Hypophosphatasia in adolescents and adults: overview of diagnosis and treatment.

This article provides an overview of the current knowledge on hypophosphatasia-a rare genetic disease of very variable presentation and severity-with a special focus on adolescents and adults. It summarizes the available information on the many known mutations of tissue-nonspecific alkaline phosphatase (TNSALP), the epidemiology and clinical presentation of the disease in adolescents and adults, and the essential diagnostic clues. The last section reviews the therapeutic approaches, including recent reports on enzyme replacement therapy (EnzRT).

The aetiology of rickets-like lower limb deformities in Malawian children.

UNLABELLED: Debilitating rickets-like lower limb deformities are common in children throughout the world, particularly in Malawi, Africa where the causes are unknown. We have identified that Blount disease and calcium deficiency rickets are the likely causes of these deformities and propose calcium supplementation as a potential treatment of Malawian rickets. INTRODUCTION: Surgical correction of rickets-like lower limb deformities is the most common paediatric operation performed at Beit Cure Orthopaedic Hospital, Malawi. The aim of this study was to investigate the aetiology of these deformities. METHODS: Children with a tibio-femoral angle of deformity >20° were enrolled (n = 42, 3.0-15.0 years). Anthropometric and early life and well-being data were collected. Early morning serum and urine samples were collected on the morning of the operation for markers of calcium and phosphate homeostasis. Knee radiographs were obtained, and the children were diagnosed with either Blount (BD, n = 22) or evidence of rickets disease (RD, n = 20). As BD is a mechanical rather than metabolic disease, BD were assumed to be biochemically representative of the local population and thus used as a local reference for RD. RESULTS: There were no differences in anthropometry or early life experiences between BD and RD. Parathyroid hormone (PTH), 1,25-dihydroxyvitamin D, total alkaline phosphatase and urinary phosphate were significantly higher and serum phosphate, 25-hydroxyvitamin D (25OHD) and tubular maximal reabsorption of phosphate significantly lower in RD than BD. There was no difference in serum calcium, fibroblast growth factor 23 or markers of iron status between groups. All children had 25OHD > 25 nmol/L. CONCLUSIONS: Vitamin D deficiency is not implicated in the aetiology of RD or BD in Malawian children. The cause of RD in Malawi is likely to be dietary calcium deficiency leading to elevated PTH resulting in increased losses of phosphate from the bone and glomerular filtrate. The causes of BD remain unclear; there was no evidence in support of previously suggested risk factors such as being overweight or starting to walk early. Prior to surgical intervention, supplementation with calcium should be considered for children with RD.

Osteogenesis imperfecta: Ultrastructural and histological findings on examination of skin revealing novel insights into genotype-phenotype correlation.

Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders of bone formation, resulting in low bone mass and an increased propensity to fracture. Over 90% of patients with OI have a mutation in COL1A1/COL1A2, which shows an autosomal dominant pattern of inheritance. In-depth phenotyping and in particular, studies involving manifestations in the skin connective tissue have not previously been undertaken in OI. The aims of the study were to perform histological and ultrastructural examination of skin biopsies in a cohort of patients with OI; to identify common and distinguishing features in order to inform genotype-phenotype correlation; and to identify common and distinguishing features between the different subtypes of OI. As part of the RUDY (Rare Diseases in Bone, Joints and/or Blood Vessels) study, in collaboration with the NIHR Rare Diseases Translational Research Collaboration, we undertook a national study of skin biopsies in patients with OI. We studied the manifestations in the skin connective tissue and undertook in-depth clinical and molecular phenotyping of 16 patients with OI. We recruited 16 patients: analyses have shown that in type 1 collagen mutation positive patients (COL1A1/ COL1A2) (n-4/16) consistent findings included: variable collagen fibril diameter (CFD) and presence of collagen flowers. Histological examination in these patients showed an increase in elastic fibers that are frequently fragmented and clumped. These observations provide evidence that collagen flowers and CFD variability are consistent features in OI due to type 1 collagen defects and reinforce the need for accurate phenotyping in conjunction with genomic analyses.

Acute bone response to whole body vibration in healthy pre-pubertal boys.

