M. L. Mariñoso

Bone remodelling in human immunodeficiency virus-1-infected patients. A histomorphometric study

The aim of this study was to identify and describe possible alterations of bone histomorphometry in patients with human immunodeficiency virus (HIV-1) infection and to assess the relation between these alterations and disease severity. Forty-four HIV-1-infected patients seen successively at our hospital were evaluated for the study. In an attempt to avoid confounding factors as far as possible, we excluded patients who fulfilled any of the following criteria: age less than 18 or greater than 40 years; recent history of extended bed rest; previous diagnosis of metabolic bone disease, renal insufficiency, or hepatic failure; clinical or echographic signs of liver cirrhosis; diabetes mellitus or previous diagnosis of other endocrine diseases; drug therapy that could act on bone metabolism; and/or moderate to severe nutritional alteration. Twenty-two patients (13 men, 9 women; age: 27.9 +/- 4.1 years, mean +/- standard deviation) were included in the study. Plasma and urine biochemistry and calcium-regulating hormones were determined. Bone mineral content was measured on vertebrae L2 to L4 and on the neck and intertrochanteric areas of the femur by dual-photon absorptiometry. A transiliac bone biopsy was performed after double-tetracycline labelling, with histomorphometric study of undecalcified bone. Serum osteocalcin was found to be lower in patients who, according to the Centers for Disease Control (CDC) classification, had greater disease severity, and showed a positive correlation with the number of CD4+ T lymphocytes. No alterations in bone densitometry were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

Detection of HPV by in situ hybridization in thin-layer (ThinPrep) cervicovaginal samples

We have studied an automated in situ hybridization (ISH) method as a possible alternative approach for detecting high-risk human papillomavirus (HPV) in monolayer (ThinPrep) cervico-vaginal samples, comparing the results with those obtained by polymerase chain reaction (PCR) using consensus primers and studying the relationship between the ISH staining pattern and the viral integration in HPV 16-positive cases. Eighty atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) cases were used for our purposes. The patients were monitored through periodic cytologies. ISH with was performed with an automated Ventana System, analysis by PCR was performed with consensus primers and integration of HPV16 was performed by realtime PCR analyzing E2 and E6 genes. Additionally, 27 HSIL cases were also studied to observe the ISH staining patterns. HPV infection was detected by ISH in 21.7% of the ASCUS cases and 55.8% of the LSIL cases. Two distinct staining patterns were observed: multipunctated (MP) and diffuse (DI). In some cases, a mixed pattern (MP + DI) was observed and these cases were considered as MP. The MP pattern increased with the degree of lesion and seemed to have a prognostic value in ASCUS/LSIL cases. The lesion in MP pattern cases persisted throughout the entire study in 77% of cases, whereas in cases with a DI staining pattern, only 41% of them showed persistence of the lesion (p < 0.001). No correlation was found between HPV integration and the ISH staining pattern. Given the lower sensitivity and negative predictive value of ISH and its incapacity to demonstrate the integration of high-risk HPV in ASCUS and LSIL cases using liquid-based cytology, we do not recommend this technique for the triage of ASCUS and LSIL cases.

Alendronate and etidronate do not regulate interleukin 6 and 11 synthesis in normal human osteoblasts in culture

Bisphosphonates exert a potent inhibitory effect on bone resorption. Several studies have been performed, with contradictory results, to ascertain whether the effect of bisphosphonates on osteoclasts could be produced, at least in part, by modulation of the synthesis of resorption-promoting factors by osteoblasts. The aim of this study was to evaluate the effect of etidronate (10-4–10-9 M) and alendronate (10-7–10-12 M) on the production of IL-6 and IL-11 using human osteoblast cultures. Cytokines were quantified by ELISA, and mRNA expression was tested. Treatment with alendronate and etidronate had no effect on the synthesis of IL-6 or IL-11, and IL-6 and IL-11 mRNA levels. These results were obtained both in nonstimulated cultures and in cultures stimulated by means of TNF-a, IL-1b, and TNF-a+IL-1b, with or without FCS. In conclusion, a possible indirect osteoclast-mediated effect of alendronate and etidronate on bone resorption would not be exerted through reduction in osteoblastic synthesis of IL-6 and IL-11.

Medullary Carcinoma of the Breast: An Ultrastructural Morphometric Study of Nine Cases

Ultrastructural and morphometric features of 10 medullary carcinomas of the breast (MC) were investigated. Cases with a long follow-up were selected by applying stringent histologic criteria. All tumors had a homogeneous appearance by light microscopy. Under transmission electron microscopy, they showed occasional intracellular lumen formation or keratinization. In one tumor squamous differentiation was prominent and diffuse. Tumors with lymph node metastases possessed over 40% more desmosomes than nonmetastatic tumors. The number of cells with three or more nucleoli per nuclear section was significantly higher in metastatic than in nonmetastatic tumors (p = .02). Classic cases of MC of the breast display a relatively uniform appearance. However, subtle differences can be identified between metastatic and nonmetastatic tumors by ultrastructural morphometry. Although these differences are not associated with changes in the outcome of patients in this study, they seem to bear some relationship to the peculiar behavior of MC.

Estudio citogenético de un carcinoma escamoso de cérvix uterino

Resumen Presentamos el caso de un carcinoma escamoso de cervix uterino en una paciente de 50 anos. En las celulas tumorales se hallo la presencia del virus del papiloma humano (VPH) 16, sin otros virus acompanantes, y el estudio citogenetico evidencio multiples alteraciones cromosomicas especialmente en los cromosomas 5 y 8 asi como multiples alteraciones cromosomicas numericas. Nuestro objetivo es enfatizar la importancia de practicar estudios citogeneticos en carcinomas escamosos de cervix a la vez que efectuar una revision de los datos existentes en la bibliografia