Factors Associated with Israeli Arab Women Anxiety and Depression During the COVID-19 Pandemic.

BACKGROUND: Reports have shown that women suffered from anxiety, stress, depression, and fatigue during the COVID-19 pandemic more than men. No study so far has examined the effect of the pandemic among the Arab minority in Israel. OBJECTIVES: To examine the associations between levels of pandemic fatigue and stress of Israeli Arab women, and their anxiety and depression, along with their socio-demographic and socio-economic characteristics. METHODS: A Cohen and Williamson questionnaire, which was based on a Likert scale, was distributed by the snowball method through social networks. Bivariate associations between the psycho-social and demographic characteristics and anxiety and depression were assessed using t-tests, chi-square tests, Z tests, and Pearson correlations. Multiple linear regressions were used to evaluate the associations with anxiety and depression, and the mediation model was examined with path analysis with bootstrapping. RESULTS: Among 2294 Israeli Arab mothers who participated in the study, 63.7% were in the clinical range for anxiety, 67.4% for depression, and 57.5% for both anxiety and depression. Low economic status, pandemic fatigue, living in closed communities, and stress were related to anxiety and depression. Pandemic fatigue was positively related to stress, which was positively related to both anxiety and depression (standardized indirect effect = 0.137, SE = 0.014, 95%CI = 0.111, 0.164, p < .001; vs. 0.133, SE = 0.013, 95%CI = 0.108, 0.160, p < .001 respectively). The contribution of stress to anxiety and depression was significantly greater than that of pandemic fatigue (Z = 19.43 and Z = 18.04, p < .001, for anxiety and depression, respectively). CONCLUSIONS: Demographic characteristics may put Arab women at a higher risk of anxiety and depression. Elevated stress alongside high fatigue may trigger mental health difficulties. The welfare of minorities should be addressed by policymakers in relation to their demographic needs.

Mapping human risk of infection with Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, in a periurban forest in France.

Lyme borreliosis is a major zoonosis in Europe, with estimates of over 26,000 cases per year in France alone. The etiological agents are spirochete bacteria that belong to the Borrelia burgdorferi sensu lato (s. l.) complex and are transmitted by hard ticks among a large range of vertebrate hosts. In Europe, the tick Ixodes ricinus is the main vector. In the absence of a vaccine and given the current difficulties to diagnose and treat chronic Lyme syndromes, there is urgent need for prevention. In this context, accurate information on the spatial patterns of risk of exposure to ticks is of prime importance for public health. The objective of our study was to provide a snapshot map of the risk of human infection with B. burgdorferi s. l. pathogens in a periurban forest at a high resolution, and to analyze the factors that contribute to variation in this risk. Field monitoring took place over three weeks in May 2011 in the suburban Sénart forest (3,200ha; southeast of Paris), which receives over 3 million people annually. We sampled ticks over the entire forest area (from 220 forest stands with a total area of 35,200m(2)) and quantified the density of questing nymphs (DON), the prevalence of infection among nymphs (NIP), and the density of infected nymphs (DIN), which is the most important predictor of the human risk of Lyme borreliosis. For each of these response variables, we explored the relative roles of weather (saturation deficit), hosts (abundance indices of ungulates and Tamias sibiricus, an introduced rodent species), vegetation and forest cover, superficial soil composition, and the distance to forest roads. In total, 19,546 questing nymphs were collected and the presence of B. burgdorferi s. l. was tested in 3,903 nymphs by qPCR. The mean DON was 5.6 nymphs per 10m(2) (standard deviation=10.4) with an average NIP of 10.1% (standard deviation=0.11). The highest DIN was 8.9 infected nymphs per 10m(2), with a mean of 0.59 (standard deviation=0.6). Our mapping and modeling revealed a strong heterogeneity of risk within the forest. The highest risk was found in the eastern part of the forest and localized patches in the northwestern part. Lyme borreliosis risk was positively associated with stands of deciduous trees (mainly oaks) and roe deer abundance. Contrary to expectations, DIN actually increased with distance from the point of introduction of T. sibiricus (i.e., DIN was higher in areas with potentially lower abundances of T. sibiricus). Thus, despite the fact that T. sibiricus is an important reservoir host for B. burgdorferi s. l., our study found that other explanatory factors played a more important role in determining the density of infected ticks. Precise mapping of the risk of exposure to Lyme borreliosis in a highly visited forest represents an important tool for targeting prevention and control measures, as well as making the general public and local health officials aware of the risks.

