Adolfo Díez

Effect of alendronate on cultured normal human osteoblasts

Alendronate is an aminobisphosphonate with a potent anti-reabsorptive action that does not appear to interfere with bone mineralization, and is even able to increase bone mineral density in osteoporotic postmenopausal women through a still not fully understood mechanism. This study was conducted to assess the direct effect of alendronate on diverse aspects of normal human osteoblast physiology. For that purpose, the in vitro effect of a wide range of concentrations [from 10(-1) to 10(-12) mol/L] of alendronate on cell viability, proliferation, collagen synthesis, and the mineral-depositing capacity of normal human osteoblasts was tested. Alendronate effects were examined at 48 and 96 h of culture in the presence or absence of fetal calf serum. In vitro alendronate affected osteoblast viability at concentrations equal to or higher than 10(-4) mol/L. At concentrations equal to or higher than 10(-3) mol/L, no viable cells were observed in cultures. In vitro alendronate at concentrations between 10(-5) and 10(-12) mol/L did not have any effect on the proliferative capacity of normal human osteoblasts determined by two different techniques: (1) tritiated thymidine incorporation to DNA and (2) cell counting. Collagen synthesis by normal human osteoblasts showed a tendency to decrease following incubation with alendronate supplemented with fetal calf serum. This decrease was only statistically significant after 96 h of culture; however, a dose-response effect could not be documented. Finally, no effect of alendronate was observed on calcium deposition in vitro by normal human osteoblasts at concentrations equal to or lower than 10(-5) mol/L. In conclusion, the present study shows that alendronate in vitro does not affect viability, proliferation, and mineral deposit capacity of normal human osteoblasts at the concentration at which it inhibits by 50% the resorptive capacity of osteoclasts that for this drug has been reported as 2 x 10(-9) mol/L.

Virologic Outcome and Predictors of Virologic Failure of Highly Active Antiretroviral Therapy Containing Protease Inhibitors

In this observational single-center cohort study outside the clinical trial setting, outcome and predictors of virologic failure of highly active antiretroviral therapy (HAART) containing a protease inhibitor were evaluated in human immunodeficiency (HIV)-infected persons. The study population consisted of 807 protease inhibitor-naive HIV-seropositive patients who initiated antiretroviral therapy with reverse transcriptase inhibitors and protease inhibitors (indinavir, nelfinavir, ritonavir) between January 1997 and January 1999. Demographic variable, plasma HIV-1 RNA levels, CD4+ T-cell count, adverse drug reactions, and adherence to HAART were assessed. Virologic treatment response was defined as a decrease in plasma HIV-1 RNA load from baseline to below 500 copies per milliliter after 12 months of therapy. Levels of HIV-1 RNA were undetectable in 43% of patients at 12 months. Factors associated with failure to suppress viral load included age 40 years or younger, baseline CD4+ T cell count less than 200 x 10(6) per liter baseline viral load greater than 4.3 log(10) per milliliter, and non-adherence to HAART. After adjustment by logistic regression, non-adherence was the only statistically significant variable associated with virologic failure (odds ratio 0.38, 95% confidence interval 0.21 to 0.67). Unselected patients in whom protease inhibitor is started in a usual clinical setting achieve viral suppression less frequently than do patients in controlled clinical trials. Failure to adherence to HAART was the strongest predictor of virologic failure.

Epidemiology of fractures of the proximal femur associated with osteoporosis in Barcelona, Spain.

SummaryWe studied the epidemiology of osteoporotic fractures of the proximal femur (cervical and trochanteric fractures) in residents of Barcelona, aged 45 years or over, for a 1-year period. Fractures resulting from metastases were not included. During the period of observation, a total of 1,358 patients with hip fractures were treated at acute-care hospitals in Barcelona. Of those, 1,163 were researched. For each case, age, sex, and home address were registered. The incidence of hip fractures per 100,000 inhabitants aged 45 years or over was 115.6 in men and 252.2 in women. Age was the most influencing factor in the occurrence of hip fractures. Women presented fractures of the proximal femur more frequently than men. The epidemiological curve was similar to that reported in other western countries, although the crude rate was lower than that found in northern countries. We conclude that osteoporotic fractures of the proximal femur are common processes in the Mediterranean area, and reflect the impact of osteoporosis in our environment; however, the lower incidence compared with northern geographical areas may probably reflect the influence of climatic, ethnic, or other factors.

Osteoblasts in HIV-infected patients: HIV-1 infection and cell function.