The skeleton responds to mechanical stimulation. We wished to ascertain the magnitude and speed of the growing skeleton's response to a standardised form of mechanical stimulation, vibration. 36 prepubertal boys stood for 10 minutes in total on one of two vibrating platforms (high (>2 g) or low (<1 g) magnitude vibration) on either 1, 3 or 5 successive days (n=12 for each duration); 15 control subjects stood on an inactive platform. Blood samples were taken at intervals before and after vibration to measure bone formation (P1NP, osteocalcin) and resorption (CTx) markers as well as osteoprotegerin and sclerostin. There were no significant differences between platform and control groups in bone turnover markers immediately after vibration on days 1, 3 and 5. Combining platform groups, at day 8 P1NP increased by 25.1% (CI 12.3 to 38.0; paired t-test p=0.005) and bone resorption increased by 10.9% (CI 3.6 to 18.2; paired t-test p=0.009) compared to baseline. Osteocalcin, osteoprotogerin and sclerostin did not change significantly. The growing skeleton can respond quickly to vibration of either high or low magnitude. Further work is needed to determine the utility of such "stimulation-testing" in clinical practice.

Parathyroid hormone changes in infants investigated for inflicted injury; an observational retrospective single centre cohort study.

BACKGROUND: Biochemical and haematological testing is recommended in the United Kingdom when inflicted injury is suspected. We examined the associations of test results with radiologically-confirmed fracture(s), and between test results, in a large retrospective observational cohort. METHODS: Infants up to age two years presenting with suspected inflicted injury, without clinically or radiologically apparent bone disease, and where a skeletal survey was undertaken during the period 1st August 2013 to 31st December 2020, were included. Biochemical parameters: corrected calcium (cCa); phosphate (P); alkaline phosphatase (ALP); parathyroid hormone (PTH); 25-hydroxyvitamin D (25D); and haematological parameters: haemoglobin (Hb); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin content (MCHC); mean corpuscular volume (MCV); platelet count were collated together with the results of the radiological assessments. FINDINGS: Of 332 eligible infants (190 male), 142 (84 male) had fracture(s) and/or intracranial injury. Mean PTH in the non-fracture group (n measured 50/190) was 27.3 ng/l; in those with intracranial injury alone (n measured 9/23) was 39.4 ng/l; in those with fracture alone (n measured 62/84) was 45.0 ng/l; and in those with fracture and intracranial injury (n measured 20/35) 51.8 ng/l. F-test of multiple means = 0.0369. There was no difference in 25D between the groups. INTERPRETATION: PTH was raised in infants who had fracture(s), intracranial injury or both. A single raised PTH may not necessarily be an indicator of prior disturbed skeletal health in these circumstances. The relevance of vitamin D status and interpretation of data from biochemical testing should be informed by the overall presentation in suspected inflicted injury cases. A single raised PTH may be a consequence of the child's injuries rather than prior disturbed bone health.

Monitoring Skull Base Abnormalities in Children with Osteogenesis Imperfecta - Review of Current Practice and a Suggested Clinical Pathway.

OBJECTIVES: In the context of a lack of national consensus on the benefits of skull base imaging in children with osteogenesis imperfecta (OI), this study aims to analyse and correlate the clinical symptoms and radiological images of children with severe OI. METHODS: A retrospective case notes and image analysis was carried out on children with complex OI between 2012 and 2018 at a specialist tertiary centre. Data were collected on patient demographic factors, clinical data, imaging findings (presence of Wormian bones, platybasia, basilar impression (McGregor's technique) and basilar invagination (McRae's technique)), and clinical features at the time of imaging. RESULTS: Of the 127 patients in the OI database, 94 were included. A total of 321 radiographs, 21 CT scans and 39 MRI scans were analysed. Average frequency of radiographs was 8 per 10 years. Of the 94 patients, 58 (62%), 10 (11%), 1 (1%) demonstrated platybasia, basilar impression, and basilar invagination, respectively. Of the radiographs analysed, platybasia, basilar impression, basilar invagination, and the presence of Wormian bones, could not be evaluated in 71 (22.3%), 48 (15.2%), 61 (19.5%) and 28 (9.4%) radiographs respectively (due to poor positioning, anatomical abnormalities, and poor image quality). Of the 140 radiographs with platybasia, 17 (12%) also demonstrated basilar impression compared to only 3 (2.9%) out of the 99 without platybasia (p = 0.03). No significant associations were seen between the presence of Wormian bones and basilar impression. Of the 39 MRIs, additional information on CSF flow rate, spinal cord signal and cerebellar morphology was reported in 14 (36%). There was a lack of concordance between MRI and matched radiographs in 7.1% (1/14) and 36% (5/14) for platybasia and basilar impression respectively, with full concordance for basilar invagination. Fewer than 5% had positive clinical symptoms/signs at the time of imaging; 2% (7/321) had macrocephaly, 0.6% (2/321) headache, all other neurological features were absent). Clinical features were not documented in >85% of patients. CONCLUSION: The apparent low prevalence of clinical symptoms and signs and of radiologically identified cranio-cervical abnormalities, suggests that current levels of serial imaging may be excessive. Until larger prospective studies clarify these issues, we suggest a clinical pathway for base of skull imaging which proposes a risk stratification approach to radiographic frequency and suggests parameters for proceeding to MRI.

