Molecular cloning, purification and characterisation of myosin of human lymphatic filarial parasite Brugia malayi.

Global efforts have been made towards development of vaccine for prevention of lymphatic filariasis. However, lack of thorough knowledge about developmental biology and pathogenesis of filarial parasite restricts us from developing an effective vaccine. A limited number of immunodominant antigens of human lymphatic filariid Brugia malayi have been characterised; however, none of these recombinant antigens so far induced significant degree of protective immunity to challenge infection. In the present study, we identified a approximately 2.0 Kb cDNA clone by immunoscreening of cDNA library of adult female Brugia malayi. The nucleotide sequence of the identified clone showed 94.3% homology with C-terminal part of myosin heavy chain gene of Brugia malayi. This cDNA insert was sub-cloned into pET28b vector and expressed in BL21(DE3). The recombinant protein was purified to near homogeneity by immobilised metal affinity chromatography (IMAC) with yield of approximately 25 mg/l. The purified protein was recognised in western blot with anti-His tag antibody as also with the antibodies present in the sera of human W. bancrofti patients of all categories and infected/immunized rodent serum demonstrating its functional role. Recombinant myosin induced marked cellular immune response as observed by lymphoproliferation assay. The present findings demonstrate the usefulness of B. malayi recombinant myosin as vaccine candidate against human lymphatic filariasis.

A histomorphometric study of cortical bone of the iliac crest in patients treated with glucocorticoids.

The effects of glucocorticoids on cancellous bone remodeling and structure are well documented but there are no reported histomorphometric studies in human cortical bone in glucocorticoid-treated patients. We have performed a histomorphometric analysis of iliac crest cortical bone in 14 patients treated with glucocorticoids, 9 females and 5 males, aged 18 to 48 years (34.1 +/- 7 years) (mean +/- standard deviation [SD]). The underlying disease was cystic fibrosis in 8 patients; asthma 3; and nephrotic syndrome; Crohn disease and inflammatory pseudotumor of the liver in one patient each. Results were compared with an age-matched control group of 10 premenopausal women and 4 men aged 22 to 38 years (30.1 +/- 4.8 years) who were not, however matched for underlying disease. Cortical bone indices were assessed by image analysis. Cortical width and area were similar in the two groups. However, cortical porosity, Haversian canal number, and density were higher in patients treated with glucocorticoids compared with controls (8.4 +/- 8.9% vs. 5.1 +/- 3.9%; P = 0.03) (45.9 +/- 23.2 vs. 31.9 +/- 24.4; P =0.003) (13.7 +/- 9.4 vs. 6.7 +/- 3.3/mm2; P = 0.00005). Haversian canal area did not differ significantly between groups. The mean wall width of the osteons, bone formation rate (microm2/microm/day) and mineral apposition rate (microm/day) were lower in treated patients compared to controls (48.8 +/- 7.1 microm vs. 59.8 +/- 12.9 microm; P = 0.01) (0.056 +/- 0.040 vs. 0.095 +/- 0.058; P = 0.05) and (0.59 +/- 0.12 vs. 0.75 +/- 0.11; P = 0.002). The proportion of canals with an eroded surface was lower in the treated compared with the control group, although this difference was not statistically significant. These results demonstrate that cortical porosity is increased in patients treated with long-term glucocorticoid therapy, due mainly to an increase in the number rather than size of Haversian canals. This may be because of increased bone resorption during the early stages of glucocorticoid therapy, in combination with long-term impairment of bone formation. Effects of the underlying disease on bone remodeling may also contributed to these changes and could not be excluded in the present study; since control subjects were not matched in terms of disease status.

The effects of hormone replacement therapy on cortical bone in postmenopausal women. A histomorphometric study.

