Expression and purification of the alpha and beta subunits of Drosophila casein kinase II using a baculovirus vector. Two recombinant baculoviruses that express the alpha and beta subunits of Drosophila melanogaster casein kinase II, respectively, have been constructed. The expressed proteins are similar to the authentic Drosophila subunits in size and are recognized by antisera raised against the Drosophila holoenzyme. Extracts derived from cells infected with the alpha subunit-expressing virus display elevated casein kinase II activity in vitro. This activity is markedly enhanced in extracts of cells infected with both viruses, or when alpha and beta subunit-containing extracts are mixed in vitro following lysis. Recombinant holoenzyme and the alpha subunit were purified to near homogeneity using phosphocellulose column chromatography. The specific activity of the purified recombinant holoenzyme was very similar to that of the native enzyme, and was fivefold higher than that of the purified free alpha subunit. The Stokes radius of the recombinant holoenzyme was estimated to be 50 A, a value similar to that reported for the native enzyme, whereas the alpha subunit demonstrated a Stokes radius of 26.5 A. Studies using sucrose density gradient centrifugation showed that, under conditions of high ionic strength, the quaternary structure of the purified holoenzyme was tetrameric (like the native enzyme), whereas the structure of the alpha subunit was monomeric. At lower ionic strength the recombinant holoenzyme had a significantly higher sedimentation coefficient, characteristic of the formation of filaments found for the native enzyme. Interestingly, the purified catalytic subunit also displayed a higher S value under conditions of low ionic strength, revealing the formation of alpha subunit aggregates.


Partial amino acid sequence of potato solanidine UDP-glucose glucosyltransferase purified by new anion-exchange and size exclusion media. Solanidine UDP-glucose glucosyltransferase (SGT) is involved in the biosynthesis of steroidal glycoalkaloids in potatoes. This enzyme is present at an extremely low level, is inherently unstable, and copurifies with the major storage protein patatin during isolation. We describe an improved method for isolating SGT from greening potato peel using two new chromatographic supports, Macro-Prep 50 Q anion-exchange and Superdex 75HR size exclusion media, under medium-pressure conditions at room temperature. The enzyme preparation was further resolved by SDS-PAGE and the proteins transferred to PVDF membrane (Immobilon-P). Two protein bands corresponding to active forms of SGT (36 and 37 kDa) were excised and cleaved with cyanogen bromide in trifluoroacetic acid. The resultant peptide mixtures were then separated by Tricine-SDS-PAGE and transferred to a PVDF membrane (Pro-Blott). The two major peptide bands observed in both digests (17 and 19 kDa) were sequenced. Identical N-terminal sequences were obtained from the 19-kDa peptides from both digests.


Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli. Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase.


Immobilized metal ion affinity chromatography. The introduction of immobilized metal ion affinity chromatography, directed toward specific protein side chains, has opened a new dimension in protein purification. This review covers the principles and practice of IMAC that can be performed under very mild, nondenaturing conditions. IMAC is particularly suitable for preparative group fractionation of complex extracts and biofluids, but can also be used in high-performance mode: "HP-IMAC." Single-step purifications of 1000-fold or more may allow isolation of a particular protein from crude extracts on a milligram or gram scale. With respect to separation efficiency, IMAC compares well with biospecific affinity chromatography, and the immobilized metal ion ligand complexes are more likely to withstand wear and tear than are antibodies or enzymes. The enormous potential of IMAC and related metal affinity techniques is only in the initial stages of being explored and exploited. Synthesis of IMA adsorbents, and various modes of performing IMAC are discussed and exemplified with selected applications. Advantages and disadvantages are listed. Effective means of counteracting the few undesirable effects that can occur are suggested.


Purification of an active species of recombinant kappa-bungarotoxin. The expression of kappa-bungarotoxin in Escherichia coli from a synthetic gene results in the production of multiple species of polypeptide. These include not only biologically active kappa-bungarotoxin but also a variety of inactive species, which include inactive monomers as well as disulfide-linked polymeric species. Identification of these species and their separation from the biologically active recombinant toxin is necessary for the use of the toxin in physiological and biochemical studies. This has been accomplished by a combination of ion-exchange and reverse-phase chromatography which results in a homogeneous toxin preparation. The active material produced is sufficient for many types of biological studies and for mutagenesis experiments directed at determining the structure function relationships of toxin interactions with the neuronal nicotinic acetylcholine receptor. In addition, the kappa-bungarotoxin produced in this manner has the distinct advantage over venom-purified kappa-bungarotoxin of not being contaminated with other venom components which could potentially affect experimental observations.


High-level expression and production of recombinant human interleukin-6 analogs. We have constructed and analyzed different mutant forms of interleukin-6 (IL-6) expressed in Escherichia coli that can be divided into two groups. The first group contains four full-length IL-6 molecules that differ in the presence of cysteine residues involved in disulfide bridges. The second group contains 22 N-terminal amino acid deletions in addition to the differences in the cysteine residues. The different IL-6 muteins were extracted and their expression levels and solubility were compared. We found that the production levels of IL-6 can be dramatically improved by deleting the first 22 N-terminal amino acids of the molecule. We have also found that the production of IL-6 containing the four cysteine residues is lower than the production of the mutant molecules that lack one or both pairs of cysteines. The yield of soluble and properly refolded IL-6 was the highest when the disulfide bond between the cysteines at positions 74 and 84 was present in the mutein form, which also lacked the 22 N-terminal amino acids.