High bone mass phenotype in a cohort of patients with Osteogenesis Imperfecta caused due to BMP1 and C-propeptide cleavage variants in COL1A1.

OBJECTIVES: Osteogenesis Imperfecta (OI) is a heterogeneous condition mainly characterised by bone fragility; extra-skeletal features in OI include blue sclerae, dentinogenesis imperfecta, skin laxity and joint hyper-extensibility. Most patients with OI are thought to have a low bone mass but contrary to expectations there are certain forms of OI with high bone mass which this study explores in further detail. METHOD: A cohort of n = 6 individuals with pathogenic variants in BMP1 and the C-propeptide cleavage variants in COL1A1 were included in this study. Detailed clinical and radiological phenotyping was done and correlated with genotype to identify patterns of clinical presentation and fracture history in this cohort of patients. This data was compared to previously reported literature in this group. RESULTS: 2 patients with BMP1 and 4 patients with pathogenic variants in C-propeptide region in COL1A1 were deep-phenotyped as part of this study and 1 patient with C-propeptide variant in COL1A1, showed low bone mineral density. In those with an elevated bone mineral density, this became even more apparent on bisphosphonate therapy. Patients in this cohort had variable clinical presentation ranging from antenatal presentation to more of an insidious course resulting in later confirmation of genetic diagnosis up to 19 years of age. CONCLUSIONS: Patients with pathogenic variants in the C-propeptide region of COL1A1/A2 and BMP1 appear to have a high bone mass phenotype with increased sensitivity to bisphosphonate therapy. It is important to closely monitor patients with these genotypes to assess their response to therapy and tailor their treatment regime accordingly.

Special report on the 2007 adult and pediatric Position Development Conferences of the International Society for Clinical Densitometry.

The International Society for Clinical Densitometry (ISCD) conducts Position Development Conferences (PDCs) for the purpose of establishing standards and guidelines in the field of bone densitometry. Topics for consideration are selected according to clinical relevance, a perceived need for standardization, and the likelihood of achieving agreement. Questions regarding nomenclature, indications, acquisition, analysis, quality control, interpretation, and reporting of bone density tests for each topic area are assigned to task forces for a comprehensive review of the scientific literature. The findings of the review and recommendations are then presented to an international panel of experts at the PDC. The expert panel votes on potential Official Positions for appropriateness, necessity, quality of the evidence, strength of the recommendation, and applicability (worldwide or variable according to local requirements). Recommendations that are approved by the ISCD Board of Directors become Official Positions. The first Pediatric PDC was 20-21 June 2007 in Montreal, QC, Canada. The most recent Adult PDC was held 20-22 July 2007, in Lansdowne, VA, USA. This Special Report summarizes the methodology of the ISCD PDCs and presents selected Official Positions of general interest.

Latent Profiles of Macronutrient Density and their Association with Mobility Limitations in an Observational Longitudinal Study of Older U.S. Adults.

