Treatable lower motor neuron disease due to vitamin D deficiency and secondary hyperparathyroidism.

Vitamin D deficiency and osteomalacia are frequently associated with muscle weakness and atrophy. We present a 78-year-old man with complaints of progressive painless weakness who was referred to us with a diagnosis of suspected motor neuron disease. Results of the neurological examination were remarkable, showing diffuse limb weakness and atrophy, rare fasciculations, normal sensory examination, no bulbar weakness, and no upper motor neuron signs. Electromyography revealed mild chronic changes, denervation and re-innervation, without fibrillations or positive waves. Serum laboratory studies showed an elevated serum parathyroid hormone and markedly reduced vitamin D level. Although the etiology of the vitamin D deficiency was not determined, the patient made a substantial clinical improvement following vitamin D therapy. Vitamin D deficiency and secondary hyperparathyroidism need to be included in the differential diagnosis of patients presenting with a progressive lower motor neuron disease.

Removal of vancomycin during plasmapheresis.

OBJECTIVE: To examine the removal of vancomycin during plasmapheresis, determine whether drug administration should be withheld prior to or a supplemental dose given after the procedure, and determine whether a redistribution phenomenon in vancomycin serum concentrations occurs after plasmapheresis. DESIGN: Prospective, cohort study. SETTING: An 800-bed, tertiary-care, teaching hospital. PATIENTS: Twelve patients receiving vancomycin as prescribed who were also undergoing therapeutic plasmapheresis. METHODS: Blood samples for determination of vancomycin concentrations were obtained from each patient immediately before, during, immediately after, and 2 hours after plasmapheresis. Vancomycin concentration in plasma removed by plasmapheresis and volume of plasma removed were measured. Patient-specific pharmacokinetic parameters were determined for each patient using serum concentration data and a one-compartment model. Percent of drug removed by plasmapheresis and percent increase in vancomycin total clearance secondary to plasmapheresis were calculated. RESULTS: A mean of 6.3% of the total body store of vancomycin was removed by plasmapheresis. Vancomycin clearance during plasmapheresis averaged 1.6 L/h, which was an average increase of 285% in the total clearance of vancomycin from the body. Nine of 10 patients had a higher observed vancomycin concentration 2 hours after plasmapheresis than that predicted by degrading the concentration observed immediately after the procedure, suggesting that redistribution in serum concentrations occurs after the procedure. CONCLUSIONS: A single one-volume plasmapheresis does not remove a clinically important amount of vancomycin; therefore, supplemental dosing after the procedure is not necessary. A redistribution phenomenon in vancomycin concentrations appears to exist after plasmapheresis. Further study is needed to determine how long the redistribution phase lasts and when vancomycin concentrations should be measured after plasmapheresis.

Effects of cell culture time and bone matrix exposure on calmodulin content and ATP-dependent cell membrane acid transport in avian osteoclasts and macrophages.

Osteoclasts mediate bone resorption by secretion at the site of bone attachment. This process depends on calmodulin concentrated at a specialized acid-secreting membrane. We hypothesized that increased calmodulin and bone attachment were required for acid secretion. We tested this by studying calmodulin, bone attachment, and membrane acid transport in osteoclasts and their precursor mononuclear cells. Osteoclasts and macrophages were isolated from medullary bone of hens; cell fractions were prepared after culturing cells with or without bone. Calmodulin was visualized by Western analysis; calmodulin mRNA was determined by Northern hybridization, and ATP-dependent membrane acid transport was assayed by acridine orange uptake. Calmodulin decreased in osteoclasts cultured without bone. Calmodulin in isolated macrophages was approximately 25% of osteoclast levels, but increased several fold by 5 days. Bone had no effect. Calmodulin mRNA was similar in osteoclasts with or without bone. However, only osteoclasts cultured with bone retained acid transport capacity. Macrophage calmodulin mRNA was not affected by bone, but increased three fold by day 5, paralleling protein production. Macrophages developed acid transport capacity at 3-5 days, but at lower levels than osteoclasts, and bone had no measurable effect. Chicken cells express 1.6 kb and inducible 1.9 kb calmodulin transcripts; in macrophages and osteoclasts, the 1.9 kb transcript predominated. We conclude that, following isolation, calmodulin levels decline in osteoclasts via a post-transcriptional mechanism. In cultured macrophages, by contrast, calmodulin mRNA, protein, and acid secretion increase with time independently of bone substrate, possibly reflecting differentiation in vitro. Increased calmodulin correlated with membrane acid transport capacity in both cell types. The macrophage findings indicate that stimuli other than bone influence acid transport capacity in this family of cells.

Avian cathepsin B cDNA: sequence and demonstration that mRNAs of two sizes are produced in cell types producing large quantities of the enzyme.

Overlapping cDNA fragments encoding avian cathepsin B were cloned from an osteoclast cDNA library and sequenced. The primary structure of the prepro enzyme deduced from this sequence has 340 amino acids. The mature portion of the enzyme is 80% identical with murine cathepsin B; regions found in other papain superfamily enzymes are conserved. In osteoclasts and cultured macrophages, which produce large quantities of cathepsin B, mRNAs of 1.8 and 2.4 kb are produced in approximately equal quantities, while cells producing smaller quantities of the enzyme produce predominantly the 2.4 kb form. This variation in mRNAs suggests transcriptional differences related to production of large quantities of the enzyme.

Calmodulin concentrated at the osteoclast ruffled border modulates acid secretion.

Osteoclasts mediate acid dissolution of bone for maintenance of serum [Ca2+] and for replacement of old bone in terrestrial vertebrates. Recent findings point to the importance of intracellular signals, particularly Ca2+, in osteoclast regulation. However, acid degradation of bone mineral subjects the osteoclast to uniquely high extracellular [Ca2+]. We hypothesized that this high calcium environment would affect calcium signalling mechanisms, and studied the calcium binding regulatory protein, calmodulin, in the osteoclast. Avian osteoclast bone resorption was inhibited 30% at 1 microM and 90% at 7 microM by the calmodulin antagonist trifluoperazine. Osteoclast bone attachment was not affected by 10 microM trifluoperazine. Quantitative immunofluorescence using fluorescein-labelled calmodulin monoclonal antibody showed a severalfold increase of calmodulin concentration in bone attached relative to plastic attached osteoclasts. Western blots confirmed this, showing two to threefold increased osteoclast calmodulin per milligram of cell protein in 3-day bone-attached vs. nonattached cells. Scanning confocal microscopy showed calmodulin polarization to areas of bone attachment. Electron micrographs with 9 nm colloidal gold labelling showed calmodulin in the acid secreting ruffled membrane. ATP-dependent acid transport in osteoclast membrane vesicles was inhibited by the calmodulin antagonist calmidazolium. This effect was reversed by addition of excess calmodulin, showing that the inhibition is specific. Vesicle acid transport inhibition reflects an approximately fourfold shift in the apparent Km for ATP of vesicular acid transport in the presence of the calmodulin antagonist. We conclude that calmodulin concentration and distribution is modified by bone attachment, and that osteoclastic acid secretion is calmodulin regulated.