1α,24(S)(OH)2D2 normalizes bone morphology and serum parathyroid hormone without hypercalcemia in 25-hydroxyvitamin D-1-hydroxylase (CYP27B1)-deficient mice, an animal model of vitamin D deficiency with secondary hyperparathyroidism.

BACKGROUND: Vitamin D compounds are effective in managing elevated PTH levels in secondary hyperparathyroidism (SHPT) of renal failure. However, undesired increases in serum calcium and phosphorus associated with compounds such as calcitriol [1,25(OH)2D3] has prompted a search for compounds with improved safety profiles. 1alpha,24(S)(OH)2D2 (1,24(OH)2D2) is a vitamin D2 metabolite with low calcium-mo bilizing activity in vivo. We studied the efficacy of 1,24(OH)2D2 in mice lacking the CYP27B1 enzyme [25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase)], a novel vitamin D deficiency model with SHPT. MATERIALS AND METHODS: 1alpha-OHase-deficient (-/-) mice and normal (+/-) heterozygous littermates re ceived 1,24(OH)2D2 (100, 300, 1000, and 3000 pg/g/day) or 1,25(OH)2D3 (30, 300, and 500 pg/g/day) for 5 weeks via daily sc injection. Control groups received vehicle. RESULTS: Vehicle-treated 1alpha-OHase-deficient mice were hypocalcemic and had greatly elevated serum PTH. 1,24(OH)2D2 at doses above 300 pg/g/day normalized serum calcium, serum PTH, bone growth plate morphology, and other bone parameters. No hy percalcemia was observed at any dose of 1,24(OH)2D2 in normal or 1alpha-OHase-deficient animals. In contrast, 1,25(OH)2D3 at only 30 pg/g/day normalized calcemia, serum PTH, and bone parameters, but at higher doses completely suppressed PTH and caused hypercalcemia in both 1alpha-OHase-deficient and normal mice. Treatment with 500 pg/g/day of 1,25(OH)2D3 also induced osteomalacia in normal animals. CONCLUSION: 1,25(OH)2D3 was maximally active at 10-fold lower doses than 1,24(OH)2D2, but induced hypercalcemia and osteomalacia at high doses. 1,24(OH)2D2 normalized serum calcium, serum PTH, and bone histomorphometry without hypercalcemia in 1alpha-OHase-deficient mice with SHPT.

Determination of hoof mass and centre of mass from morphological measurements.

Determination of net joint moments and powers requires accurate measurements of mass and centre of mass (COM) for the limb segments. The objective of this research was to develop regression equations for calculating mass and COM of the hoof segment that are applicable over a wide range of hoof morphologies. Ninety-one hooves (mean +/- s.d. 0.99 +/- 0.50 kg) were weighed and the 3-dimensional COM determined using a mass balance technique. Ten morphological variables were measured and used as independent variables in linear regressions with dependent variables of mass and centre of mass. The regression equation for mass was dependent upon coronet circumference, lateral height, solar length, solar width, heel height, toe height and base circumference (r2 = 0.978). Mediolateral COM was dependent only on solar length (r2 = 0.874). Dorsopalmar COM depended only upon solar length (r2 = 0.792) and vertical COM was defined only by lateral height (r2 = 0.377). Midlateral COM, measured at the midhoof wall along the tubules, was found to depend on coronet circumference, lateral height, toe length and toe height (r2 = 0.414). The large sample size and the inclusion of a variety of hoof masses and morphologies allow these results to be generalised to a wide range of the equine population. It was concluded that the mass and COM of the hoof segment can be estimated to a high degree of accuracy based on easily obtained morphological measurements.

Targeted inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets.

Pseudovitamin D-deficiency rickets is caused by mutations in the cytochrome P450 enzyme, 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-OHase). Patients with the disease exhibit growth retardation, rickets, and osteomalacia. Serum biochemistry is characterized by hypocalcemia, secondary hyperparathyroidism, and undetectable levels of 1alpha,25-dihydroxyvitamin D(3). We have inactivated the 1alpha-OHase gene in mice after homologous recombination in embryonic stem cells. Serum analysis of homozygous mutant animals confirmed that they were hypocalcemic, hypophosphatemic, hyperparathyroidic, and that they had undetectable 1alpha,25-dihydroxyvitamin D(3). Histological analysis of the bones from 3-week-old mutant animals confirmed the evidence of rickets. At the age of 8 weeks, femurs from 1alpha-OHase-ablated mice present a severe disorganization in the architecture of the growth plate and marked osteomalacia. These results show that we have successfully inactivated the 1alpha-OHase gene in mice and established a valid animal model of pseudovitamin D-deficiency rickets.