Investigations of the actions of estrogen on the skeleton have mainly focused on cancellous bone and there are no reported histomorphometric studies of the effects of oestrogen on cortical bone in humans. The aim of this study was to investigate the effects of both conventional hormone replacement therapy (HRT) and high-dose oestradiol on cortical bone in postmenopausal women. Transiliac biopsies were obtained from nine postmenopausal women aged 54-71 yr before and after 2 yr (mean, 23.5 months) of conventional HRT and in seven postmenopausal women aged 52-67 yr after long-term, high-dose oestradiol implant therapy (at least 14 yr). Indices of bone turnover, remodeling, and cortical structure were assessed by image analysis. Cortical width was highest in the women treated with high-dose oestrogen therapy (2.29 +/- 0.78 mm; mean +/- SD) and lowest in untreated women (1.36 +/- 0.60 mm; P=0.014). The proportion of canals with an eroded surface was significantly lower in the high-dose oestrogen group than in women before or after conventional HRT (3.03 +/- 3.7% vs. 11.1 +/- 7.1% and 10.5 +/- 8.6%; P=0.017 and 0.05, respectively). Bone formation rate (microm2/microm/day) in untreated women was significantly higher than in the high-dose oestrogen group (0.121 +/- 0.072 vs. 0.066 +/- 0.045, respectively; P=0.05), values in women treated with conventional HRT being intermediate. Our results provide the first histomorphometric evidence in postmenopausal women of dose-dependent oestrogen-induced suppression of bone turnover in iliac crest cortical bone. There was also a trend toward higher wall width with increasing dose of oestrogen, consistent with the previously reported anabolic effect in cancellous bone.

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.

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.

Bone structure in patients with low bone mineral density with or without vertebral fractures.

Vertebral fractures (VFX) are caused by low bone mass and microstructural deterioration of bone tissue. The latter is not well defined. We investigated bone structure in transiliac biopsy specimens from 88 volunteers. Biopsy specimens were obtained at baseline in the Multiple Outcomes of Raloxifene Evaluation trail, a prospective study in osteoporotic (BMD < or = -2.5 T score) postmenopausal women without or with VFX on standardized lateral spinal radiographs. Bone biopsy specimens were embedded in methylmethacrylate (MMA). Histomorphometry was done in 8 microns (U.S.A.) or 5 microns (Europe) Goldner stained sections. Vertebral fracture status (yes/no) was the outcome variable in logistic regression models adjusted for age and biopsy specimen origin (U.S.A. vs. Europe). Patients with and without VFX (26/62) were similar regarding age (69.2 +/- 5.2 years vs. 67.3 +/- 6.7 years), bone volume (BV/TV; 17.7 +/- 4.7% vs. 19.0 +/- 5.8%), and bone surface (BS/TV; 2.7 +/- 0.6 mm2/mm3 vs. 2.8 +/- 0.6 mm2/mm3). A lower cortical thickness (C.Th; 652 +/- 267 microns vs. 822 +/- 325 microns), total strut length (TSL; 826 +/- 226 microns/mm2 vs. 922 +/- 256 microns/mm2), node-to-loop (Nd-Lp) strut length (10.1 +/- 10.3% vs. 15.0 +/- 13.6%), together with a higher node-to-terminus (Nd-Tm) strut length (45.6 +/- 9.7% vs. 39.1 +/- 9.3%) were each associated with prevalent VFX (0.01 < p < 0.10). Differences in BV/TV did not explain these associations. In conclusion, cortical thinning and disruption of trabecular lattice are possible pathogenic mechanisms in patients with VFX.

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.

Mechanism of bone loss after liver transplantation: A histomorphometric analysis.

Organ transplantation is associated with increased bone loss and high fracture risk, but the pathophysiological mechanisms responsible have not been established. We have performed a histomorphometric analysis of bone remodeling before and 3 months after liver transplantation in 21 patients (14 male, 7 female) aged 38-68 years with chronic liver disease. Eight-micrometer undecalcified sections of trans-iliac biopsies were assessed using image analysis. Preoperatively, bone turnover was low with a tendency toward reduced wall width and erosion depth. The bone formation rate increased from 0.021 +/- 0.016 (mean +/- SD) to 0.067 +/- 0.055 microm2/microm/day after transplantation (p < 0.0002) and activation frequency from 0.24 +/- 0.21/year-1 to 0.81 +/- 0. 67/year-1 (p < 0.0001). No significant change was observed in wall width, but there was a trend toward an increase in indices of resorption cavity size. There was a small increase in osteoid seam width postoperatively (p< 0.02) and decrease in mineralization lag time (p < 0.001). No significant changes in indices of cancellous bone structure were observed in the postoperative biopsies. These results demonstrate a highly significant and quantitatively large increase in bone turnover in the first 3 months after liver transplantation. Although no significant disruption of cancellous bone structure was demonstrated during the time course of the study, the observed changes in bone remodeling predispose to trabecular penetration and may thus result in long-term adverse effects on bone strength.