Expression and purification of the cytoplasmic tail of an endocytic receptor by fusion to a carbohydrate-recognition domain. Gene fusion has been used to produce the cytoplasmic domain of an endocytic receptor. DNA sequences coding for the 52 COOH-terminal amino acids of the mannose receptor from human macrophages, including the 41-amino acid cytoplasmic tail, were fused to the codons specifying the carbohydrate-recognition domain (CRD) of rat mannose-binding protein. The fusion protein was expressed in Escherichia coli and purified in one step on mannose-Sepharose, making use of the carbohydrate-binding activity of the CRD. The tail peptide was released from the fusion protein using endoproteinase Arg-C. This method provides an alternative to chemical synthesis for the production of midlength peptides.


Production, purification, and characterization of human recombinant IL-8 from the eucaryotic vector expression system baculovirus. The cDNA for the human chemotactic interleukin, IL-8, was subcloned from a bacterial source into the eucaryotic vector expression system baculovirus. Recombinant human IL-8 (rhIL-8) was synthesized and secreted from Sf9 cells derived from Spodoptera frugiperda following infection of a recombinant virus harboring the full-length IL-8 structural gene. Infected Sf9 cells produced rhIL-8 in a range from 0.5 to 2.0 mg of rhIL-8/liter of postinfection cell culture media. The recombinant interleukin was purified (greater than 600-fold) to homogeneity using preparative HPLC. rhIL-8 retained all of the physical, immunological, and biochemical properties observed for the natural product, monocyte-derived IL-8. rhIL-8 was assessed for biological efficacy by three criteria: (a) ability to induce chemotaxis in human neutrophils, (b) ability to induce oxygen burst metabolism, and (c) ability to be recognized by purified rabbit antibody generated against monocyte-derived IL-8. Antibody generated against monocyte-derived IL-8 recognized rhIL-8 isolated during all stages of the purification protocol. rhIL-8 was strongly chemotactic for human neutrophils and exhibited a chemotactic index comparable to that reported for other strong chemotactic peptides. rhIL-8 was identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single silver-stained band having an estimated molecular weight of 9.2 kDa and displayed amino acid residue molar abundance homology predicted for the mature form of the interleukin. Baculovirus vector expression coupled to preparative HPLC proved to be a very efficient method for large-scale recombinant interleukin production.


Expression and purification of recombinant and native calreticulin. Calreticulin is a 60-kDa Ca(2+)-binding protein of the endo(sarco)plasmic reticulum membranes of a variety of cellular systems. The protein binds approximately 25 mol of Ca2+ with low affinity and approximately 1 mol of Ca2+ with high affinity and is believed to be a site for Ca2+ binding/storage in the lumen of the endo(sarco)plasmic reticulum. In the present study, we describe purification procedures for the isolation of recombinant and native calreticulin. Recombinant calreticulin was expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and was purified to homogeneity on glutathione-Sepharose followed by Mono Q FPLC chromatography. A selective ammonium sulfate precipitation method was developed for the purification of native calreticulin. The protein was purified from ammonium sulfate precipitates by diethylaminoethyl-Sephadex and hydroxylapatite chromatography procedures, which eliminates the need to prepare membrane fractions. The purification procedures reported here for recombinant and native calreticulin yield homogeneous preparations of the proteins, as judged by the HPLC reverse-phase chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified native and recombinant calreticulin were identified by their NH2-terminal amino acid sequences, by their Ca2+ binding properties, and by their reactivity with anticalreticulin antibodies.


Construction of a high-copy "ATG vector" for expression in Escherichia coli. We report the construction of an inducible, high-copy plasmid for the expression of foreign proteins in Escherichia coli. This plasmid, pPB1, combines the trc promoter, beta-galactosidase translation start site, and polylinker of pKK233-2 with the origin of replication region of pUC19. Replacement of the origin of replication of pKK233-2 results in a threefold increase in plasmid copy number of pPB1 compared with pKK233-2. Subclones of the cDNA for rabbit muscle fructose-1,6-bisphosphate aldolase (E.C. 4.1.2.13) in the two expression plasmids exhibit a comparable difference in copy number. An increase in protein expression measured by SDS-PAGE and aldolase specific activities reflects the increased copy number. Specific activities of aldolases in bacterial extracts differ approximately sixfold between the two expression plasmids in E. coli JM83. Aldolase A can compose up to 40% of the total protein in E. coli JM83 when expressed in pPB1, from which more than 100 mg of purified enzyme can be obtained per liter culture.


Overexpression and purification of the separate tryptophan synthase alpha and beta subunits from Salmonella typhimurium. To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta subunits, respectively. The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit. We also report new and efficient methods for purifying the individual alpha and beta subunits. Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase alpha 2 beta 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts. The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes. Purification of the alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified alpha and beta subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis. The procedures developed can be applied to the expression and purification of mutant forms of the separate alpha and beta subunits. The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.