What lies behind the low rates of vaccinations among nurses who treat infants?

BACKGROUND: In most countries rates of immunizations of health care workers with recommended vaccines are not satisfactory. OBJECTIVES: To identify reasons behind the low rates of compliance of Israeli nurses in Mother and Child Healthcare Centers (MCHC) with an official request for pertussis vaccination. METHODS: Three focus groups were conducted. Qualitative analysis identified themes that could explain the nurses' non-compliance. RESULTS: Trust in health authorities was low, mainly following the A/H1N1 purported influenza pandemic. In addition, nurses did not see the importance of being role models for the public and demanded the autonomy to decide whether to receive vaccinations. The nurses differentiated between their role as nurses and their personal life, expressed fear of new vaccines and exhibited low levels of risk perception. Misconceptions regarding vaccinations were expressed by the nurses. CONCLUSIONS: Antivaccinationist ideas were expressed by MCHC nurses and these attitudes may have led to non-compliance with vaccination guidelines.

Osteoclast formation and bone resorption are inhibited by megakaryocytes.

It has been previously reported that addition of megakaryocytes (MKs) to osteoblasts in vitro results in increased osteoblastic collagen and osteoprotegerin (OPG) production, suggesting a role for MKs in bone formation. To further investigate this role, we have studied the effects of MKs on osteoclast formation and activity. Human osteoclasts were generated from CD14 monocytes isolated from peripheral blood and cultured in the presence of M-CSF and sRANKL on dentine and calcium phosphate substrates. MKs were generated from CD34+ cells isolated from either human peripheral blood or cord blood and cultured in liquid medium for 6 days, after which time maturing MKs (CD61-positive cells) were isolated and added to monocyte cultures. After 6 and 9 days of culture, the number of osteoclasts identified morphologically and by TRAP staining was counted. Cells were removed and the area of resorption was identified by von Kossa staining and quantitatively assessed by image analysis. The addition of MKs to osteoclast cultures at day 0 inhibited the number of osteoclasts formed 1.9-fold (p>0.003), whereas addition at 3 days had no effect on osteoclast number. The presence of MKs inhibited resorption 8.7-fold when co-cultured with osteoclasts from day 0 (p>0.004), but only by 3.1-fold when co-cultured from day 3 (p>0.01). In dose-response experiments, it was found that 1-10% of MKs added to monocyte cultures elicited the greatest inhibition of resorption. Similar osteoclast cultures were treated with CD61-negative cells (non-MKs) to confirm that the inhibition of osteoclast formation and activity was specifically due to MKs. Experiments with a cell-impermeable membrane indicated that both cell to cell contact and release of soluble factor(s) were involved in mediating these effects. These results show that MKs inhibit osteoclast formation and activity. The most pronounced effects were seen when MKs and osteoclasts were co-cultured from day 0, suggesting that MKs act primarily on osteoclast precursors.

Dairy management practices associated with incidence rate of clinical mastitis in low somatic cell score herds in France.

An epidemiological prospective study was carried out in French dairy herds with Holstein, Montbéliarde, or Normande cows and with low herd somatic cell scores. The objective was to identify dairy management practices associated with herd incidence rate of clinical mastitis. The studied herds were selected on a national basis, clinical cases were recorded through a standardized system, and a stable dairy management system existed. In the surveyed herds, mean milk yield was 7420 kg/cow per yr and mean milk somatic cell score was 2.04 (132,000 cells/mL). Overdispersion Poisson models were performed to investigate risk factors for mastitis incidence rate. From the final model, the herds with the following characteristics had lower incidence rates of clinical mastitis: 1) culling of cows with more than 3 cases of clinical mastitis within a lactation; 2) more than 2 person-years assigned to dairy herd management; 3) balanced concentrate in the cow basal diet. Moreover, herds with the following characteristics had higher incidence rates of clinical mastitis: 1) milking cows loose-housed in a straw yard; 2) no mastitis therapy performed when a single clot was observed in the milk; 3) clusters rinsed using water or soapy water after milking a cow with high somatic cell count; 4) 305-d milk yield >7435 kg; 5) herd located in the South region; 6) herd located in the North region; 7) cows with at least 1 nonfunctional quarter; and 8) premilking holding area with a slippery surface. The underlying mechanisms of some highlighted risk factors, such as milk production level and dietary management practices, should be investigated more thoroughly through international collaboration.