The effects of long-term hormone replacement therapy on bone remodeling in postmenopausal women.

Hormone replacement therapy prevents menopausal bone loss and reduces the risk of fragility fracture, but its effects on bone remodeling have not been clearly established. We studied the effects of long-term hormone replacement therapy on bone turnover and remodeling balance in 22 postmenopausal women with osteopenia or osteoporosis. Iliac crest biopsies were obtained before and after treatment (mean 23.5 months) after double tetracycline labeling and subjected to histomorphometric analysis. Post-treatment biopsies showed a significant reduction in bone formation rate at tissue level and activation frequency (p = 0.002 and 0.01, respectively). There was also a reduction in mineral appositional rate (p = 0.0002) and osteoid seam width (p = 0.01), but no significant change in mineralization lag time or osteoid maturation period. Wall width showed a significant decrease after treatment (p = 0.03) and there was a consistent trend toward a reduction in resorption cavity dimensions with a statistically significant decrease in the resorption cavity length (p = 0.03). These results confirm the previously reported reduction in bone turnover in postmenopausal women treated with hormone replacement therapy. Post-treatment biopsies also showed a reduction in resorption cavity size and a decrease in wall width, the latter possibly reflecting a compensatory change in response to the reduction in erosion depth. Our data do not provide any evidence that conventional hormone replacement therapy has anabolic effects at the level of the bone remodeling unit and indicate that its beneficial skeletal effects result from suppression of bone turnover and a reduction in the size of resorption cavities.

Effects of hormone replacement therapy on cancellous bone microstructure in postmenopausal women.

Menopausal bone loss is associated with disruption of cancellous bone architecture which has adverse mechanical effects and is believed to be irreversible. The aim of this study was to examine the effects of long-term hormone replacement therapy on cancellous bone structure in women with postmenopausal osteoporosis. Iliac crest biopsies from 22 women with osteopenia or osteoporosis were obtained before and after hormone replacement therapy (mean duration 23.5 months). Cancellous bone architecture was assessed by strut analysis, trabecular bone pattern factor, and marrow star volume. Post-treatment biopsies showed no significant changes in any of the structural indices assessed. Our results suggest that hormone replacement therapy preserves existing cancellous bone structure but provide no evidence that this treatment is able to reverse structural disruption in women with postmenopausal osteopenia or osteoporosis.

Reduced bone formation in patients with osteoporosis associated with inflammatory bowel disease.

The pathophysiology of bone loss associated with inflammatory bowel disease has not been clearly defined. In this study we have performed a detailed histomorphometric analysis of iliac crest bone obtained from 19 patients with inflammatory bowel disease in whom a diagnosis of osteoporosis had been made. Eleven subjects were receiving prednisolone at the time of their biopsy. Comparison with control values demonstrated a highly significant reduction in trabecular bone area in the patient group (p < 0.001). Wall width, adjusted appositional rate and bone formation rate were all significantly reduced in the patient group (p < 0.001) and the formation period was significantly increased (p < 0.001). Resorption cavities were slightly smaller in the patient group, differences in maximum cavity depth and cavity length achieving statistical significance (p < 0.005 and p < 0.05 respectively). The mineral appositional rate was significantly reduced in the patients with inflammatory bowel disease (p < 0.001) and the mineralization lag time significantly increased (p < 0.001); however, osteoid area, perimeter and seam width were not significantly different from controls. These results demonstrate that osteoporosis associated with inflammatory bowel disease is characterized by reduced bone formation at the cellular and tissue level; the proportionately greater change in wall width than in resorption cavity depth is consistent with a negative remodelling balance. Although none of the patients had osteomalacia as defined by the criteria of increased osteoid seam width and mineralization lag time, the higher mineralization lag time in the patient group indicates a mild mineralization defect.

