequently among injecting drug users (4 of 12, 33%) and female commercial sex workers (2 of 8, 25%) engaging in high-risk behaviors. The recombinant nature of these HIV-1 strains was verified in three specimens, indicating the presence of new forms of HIV-1 intersubtype C/B' and C/B'/E recombinants with different recombination breakpoints. The data suggest that multiple subtypes of B', C, and CRF01_AE are cocirculating in central Myanmar, leading to the evolution of new forms of intersubtype recombinants among the risk populations exhibiting one of the highest HIV infection rates in the region.

Asunto(s)

Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/genética , Recombinación Genética , Proteínas Virales , Adolescente , Adulto , Secuencia de Aminoácidos , Femenino , Productos del Gen gag/química , Productos del Gen gag/genética , Antígenos VIH/química , Antígenos VIH/genética , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Masculino , Datos de Secuencia Molecular , Mianmar/epidemiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Filogenia , Análisis de Secuencia de ADN , Productos del Gen gag del Virus de la Inmunodeficiencia Humana

RESUMEN

To investigate the molecular epidemiology of a recent HIV-1 outbreak in northern Vietnam and its relation to the epidemic in surrounding areas, we analyzed 17 HIV-positive blood specimens from 3 heterosexuals, 2 sexually transmitted disease patients, and 12 injecting drug users (IDUs), collected in 4 provinces near Hanoi in 1998. These were compared with the specimens from Ho Chi Minh City (n = 10) and An Giang Province (n = 10) in southern Vietnam and with published sequences from neighboring countries. Genetic subtyping based on the env C2/V3 sequences revealed that HIV-1 subtype E predominated throughout Vietnam in all risk populations; the exception was one typical United States-European-type HIV-1 subtype B detected in a patient in Ho Chi Minh City, the first case of HIV infection identified in Vietnam in 1990. The HIV-1 subtype E sequences identified in 9 of the 12 IDUs from northern provinces were closely related phylogenetically to those in IDUs in nearby Guangxi Province of China, and also shared a common amino acid signature downstream of the env V3 loop region. The low interperson nucleotide diversity among IDUs in northern Vietnam supports the view that HIV-1 subtype E was introduced recently among IDUs in northern Vietnam. These data indicate a linkage between HIV-1 circulating among IDUs in northern Vietnam and southern China, and suggest recent transborder introductions as the likely source of HIV-1 subtype E in northern Vietnam.

Asunto(s)

Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/genética , Abuso de Sustancias por Vía Intravenosa/virología , Adolescente , Adulto , Secuencia de Aminoácidos , China/epidemiología , Brotes de Enfermedades , Femenino , Genes gag/genética , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/complicaciones , VIH-1/clasificación , Infecciones por HTLV-II/virología , Humanos , Técnicas para Inmunoenzimas , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Prevalencia , Factores de Riesgo , Análisis de Secuencia de ADN , Abuso de Sustancias por Vía Intravenosa/complicaciones , Vietnam/epidemiología

RESUMEN

We previously described a Sendai virus (SeV)-based expression system for the recombinant gp120 of HIV-1 subtype B (rgp120-B), which has permitted the production of antigenetically and functionally authentic gp120 at a concentration as high as 6 microg/ml of culture supernatant (Yu D et al.: Genes Cells 1997;2:457-466). Here the same procedure was successfully applied to the production of HIV-1 subtype E gp120 (rgp120-E). The remarkable production of the proteins by the SeV expression system enabled us to use crude culture supernatants for serological and functional studies of gp120s. The immunological authenticity of rgp120-E was verified by patient sera and anti-V3 loop monoclonal antibodies specific for HIV-1 subtypes B and E. CD4-binding properties were corroborated by FACS analyses. The rgp120s were then used in an enzyme immunoassay (rgp120-EIA) to detect antibodies in the sera of HIV-1-infected individuals, and the performance was assessed in comparison with a conventional V3 loop peptide EIA (V3-EIA). The initial evaluation of a serum panel (n = 164) consisting of 76 subtype E and 88 subtype B sera revealed that the rgp120-EIA was nearly 1000-fold more sensitive than the V3-EIA and was able to detect subtype-specific antibody with 100% sensitivity and with a complete correlation with the genotypes, whereas the V3-EIA failed to detect 9 and 24% of the same subtype E and B sera, respectively. Furthermore, a study employing a panel of 28 international sera with known genotypes (HIV-1 subtypes A through F) confirmed the remarkable specificity of this method. An EIA reactivity higher than 1.0 was an unambiguous predictor of HIV-1 subtype E and B infections. The data imply the presence of strong subtype-specific epitopes for antibody bindings to these rgp120s.