Megakaryocytes modulate osteoblast synthesis of type-l collagen, osteoprotegerin, and RANKL.

We have previously reported evidence that megakaryocytes may play a role in bone remodeling, possibly by interactions with cells at the bone surface. To investigate the direct effects of megakaryocytes on osteoblasts, maturing megakaryocytes (CD61 positive cells) were isolated and added to cultures of human osteoblasts. Osteoblasts alone and osteoblasts treated with CD61-negative (non-megakaryocytic) cells were used as control cultures. After 48 h in culture, megakaryocytes were removed and osteoblasts immunolocalized for type-1 collagen, osteoprotegerin (OPG), and RANKL expression. Similar cultures were used for RNA extraction with mRNA for Col 1A1, OPG, and RANKL in osteoblasts measured quantitatively by RT-PCR. Osteoblasts cultured alone showed high levels of expression of collagen with 74% (+/-7) of cells staining positively. When cultured with megakaryocytes, the number of positively staining cells remained similar but the intensity of expression was increased 1.54-fold (P < 0.02). OPG was expressed by 32% (+/-6.3) of osteoblasts increasing to 51% (+/-5.5) when cultured in the presence of megakaryocytes (P < 0.01) with a 1.63-fold increase in intensity of expression (P < 0.01). In contrast, osteoblasts cultured with megakaryocytes showed suppression of RANKL expression; 35.6% (+/-5.8) of osteoblasts cultured alone stained positively decreasing to 24.3% (+/-5.3) with a 1.6-fold diminished intensity of expression (P < 0.02). Osteoblasts co-cultured with CD61-negative cells showed no differences in collagen, OPG, or RANKL expression levels compared to osteoblasts cultured alone. mRNA data supported these findings with a 3.1-fold increase in Col 1A1 expression in megakaryocyte-treated cultures compared to controls (P < 0.02). Low-level OPG mRNA expression increased 8.14-fold in osteoblasts cultured in the presence of megakaryocytes (P < 0.01), while RANKL expression was suppressed 3.3-fold (P < 0.02). These results demonstrate that in vitro, megakaryocytes have direct effects on osteoblastic production of factors affecting both bone formation and resorption. These data provide further evidence that megakaryocytes may play an important role in bone remodeling.

Synthesis of osteoprotegerin and RANKL by megakaryocytes is modulated by oestrogen.

To investigate the mechanisms by which megakaryocytes (MKs) may influence bone remodelling, CD34(+) cells were cultured for 6, 9 and 12 d with or without 17beta-oestradiol (E) and immunolocalized for osteoprotegerin (OPG), receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and CD61. Specific protein expression was measured quantitatively by image analysis. Fluorescence-based immunocytochemistry was used to co-localize OPG and RANKL with CD61. OPG and RANKL mRNA was assessed in CD61(+) cells with or without E at 24 and 48 h. At 6 d, OPG and RANKL expression was unchanged by E treatment. At 9 d, the E-treated cultures with maturing MKs showed a 1.72-fold (P < 0.01) increase in OPG expression and a 1.8-fold (P < 0.01) reduction in RANKL. Maximal OPG expression was seen at 12 d with a threefold induction of expression (P < 0.001), whilst RANKL levels were further suppressed by 2.3-fold compared with controls (P < 0.001). CD61 co-localized with OPG and RANKL. mRNA data were consistent with that of protein, with a 90-fold induction in OPG expression and a 34-fold suppression of RANKL expression by E (P < 0.001). Thus, E stimulates megakaryocytopoiesis and modulates OPG and RANKL expression, providing evidence that MKs may play a role in bone remodelling and, in particular, in E-induced changes in osteoclastogenesis and bone resorption.

Joint erosion in rheumatoid arthritis: interactions between tumour necrosis factor alpha, interleukin 1, and receptor activator of nuclear factor kappaB ligand (RANKL) regulate osteoclasts.