BackgroundHIV-infected patients have been shown to have a severe alteration in osteoblast function that appears to be related to the infection. ObjectiveTo determine whether normal human osteoblasts express CD4, whether osteoblasts from patients with HIV infection are infected by HIV-1 and whether osteoblast dysfunction observed in vivo also occurs in vitro. MethodsOsteoblast cultures from bone marrow biopsies of HIV-infected patients (n = 14) and control patients (n = 10) were used in a cross-sectional study and a case–control prospective study. Expression of CD4 was analysed using flow cytometry and reverse transcriptase polymerase chain reaction; the presence of HIV-1 particles was determined by measuring p24 antigen in the supernatants of osteoblast cultures and viral DNA or RNA in the osteoblasts using the polymerase chain reaction. Osteoblast function was assessed by measuring cell proliferation, type I collagen and osteocalcin synthesis. ResultsIn human osteoblasts, CD4 expression could not be determined using flow cytometry, although low levels of mRNA coding for CD4 were detected. HIV infection was not observed in osteoblast cultures from HIV-infected patients nor was there any alteration in replication and synthesis of type I collagen, although osteocalcin synthesis was increased. ConclusionsIt is unlikely that HIV-1 infects human osteoblasts in vivo; therefore, the hypothesis that these cells could act as local HIV-1 reservoirs should be reconsidered.

Acute Effects of Ethanol on Mineral Metabolism and Trabecular Bone in Sprague-Dawley Rats

Abstract. In order to assess the effects of acute ethanol intoxication on bone, 45 female Sprague-Dawley rats were studied. Five rats were sacrificed at baseline. The remainder received either ethanol (2 g/kg of body weight) intraperitoneally or isotonic saline. Rats were sacrificed in groups of 10 (5 intoxicated and 5 placebo) at 1, 4, 8, and 24 hours after injection. At the time of sacrifice, a blood sample was obtained and the 4th vertebra was excised for histomorphometric analysis of undecalcified bone. Effect of ethanol was assessed by an analysis of variance test using a Scheffé procedure. In ethanol-treated rats we observed (mean ± SD, ethanol versus controls, maximum difference point, P value) a significant decrease in osteiod surface with osteoblasts (42.86 ± 15.61% versus 64.57 ± 6.24%, P < 0.05); osteoclast number (0.05 ± 0.02 n/mm2 versus 0.17 ± 0.09 n/nm2, P < 0.05), and osteocalcin (36.9 ± 2.21 ng/ml versus 45.8 ± 5.1 ng/ml, P < 0.05). Osteoclast surface was initially reduced (0.129 ± 0.09% versus 0.425 ± 0.26%, P < 0.01) but showed a subsequent increase (0.765 ± 0.24% versus 0.226 ± 0.17%, P < 0.01) attributable to alcohol. There was also a significant decrease in serum Ca (8.51 ± 0.23 mg/dl versus 9.10 ± 0.29 mg/dl, P < 0.01) and parathyroid hormone values (23.51 ± 5.72 pg/ml versus 76.39 ± 11.66 pg/ml, P < 0.001). We conclude that acute alcohol intoxication in rats induces early striking changes in bone histology and analytical parameters, not completely reversed after 24 hours. These data are consistent with a toxic effect induced by alcohol on bone.

Bone remodeling alterations in myelodysplastic syndrome

There is a close relationship between hematopoietic bone marrow and bone cells. Thus, the profound derangement of hematopoiesis in myelodysplastic syndromes (MDS) might be expected to affect bone cell function. We studied the dynamic histomorphometric changes in bone in 22 MDS patients to examine this relationship and analyze the influence of hematological disease on bone remodeling. Bone-regulating hormones and histomorphometry of undecalcified transiliac bone biopsies, after double tetracycline labeling, were studied. Serum calcium, phosphorus, creatinine, alkaline phophatase, osteocalcin, iPTH, 25(OH)D3, 1,25(OH)2D3, hydroxyprolinuria, and calcium/creatinine ratio in urine were normal compared with controls. Histomorphometry showed a significant decrease in osteoblast surface (Ob.S/BS) (0.30 +/- 0.40 vs. 0.8 +/- 1.1, p = 0.031), wall thickness (W.Th), (22.03 +/- 5.5 vs. 31.8 +/- 5.8, p < 0.005), osteoclast number (N.Oc/T.Ar) (0.004 +/- 0.01 vs. 0.017 +/- 0.01, p = 0.03), mineral apposition rate (MAR) (0.16 +/- 0.15 vs. 0.53 +/- 0.19, p < 0.005), bone formation rate, surface referent (BFR/BS) (0.004 +/- 0.10 vs. 0.016 +/- 0.016, p = 0.009), and activation frequency (Ac.f) (0.06 +/- 0.07 vs. 0.21 +/- 0.23, p = 0.008). An increase in mineralization lag time (MLT) (119.2 +/- 78.6 vs. 29.6 +/- 77, p < 0.005), (mean +/- SD, unpaired Student t-test) was observed. Bone volume (BV/ TV), eroded surfaces (ES/BS), and osteoid thickness (O.Th) remained unchanged. This picture of adynamic bone with decreased mineral apposition rate and markedly decreased osteoclast number is a characteristic finding in MDS patients. Thus, bone histomorphometric finding in MDS patients show the relationships and interactions between hematopoietic and bone cells.

