Laurence J. Fraher

Mineral homeostasis and bone mass at diagnosis in children with acute lymphoblastic leukemia

OBJECTIVE To determine whether the osteopenia and unusual fractures observed in children with acute lymphoblastic leukemia (ALL) were related to the disease rather than to its treatment. DESIGN Prospective analysis of the bone and mineral status in 40 consecutive children with ALL seen in a pediatric tertiary-care referral center. METHODS Biochemical indicators of mineral, endocrine, and vitamin D status were measured before initiation of therapy. Bone mass was determined radiographically and by dual-photon absorptiometry of the lumbar region of the spine (L2-L4). Correlations between clinical observations, leukemia variables, bone mass, and biochemical assessment were determined. RESULTS At the time of diagnosis musculoskeletal pain was present in 36% of patients and was more common in children with CD10-positive leukemia and leukocyte counts less than 20 x 10(9) cells/L. Radiographic evidence of osteopenia and fractures was observed in 13% and 10% of children, respectively. The mean bone mineral content was normal. Bone mass measurement z scores correlated with plasma 1,25-dihydroxyvitamin D3 concentrations (r = 0.43, p < 0.05). Plasma calcium, magnesium, phosphorus, and 25-hydroxyvitamin D3 levels were normal. Low plasma osteocalcin (mean +/- SD, 1.6 +/- 1.6 nmol/L) and 1,25-dihydroxyvitamin D3 (33.4 +/- 26.4 pmol/L) values were observed. Parathyroid hormone levels were low in 14% of children. Hypercalciuria was detected in 64% of children. Urinary deoxypyridinoline was lower (p < 0.01) than in age-matched control subjects. Histomorphometric measurements of iliac bone showed abnormalities in mineralization in the biopsy specimens from three of nine children. CONCLUSION Most children with ALL have alterations in bone metabolism and bone mass when first examined. These data suggest defective mineralization as the mechanism for decreased bone mass and implicate the leukemic process as causative.

Surfactant protein A inhibits T cell proliferation via its collagen-like tail and a 210-kDa receptor

Investigation of possible mechanisms to describe the hyporesponsiveness of pulmonary leukocytes has led to the study of pulmonary surfactant and its constituents as immune suppressive agents. Pulmonary surfactant is a phospholipid-protein mixture that reduces surface tension in the lung and prevents collapse of the alveoli. The most abundant protein in this mixture is a hydrophilic molecule termed surfactant-associated protein A (SP-A). Previously, we showed that bovine (b) SP-A can inhibit human T lymphocyte proliferation and interleukin-2 production in vitro. Results presented in this investigation showed that different sources of human SP-A and bSP-A as well as recombinant rat SP-A inhibited human T lymphocyte proliferation in a dose-dependent manner. A structurally similar collagenous protein, C1q, did not block the in vitro inhibitory action of SP-A. The addition of large concentrations of mannan to SP-A-treated cultures also did not disrupt inhibition, suggesting that the effect is not mediated by the carbohydrate recognition domain of SP-A. Use of recombinant mutant SP-As revealed that a 36-amino acid Arg-Gly-Asp (RGD) motif-containing span of the collagen-like domain was responsible for the inhibition of T cell proliferation. A polyclonal antiserum directed against an SP-A receptor (SP-R210) completely blocked the inhibition of T cell proliferation by SP-A. These results emphasize a potential role for SP-A in dampening lymphocyte responses to exogenous stimuli. The data also provide further support for the concept that SP-A maintains a balance between the clearance of inhaled pathogens and protection against collateral immune-mediated damage.

Fetal endocrine responses to chronic placental embolization in the late-gestation ovine fetus * ** * **

OBJECTIVE The purpose of this study was to examine the effect of chronic fetal placental embolization on the fetal corticotropin, cortisol, and catecholamines concentrations and on myometrial contractility pattern. STUDY DESIGN Fourteen fetal sheep were studied (seven embolized, seven controls) for 10 days between 0.84 and 0.91 of gestation. Daily injections of nonradioactive microspheres were performed to decrease fetal arterial oxygen content by 30% to 35% of the preembolization value. Umbilical artery Doppler flow velocity waveforms were measured daily. RESULTS Chronic fetal placental embolization produced progressive fetal hypoxemia (p < 0.001) with changes in umbilical artery Doppler flow velocity waveforms indicative of a 25% increase in placental vascular resistance (p < 0.01). In response to chronic fetal hypoxemia there was a progressive increase in baseline fetal plasma norepinephrine concentration (p < 0.001). There was a transient fourfold to fivefold increase in baseline fetal plasma cortisol levels concomitant with a significant decrease in baseline immunoreactive corticotropin between days 7 and 9 of embolization (both p < 0.05), with a return to control values by day 10. There was a 57% increase in myometrial contracture frequency in the embolized group when compared with controls (p = 0.001). CONCLUSIONS During repetitive chronic placental damage that led to fetal hypoxemia, the fetal endocrine environment changed with time in a direction that would prevent the onset of premature activation of the hypothalamic-pituitary-adrenal axis and premature delivery.