OBJECTIVES: Our first objective was to estimate empirically-derived subgroups (latent profiles) of observed carbohydrate, protein, and fat intake density in a nationally representative sample of older U.S. adults. Our second objective was to determine whether membership in these groups was associated with levels of, and short term change in, physical mobility limitations. DESIGN AND SETTING: Measures of macronutrient density were taken from the 2013 Health Care and Nutrition Study, an off-year supplement to the Health and Retirement Study, which provided indicators of physical mobility limitations and sociodemographic and health-related covariates. PARTICIPANTS: 3,914 community-dwelling adults age 65 years and older. MEASUREMENTS: Percent of daily calories from carbohydrate, protein, and fat were calculated based on responses to a modified Harvard food frequency questionnaire. Latent profile analysis was used to describe unobserved heterogeneity in measures of carbohydrate, protein, and fat density. Mobility limitation counts were based on responses to 11 items indicating physical limitations. Poisson regression models with autoregressive controls were used to identify associations between macronutrient density profile membership and mobility limitations. Sociodemographic and health-related covariates were included in all Poisson regression models. RESULTS: Four latent subgroups of macronutrient density were identified: "High Carbohydrate", "Moderate with Fat", "Moderate", and "Low Carbohydrate/High Fat". Older adults with the lowest percentage of daily calories coming from carbohydrate and the greatest percentage coming from fat ("Low Carbohydrate/High Fat") were found to have greater reported mobility limitations in 2014 than those identified as having moderate macronutrient density, and more rapid two-year increases in mobility limitations than those identified as "Moderate with Fat" or "Moderate". CONCLUSION: Older adults identified as having the lowest carbohydrate and highest fat energy density were more likely to report a greater number of mobility limitations and experience greater increases in these limitations than those identified as having moderate macronutrient density. These results suggest that the interrelation of macronutrients must be considered by those seeking to reduce functional limitations among older adults through dietary interventions.

Compound heterozygous variants in NBAS as a cause of atypical osteogenesis imperfecta.

BACKGROUND: Osteogenesis imperfecta (OI), the commonest inherited bone fragility disorder, affects 1 in 15,000 live births resulting in frequent fractures and reduced mobility, with significant impact on quality of life. Early diagnosis is important, as therapeutic advances can lead to improved clinical outcome and patient benefit. REPORT: Whole exome sequencing in patients with OI identified, in two patients with a multi-system phenotype, compound heterozygous variants in NBAS (neuroblastoma amplified sequence). Patient 1: NBAS c.5741G>A p.(Arg1914His); c.3010C>T p.(Arg1004*) in a 10-year old boy with significant short stature, bone fragility requiring treatment with bisphosphonates, developmental delay and immunodeficiency. Patient 2: NBAS c.5741G>A p.(Arg1914His); c.2032C>T p.(Gln678*) in a 5-year old boy with similar presenting features, bone fragility, mild developmental delay, abnormal liver function tests and immunodeficiency. DISCUSSION: Homozygous missense NBAS variants cause SOPH syndrome (short stature; optic atrophy; Pelger-Huet anomaly), the same missense variant was found in our patients on one allele and a nonsense variant in the other allele. Recent literature suggests a multi-system phenotype. In this study, patient fibroblasts have shown reduced collagen expression, compared to control cells and RNAseq studies, in bone cells show that NBAS is expressed in osteoblasts and osteocytes of rodents and primates. These findings provide proof-of-concept that NBAS mutations have mechanistic effects in bone, and that NBAS variants are a novel cause of bone fragility, which is distinguishable from 'Classical' OI. CONCLUSIONS: Here we report on variants in NBAS, as a cause of bone fragility in humans, and expand the phenotypic spectrum associated with NBAS. We explore the mechanism underlying NBAS and the striking skeletal phenotype in our patients.

Male sex and low physical activity are associated with reduced spine bone mineral density in survivors of childhood acute lymphoblastic leukemia.