Thyrotropin modulation of epidermal growth factor (EGF) binding to receptors on cultured thyroid cells. Previous studies had shown that epidermal growth factor (EGF) will stimulate growth of cultured thyroid cells in vitro, and TSH will stimulate total assayable EGF receptor in cultured porcine thyroid cells. In this study, we report the effect of TSH on EGF binding to human thyroid cells. Addition of bTSH (1 mU/mL) in binding buffer during receptor assay stimulated specific EGF binding to cells, with an increase of 44% observed over the control after 1 h incubation at 37 degrees C. Affinity crosslinking of the [125I]EGF-receptor complex showed a single labeled band with molecular size of 170 kD. No additional band was detected in the presence of TSH. Preincubation of cells with chloroquine, which inhibits lysosomal degradative enzyme activity, caused a continuous accumulation of bound EGF over a 4 h study period at 37 degrees C, and TSH stimulated an increase in internalized EGF. In the presence of chloroquine, total specific bound EGF was linearly correlated to incubation time up to 4 h and can be expressed as Bound = slope*time+intercept (time0) Addition of TSH during the binding assay significantly increased the value of the slope when compared to control (p < 0.002). The rate at which prebound [125I]EGF was released into medium was not affected by the presence of TSH, indicating that TSH-enhanced binding may not be attributed to a reduction in EGF degradation. Coincubation of thyroid cells with EGF at 0 and 1 ng/mL and increasing concentrations of TSH (0-10 mU/mL) indicated that EGF stimulated thymidine incorporation, although TSH failed to synergistically enhance EGF-stimulated cell growth.(ABSTRACT TRUNCATED AT 250 WORDS)


Peroxidase and coupling activities of thyroid peroxidase in benign and malignant thyroid tumor tissues. The coupling activity of thyroid peroxidase (TPO) in thyroid glands from patients with benign adenoma, papillary carcinoma, and diffuse goiter (Graves' disease) was measured for the first time, in addition to the peroxidase activity of these tissues. The peroxidase activity of TPO in the mitochondria-microsomes fraction was measured with guaiacol or iodide as the second substrate. In the case of papillary carcinoma, the mean protein-based specific activity obtained by the guaiacol assay was about 1/7 of that of diffuse goiter. The iodide oxidation activity of carcinoma was very low, about 1/25 [corrected] of that in diffuse goiter and 1/70 of that in adenoma. The peroxidase activity in adenoma was almost similar in the guaiacol oxidation assay and approximately one half in the iodide oxidation assay as compared with that in diffuse goiter. There was a close correlation between the guaiacol and iodide oxidation assays in individual patients with adenoma and diffuse goiter, but not in patients with papillary carcinoma. The coupling activity of TPO was measured with thyroglobulin purified from pooled toxic diffuse goiters and chemically iodinated to contain little additional T3 and T4. The specific coupling activity of TPO in mitochondria-microsomes from carcinoma was significantly lower (about 1/5) than that of diffuse goiter, and the activity in adenoma was not significantly different (about 1/2) from that of diffuse goiter. The data of coupling activities has a close correlation with that of peroxidase activities in individual patients with adenoma but not in patients with carcinoma. Based on these findings, the qualitative abnormality of TPO and its relation to the cold 123I scintigram in thyroid tumors are discussed.


Significance of antibodies reactive with a 64 kDa eye muscle membrane antigen in patients with thyroid autoimmunity. SDS-polyacrylamide gel electrophoresis and Western blotting for antibodies reactive with a 64 kDa protein in pig eye muscle membrane was carried out in patients with lid lag and retraction, but no other signs of ophthalmopathy, associated with thyroid disease or nonimmunologic goiter and in patients with Graves' hyperthyroidism without ophthalmopathy who were studied prospectively to determine the relationship of eye muscle antibodies to clinical features of the ophthalmopathy as they appeared in this group of predisposed patients. Seventy-one percent of euthyroid patients with lid lag and retraction but no established ophthalmopathy had detectable serum antibodies to a 64 kDa eye muscle membrane protein. Much smaller proportions had antibodies to proteins of other MW. In normal subjects with previously detectable antibodies to a 64 kDa protein, serum titers, determined by carrying out immunoblotting at serum dilutions of 1:25-1:6400, were low (< or = 1:100) in all cases tested. On the other hand, titers were higher (1:200-1:6400) in 16 of 22 patients with established ophthalmopathy and in 5 of 7 patients with lid lag and retraction tested. Titers tended to be lower in patients with ophthalmopathy of > or = 3 years duration than in those of < or = 1 year duration. Antibody titers were low (1:25) in 6 of 7 patients with Graves' hyperthyroidism without evident eye disease tested. Antibodies to a 64 kDa eye muscle membrane protein were predictive of the development of ophthalmopathy in patients with Graves' hyperthyroidism studied prospectively for periods of 8-42 months.(ABSTRACT TRUNCATED AT 250 WORDS)


Relapse of Graves' disease following development of a pheochromocytoma. Catecholamines stimulate thyroid hormone synthesis as well as release of thyroid hormone and cause immunologic disturbances that possibly contribute to the manifestations of Graves' disease. This has led to repeated speculations about the possible role of catecholamines in the initiation and maintenance of hyperthyroidism. We describe a patient with Graves' disease who was treated with antithyroid drugs for 2 years. After withdrawal of antithyroid drugs, the patient was in remission for 5 years. After the antithyroid drug treatment and the long remission, the probability of relapse of Graves' disease was very low. Nonetheless, a relapse did occur. Two years after subtotal thyroid resection, further investigation because of persistent hypertension revealed a pheochromocytoma. Retrospective anamnestic data suggest that this pheochromocytoma had been present 2 years before the patient's relapse of Graves' disease. This sequence of diseases has not been described previously. The low probability for a Graves' disease relapse in this patient and the association of this patient's relapse with the manifestation of a pheochromocytoma suggest a possible etiologic role of excess catecholamine production in the relapse of Graves' disease.