BACKGROUND: Osteoclasts, specialised bone resorbing cells regulated by RANKL and M-CSF, are implicated in rheumatoid joint erosion. Lymphocyte-monocyte interactions activate bone resorption, this being attributed to tumour necrosis factor alpha (TNFalpha) and interleukin 1 beta (IL1beta) enhanced osteoblast expression of RANKL. In animal studies, TNF potently increases osteoclast formation in the presence of RANKL. RANKL-independent osteoclastogenesis also occurs, though IL1 is required for resorptive function in most studies. These inflammatory cytokines have a pivotal role in rheumatoid arthritis, OBJECTIVE: To study the interactions of TNFalpha and IL1beta with RANKL, particularly the time course of the interactions and the role of lymphocytes. METHOD: Cultures of lymphocytes and monocytes (osteoclast precursors) or of purified CD14(+) cells alone (osteoclast precursors) were exposed to various combinations of TNFalpha, RANKL, and IL1beta or the inhibitors osteoprotegerin, IL1 receptor antagonist, or neutralising antibodies to RANKL or to IL1. Osteoclastogenesis and resorptive activity were assessed on microscopy of dentine slices. RESULTS: TNFalpha potently increased osteoclast proliferation/differentiation in the presence of RANKL. This effect was greatest when RANKL was present before but not after exposure of osteoclast precursor cells to TNFalpha. The resorptive activity of osteoclasts generated by TNFalpha in the absence of RANKL was critically dependent upon IL1, which was expressed by lymphocyte-monocyte interaction. CONCLUSION: TNFalpha potently enhances RANKL mediated osteoclast activity. Interactions between TNFalpha and IL1 also result in osteoclastic activity independently of RANKL. These findings will inform therapeutic approaches to the prevention of joint erosion in rheumatoid arthritis.

The effects of estrogen on osteoprotegerin, RANKL, and estrogen receptor expression in human osteoblasts.

Estrogen is essential for bone growth and development and for the maintenance of bone health in adulthood. The cellular responses of osteoblasts and osteoclasts to estrogen are initiated via two high-affinity receptors (ERs). Osteoblasts synthesize RANKL (receptor activator of NF-kappaB ligand), necessary for osteoclast formation and function, and osteoprotegerin (OPG), its decoy receptor. To investigate the effects of estrogen on the expression of OPG, RANKL, and ERs in human osteoblasts, cells were cultured with physiological (10(-10) M) and high-dose (10(-7) M) 17beta-estradiol for 24 and 48 h. Proteins and corresponding mRNA levels were quantitatively determined by immunocytochemistry and RT-PCR. OPG expression was significantly increased three- and sevenfold at 24 h with 10(-10) M (P < 0.05) and 10(-7) M (P < 0.01) estradiol, respectively, compared to untreated cells. Similar but smaller increases were seen at 48 h (P < 0.05). Osteoblasts treated with estradiol demonstrated increased RANKL protein expression at 24 h (P < 0.05), but this was not maintained at 48 h. ERalpha expression was significantly increased by high-dose estradiol (P < 0.01) at 24 h and dose-dependently increased at 48 h (P < 0.01), while ERbeta was only increased at 24 h (P < 0.01). The estrogen-induced protein expression of ER, OPG, and RANKL was abrogated when cells were cultured in the presence of the estrogen antagonist ICI 182780. mRNA levels at 24 h demonstrated a significant suppression of RANKL with the low-dose but not the high dose. ERalpha mRNA but not ERbeta expression was up-regulated by estrogen. Our results suggest that estrogen may exert its anti-resorptive effects on bone, at least in part, by stimulating ER and OPG expression in osteoblasts.

Reduced bone formation in UK Gulf War veterans: a bone histomorphometric study.