Deficient mineralization of intramembranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D.

The 25-hydroxyvitamin D-24-hydroxylase enzyme (24-OHase) is responsible for the catabolic breakdown of 1,25-dihydroxyvitamin D [1,25(OH)2D], the active form of vitamin D. The 24-OHase enzyme can also act on the 25-hydroxyvitamin D substrate to generate 24,25-dihydroxyvitamin D, a metabolite whose physiological importance remains unclear. We report that mice with a targeted inactivating mutation of the 24-OHase gene had impaired 1,25(OH)2D catabolism. Surprisingly, complete absence of 24-OHase activity during development leads to impaired intramembranous bone mineralization. This phenotype was rescued by crossing the 24-OHase mutant mice to mice harboring a targeted mutation in the vitamin D receptor gene, confirming that the elevated 1,25(OH)2D levels, acting through the vitamin D receptor, were responsible for the observed accumulation of osteoid. Our results confirm the physiological importance of the 24-OHase enzyme for maintaining vitamin D homeostasis, and they reveal that 24,25-dihydroxyvitamin D is a dispensable metabolite during bone development.

Heart, brain, and body wall defects in mice lacking calreticulin.

Calreticulin is a ubiquitously expressed protein, which has been implicated in a large number of cellular functions, including calcium storage and signaling, protein folding, and cell attachment. To examine the role of calreticulin during in vivo development, mice deficient in calreticulin were generated by targeted inactivation of the calreticulin gene. Calreticulin-deficient mutants die in utero, mostly in late gestation. Half of these embryos had decreased cardiac cell mass, associated with increased apoptosis of cardiac myocytes. In vitro differentiation cultures of calreticulin-deficient embryonic stem cells resulted in fewer embryoid bodies with contractile activity than cultures derived from calreticulin +/- stem cells (P < 0.001). Sixteen percent of the mutants exhibited exencephaly secondary to a defect in neural tube closure. Embryos surviving until Embryonic Day 16.5 had omphalocele. Lack of calreticulin did not influence survival of embryonic fibroblasts under various endoplasmic reticulum stress conditions. However, calreticulin did influence cell migration in a calcium- and substrate-dependent manner. We conclude that calreticulin is not essential during the early stages of embryonic development, but is important for the development of heart and brain and for ventral body wall closure. The observed abnormalities are compatible with a role of calreticulin in the modulation of cellular calcium signaling.

Interpretive algorithms for the symptom-limited exercise test: assessing dyspnea in Persian Gulf war veterans.

Interpretation of symptom-limited exercise testing requires analysis of a large body of simultaneously recorded cardiopulmonary data. Karlman Wasserman has recommended an algorithmic approach to interpretation (WA) that leads to a dichotomous choice between pulmonary and cardiovascular impairment. An alternative algorithm published by William Eschenbacher (EA) provides for concurrent assessment of cardiovascular and pulmonary exercise impairment. We analyzed a group of 29 individuals referred to the Pulmonary Physiology Laboratory at the Washington Veterans Affairs Medical Center for evaluation of dyspnea following service in the Persian Gulf War to assess the concordance of the two algorithms in determining the cause of dyspnea and exercise impairment in these individuals. They each performed a progressive, ramped, symptom-limited exercise test on a bike for a minimum of 6 min. Exercise measurements were analyzed by both interpretive algorithms. Concordance was found in 28% of tests. The greatest discordance occurred in identifying pulmonary limitation. Eleven had pulmonary limitation by EA; of these, WA found 1 to have pulmonary limitation, 5 to be normal, 4 indeterminate, and 1 musculoskeletal limitation. Of the 11 with pulmonary limitation by EA, but not by WA, 5 had abnormal resting pulmonary function measurements. Analysis of the differences between these two interpretive approaches is given. The EA algorithm may be more sensitive for detecting exercise impairment of pulmonary origin, but its specificity remains to be determined.

Pseudo-vitamin D deficiency: absence of 25-hydroxyvitamin D 1 alpha-hydroxylase activity in human placenta decidual cells.