Longitudinal assessment of growth, mineral metabolism, and bone mass in pediatric Crohn's disease.

Summary In children with inflammatory bowel disease, controversy continues about the use of long-term alternate day prednisone therapy (ADP) to suppress disease activity and to encourage appetite and growth. One possible side effect of both disease process and prednisone therapy is risk of development of osteoporosis. To evaluate this risk factor, growth, biochemical indices of mineral and vitamin D status, and bone mass were measured in nine adolescents with Crohns disease (CD) who were treated with ADP (0.3 mg/kg >3 months per year) compared with eight adolescents treated with minimal ADP exposure (<3 months per year). Single photon densitometry was used to measure bone mineral mass at the 1/3 distal radius three times over 2 years. Mean age of the 17 CD boys was 13.9 ± 2.1 years at baseline. CD patients had lower bone BMC/BW mineral content/bone width (BMC/BW) compared with age- and height-matched normal boys at all times. The difference was less when compared to height-matched normal values as CD patients were shorter than healthy reference boys. Plasma 1,25-dihydroxyvitamin D, alkaline phosphatase, and parathyroid hormone significantly increased with treatment of disease but there were no differences between treatment groups. CD patients treated with ADP had similar heights and weights at baseline and demonstrated similar linear growth over 2 years (9.1 cm/2 years) to CD patients without ADP (10.3 cm/2 years). In both groups, BMC/BW increased significiantly from year 1 to year 2, but absolute values for bone mass did not differ between the groups. These data suggest that over a 2-year treatment period male CD patients with chronic low-dose ADP exposure achieve linear growth rates and maintain bone mineralization at least as well as male CD patients who do not receive ADP.

Biochemical responses to sequential human parathyroid hormone (1–38) and calcitonin in osteoporotic patients

Parathyroid hormone (PTH) has been proposed as a skeletal activator for cyclical protocols of treatment for osteoporosis; among several potential drugs that might serve to depress the subsequent phase of osteoclastic bone resorption, calcitonin is the most selective. Twenty patients aged 50–78 years were enrolled in a study of their biochemical responses during a 14-day activation cycle with synthetic hPTH 1–38, given as a subcutaneous injection of 400 IU/day; half the patients were randomly allocated to receive a subsequent 56-day depressor cycle with calcitonin in a dose of 100 U/day, while the remainder received no further treatment. All patients received an initial 24-h intravenous infusion of hPTH 1–38 (0.5 U/kg/h) to evaluate the PTH-dependent renal synthesis of 1,25(OH)2D. Serum calcium increased from 2.20 ± 0.07 mmol/l to 2.56 ± 0.16 (P < 0.005) during PTH infusion, but was not significantly different from baseline during intermittent treatment. Baseline concentrations of serum 1,25(OH)2D were 22.8 ± 8.2 pg/ml, increased to 52.2 ± 25.1 (P < 0.005) during infusion and remained significantly higher than baseline after 14 days intermittent therapy (33.1 ± 19.4, P < 0.05). Gastrointestinal absorption of 45Ca, as represented by alpha (peak fractional absorption/h), increased from 0.397 ± 0.173 to 0.552 ± 0.210 (P < 0.01) during hPTH 1–38 therapy and was moderately correlated with the increment in serum 1,25(OH)2D levels (r = 0.5, P < 0.03). Daily calcium excretion was significantly increased above baseline during hPTH 1–38 therapy, but there were no correlations between changes in urinary calcium, alpha or serum 1,25(OH)2D levels. Baseline fasting urinary excretion of OH-proline increased during hPTH 1–38 treatment from 30.5 ± 13.9 mol/mmol creatinine to 43.4 ± 17.5 immediately after hPTH 1–38 infusion (P < 0.025), and mean excretion was persistently higher than baseline during intermittent treatment; the increased urine calcium and OH-proline excretion are consistent with PTH-induced activation of bone resorption. Serum alkaline phosphatase and osteocalcin levels increased significantly during a 90-day period of observation after the hPTH 1–38 cycle, which is consistent with increased osteoblast activity in association with coupled bone formation. In those patients subsequently receiving calcitonin, biochemical changes in serum alkaline phosphatase and osteocalcin were significantly blunted in comparison to the findings seen in those treated with hPTH 1–38 alone; urine OH-proline excretion was 25% lower at the end of calcitonin therapy, compared to the group treated with hPTH 1–38 alone. These preliminary findings may form the basis for designing further ADFR protocols utilizing PTH peptides and calcitonin to treat osteoporosis.