Survivors of acute lymphoblastic leukemia (ALL) are at risk of osteoporosis and obesity. We studied bone mineral density (BMD), percent of fat mass (%FM), and activity levels in survivors of ALL treated without radiotherapy. Lumbar and total areal BMD (g/cm2) and %FM were measured in 28 survivors (aged 5.7-14.7 years) of childhood ALL by dual-energy X-ray absorptiometry (DXA) scan (GE Lunar, Prodigy) an average of 5 years after completion of chemotherapy (UK Medical Research Council randomized trial protocol XI [UKALL XI]). One boy fractured his arm during treatment. Apparent volumetric lumbar BMD (BMD(vol); g/cm3) was calculated and %FM was adjusted for sex and age (%FM(adj)). Physical activity was measured by accelerometer and questionnaire. The results were compared with 28 sex- and age-matched healthy controls. Total body and lumbar areal BMD (g/cm2) were not different between the ALL group and the control group. However, mean lumbar BMD(vol) in survivors of ALL was significantly lower than in controls (0.303 +/- 0.036 g/cm3 vs. 0.323 +/- 0.03 g/cm3; p < 0.01), which mostly was caused by the difference in boys (0.287 +/- 0.032 g/cm3 vs. 0.312 +/- 0.027 g/cm3; p < 0.05). Weekly activity score by questionnaire was significantly lower in the ALL group than in the control group (geometric mean 50 vs. geometric mean 74; p < 0.05). Male gender, low activity levels and an intravenous (iv) high dose of methotrexate were associated with low lumbar BMD(vol). Patients who received an iv high dose of methotrexate (n = 18) had significantly higher %FM(adj) than those with intrathecal methotrexate only (n = 10; 141 +/- 70% vs. 98 +/- 37%;p < 0.05). In conclusion, male survivors of childhood ALL have reduced lumbar BMD(vol), whereas no such difference was seen in girls. Overall, survivors of ALL were physically less active than their healthy controls and lower activity correlated with lower lumbar BMD(vol) and higher %FM(adj).

Type V osteogenesis imperfecta: a new form of brittle bone disease.

Osteogenesis imperfecta (OI) is commonly subdivided into four clinical types. Among these, OI type IV clearly represents a heterogeneous group of disorders. Here we describe 7 OI patients (3 girls), who would typically be classified as having OI type IV but who can be distinguished from other type IV patients. We propose to call this disease entity OI type V. These children had a history of moderate to severe increased fragility of long bones and vertebral bodies. Four patients had experienced at least one episode of hyperplastic callus formation. The family history was positive for OI in 3 patients, with an autosomal dominant pattern of inheritance. All type V patients had limitations in the range of pronation/supination in one or both forearms, associated with a radiologically apparent calcification of the interosseous membrane. Three patients had anterior dislocation of the radial head. A radiodense metaphyseal band immediately adjacent to the growth plate was a constant feature in growing patients. Lumbar spine bone mineral density was low and similar to age-matched patients with OI type IV. None of the type V patients presented blue sclerae or dentinogenesis imperfecta, but ligamentous laxity was similar to that in patients with OI type IV. Levels of biochemical markers of bone metabolism generally were within the reference range, but serum alkaline phosphatase and urinary collagen type I N-telopeptide excretion increased markedly during periods of active hyperplastic callus formation. Qualitative histology of iliac biopsy specimens showed that lamellae were arranged in an irregular fashion or had a meshlike appearance. Quantitative histomorphometry revealed decreased amounts of cortical and cancellous bone, like in OI type IV. However, in contrast to OI type IV, parameters that reflect remodeling activation on cancellous bone were mostly normal in OI type V, while parameters reflecting bone formation processes in individual remodeling sites were clearly decreased. Mutation screening of the coding regions and exon/intron boundaries of both collagen type I genes did not reveal any mutations affecting glycine codons or splice sites. In conclusion, OI type V is a new form of autosomal dominant OI, which does not appear to be associated with collagen type I mutations. The genetic defect underlying this disease remains to be elucidated.

Bone mineralization and turnover in preterm infants at 8-12 years of age: the effect of early diet.