Asunto(s)

Anticuerpos Antivirales/sangre , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/inmunología , Respirovirus/genética , Secuencia de Bases , Western Blotting , Clonación Molecular , Cartilla de ADN , Citometría de Flujo , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/diagnóstico , Infecciones por VIH/inmunología , Humanos , Técnicas para Inmunoenzimas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad

Asunto(s)

Genes env/genética , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/genética , Adulto , Secuencia de Aminoácidos , Cambodia , ADN Viral/análisis , Femenino , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN

RESUMEN

To study the molecular epidemiology of HIV-1 spread in Myanmar and the interplay with the epidemic in surrounding Southeast Asian countries, we determined the HIV-1 subtypes prevailing in Myanmar. Thirty HIV-positive blood specimens were sampled in the capital city, Yangon, and an additional 459 sera were collected nationwide in 1995. Genetic subtyping based on the env C2/V3 sequence and serologic data, using a V3 peptide enzyme immunoassay (PEIA), revealed three patterns of HIV spread in different geographic regions in Myanmar: (1) in the capital city, Yangon, HIV-1 subtype B' ("Thai-B" cluster within subtype B) predominated both in IDUs and heterosexuals; (2) in the cities near the border with Thailand, including Tachelaik and Kawthaung, where heterosexual transmission is a major pathway of HIV-1 spread, HIV-1 subtype E was predominantly distributed among the commercial sex workers and heterosexuals; (3) in central and northeast Myanmar, both HIV-1 subtypes B' and E occurred in a mixed distribution, without showing any significant segregation by risk group. In addition, the PEIA data implied the occurrence of other subtype(s) in these areas. The interperson nucleotide sequence variations in env C2/V3 regions of B' and E, prevailing in Yangon, were 6.7 +/- 2.1 and 7.1 +/- 0.7%, respectively. They were similar to those levels observed in Thailand. These findings are consistent with the view that HIV spread in Myanmar might have taken place at about the same time as that in Thailand, and that multiple entries and exchanges of HIV-1 with neighboring countries are important factors contributing to the current distribution of subtypes in Myanmar.

Asunto(s)

VIH-1/clasificación , Secuencia de Aminoácidos , ADN Viral , Femenino , Heterogeneidad Genética , Infecciones por VIH/epidemiología , Seropositividad para VIH/virología , VIH-1/genética , Humanos , Masculino , Epidemiología Molecular , Datos de Secuencia Molecular , Mianmar/epidemiología , Alineación de Secuencia , Análisis de Secuencia de ADN , Serotipificación , Tailandia/epidemiología

RESUMEN

A 60-year-old man with portal hypertensive gastropathy due to type C liver cirrhosis developed severe bone pains, marked hypophosphatemia with inappropriately increased urinary excretion of phosphate (%TRP; 9.6%), and hyperalkaline phosphatasia, after intravenous administration of saccharated ferric oxide (SFO) at a dose of 80-240 mg/week over a period of more than 5 years. The total iron infused was estimated to be more than 25 g. On a diagnosis of SFO-induced osteomalacia, the infusion of iron was immediately discontinued, and phosphate and vitamin D2 (1000 IU/day) were administered. Serum levels of 25-OHD2 increased after 1 week, whereas levels of 1,25-(OH)2D2 did not increase until 3 months later, accompanied by improvement of renal tubular reabsorption of phosphate and gradual improvement of the bone pains. The patient has been doing well for the last 2 years, with normal serum levels of phosphate, calcium, and alkaline phosphatase, without any supplementation of phosphate, vitamin D, or iron-containing agents. In primary culture of neonatal mouse renal tubules, in which 1,25-(OH)2D3 was produced from 25-OHD3 in response to PTH, SFO significantly inhibited PTH-induced production of 1,25-(OH)2D3 at 30 mumol/L, which is attainable in the urine of patients receiving a therapeutic intravenous dose of SFO. Furthermore, SFO decreased the calcium content and inhibited 45Ca incorporation in cultured fetal mouse parietal bones at 3 mumol/L. Such SFO concentration may be transiently observed in the plasma of patients receiving excessive intravenous doses of SFO for a prolonged period. These in vitro findings together with the clinical observations suggest that SFO, after filtration through the glomerulus and reabsorption in the proximal renal tubules, impaired proximal renal tubular function, such as tubular reabsorption of phosphate and 1 alpha-hydroxylase activity, leading to hypophosphatemic osteomalacia. Furthermore, it is highly likely that SFO in the peripheral blood, when transferrin is saturated with iron, may impair bone formation and aggravate osteomalacia. Although SFO-induced osteomalacia is reversible simply by discontinuation of the agent, excessive and prolonged administration of SFO should be avoided.