Fetal thyroid function.

Fetal thyroid function. Cordocentesis has permitted the study of fetal thyroid function in utero. In normal fetuses, fetal TSH, TBG, and thyroid hormone concentrations increase progressively throughout intrauterine life. Fetal TSH concentrations are always high compared to nonpregnant adult values. TBG concentrations reach adult levels at term. TT4 and FT4 concentrations reach adult levels at approximately 36 weeks gestation, but TT3 and FT3 are always below adult concentrations. There are no significant associations between fetal and maternal concentrations of TSH, TBG, or thyroid hormones. The maternal administration of TRH from at least 25 weeks gestation stimulates the fetal pituitary gland to produce TSH. The response is rapid, unrelated to gestational age, and much greater than that of the mother. These findings suggest that in intrauterine life there is independent and autonomous maturation of the pituitary, thyroid, and liver. The fetal pituitary is able to respond to the maternal administration of TRH and appears to be more sensitive than in the adult. In small-for-gestational-age fetuses, the concentrations of TSH are higher and the concentrations of TT4 and FT4 are lower than in appropriately grown fetuses. The degrees of elevation of TSH and fall in thyroid hormones are significantly related to the degree of fetal hypoxemia and acidemia, respectively. Although the low concentrations of thyroid hormones may have some beneficial effects by reducing oxygen requirements, they may adversely affect brain development.


Diagnosis and management of Graves' disease in pregnancy. Most of the hyperthyroidism seen in association with pregnancy is Graves' disease. The best treatment is prevention. For most patients there is an opportunity to treat the hyperthyroidism decisively with radioiodine or surgery before the patient becomes pregnant. Pregnancy complicated by hyperthyroidism is often a consequence of the conscious decision to treat hyperthyroidism in women in the childbearing years with antithyroid drugs. Propylthiouracil (PTU) is the preferred treatment for hyperthyroidism in pregnancy, but it does cross the placenta and can induce fetal goiter, with mental and physical retardation. Hence, the lowest possible PTU dose should be used. One should aim for high normal or slightly elevated thyroid function in the mother. Patients should be followed at 3-week intervals if progress is satisfactory, more often otherwise. Thyroid function should be monitored by the free T4 assay. PTU dosage should be reduced progressively in anticipation of the customary steady amelioration in the hyperthyroidism that occurs in later stages of pregnancy. Since pregnant hyperthyroid patients are sometimes irresponsible and continue PTU without supervision, PTU prescriptions should be limited to the amount required for the time until the next scheduled visit. For about one third of patients, PTU can be discontinued in the second half of the pregnancy. After the pregnancy is terminated, persistent or recurrent hyperthyroidism should be treated definitively to prevent another episode of pregnancy complicated by hyperthyroidism.


The fetus in maternal hyperthyroidism.

The fetus in maternal hyperthyroidism. A recent article in the New England Journal of Medicine reported the successful diagnosis and treatment of fetal goitrous hypothyroidism in a mother with Graves' disease. The fetus is being recognized as an important patient in its own right in terms of thyroid disease. The fetal thyroid system develops independently of the normal maternal thyroid axis. Presence of feedback suppression of TSH by T4 has been demonstrated in a 35-week fetus. Information learned from congenital hypothyroidism suggests that lack of fetal thyroid hormones may have a negative impact on the developing fetal brain with lack of normal myelination. It is uncertain at what gestational age the fetus and the developing central nervous system become adversely affected by thyroid hormone deficiency. Since congenital hypothyroidism is sporadic and since there is no current method for easily screening all pregnancies for hypothyroidism, the thrust in fetal diagnosis and therapy has been in those pregnancies suspected of having a hypothyroid fetus when a fetal goiter is detected by ultrasonography or in a hyperthyroid mother who may be on antithyroid therapy. Intraamniotic injections of L-thyroxine have proven successful for fetal therapy. Amniotic fluid TSH may prove useful in the diagnosis and treatment of a hypothyroid fetus. Previous studies have suggested that the period of thyroxine dependency of the fetal central nervous system is limited predominantly to the last 4-8 weeks of gestation. Fetal hyperthyroidism due to transplacental transmission of thyroid-stimulating immunoglobulins may occur in a mother with a history of hyperthyroidism due to Graves' disease.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunosuppressive treatment of Graves' ophthalmopathy. Glucocorticoids and retrobulbar irradiation are the most employed immunosuppressive treatment modalities in Graves' ophthalmopathy. The response rate is approximately 60%. Efficacy is good for improvement of appearance and visual acuity, moderate for correction of extraocular muscle dysfunction, and poor for reduction of proptosis. Immunosuppression seldom cures the eye disease. Its main advantage is to stabilize the eye disease by inactivating the inflammation of orbital tissues, thereby permitting corrective eye surgery to be performed at an earlier time. Future developments in immunosuppression aim at reduction of side effects and enhancement of efficacy. Alternative treatment schedules (e.g., methylprednisolone pulses, intravenous immunoglobulin) may have equal efficacy but less side effects than classic high-dose oral steroids. Efficacy can be improved by restriction of immunosuppression to patients with active eye disease who are more likely to respond. Disease activity might well be the main determinant of therapeutic outcome of immunosuppression in Graves' ophthalmopathy.