Our previous studies raised two hypotheses: first that suboptimal early nutrition and second that human milk have enhancing effects on long-term bone mineralization. To test these hypotheses experimentally, we measured whole body and regional bone mineral content (BMC) and bone mineral density (BMD), using dual-energy X-ray absorptiometry and single-photon absorptiometry, and bone turnover at 8-12 years in 244 preterm children (128 boys) who participated in a prospective randomized study of diet during the neonatal period. Dietary randomizations studied were: banked human milk (BBM, n = 87) versus preterm formula (PTF, n = 96) as the sole diet or as a supplement to mother's expressed breast milk (EBM); PTF (n = 25) versus term formula (TF, n = 36) as sole diet. Ninety-five term children of the same age were also studied. First, preterm children were shorter and lighter than term children (height SD scores -0.49 (1.1) vs. +0.22 (0.9), weight SD scores -0.41 (1.2) vs. +0.38 (1.0)) and had significantly lower whole-body BMC than their peers; decrements were also evident at some regional sites. These differences disappeared after adjusting for bone area, body size, and pubertal status. Second, children previously randomized to BBM versus PTF or TF versus PTF showed no significant differences in anthropometry, BMC, BMD, or osteocalcin (OC). Third, there was no independent effect of the proportion of EBM on BMC, BMD, or OC and no interaction between randomized diet and the amount of EBM received. Fourth, plasma OC was significantly higher in preterm children than in term children (12.4 vs. 11.0 ng/ml, p < 0.005) and in preterm children who had received a low-nutrient (BBM/TF) as opposed to a high-nutrient diet (PTF) during the neonatal period (12. 9 vs. 11.9 ng/ml, p = 0.03). In conclusion, preterm children are shorter, lighter, and have lower bone mass than their peers at age 8-12 years. The lower BMC is, however, appropriate for the bone and body size achieved. Despite large differences in early mineral intake, early diet does not affect bone mass in preterm children, and fresh human milk has no specific effect. However, poor nutrition during the neonatal period may result in higher bone formation rates during childhood.

Pamidronate treatment of severe osteogenesis imperfecta in children under 3 years of age.

Severe osteogenesis imperfecta (OI) is a hereditary disorder characterized by increased bone fragility and progressive bone deformity. Cyclical pamidronate infusions improve clinical outcome in children older than 3 yr of age with severe OI. Because earlier treatment may have potential to prevent deformities and improve functional prognosis in young children, we studied nine severely affected OI patients under 2 yr of age (2.3-20.7 months at entry) for a period of 12 months. Pamidronate was administered i.v. in cycles of 3 consecutive days. Patients received four to eight cycles during the treatment period, with cumulative doses averaging 12.4 mg/kg. Clinical changes were evaluated regularly during treatment, and radiological changes were assessed after 6-12 months of treatment. The control group consisted of six age-matched, severely affected OI patients, who had not received pamidronate treatment. During treatment bone mineral density (BMD) increased between 86-227%. The deviation from normal, as indicated by the z-score, diminished from -6.5 +/- 2.1 to -3.0 +/- 2.1 (P < 0.001). In the control group the BMD z-score worsened significantly. Vertebral coronal area increased in all treated patients (11.4 +/- 3.4 to 14.9 +/- 1.8 cm2; P < 0.001), but decreased in the untreated group (P < 0.05). In the treated patients, fracture rate was lower than in control patients (2.6 +/- 2.5 vs. 6.3 +/- 1.6 fractures/year; P < 0.01). No adverse side-effects were noted, apart from the well known acute phase reaction during the first infusion cycle. Pamidronate treatment in severely affected OI patients under 3 yr of age is safe, increases BMD, and decreases fracture rate.

Perinatal metabolism of vitamin D.

During pregnancy, maternal serum concentrations of 25-hydroxyvitamin D, the circulating form of vitamin D, correlate with dietary vitamin D intake. Maternal serum concentrations of 1,25-dihydroxyvitamin D, the hormonal circulating and active form of vitamin D, are elevated during pregnancy; 1,25-dihydroxyvitamin D is synthesized mainly by the decidual cells of the placenta and allows for increased calcium absorption. The fetus is entirely dependent on the mother for its supply of 25-hydroxyvitamin D, which is believed to cross the placenta. Hypocalcemia and increased parathyroid hormone secretion induce synthesis of 1,25-dihydroxyvitamin D after birth in both full-term and preterm neonates. Nevertheless, serum concentrations of 25-hydroxyvitamin D are a rate-limiting factor in the synthesis of 1,25-dihydroxyvitamin D. In vitamin D-replete infants, circulating 1,25-dihydroxyvitamin D concentrations are higher than those observed in older infants. In countries where dairy products are not routinely supplemented with vitamin D, maternal vitamin D supplementation during pregnancy is necessary. However, there is no indication for the use of pharmacologic doses of vitamin D or its metabolites in the perinatal period.