Asunto(s)

Desarrollo Óseo/efectos de los fármacos , Ergocalciferoles/sangre , Compuestos Férricos/efectos adversos , Túbulos Renales/efectos de los fármacos , Osteomalacia/inducido químicamente , Fosfatasa Alcalina/sangre , Animales , Calcitriol/metabolismo , Calcio/sangre , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Ergocalciferoles/administración & dosificación , Ergocalciferoles/farmacología , Ergocalciferoles/uso terapéutico , Compuestos Férricos/administración & dosificación , Compuestos Férricos/química , Hemorragia Gastrointestinal/tratamiento farmacológico , Hemorragia Gastrointestinal/etiología , Humanos , Hipertensión Portal/complicaciones , Hipertensión Portal/metabolismo , Hipertensión Portal/fisiopatología , Hipofosfatemia/inducido químicamente , Hipofosfatemia/patología , Inyecciones Intravenosas , Túbulos Renales/citología , Túbulos Renales/metabolismo , Cirrosis Hepática/complicaciones , Cirrosis Hepática/fisiopatología , Masculino , Ratones , Persona de Mediana Edad , Osteomalacia/diagnóstico , Osteomalacia/tratamiento farmacológico , Dolor/tratamiento farmacológico , Hormona Paratiroidea/farmacología , Hueso Parietal/citología , Hueso Parietal/embriología , Hueso Parietal/metabolismo , Fosfatos/administración & dosificación , Fosfatos/sangre , Fosfatos/uso terapéutico , Fosfatos/orina

RESUMEN

Using a bone organ culture system that shows mineralization in vitro, we investigated whether 17 beta-estradiol dose-dependently increases calcium content in cultured calvarial bones in serum-free medium. Fetal mouse parietal bones (3 x 3 mm) were cultured in phenol red-free BGJ medium containing phosphate (3-4 mmol/L), calcium (1-1.25 mmol/L), insulin (6 micrograms/ML), and transferrin (6 micrograms/mL) for 4-5 days. Under these culture conditions, the calcium content of the cultured bones (at dissection 34.0 +/- 4.6 micrograms/bone [mean +/- SD], n = 50) increased by 15-20 micrograms during 4-5 days of culture. 17 beta-Estradiol increased the calcium content significantly at 10(-12) to 10(-11) mol/L, but not at lower (10(113) mol/L) or higher (10(-10) to 10(-9) mol/L) concentrations. 17 alpha-Estradiol had no effect. The stimulatory effect of 17 beta-estradiol was completely inhibited by the antiestrogen agent ICI-182,780. The anabolic effect of 17 beta-estradiol was elicited not only in bones from females but also in those from males. 17 beta-Estradiol had no significant effect on 45Ca release from prelabeled parietal bones. Furthermore, light- and electron-microscopic examinations revealed that bone mineralization proceeded through formation of matrix vesicles, without any metastatic or dystrophic calcification. These in vitro findings suggest that 17 beta-estradiol elicits small, but reproducible, direct effects on calcium content in the parietal bones not only in female but also in male fetal mice at physiological-free E2 concentrations (10(-12)-10(-11) mol/L), which is attainable in serum of normal human subjects. In contrast to in vivo studies, pharmacological doses of 17 beta-estradiol had no anabolic effect on parietal bones. The mechanism of such a biphasic effect of estrogens remains to be elucidated.

Asunto(s)

Calcio/metabolismo , Estradiol/farmacología , Hueso Parietal/efectos de los fármacos , Análisis de Varianza , Animales , Calcificación Fisiológica/efectos de los fármacos , Medio de Cultivo Libre de Suero , Estradiol/fisiología , Femenino , Feto/efectos de los fármacos , Feto/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Técnicas de Cultivo de Órganos , Hueso Parietal/embriología , Hueso Parietal/metabolismo , Fosfatos/farmacología

RESUMEN

Vascular endothelial growth factor (VEGF), a secreted endothelial cell-specific mitogen, is produced in endocrine organs and regulated by trophic hormones. Because angiogenesis and osteogenesis are closely regulated, we studied whether human osteoblast-like cells produce VEGF, and if so, what factors regulate VEGF mRNA expression. Human osteoblast-like cells (HObLC) derived from trabecular bone explants were cultured in alpha-MEM supplemented with 10% fetal calf serum. Northern blot analysis revealed that HObLC expressed VEGF mRNA, as did several human osteosarcoma cells. 1,25-(OH)2D3 increased the steady-state levels of VEGF mRNA in a time- and concentration-dependent manner in HObLC and one of the osteosarcoma cell lines, SaOS-2, accompanied by an increase in the concentration of immunoreactive VEGF in the conditioned medium. PTH and IGF-I also increased the level of VEGF mRNA in HObLC and SaOS-2 cells. Furthermore, 12-O-tetradecanoylphorbol ester stimulated VEGF mRNA in a time-and concentration-dependent manner. The VEGF mRNA expression induced by 1,25-(OH)2D3 was completely inhibited by H-7, but only partially by staurosporine. We have demonstrated that PTH, IGF-I, and most potently 1,25-(OH)2D3 stimulate the mRNA expression and secretion of VEGF in human osteoblast-like cells, suggesting that one of the anabolic effects of 1,25-(OH)2D3 on skeletal tissue may be mediated by VEGF produced by osteoblasts.