AIMS: Gulf War veterans report a high prevalence of musculoskeletal symptoms. The aim of this study was to establish whether there were abnormalities in bone turnover and remodelling in a group of symptomatic subjects who had served in the Gulf War. METHODS: Iliac crest bone biopsies were obtained from 17 Gulf War veterans who were seeking litigation and compared with those of 13 age and sex matched healthy controls. Bone histomorphometry was performed using image analysis. RESULTS: Cancellous bone area was significantly lower in Gulf War veterans than in control subjects (p = 0.027) and this was associated with a significantly reduced mineral apposition rate (p = 0.002), mean wall width (p < 0.0001), and bone formation rate at the tissue level (p < 0.0001). CONCLUSIONS: These results demonstrate that in this group of Gulf War veterans there was a significant reduction in bone formation at both the cellular and tissue level and this was associated with a reduction in cancellous bone area. The cause of these abnormalities is unknown but might be related to potentially harmful exposures during service in the Gulf War or to changes in life style as a result of chronic ill health. The clinical relevance of the observed reduction in bone formation remains to be established.

Mechanisms by which high-dose estrogen therapy produces anabolic skeletal effects in postmenopausal women: role of locally produced growth factors.

Conventional hormone replacement therapy acts primarily by preserving bone, but cannot restore lost bone in women with established osteoporosis. Studies in rodents have shown that high doses of estrogens have anabolic skeletal effects, and recent observations in a group of women treated long term with high doses of estrogen indicated that similar effects occur in humans. This study examines the hypothesis that locally produced growth factors, including transforming growth factor-beta (TGF-beta) and platelet-derived growth factors (PDGFs), are involved in mediating the anabolic effects of high-dose estrogen. Transiliac-crest bone biopsies were taken from ten women, aged 52-67 years (mean 58 years), who had been treated with high-dose estrogen for 15 years. Control samples were obtained from four age-matched postmenopausal women not receiving estrogen therapy. TGF-betas and PDGFs were analyzed for mRNA and protein expression by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Results showed both TGF-beta1 and -beta2 mRNA, expressed as a ratio to GAPDH, were increased in the estrogen-treated group with an eightfold increase for TGF-beta1 (0.258 +/- 0.246 [mean +/- SD] vs. 0.032 +/- 0.053 in the control group, p = 0.02) and a twofold increase for TGF-beta2 (p = n.s.). TGF-beta3 analysis showed only negligible amounts in both groups. Protein expression levels for TGF-beta1, -beta2, -betaRI and -RII were higher in the estrogen-treated group than in controls, the most marked effects being seen for TGF-beta1. PDGF-A protein expression was also significantly higher in osteoblasts and osteocytes in women treated with estrogen, whereas PDGF-B showed only modest differences. The percentage of bone surface occupied by osteoclasts, as determined by tartrate-resistant acid phosphatase (TRAP) staining, was significantly reduced in the estrogen-treated group (p = 0.001). These results demonstrate that high-dose estrogen therapy is associated with increased TGF-beta, TGF-betaR, and PDGF synthesis and decreased osteoclast activity, consistent with the hypothesis that these growth factors may mediate the actions of estrogen in bone.

Colocalization of glucocorticoid and mineralocorticoid receptors in human bone.

Osteoporosis is a poorly understood but common complication of glucocorticoid therapy. The actions of glucocorticoids are mediated via glucocorticoid receptors (GRs), but in vitro, glucocorticoids also can bind to mineralocorticoid receptors (MRs). It is not known if MR protein is present in human bone and little is known of GR isoform expression (GRalpha and GRbeta). GR and MR protein expression and possible sites of action were investigated in neonatal rib and adult iliac crest biopsy specimens using antibodies specific for MR, GRalpha, and GRalphabeta. Colocalization [MR GRalpha] [MR GRalphabeta] was performed using fluorescent-conjugated secondary antibodies. GRalpha, GRbeta, and MR show distinct but overlapping patterns of expression, suggesting important functions for each receptor type. Osteoclasts showed no staining for GRalpha but strong staining for GRalphabeta, indicating expression of GRbeta and a specific role in addition to antagonizing the transcriptional activity of GRalpha. MR also was observed in osteoclasts and colocalized with GRalphabeta. Coexpression of MR, GRalpha, and GRalphabeta was seen in osteoblasts. Reverse-transcription-polymerase chain reaction (RT-PCR) of cultured osteoblast RNA confirmed expression of both GRalpha and GRbeta. Osteocytes stained with MR, GRalpha, and GRalphabeta antibodies but to a lesser degree than osteoblasts. In the neonatal rib cartilage, staining for GRalpha, GRalphabeta, and MR was present in approximately one-half of the resting and hypertrophic chondrocytes and in most of proliferating chondrocytes and chondrocytes within the mineralizing matrix. Identification of MR raises the possibility that the physiological and pharmacologic effects of glucocorticoids on bone may be mediated via MR as well as GR and that GRalpha, GRbeta, and MR synergize to influence corticosteroid metabolism in human bone.