Pseudo-Vitamin D Deficiency Rickets (PDDR), an heritable defect in renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity, leads to insufficient synthesis of 1 alpha, 25-dihydroxyvitamin D (calcitriol) and the early onset of severe rickets which can only be offset by replacement therapy with calcitriol. The nature of the underlying mutation remains unknown. Hydroxylation of 25-hydroxyvitamin D in the 1 alpha-position is not restricted to the renal tubular cells. We have previously shown that human decidual cells do produce calcitriol and that the enzyme activity was regulated by feedback mechanisms. We now demonstrate that cells isolated from the decidua of PDDR patients lack that function making them likely targets for the mutation. This suggests that the decidual and renal enzymes (or a controller of their activity) are encoded by the same gene. Thus the PDDR placenta represents a source of mutant cells for further investigation of the PDDR molecular defect.

Rat kidney 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylases: evidence for two distinct gene products.

Total RNA was isolated from kidneys of Sprague-Dawley rats. Oligo (dT)-primed single-stranded cDNA was obtained by the reverse transcriptase reaction from which a 285 bp cDNA probe coding for 25-hydroxyvitamin D3-24-hydroxylase [25(OH)D3-24-OHase] was generated by the polymerase chain reaction. Northern blotting performed with kidney poly (A)+ RNA isolated from rats (1) fed a standard diet, (2) depleted in D3 and hypocalcemic, and (3) fed a standard diet and injected intraperitoneally with 50,000 IU of vitamin D3 for 5 days showed that the transcript for 24-OHase was weakly expressed in control, and highly induced in vitamin D3-treated animals. No transcript could be elicited in vitamin D-depleted hypocalcemic animals in which 25(OH)D3-1 alpha-OHase was maximally induced. The data show that 24-OHase is independently regulated of 1 alpha-OHase, strongly suggestive of the enzymes being encoded by two distinct genes.

Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4.

T cells express T-cell antigen receptors (TCR) for the recognition of antigen in conjunction with the products of the major histocompatibility complex. They also express two key surface coreceptors, CD4 and CD8, which are involved in the interaction with their ligands. As CD4 is expressed on the early haemopoietic progenitor as well as the early thymic precursor cells, a role for CD4 in haemopoiesis and T-cell development is implicated. Thymocytes undergo a series of differentiation and selection steps to become mature CD4+8- or CD4-8+ (single positive) T cells. Studies of the role of CD4+ T cells in vivo have been based on adoptive transfer of selected or depleted lymphocytes, or in vivo treatment of thymectomized mice with monoclonal antibodies causing depletion of CD4+ T cells. In order to study the role of the CD4 molecule in the development and function of lymphocytes, we have disrupted the CD4 gene in embryonic stem cells by homologous recombination. Germ-line transmission of the mutation produces mutant mouse strains that do not express CD4 on the cell surface. In these mice, the development of CD8+ T cells and myeloid components is unaltered, indicating that expression of CD4 on progenitor cells and CD4+ CD8+ (double positive) thymocytes is not obligatory. Here we report that these mice have markedly decreased helper cell activity for antibody responses, although cytotoxic T-cell activity against viruses is in the normal range. This differential requirement for CD4+ helper T cells is important to our understanding of immune disorders including AIDS, in which CD4+ cells are reduced or absent.

Influence of calcitriol on prolactin and prostaglandin production by human decidua.

Receptors for calcitriol are described in human decidua. They exhibit a dissociation constant of 35 +/- 6 pM and they are at concentrations similar to those found in other tissues (0.45 +/- 0.04 fmol/micrograms DNA). They are highly specific for calcitriol since neither of the other vitamin D3 derivatives nor the steroid hormone tested displaced labeled calcitriol from the receptor. Also, calcitriol at concentrations of 10(-13) to 10(-11) M stimulates prolactin secretion by dispersed decidual cells. At these concentrations, however, the hormone has no effect on prostaglandin production. The specificity of calcitriol action was further examined by studying the effect of estrogen, progesterone, testosterone, dexamethasone and cortisol, all at 10(-7) M, on the secretion of prolactin and prostaglandins. Under the conditions used in this study, the steroids have no effect on prolactin secretion; but dexamethasone significantly inhibits prostaglandin F2 alpha output by the cells. Taken together with previous studies from our laboratory demonstrating that decidua can synthesize calcitriol the present study indicates that this hormone has an autocrine effect on human decidual cells.