Variations in thyroid hormone transport proteins and their clinical implications. Variations in major thyroid hormone transport proteins may be inherited or acquired and may be associated with changes in serum concentration of the proteins or their affinity for thyroid hormones. These variations most frequently involve thyroxine-binding globulin (TBG), but changes in transthyretin and albumin are also observed. The consequent alteration of thyroid hormone-binding capacity in serum is associated with variations in total thyroid hormone concentration. Increased serum total thyroid hormone levels are found in subjects with TBG excess, familial dysalbuminemic hyperthyroxinemia, and transthyretin-associated hyperthyroxinemia. Conversely, diminished serum thyroid hormone values are observed in subjects with TBG deficiency, and decreased concentration or affinity of transthyretin and albumin is not associated with variations in serum concentrations of thyroid hormones. The transport protein-associated variations in serum total thyroid hormone concentrations do not reflect a change in thyroid status. Euthyroidism can be easily established in subjects with transport protein abnormalities by the normal free thyroid hormone and TSH concentrations. It is, however, crucial to select methods for free thyroid hormone measurement that are not affected by abnormalities of transport proteins. Some assays, such as the analog method, often provide artifactual and misleading results, which may lead to inappropriate and even detrimental treatments. The evolutionary advantage of TBG (and albumin) in terms of thyroid homeostasis still remains to be elucidated.


Thyroid hormone suppressive therapy of sporadic nontoxic goiter. Thyroid hormone has been used to reduce the size of sporadic nontoxic goiter since 1894. Noncontrolled clinical studies suggest that about two thirds of goiters respond to therapy, and a recent randomized clinical trial confirms the efficacy of suppressive therapy for sporadic nontoxic goiter. Efficacy is at least partly correlated with suppression of pituitary TSH production, response is usually evident by 3 months, relapse occurs when therapy is withdrawn, and nodular goiters may be less responsive than diffuse goiters. Some, but not all, series suggest that postoperative use of thyroid hormone suppressive therapy prevents recurrence of benign goiter. Three recent randomized trials suggest that thyroid hormone administered for 6 months to 3 years does not reduce the size of solitary thyroid nodules. About one third of nodules regressed in both treatment and placebo groups. Long-term studies are needed to define the effects of thyroid hormone suppressive therapy on the growth of goitrous lesions based on their underlying pathophysiology.


Prenatal diagnosis and caring.

Prenatal diagnosis and caring. Although the science of prenatal diagnosis is rapidly expanding, the art of caring for these patients is poorly understood and taught. Prenatal diagnosis programs must acknowledge the psychosocial consequences of electing to undergo prenatal testing, receiving either normal or abnormal test results, and choosing to continue or terminate a pregnancy with an identified fetal defect. There are suggestions from the literature of interventions to assist families under these circumstances (Table 3). Prospective studies of patient adaptation are needed; however, on the basis of the retrospective studies and common sense, a reasonable plan for patient support and care can be established. Prenatal diagnosis programs must demand not only excellent medical knowledge and technical expertise but also compassionate and continued care for the patient, her partner, and family.


Women's health care: rights and responsibilities. We recently participated in efforts to implement community-based women's health programs in regions of the world that are undergoing broad reforms as one form of government is replaced by another. In the course of these activities, we have had reason to review the rich and voluminous literature on the pivotal contributions by patients, providers, and society that result in optimal health for women. This article reflects our perspective of that literature and a framework to achieve that goal.


Specialists and generalists in obstetrics and gynecology: conflicts of interest in referral and an ethical alternative. Referral patterns of physicians have a direct impact upon the care of patients, particularly in obstetrics and gynecology. The choice of referral is influenced by the history of specialization, physician altruism, and intricate patterns of financial conflicts of interest. The conflicts of interest are further obscured by the lack of clear definition of roles and responsibilities for generalist, specialist, and subspecialist. Alternate patterns for referral based on financial incentives or directed referral care plans are reviewed to examine the potential conflicts of interest. An ethics-based format for referral is outlined to address these conflicts of interest.


The effects of woman abuse on health care utilization and health status: a literature review. Estimates of the physical abuse of women by husbands or boyfriends in the United States range from 85 per 1000 couples to 113 per 1000 couples per year. Victims of abuse are much more likely than nonvictims to have poor health, chronic pain problems, depression, suicide attempts, addictions, and problem pregnancies. Abused women use a disproportionate amount of health care services, including emergency room visits, primary care, and community mental health center visits. Despite its high prevalence and the disproportionate use of health care services it causes, woman abuse is rarely recognized by health care providers. Even when the abuse is recognized, health care professionals often provide inappropriate or even harmful treatment. Because many abused women pass through the health care system, it is important that providers learn how to identify those who are abused, treat all the effects of the abuse, and make appropriate referrals.