The addition of a raloxifene analog (LY117018) allows for reduced PTH(1-34) dosing during reversal of osteopenia in ovariectomized rats.

To test the hypothesis that an antiresorptive agent might reduce the dosing requirement for an anabolic drug during reversal of osteopenia due to estrogen deficiency, the following experiment was conducted in 6‐month‐old female rats. Ovariectomy or sham surgery was performed and the following six experimental groups were studied. Untreated (SHAM) or ovariectomized (OVX) animals served as control groups. Four weeks post‐OVX, osteopenic rats (now 7 months old), were treated in one of four experimental protocols: human parathyroid hormone (hPTH(1–34), 80 μg/kg/day, given by subcutaneous injection 5 days/week; a selective estrogen receptor modulator (SERM), raloxifene analog LY117018 (RA), 3 mg/kg/day, given by gavage 5 days/week; and two combinations of LY117018 at the same dose and frequency with hPTH(1–34) (same dose, 5 times/week) and a reduced dosing interval of hPTH(1–34) (same dose, 2 times/week). After 12 weeks of treatment, the four experimental groups were sacrificed at age 10 months. SHAM and OVX controls were also studied at 7 and 10 months of age. Bone mineral density (BMD) was measured by dual‐energy X‐ray absorptiometry at four skeletal sites: two mixed cortical/trabecular sites (femur and tibia) and two predominantly trabecular sites (lumbar spine and pelvis). The differences in BMD were consistent at all four sites. RA alone maintained BMD at all skeletal sites, but the results were not significantly improved over OVX controls, at age 10 months. hPTH(1–34) injections given 5 days/week resulted in BMD increments significantly higher than in either OVX or SHAM controls (p < 0.001). While the RA did not enhance the anabolic effects of full doses of hPTH(1–34), the addition of RA treatment to twice‐weekly hPTH(1–34) dosing resulted in BMD increments at all four skeletal sites that were similar to the more intensive anabolic regimen of hPTH(1–34) therapy given 5 times/week. Therefore, an antiresorptive agent such as SERMs may potentially reduce the pharmacologic doses of PTH needed to reverse estrogen deficiency‐induced osteopenia.

Assessment of Maintenance Therapy With Reduced Doses of PTH(1-34) in Combination With a Raloxifene Analogue (LY117018) Following Anabolic Therapy in the Ovariectomized Rat

This experiment was designed to evaluate the ability of a raloxifene analogue (RA), LY117018, with or without reduced dosing of human parathyroid hormone (hPTH)(1-34) to maintain gains in bone mass after a fully anabolic treatment regimen given to aging osteopenic rats. Six-month-old rats were ovariectomized (ovx) or sham-operated (sham). After 1 month, ovx rats were treated with an anabolic regimen consisting of subcutaneous hPTH(1-34) 80 microg/kg/day and oral raloxifene 3 mg/kg/day, each given 5 days/week for 3 months. Thereafter, the treated ovx rats went on to an 8 week maintenance phase of treatment with either RA alone at the same dose, hPTH(1-34) at a reduced dosing interval (twice a week), or a combination of the two. Bone mineral density (BMD) was measured ex vivo at four skeletal sites, lumbar spine (L2-4), proximal hemipelvis, whole femur, and tibia, by dual-energy X-ray densitometry. All four sites showed a similar pattern of response. After the 3 month anabolic phase, the sham group had significantly higher BMD values than ovx rats at all skeletal sites (p < or = 0.002). The ovx rats treated with PTH + RA during the anabolic phase of the protocol had significantly higher BMD than the sham group in the femur, tibia, and spine (p < or = 0.02) and higher but not significantly different values in the pelvis. Following the 2 month maintenance phase, comparisons were made with the PTH-RA group at the end of the anabolic phase. Decrements in BMD were seen in all three maintenance therapy groups, but they were not statistically significant in the RA plus reduced PTH dose group. However, reduced hPTH(1-34) dosing and RA alone resulted in significant reductions of bone mass measurements at several skeletal sites during the maintenance phase. We conclude that the raloxifene analogue LY117018 may be useful in maintaining bone mass in aging ovx rats following anabolic therapy with hPTH(1-34) and raloxifene analogue, but that this strategy only allows for dose reduction of hPTH(1-34) rather than its discontinuation.