Kinetics and regulation of 25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from human term decidua.

Kinetics and regulation of 25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from term human decidua were studied. The production of 1 alpha,25-dihydroxycholecalciferol (calcitriol) was linear with time for up to 6 h and was directly proportional to the number of cells up to 20 X 10(6)/dish at a substrate concentration of 100 nM. Under these conditions the apparent Km was 88 nM and the Vmax 3.0 pmol/10(6) cells. The production of [3H]calcitriol was inhibited by 0.1 nM (P less than 0.01) and 1 nM (P less than 0.005) unlabeled calcitriol. Unlike the kidney enzyme and for reasons that remain unclear, neither inorganic phosphate salts nor parathyroid hormone had any acute effect on the calcitriol production. Further studies are required to delineate the regulatory mechanism of this enzyme.

Endoscopic removal of a bronchial lipoma with the Neodymium-YAG laser.

A 56-year-old man presented with right middle and lower lobe pneumonia with empyema. Bronchoscopy and endobronchial biopsy revealed a lipoma in the bronchus intermedius. Removal of the lipoma was achieved with the Neodymium-Yttrium-Aluminum-Garnet (Nd:YAG) laser through a fiberoptic bronchoscope. The present case represents the first report of endobronchial lipoma with the Nd:YAG laser in the United States.

Nasopulmonary intubation with feeding tubes: case reports and review of the literature.

Pulmonary aspiration of regurgitated feeding material is a recognized complication of enteral nutrition by feeding tube. Nasopulmonary intubation with feeding tubes has been reported infrequently. We present two patients with such complications. In one, nasopulmonary intubation resulted in a pneumothorax. In the other unrecognized misplacement resulted in pneumonia and hydrothorax. Similar cases in the literature are discussed. Common features of this complication are: (1) patients who are obtunded, debilitated, or without gag reflex; (2) previous successful insertion of a tube; (3) successful passage past endotracheal tubes and tracheostomies; and (4) positive auscultation of insufflated air over the left upper quadrant. Complete prevention of this complication probably is not possible, but health care personnel should be aware of this potential problem in the patient at risk.

In vitro metabolism of 25-hydroxycholecalciferol by isolated cells from human decidua.

An increase in maternal serum levels of 1 alpha, 25-dihydroxycalciferol during pregnancy has been linked to enhancement of intestinal calcium absorption. Several sites of its synthesis have been proposed in different species, human decidua being one of them. Collagenase-dispersed decidual cells isolated from term placenta were fractionated on a Percoll gradient. The isolated cells were set in culture in the presence of 6 nM [3H]cholecalciferol. Two cell populations of similar morphology hydroxylated the substrate, yielding a compound that had a mass spectrum identical to and that comigrated with authentic 1 alpha, 25-dihydroxycholecalciferol in four chromatographic systems and bound to a specific rachitic chick receptor. These preparations, thus, provide a potential system by which the kinetics and regulation of the synthesis of the hormonal form of vitamin D by human placenta can be studied in vitro.

Laser therapy in patients with primary lung cancer.

Twenty-eight therapeutic bronchoscopic procedures with laser were carried out on 20 patients with primary lung cancer. Indications for laser bronchoscopic study were nonresectable, endoscopically visible tumor in patients who had any of the following complications after chemotherapy and/or radiation therapy: increasing dyspnea, postobstructive pneumonia, atelectasis, or hemoptysis from an endoscopically visible site. Symptomatic benefit was noted by 15 of 20 patients after laser therapy. There were two procedure-related deaths. Our conclusions regarding selection of patients for laser therapy of endotracheal and endobronchial lesions are given.

Kinetics of liver microsomal cholecalciferol 25-hydroxylase in vitamin D-depleted and -repleated rats.

Kinetics of vitamin D-depleted and -repleted rat liver microsomal cholecalciferol 25-hydroxylase were studied. Anaerobiosis, CO, omission of a NADPH-generating system and addition of detergents all decreased the activities, showing that the hydroxylase behaves like a cytochrome P-450-dependent enzyme. An apparent Km of 0.18 micrometer and Vmax. of 32pmol/min per g of tissue were found for vitamin D-deficient animals. Although both apparent Km and Vmax. were significantly altered in vitamin D-repleted animals no inhibition of the enzyme was elicited. These latter results show that at normal vitamin D intake, rat liver cholecalciferol 25-hydroxylase is not feedback-inhibited.