Regulation of interleukin 4 production and of interleukin 4-producing cells. Interleukin 4 (IL-4) is a cytokine with a broad range of functions on immune cells including the stimulation of IgE synthesis by B cells. IL-4 is synthesized by freshly isolated T cells following appropriate stimulation with either lectins or antibodies against selected surface antigens. IL-4 production has also been observed by freshly isolated FceR+ non-T, non-B cells. With T cell clones, IL-4 is produced by clones of the Th2 and Th0, but not of the Th1 type. The molecular basis for restricted cytokine production by such clones as well as the signals responsible for differentiation into either phenotype are only at the beginning to be understood.


Human TH1 and TH2 subsets: regulation of differentiation and role in protection and immunopathology. A large body of evidence has accumulated suggesting the existence of human TH1 and TH2 subsets, reminiscent of those described for mouse T cells. Human TH1 cells develop in response to intracellular bacteria and viruses, do not provide help for IgE synthesis and are cytolytic. Human TH2 cells develop in response to allergens and helminth components, provide help for IgM, IgG, IgA, and IgE synthesis and lack cytolytic potential. The cytokine profile of 'natural' immunity (high IFN-gamma and no IL-4 production) evoked by intracellular bacteria and viruses that activate both macrophages and NK cells, probably determines the phenotype of the subsequent specific TH1 response. Absence or low concentration of IFN-gamma and early production of IL-4 by non-T cells or by T cells themselves, that occur in response to allergens and helminth components, probably favor the development of TH2 cells. TH1 and TH2 cells play different roles not only in protection against exogenous offending agents, but also in immunopathology. TH1 cells are involved in contact dermatitis, organ-specific autoimmunity and allograft rejection. TH2 cells are responsible for the initiation of the allergic cascade.


Inflammatory cytokines and enzymes in synovial fluid of patients with rheumatoid arthritis and other arthritides. Cytokines and lysosomal enzymes, which are produced by inflammatory cells, play a role in inflammation. We have found that synovial fluid (SF) in rheumatoid and septic arthritis contained a large number of white blood cells (WBCs) and high levels of cytokines and enzymes, while in contrast the SF of osteoarthritis and traumatic arthritis did not contain significant amounts. Measurements of WBCs, cytokines and enzymes in SF are useful for evaluating clinical disease activity. Assays for WBCs and enzymes are simple and rapid when compared to those for cytokines.


Effect of antigens from Nippostrongylus brasiliensis and cytokines on the ongoing IgE synthesis in vitro. The modulation of an ongoing IgE-mediated immune response induced by the infection of mice with the nematode Nippostrongylus brasiliensis (N.b.) was investigated in vitro. In previous experiments antigens derived from the homogenate of adult worms (WH) and third stage larvae (LH) were characterized by immunoblotting. Our results demonstrate that only antigens of the WH were recognized by IgE antibodies. The effect of worm antigens and cytokines (IL-2, IL-4, IL-5, IFN gamma) on the IgE and IgG synthesis in different culture systems was studied. The IgG synthesis of B cells was stimulated by WH or WH/cytokines. The IgE production of B cells was enhanced only by WH/cytokines or when T cells were present. Individual antigen fractions (WH 5, 8, 9, 10, 13) increased the IgE production, which was enhanced in the presence of IL-4 2- to 3.5-fold) but had no significant effects on the IgG production.


Immunochemical properties of anti-DNA antibodies in the sera of patients with Escherichia coli bacteremia. To assess the role of infection in anti-DNA antibody production, the DNA-binding activity of sera from patients with Escherichia coli bacteremia was analyzed. Among 8 patients with bacteremia documented by blood culture, 5 demonstrated increased levels of antibodies to single-stranded DNA from E. coli as measured by enzyme-linked immunosorbent assay. Sera from these patients also reacted with single-stranded DNA from other bacterial and mammalian species as well as certain synthetic polynucleotides including poly-dT and poly-dC. The isotype distribution of these antibodies and their avidity as assessed by competition enzyme-linked immunosorbent assay resembled, moreover, responses of patients with systemic lupus erythematosus. These results suggest that, during the course of infection with E. coli, some patients may produce antibodies with immunochemical properties similar to those arising in systemic lupus erythematosus.