Randomized outcome trial of human milk fortification and developmental outcome in preterm infants.

Despite potential benefits, human milk may fail to meet preterm infants' nutrient requirements. We tested the hypothesis that fortified breast milk, fed alone or with preterm formula, would improve neurodevelopment and growth at 18-mo follow-up without adverse short-term clinical or biochemical consequences. Two hundred seventy-five preterm infants from two medical centers (birth weight < 1850 g; mean gestation 29.8 +/- 2.7 wk) whose mothers chose to provide breast milk were randomly assigned to receive for a mean of 39 d a multinutrient fortifier or control supplement containing phosphate and vitamins. Breast milk comprised 47.6% and 46.4% of enteral intake in fortified and control groups, respectively; preterm formula supplements were used when insufficient breast milk was available. Overall, there were no significant growth advantages with fortification; although, when breast milk exceeded 50% of intake, fortification promoted faster weight gain (an advantage of 1.6 g.kg-1.d-1; 95% CI: 0.1, 3.1; P < 0.05). Compared with control infants, the fortified group showed 1) higher plasma urea from week 2 (P = 0.04), 2) higher plasma calcium (mean 2.34 +/- 0.01 compared with 2.27 +/- 0.02 mmol/L; P = 0.003), 3) a greater rise in alkaline phosphatase by week 6 (P = 0.04), 4) more clinical infections (suspected plus proven; 43% compared with 31%, P = 0.04), 5) a nonsignificantly increased incidence of necrotizing enterocolitis (5.8% compared with 2.2%, P = 0.12), and 6) higher white cell and platelet counts. Developmental scores at 18 mo were slightly but not significantly higher in the fortified group. This study confirmed that breast milk fortifiers can improve short-term growth (when breast milk intakes are high); but beneficial effects on long-term development remained unproven. Future research is required to evaluate potential adverse consequences and explore more optimal fortification strategies.

Chemically modified tetracyclines act through multiple mechanisms directly on osteoclast precursors.

Chemically modified tetracyclines (CMTs) are thought to inhibit bone resorption primarily through their ability to inhibit matrix metalloproteinases (MMPs). We have previously demonstrated that some tetracycline compounds (TCs) induce apoptosis in mature rabbit osteoclasts and inhibit osteoclastic resorption in mouse osteoblast/marrow co-cultures in vitro. In this report, we now show that non-antibiotic analogues of doxycycline (CMT-3) and minocycline (CMT-8) are potent inhibitors of osteoclastogenesis in vitro from human peripheral blood mononuclear cells (PBMC) stimulated with macrophage colony stimulating factor (MCSF) and receptor activator of NF-kappaB ligand (RANKL), through an action that is independent of osteoblast-osteoclast interactions. Osteoclast formation over 20 days was completely abrogated when CMT-3 or CMT-8 were included in PBMC cultures at a concentration of 250 ng/ml, although doxycycline at this concentration reduced osteoclast formation to ca. 50% of control. CMT-3 and CMT-8 also significantly induced apoptosis over 24 h in mature osteoclasts generated over 20 days when added to cultures at 5 microg/ml or more. In a time-course experiment, apoptosis was evident after a delay of 1-2 h following treatment of mature osteoclasts with CMT-3 at 20 microg/ml. The broad-spectrum MMP inhibitor BB94 (Batimastat) did not recapitulate the apoptosis induced by CMT-3, even at a concentration where MMP-13 activity was completely inhibited. There was no evidence for an anabolic effect of any of the TCs on osteoblast lineage cells in a calcifying fibroblastic colony (CFU-f) formation assay, where CMT-3 partially inhibited CFU-f formation at 5 microg/ml. Our data indicate that inhibition of osteoclast formation and induction of osteoclast apoptosis are pharmacologically significant actions of CMTs in inhibiting bone resorption, and that osteoclast apoptosis cannot be attributed to the ability of CMTs to inhibit MMPs or to actions mediated by osteoblastic lineage cells.