Estrogen receptors alpha and beta are differentially expressed in developing human bone.

Estrogen plays an essential role in the development and maintenance of the skeleton; its effects are mediated via interactions with two estrogen receptor (ER) subtypes, alpha and beta. The aim of this study was to establish the cellular distribution of ERalpha and ERbeta in neonatal human rib bone. ERalpha and ERbeta immunoreactivity was seen in proliferative and prehypertrophic chondrocytes in the growth plate, with lower levels of expression in the late hypertrophic zone. Different patterns of expression of the two ERs were seen in bone. In cortical bone, intense staining for ERalpha was observed in osteoblasts and osteocytes adjacent to the periosteal-forming surface and in osteoclasts on the opposing resorbing surface. In cancellous bone, ERbeta was strongly expressed in both osteoblasts and osteocytes, whereas only low expression of ERalpha was seen in these areas. Nuclear and cytoplasmic staining for ERbeta was apparent in osteoclasts. These observations demonstrate distinct patterns of expression for the two ER subtypes in developing human bone and indicate functions in both the growth plate and mineralized bone. In the latter, ERalpha is predominantly expressed in cortical bone, whereas ERbeta shows higher levels of expression in cancellous bone.

Tie2 ligands angiopoietin-1 and angiopoietin-2 are coexpressed with vascular endothelial cell growth factor in growing human bone.

Angiogenesis is essential for bone growth and repair. Recent studies have shown that the endothelial-specific mitogen vascular endothelial growth factor (VEGF) is a key regulator of vascular invasion into the growth plate in infant and adolescent animals. In order to identify mechanisms regulating VEGF-induced angiogenesis in growing bone, we have investigated the expression of the angiopoietins (Ang-1 and Ang-2) in human neonatal ribs. Ang-1 and Ang-2 exhibited similar patterns of staining in the growing rib. In the cartilage, expression of Ang-1 and Ang-2 increased with chondrocyte maturation. Ang-1, Ang-2, and VEGF were not detected in the resting zone except adjacent to vascular canals, and maximum expression was detected at the cartilage bone interface. In the cartilage, Ang-2 was more highly expressed than Ang-1 or VEGF, with staining observed in the proliferating, hypertrophic, and mineralized zones. In the bone, Ang-1, Ang-2, and VEGF were detected in modeling and remodeling sites. Ang-1 was detected in the majority of osteoblasts, osteoclasts, and in some marrow space cells. Ang-2 was expressed at variable levels by osteoblasts and osteoclasts in modeling and remodeling bone. VEGF was detected in cells at bone surfaces and in the marrow spaces. Strong staining for VEGF was observed in osteoblasts and osteoclasts in modeling and remodeling bone. In the perichondrium, Ang-1, Ang-2, and VEGF were most highly expressed adjacent to the hypertrophic zone and at sites of bone collar formation. In the periosteum, Ang-1, Ang-2, and VEGF expression colocalized with alkaline phosphatase expression. These observations provide the first evidence for the expression of the angiopoietins in growing human bone in vivo. The distribution of Ang-1, Ang-2, and VEGF indicate these factors may play key roles in the regulation of angiogenesis at sites of endochondral ossification, intramembranous ossification, and bone turnover in the growing human skeleton.

Megakaryocyte population in human bone marrow increases with estrogen treatment: a role in bone remodeling?