Combined inter-observer and inter-method variation in bone histomorphometry.

Manual methods for the measurement of bone biopsies have largely been superseded by semi-automatic computerised techniques. Histomorphometrists often use control data obtained by other observers using different methods, thus combining inter-observer and inter-method variation. We have examined the combined effect of inter-method and inter-observer variation on measurements of bone area, osteoid perimeter, and osteoid width in iliac crest biopsies from healthy subjects, one observer using the manual grid system and the other using a semi-automated technique. Inter-observer and inter-method variation were independently determined, and the proportion of each expressed as a percentage of combined error. Our results indicate that the combination of inter-method and inter-observer variation causes significant differences in the values obtained for osteoid perimeter, whereas inter-method variation is mainly responsible for differences in osteoid width values; differences in bone area are largely due to inherent sampling variation. These variations indicate that caution is required when comparison is made with control data from other sources, especially if different techniques are employed.

Low prevalence of osteomalacia in elderly patients with hip fracture.

In order to establish the prevalence of osteomalacia in elderly patients with hip fracture, trans-iliac biopsies were obtained from 49 patients, aged 67-92 years, admitted to Cardiff Royal Infirmary with hip fracture. Undecalcified sections were quantitatively assessed and the diagnosis of osteomalacia was made when there was an increase in mean osteoid seam width (greater than 15 microns) associated with a reduction in calcification fronts (less than 60% of osteoid-covered surfaces). Osteomalacia was present in only one patient; in the remaining patients, osteoid surface extent, volume and mean seam width were within normal limits. Thus osteomalacia, when defined by rigorous histomorphometric criteria, was rare in these elderly subjects with hip fracture.

Reduced bone formation in non-steroid treated patients with rheumatoid arthritis.

The cellular basis of trabecular bone loss in rheumatoid arthritis was investigated in 45 non-steroid treated patients. Mean wall thickness, an indicator of the amount of bone formed per remodelling unit, mean interstitial bone thickness, which is related to resorption depth, and the extent of trabecular surface covered by osteoid, which reflects the number of remodelling units, were assessed in iliac crest biopsy specimens. The mean wall thickness was significantly reduced in the patient group when compared with controls matched for age and sex (mean (SD) 39.8 (5.4) v 51.6 (9.7) microns). There was no significant difference between patients and controls in the mean interstitial bone thickness (51.0 (26.4) v 61.4 (31.9) microns) or osteoid surface (16.7 (11.4) v 21.0 (10.5)%). These results show that reduced bone formation at the remodelling unit level is the predominant mechanism of bone loss in rheumatoid arthritis.

Measurement of mean trabecular plate thickness by a new computerized method.

Mean trabecular plate thickness (MTPT) has been measured by a new direct computerised method on an IBAS II image analyser in iliac crest bone from 56 normal subjects. The new method has been shown to be accurate and reproducible; apart from some initial editing of the image, it is carried out automatically without any need for observer interaction. A close correlation was found between values for MTPT obtained using the new direct method and values obtained by calculation based on area and perimeter measurements made on the IBAS II (r = 0.98).

Direct and indirect measurements of osteoid seam width in human iliac crest trabecular bone.

The osteoid thickness index, calculated from the relative osteoid volume and surface, has been compared with the mean osteoid seam width, measured directly, in iliac crest trabecular bone from 57 normal subjects and 33 patients with privational or malabsorption metabolic bone disease. In normal biopsies the osteoid thickness index overestimated mean osteoid seam width by a variable amount and the two variables were only weakly correlated (r = 0.32, P less than 0.01). In patients with hyperosteoidosis there was a stronger correlation between the osteoid thickness index and the true mean seam width (r = 0.89, P less than 0.001). Examination of the mean width of individual seams pooled from 15 randomly selected patients in each group revealed a skewed distribution with thin seams predominating, especially in normal biopsies. Median seam width was significantly lower than mean seam width in both groups studied. We conclude that osteoid thickness index is an inaccurate method of predicting the mean osteoid seam width, especially in biopsies with normal osteoid amount. Median values of osteoid seam width are more representative of average seam width, both in normal and abnormal biopsies.