In Vitro Synthesis of Type I Collagen: Quantification of Carboxyterminal Propeptide of Procollagen Type I versus Tritiated Proline Incorporation

Abstract. Radioimmunoassay of the carboxyterminal propeptide of human type I procollagen has been recently introduced to measure in vitro synthesis of type I collagen by osteoblasts and fibroblasts. However, it has not been assessed whether the equivalent results are obtained with this new assay and with tritiated proline incorporation to collagen protein. To this purpose, both methods were used to quantify synthesis of type I collagen in normal human osteoblast cultures to which fetal calf serum and human serum were added in order to stimulate protein synthesis. A positive correlation in the results obtained by both methods was obtained (r = 0.95, P= 0.0001). Given the technical advantages of detection of levels to carboxyterminal propeptide of type I procollagen, we consider that this is the technique of choice for the quantification of in vitro synthesis of type I collagen by normal human osteoblasts.

Long-standing remission after 25-OH D3 treatment in a case of chronic myelomonocytic leukaemia

Summary This report describes a case of chronic myelomonocytic leukaemia (CMML) in whom a complete remission was achieved and sustained 15 months after treatment with 25‐OH vitamin D3. No side‐effects were observed. Although vitamin D3 has been used in the treatment of myelodysplastic syndromes, to our knowledge this is the first case of longstanding remission in a patient with CMML. This ‘differentiation therapy’ might be considered as a possible alternative in the management of this disease.

Tratamiento de la osteoporosis con calcio y vitamina D. Revisión sistemática

Fundamento Revision sistematica de la eficacia del calcio y la vitamina D en el tratamiento de la osteoporosis. Material y metodo Revision de la base de datos MEDLINE entre 1966 y mayo de 1998, mediante las palabras clave: osteoporosis, calcium, vitamin D (y terminos relacionados) y ensayo clinico alea-torizado. Revision de las ediciones electronicas de Best Evidence, Cochrane Library, resumenes de congresos y bibliografia de dos libros de texto. Revision ascendente de la bibliografia. Todas las revisiones se realizaron de forma independiente por dos de los autores. Tabulacion de los parametros de diseno y resultados principales de los ensayos identificados a partir de los articulos primarios. Calificacion metodologica de los estudios por parte de 2 observadores independientes. Tabulacion de los resultados y juicio evaluativo. Resultados Se han incluido 11 estudios sobre calcio, ocho sobre vitamina D y 12 sobre calcitriol y otros derivados de la hormona. Los estudios con calcio son fundamentalmente sobre poblacion no clinica y en tres se analiza eficacia sobre tasa de fracturas. Los resultados fueron positivos en poblaciones con ingestion basal baja y aporte elevado del farmaco. Los ensayos con vitamina D se realizaron sobre poblacion no clinica o ingresados en instituciones. Los ensayos con calcitriol se llevaron a cabo fundamentalmente en pacientes con fractura osteoporotica y tuvieron peores puntuaciones de validez metodologica. La heterogeneidad de los estudios impidio un metaanalisis de los diferentes tratamientos. En los estudios de calcio se observo de forma mas uniforme la efica-cia clinica. La concordancia entre observadores fue buena (? = 0,81) y no hubo correlacion significativa entre tamanos muestrales y efecto en los diferentes estudios. Conclusiones El tratamiento con calcio es eficaz en poblacion con una ingestion baja de calcio que recibe aportes importantes. La vitamina D es eficaz fundamentalmente, asociada a calcio, en poblacion deficitaria. El calcitriol y otros derivados de la hormona tienen una eficacia mas controvertida.

Osteoblastic Proliferation in Bone Biopsies from Patients with End-Stage Chronic Renal Failure

Osteoblasts have traditionally been considered to be terminally differentiated cells and therefore unable to divide. Data in recent years, however, indicate that cellular differentiation does not usually preclude preservation of proliferative ability and that most differentiated cells are able to divide under adequate stimuli. The aim of this study was to assess whether cubic osteoblasts undergo proliferation during the formation phase of the remodeling cycle under a stimulus that increased bone turnover. For that purpose, the osteoblastic proliferation index (OPI) was analyzed by DNA image cytometry in transiliac bone biopsies from 33 patients with chronic renal failure (23 men, 10 women; mean age 50.4 ± 15.1 years) who have been classified into low (n = 13), normal (n = 15), and high (n = 15) bone turnover according to activation frequency (Ac.f). OPI was significantly higher (p < 0.002) in the high bone turnover group (13.90 ± 4.72%) compared with the low (2.38 ± 4.13%) and normal turnover groups (2.84 ± 4.04%). There was a positive correlation between OPI and the following histomorphometric parameters: bone formation rate, surface referent (r = 0.76, p = 0.00001), activation frequency (r = 0.73, p = 0.00001), mineral apposition rate (r = 0.73, p = 0.00001), bone formation rate, volume referent (r = 0.71, p = 0.00001), and mineralizing surface (r = 0.62, p = 0.0001). This study shows that a rise in bone turnover is associated with a marked increase of bone‐forming cell proliferation in patients with end‐stage chronic renal failure. From this finding, it may be concluded that cubic osteoblasts do not behave as “terminally differentiated” cells in vivo, because a high proportion of them are still able to divide.