Skeletal effects of conventional hormone replacement therapy (HRT) are predominately antiresorptive, while high doses of estrogen have anabolic effects. The mechanisms mediating these effects are unclear but may involve cells in the bone marrow. We have investigated the in vivo effects of estrogen on the megakaryocyte (MK) population in bone marrow in 10 postmenopausal women before and after 2 years of conventional HRT, in 11 women after long-term, high-dose estradiol therapy, and in 2 premenopausal and 4 postmenopausal women who had received no previous estrogen treatment. Transiliac crest biopsies were halved and either decalcified and paraffin wax embedded for immunolocalization studies or dehydrated and embedded in LR White resin for histology. MKs were identified morphologically, and the bone marrow cell population and MK number quantified by cell counting in a defined area of view (1 mm(2)) from 5 randomly selected fields of bone marrow. Compared with pretreatment values, significantly higher MK numbers were found after conventional HRT treatment (before treatment, mean +/- SEM; 7.3 +/- 1.1 vs. after treatment, 18.0 +/- 1.6/5 mm(2); p < 0.0001), while the greatest MK number was associated with long-term, high-dose estradiol treatment (32.8 +/- 2.1/5 mm(2); p < 0.0001). Total bone marrow cell number did not differ significantly between groups. Immunolocalization studies revealed more intense estrogen receptor (ER)beta expression in MKs in the high-dose estradiol-treated group but similar levels of weak ERalpha staining in MKs in the control and high-dose estrogen-treated groups. Positive immunoreactivity for transforming growth factor (TGF)beta1, 2, and 3 and TGFbeta receptor I, II, and III was detected in MKs, with more intense staining being demonstrated in the high-dose estradiol-treated group, particularly for TGFbeta2 and TGFbetaRI and II. Our results demonstrate an increase in the MK population in bone marrow from women treated with estrogen. The ability of MKs to express ERs and synthesise TGFbeta, a potent mitogen in osteoblast differentiation, suggests that these cells may play a role in mediating estrogen-induced effects on bone.

Reduced bone formation after exposure to organophosphates.

Bone histomorphometric analysis in 24 agricultural workers with chronic organophosphate exposure showed significantly lower bone formation at tissue and cellular level than in healthy controls.

Bone remodeling and structure in postmenopausal women treated with long-term, high-dose estrogen therapy.

Conventional hormone replacement therapy preserves bone mass predominantly by reducing bone turnover but does not exert significant anabolic skeletal effects. In contrast, high doses of estrogen have been shown to increase bone formation in animals and we have recently reported high bone mineral density values in women treated long-term with estradiol implant therapy. The aim of this study was to investigate the mechanisms by which high doses of estrogen may increase bone mass in postmenopausal women. Iliac crest biopsies were obtained from 12 women who had received long-term treatment with estradiol implants (at least 14 years), on demand, following hysterectomy and bilateral salpingo-oophorectomy. Indices of bone turnover, remodeling balance and cancellous bone structure were assessed by image analysis and compared with those of premenopausal women. Mean wall width was significantly higher in women treated with estradiol therapy than in premenopausal women (44. 8 +/- 4.8 vs 38.8 +/- 2.8 mm; mean +/- SD; p = 0.001) and eroded cavity area was significantly lower in the implant-treated women (3612 +/- 956 vs 5418 +/- 1404 mm(2); p = 0.001). Bone formation rate at tissue level and activation frequency were lower in the women treated with implants, although the differences were not statistically significant. Indices of cancellous bone structure were generally similar between the two groups. These results provide the first direct evidence that high-dose estrogen therapy produces anabolic skeletal effects in postmenopausal women and indicate that these are achieved by stimulation of osteoblastic activity.

Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage.

Angiogenesis is essential for the replacement of cartilage by bone during growth and repair. In order to obtain a better understanding of the mechanisms regulating vascular invasion at sites of endochondral ossification we have investigated the expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), by chondrocytes in human neonatal growth plates. VEGF was absent from chondrocytes in the resting zone and only weakly expressed by occasional chondrocytes in the proliferating region. In the hypertrophic zone the number of chondrocytes stained and the intensity of staining for VEGF increased with chondrocyte hypertrophy, maximum expression of VEGF being observed in chondrocytes in the lower hypertrophic and mineralised regions of the cartilage. These observations provide the first demonstration of the presence of VEGF in situ in developing human bone and are consistent with in vitro observations demonstrating the upregulation of proangiogenic growth factor production with increasing chondrocyte hypertrophy. The presence of numerous small blood vessels and vascular structures in the subchondral region where VEGF expression was maximal indicates that VEGF produced by hypertrophic chondrocytes may play a key role in the regulation of vascular invasion of the growth plate.