Asunto(s)

Calcitriol/farmacología , Factores de Crecimiento Endotelial/genética , Linfocinas/genética , Osteoblastos/efectos de los fármacos , ARN Mensajero/metabolismo , Autorradiografía , Northern Blotting , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Factores de Crecimiento Endotelial/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Linfocinas/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteosarcoma/genética , Osteosarcoma/patología , Hormona Paratiroidea/farmacología , ARN Mensajero/genética , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Interleukin-4 (IL-4) potently inhibits bone resorption by preventing the differentiation of osteoclast precursors to osteoclasts. To elucidate the role of IL-4 in bone formation, we studied the effects of human IL-4 on human osteoblast-like cells obtained from trabecular bone, which showed increased osteocalcin production in response to 1,25-(OH)2D3 in more than 10 passages. IL-4 stimulated the proliferation of osteoblast-like cells in a concentration-dependent manner, showing the minimal and maximal stimulatory effects at 10 pg/ml and 100-1000 pg/ml, respectively. IL-4 also stimulated the expression of alkaline phosphatase mRNA (1.7-fold) and the enzyme activity to the same extent at 10-100 pg/ml. Furthermore, IL-4 stimulated collagen type I mRNA expression in human osteoblast-like cells. The cytokine did not affect osteocalcin production in a short culture period (3 days). These in vitro findings suggest that IL-4, a bone-resorption-inhibitory cytokine produced by activated T cells in bone marrow, may exert an anabolic effect on osteoblast-like cells in trabecular bone through a paracrine mechanism.

Asunto(s)

Fosfatasa Alcalina/metabolismo , Colágeno/metabolismo , Interleucina-4/farmacología , Osteoblastos/efectos de los fármacos , Adulto , Anciano , Fosfatasa Alcalina/genética , Northern Blotting , Desarrollo Óseo/efectos de los fármacos , Calcitriol/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Colágeno/genética , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Hibridación de Ácido Nucleico , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes/farmacología

RESUMEN

Malignancy-associated hypercalcemia is mainly caused by excessive production of parathyroid hormone-related protein (PTHrP) by the tumor. Using anti-PTHrP-(1-34) monoclonal murine antibody (anti-PTHrP MoAb), we studied whether repeated injection of the homologous antibody would continuously decrease the serum calcium concentration in hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors, leading to prolongation of their survival time. Daily SC injections of anti-PTHrP MoAb decreased the serum calcium concentration almost to within the normal range in nude mice bearing transplanted human PTHrP-producing tumors (T3M-1, EC-GI, PC-3, and FA-6) but not in a nude mouse bearing a transplanted parathyroid carcinoma. The antibody did not affect FA-6 tumor growth either in vitro or in vivo. Pancreatic carcinoma cells (FA-6), which caused the most severe hypercalcemia, were inoculated into 6-week-old nude mice. When severe hypercalcemia (approximately 19 mg/dl) had developed, daily SC injection of anti-PTHrP MoAb was started. Within 18 days of this time point, all untreated tumor-bearing mice (n = 10) died of hypercalcemia and cachexia, whereas all the treated mice (n = 10) showed an increase in body weight and survived for at least 25 days. Histologic examination of the treated mice revealed a marked decrease in osteoclastic bone resorption, without toxicologic findings in the kidney and liver. These results suggest that passive immunization against PTHrP can continuously ameliorate the hypercalcemia and markedly prolong the survival time of severely hypercalcemic, tumor-bearing mice. If a human monoclonal antibody against PTHrP-(1-34) could be developed, then passive immunization would be potentially one of the most effective therapies for patients with malignancy-associated hypercalcemia due to excessive production of PTHrP.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Calcio/sangre , Hipercalcemia/terapia , Inmunización Pasiva , Proteína Relacionada con la Hormona Paratiroidea , Neoplasias de las Paratiroides/metabolismo , Fragmentos de Péptidos/biosíntesis , Biosíntesis de Proteínas , Animales , Calcio/orina , División Celular/efectos de los fármacos , Humanos , Hipercalcemia/etiología , Interleucina-1/biosíntesis , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Hormona Paratiroidea/biosíntesis , Hormona Paratiroidea/orina , Neoplasias de las Paratiroides/patología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/orina , Proteínas/inmunología , Teriparatido , Células Tumorales Cultivadas