Characteristics of long-term human thyroid peroxidase autoantibody secretion in scid mice transplanted with lymphocytes from patients with autoimmune thyroiditis. We have explored scid mice as an in vivo model to study lymphocyte function and autoantibody production in patients with autoimmune thyroiditis and thyroid peroxidase (hTPO) autoantibodies. Patient's peripheral blood mononuclear cells (PBMC) were transplanted into scid mice via intraperitoneal injections and human immunoglobulin G (hIgG) and thyroid autoantibody levels in the murine sera were monitored for a minimum of 3 months after transplantation. Human IgG reached maximum serum levels of > 3,000 micrograms/ml (mean +/- SEM = 1,199 +/- 354 micrograms/ml) after an average of 6.5 weeks. In reconstituted mice (hereafter named At-Scid-hu) substantial titers of anti-hTPO of up to 0.51 (ELISA index, normal range < 0.02) were observed over a period of 1-2 months, followed by a gradual decline. Immunization of AT-Scid-hu mice with immunogenic, recombinant human hTPO (rec-hTPO) failed to enhance hTPO-Ab levels. Furthermore, there was no correlation between the magnitude of human IgG in the murine serum and concomitant levels of anti-hTPO. Murine thyroid function was unaffected by the transplantation of PBMC, as evidenced by normal serum thyroxine (T4) levels, and lack of specific pathologic changes in the thyroid. These data indicate, for the first time, the potential for longer-term human thyroid autoantibody secretion in the scid mouse reconstitution model allowing for further investigation of the regulatory factors inpinging on the human B cells surviving in the murine environment.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression and thrombin cleavage of Poa p IX recombinant allergens fused to glutathione S-transferase. The high-level expression and purification of Poa p IX recombinant grass pollen allergens were examined utilizing a modified pGEX plasmid, designated as pGEX 2T-1. This vector permits frame-1 ligation of lambda gt11 cDNA inserts and cleavage of the recombinant allergenic protein from the fusion partner glutathione S-transferase. The expression of the fusion proteins in water-soluble form varied among the transformants of the same bacterial strain and also between different host strains. Purification of the fusion proteins by affinity chromatography employing glutathione agarose gel revealed that proteases in the bacterial lysate bound to the gel and were co-eluted with the fusion proteins. These proteases, which specifically degraded the recombinant proteins to varying degrees, were inhibited by both of the inhibitors, phenylmethylsulfonyl fluoride and aprotinin. Cleavage by thrombin of the fusion proteins indicated that the structure of the individual protein affected the thrombin accessibility to the cleavage site. Increased concentration of thrombin partly compensated this effect, but resulted in a broader specificity of the enzyme. By contrast, cleavage of the fusion protein when it was still attached to the glutathione gel was convenient and led to purification of the product devoid of proteolytic activity. Since almost all the recombinant allergens have been cloned in lambda gt11 vector, the pGEX 2T-1 vector reported herein will facilitate the synthesis, purification of the corresponding allergenic proteins or their peptides in soluble and biologically active forms.


Pharmacological modulation of the late eosinophilia induced by antigen in actively sensitized rats. The intrathoracic injection of ovalbumin (12 micrograms/cavity) into actively sensitized rats led to a long-lasting eosinophil recruitment, which appeared 24 h after stimulation. In this study, pharmacological antagonists were used in order to evaluate the potential involvement of arachidonic acid metabolites and PAF-acether in the pleural eosinophil accumulation by antigen. Administration of the cyclooxygenase inhibitor indomethacin (2 mg/kg, i.p.), 1 h before the antigen challenge, failed to modify the 24-hour eosinophilia. In contrast, the dual cyclooxygenase and lipoxygenase inhibitor BW 755C and the more selective inhibitor BW A4C (5 and 10 micrograms/cavity, i.t.), injected 1 h before the antigen, were effective. Similarly, the PAF-acether antagonists BN 52021 and WEB 2086 (20 mg/kg, i.p.) abrogated the eosinophil accumulation, which was also sensitive to the topical treatment with the glucocorticoid dexamethasone (5 and 10 micrograms/cavity). Our findings suggest that the antigen-induced eosinophil mobilization is dependent on lipoxygenase derivatives and PAF-acether, but not on prostaglandins.


Expression of birch pollen-specific IgE-binding activity in seeds and other plant parts of birch trees (Betula verrucosa Ehrh.). Dot immunoblotting of crude extracts of various aerial parts of birch trees, using patient serum rich in birch pollen IgE, showed IgE-binding activity in leaves, buds, twigs, seeds, bark, and old male catkins. Seed extracts analysed by SDS-PAGE, electroblotting to nitrocellulose and immune detection using isotope-conjugated anti-IgE verified the presence in seeds of an IgE-binding protein of an approximate molecular weight of 12 kD, distinct from the major allergen (molecular weight 17 kD) of Betula verrucosa pollen. The allergen of birch seeds was readily leachable from the seeds. Many of the birch plant part extracts were active in RAST inhibition using birch pollen RAST discs, but showed low potency relative to the allergenicity of birch pollen allergens.


Differentiation of coeliac disease and other malabsorption diseases using specific serum antigliadin IgG subclass profiles and IgA1 levels. The differential diagnostic potential of serum gliadin-specific IgG subclass antibodies was assessed by comparing the antigliadin IgG1, 2, 3, 4 profile at different stages of coeliac disease with that of gastro-intestinal infection and also conditions associated with increased intestinal permeability. The IgG subclass profile of untreated coeliac disease was found to be the same as in healthy controls (IgG1 approximately IgG2 > IgG3 > IgG4), with only the magnitude of the individual subclass responses being increased in coeliac patients. Coeliac adults and children on gluten-free diets had different antigliadin IgG subclass profiles with IgG2 being elevated. Increased intestinal permeability or recent gastro-intestinal infection did not alter the antigliadin subclass profile from that observed in healthy individuals. Assessment of the diagnostic potential of antigliadin IgA1 and IgG1-4 measurements in screening for coeliac disease demonstrated that measurement of subclasses of gliadin-specific IgA and IgG was less sensitive and specific compared with the combined use of total antigliadin IgA and IgG. Therefore it is suggested that IgG subclasses should not be used for routine screening for coeliac disease.