Measurement of total resorption surface in iliac crest trabecular bone in man.

Total resorption surface has been measured under ordinary light and polarized light in trabecular iliac crest bone from 57 healthy subjects and 40 patients with privational or malabsorption metabolic bone disease. Results obtained with the two methods were similar, although values for total resorption surface measured under polarized light were usually lower than those obtained under ordinary light in both groups of subjects studied. This most likely reflects the greater accuracy in the microscopic identification of resorption surface under polarized light.

Value of the history in diagnosis of histological osteomalacia among Asians presenting to the NHS.

A questionnaire screening for osteomalacia was used to obtain histories from 53 consecutive Asian patients referred to a gastroenterology unit. 15 of the patients were found to have osteomalacia on subsequent histology. The questions that best distinguished between osteomalacic and non-osteomalacic patients were identified by discriminant analysis. These questions were used to derive discriminant functions by which a second sample of 45 Asian patients presenting to other parts of the National Health Service were classified as osteomalacic or non-osteomalacic. Bone histology showed false-negative and false-positive rates of 10% and 11%; the predictive value of the negative result was 97%. A short questionnaire would be a cheaper and more convenient method than biochemical screening for osteomalacia case-finding among Asian patients presenting to the NHS.

Histomorphometric analysis of dynamic parameters of trabecular bone formation in the iliac crest of normal British subjects.

Some dynamic parameters of bone formation in trabecular iliac crest bone have been measured in a group of normal British subjects of both sexes over a wide age range. There was a significant age-related decrease in mean wall thickness. When either double plus single or double only tetracycline-labeled surfaces were used to represent actively mineralizing surfaces, there was a significant age-related decrease in the bone formation rate at the basic multicellular unit level. Osteoid maturation period showed a significant age-related increase when calculated using double plus single labeled surfaces. There was no significant change with age in fractional labeled surfaces, mean osteoid seam width, bone formation rate at tissue level, or bone formation period. The mean osteoid seam width and osteoid maturation period were significantly higher in males than in females.

Value of plasma calcium, phosphate, and alkaline phosphatase measurements in the diagnosis of histological osteomalacia.

Plasma calcium and phosphate concentrations and alkaline phosphatase activities were examined retrospectively in 50 patients with histologically proven osteomalacia and 50 age- and sex-matched control subjects with normal bone histology. An abnormal plasma alkaline phosphatase activity was more useful than an abnormal plasma calcium or phosphate concentration in distinguishing between normal and osteomalacic subjects, producing a false-negative rate of 14% and a false-positive rate of 8%. False-negative and false-positive rates of 10% and 8% respectively were obtained when the presence of an abnormality in any one of the three biochemical measurements was used as a predictor of histological osteomalacia. When discriminant analysis was applied to plasma calcium, phosphate and alkaline phosphatase together a false-negative rate of 12% and a false-positive rate of 0% was obtained.Sixty-two patients in whom a diagnosis of osteomalacia was suspected were investigated prospectively, using both single biochemical abnormalities and the classification functions derived from the discriminant analysis of all three biochemical measurements to predict the presence or absence of histological osteomalacia. Plasma alkaline phosphatase activity gave false-negative and false-positive rates of 10% and 32% respectively but was a more reliable predictor of abnormal bone histology than were plasma calcium or plasma phosphate concentrations or the presence of an abnormality in any one of the three measurements. Discriminant analysis using plasma calcium, phosphate and alkaline phosphatase together produced a false-negative rate of 16% and a false-positive rate of 10%. We conclude that plasma alkaline phosphatase activity is the best single routine biochemical screening test for osteomalacia, although a high false-positive rate may occur. Direct discriminant analysis of plasma calcium, phosphate and alkaline phosphatase together provides a more sensitive method of detecting histological osteomalacia which should be useful in determining the prevalence of osteomalacia within high-risk populations.