Effect of intermittent umbilical cord occlusion on fetal respiratory activity and brain adenosine in late-gestation sheep

It was hypothesized that intermittent umbilical cord occlusion (UCO) would inhibit ovine fetal breathing movements (FBM) in association with increased cerebral adenosine levels. To test this hypothesis, on two successive days during late gestation (133-134 days; term = 146 days), microdialysis samples were collected from the brains of 10 chronically instrumented fetal sheep during 2-h periods of complete UCO induced every 30 min (Day 1: 2-min UCOs; Day 2: 4-min UCOs). Control fetuses (n = 10) underwent no UCO. Tracheal pressure was measured throughout. This regimen resulted in a decrease in fetal arterial PO2 (PaO2) during each UCO to 7.3 +/- 0.8 mmHg (P<0.01; Day 1) and 8.4 +/- 1.1 mmHg (P<0.01; Day 2). Throughout each UCO period, fetal arterial pH (pHa) decreased to 7.28 +/- 0.02 (P<0.01; Day 1) and 7.11 +/- 0.07 (P<0.01; Day 2). The hourly incidence of FBM decreased significantly only on Day 2, from 38.6 +/- 4.1% to 4.1 +/- 1.6% (P<0.01). The frequency of deep isolated inspiratory efforts increased from 4.7 +/- 2.0 h(-1) to 17.6 +/- 6.1 h(-1) (P<0.05; Day 1) and from 2.2 +/- 0.9 h(-1) to 33.6 +/- 4 h(-1) (P<0.01; Day 2). The amplitude of both FBM and deep isolated inspiratory efforts increased during the UCO periods on both days. The concentration of cerebral extracellular fluid (ECF) adenosine during UCO increased by 219 +/- 215% (P<0.05; Day 1) and 172 +/- 107% (P<0.05; Day 2) over the baseline periods. In conclusion, the severity of the inhibitory effect of repeated UCO on FBM depends, in part, on the length of the occlusions. The inhibition of FBM during intermittent UCO may be mediated by the increase in ECF adenosine in the fetal brain. Furthermore, FBM and deep isolated inspiratory efforts appear to be regulated by different mechanisms.

Immunoreactive parathyroid hormone-related protein: its association with preterm labor

OBJECTIVE Parathyroid hormone-related protein (PTHrP) is a 141 amino acid protein which contains a 1-36 N-terminal domain resembling parathyroid hormone which has smooth muscle relaxant activity and a mid (67-86) domain which reportedly alters placental calcium transport. Using specific antibodies to these regions of PTHrP, the objective of this study was to determine changes in the levels and localization of the peptides in placenta and membranes that might be indicative of their biological activity and role during term and preterm labor. STUDY DESIGN Placenta and fetal membranes were collected from patients with preterm delivery (PTL) (n = 16), term cesarean section in the absence of labor (n = 10) and term vaginal delivery (n = 5). Immunohistochemistry was performed with specific antisera visualized by the avidin-biotin peroxidase method and the staining intensity was quantified with an image analysis system MCID. RESULTS Immunoreactive (ir)-PTHrP(1-34) and ir-PTHrP(67-86) were localized to the amnionic epithelium chorionic trophoblasts, decidual cells and placental syncytiotrophoblast. Intense immunostaining was observed for ir-PTHrP(67-86) but not for ir-PTHrP(1-34) in the endothelial lining of the villous capillaries. Ir-PTHrP(1-34) staining was lower in placenta and fetal membranes of PTL patients compared with term cesarean section in the absence of labor (P < 0.05 Mann-Whitney test). In contrast, there was no difference in ir-PTHrP [67-86] staining intensity between delivery categories. CONCLUSION These results showing differential localization of PTHrP(1-34) and PTHrP(67-86) suggest cell specific processing of PTHrP precursor in the human placenta. Moreover, the changes in ir-PTHrP(1-34) but not ir-PTHr(67-86) with labor are indicative of a particular role for this peptide in the delivery process.

Effects of alveolar surfactant aggregates on T-lymphocyte proliferation

The effects of alveolar large aggregate (LA) and small aggregate (SA) surfactant subfractions isolated from healthy adult rats on mitogen-stimulated proliferative responses of human peripheral blood mononuclear cells (PBMC) was examined. Various concentrations of total surfactant suppressed proliferation of stimulated lymphocytes by up to 95% of mitogen-stimulated cells alone. LA subfractions of total surfactant had no effect on proliferation, whereas SA significantly enhanced the lymphocyte proliferation at lower concentrations (7.8 microg/ml) compared to mitogen-stimulated cells alone. Higher concentrations of SA (62.5 microg/ml) inhibited lymphocyte proliferation. This concentration-dependent effect of SA on proliferation of PBMC was also present when cells were stimulated with various lectins including anti-CD3, concanavalin A and phytohemagglutinin. Analysis of the supernatant of mitogen-stimulated cell cultures treated with inhibitory concentrations of SA showed decreased amounts of interleukin (IL)-2, compared to cells alone, which could be reversed by adding exogenous IL-2 to the cell cultures with the SA. These results suggest that alveolar surfactant subfractions have distinct functions within the alveoli, both biophysically and with respect to their effects on the hosts immunomodulatory responses.