SO2-induced enhancement of inhalative allergic sensitization: inhibition by anti-inflammatory treatment. Epidemiological studies have shown a relationship between air pollution and allergic airway disease. In a previous study we have found that exposure to SO2 enhances allergic sensitization to inhaled ovalbumin (OA) in the guinea pig. We have now investigated the influence of pre-treatment with anti-inflammatory drugs on SO2-induced enhancement of allergic sensitization in this model. Four groups of 6 guinea pigs each were exposed to 5 ppm SO2 on 5 consecutive days over 8 h per day with intermittent inhalation of OA, while the air-control group was exposed to clean air and OA. During the period of SO2 exposure and sensitization three experimental groups were treated with indomethacin (group I), methylprednisolone (group M) and nebulized nedocromil sodium (group N), while the control group remained untreated. Guinea pigs were investigated for sensitization to OA by specific bronchial provocation tests using body plethysmographic measurement of compressed air (CA) and by measurement of specific antibody response in serum. While in the SO2-exposed control group 5 of 6 animals reacted to specific bronchial provocation testing (CA median 0.15 ml, range 0-0.175 ml), only 1 animal was sensitized in group M (CA 0 ml, 0-0.125, p < 0.05), whereas no bronchial reactions were seen in groups I and N (CA 0 ml, 0-0.05, p < 0.025). Specific IgG antibody titres increased in the control group (median 43 EU-->85 EU), but not in the treatment groups (medians group I 35 EU-->35 EU, group M 30-->35 EU, group N 64-->50 EU).(ABSTRACT TRUNCATED AT 250 WORDS)


Mast-cell-derived mediators induce epidermal cell proliferation: clue for lichenified skin lesion formation in atopic dermatitis. Mast-cell-derived mediators showed mitogenic activities on mouse-transformed epidermal cell line Pam 212 cells. These activities were eluted into the low-molecular-weight fractions below a molecular weight of 10 kD on a high performance liquid chromatography TSK 2000G column, and were partially abrogated by antihistamines or anticytokine antibodies, including anti-IL1 alpha, -IL1 beta or IL6 antibodies. Pretreatment of mast cell lines with sodium butyrate enhanced the production of these factors. Calcium ionophore or Concanavalin A (ConA) stimulated mast cells to generate factor production. These results suggest that mast-cell-derived mediators might play some role in epidermal hyperplasia seen in lichenified lesions in atopic dermatitis.


Heparin-containing mast cells in the jejunal mucosa of normal and parasitized Mongolian gerbils, Meriones unguiculatus. Histochemical stainability of mast cells in the small intestine of Mongolian gerbils, Meriones unguiculatus, was examined before and after infection with Nippostrongylus brasiliensis, a well-known intestinal helminth inducing intestinal mastocytosis in rats and mice. Mast cells seen in the small intestine of Mongolian gerbils were exclusively formalin-resistant and were strongly stained by berberine sulfate, regardless of whether the animals were normal or infected with N. brasiliensis. Furthermore, they were, either normal or induced by infection, only seen in the lamina propria and submucosa but never seen in the jejunal epithelium. Such histochemical properties and distribution of intestinal mast cells of Mongolian gerbils were different from previously known characteristics of mast cells in the small intestine of rats and other animal species.


[Quantitative and qualitative anomalies of sperm in African individuals infected with HIV]. Semen from 16 HIV1-infected african individuals (12 at stage II/CDC; 4 with pre-AIDS) has been analyzed both quantitatively (volume; number of spermatozoa) and qualitatively (motility). 12 healthy HIV1-seronegative individuals were recruited as controls. The HIV1-infected patients showed generally abnormalities of the semen: oligospermia (7 cases), azoospermia (3 cases), or asthenospermia (10 cases). These abnormalities were more marked in patients at advanced stages of the HIV infection. In comparison with the control group, the HIV1-infected individuals had more frequently a past history of sexually transmitted diseases. These abnormalities in the composition of the semen in HIV1-infected african individuals could affect the fertility of these men; that could indirectly restrict the risk of congenitally HIV contaminated child.


[Cutaneous ulcer from Mycobacterium ulcerans. Apropos of 1 case in French Guiana]. Skin ulcer due to Mycobacterium ulcerans is presented. The patient come from East of French Guyana. Growth of this mycobacteria is obtained with diphasic Lowenstein medium at 30 degrees C. Diagnostic of M. ulcerans results from mycolic acids study.


[Where is the diagnosis of anthrax?].

[Where is the diagnosis of anthrax?]. The large use of antibiotics entailed a dizzy fall of the bacteriological positivity in diagnosis of acute anthrax, from 98-100% to 25% in average. Common serological methods are of low sensitivity and not so confluent. The more sophisticated methods (ELISA, EITB) are of a much better sensitivity but their employment in field epidemiological investigations is difficult and does not be suitable for a retrospective diagnosis. These inconveniences can be successfully surmounted by use of the anthraxin skin test. This test allows to diagnose acute anthrax in 98-100% of cases, and retrospectively (from 45 days to 30 years after recovery) in an average of 86.2% of cases. This skin test is simple for use, and does not requires a special medical competence.


[Stability and chemosensitivity of Plasmodium falciparum to chloroquine in 1990 and 1991 in Ankazobe, a village in high plateau Madagascar]. The in vivo and in vitro response of Plasmodium falciparum to chloroquine was conducted in Ankazobe, a village located in the high plateau area. These studies confirmed the low level of chloroquine-resistance. The in vivo data indicate the absence of increase resistance during the 2 years study. Chloroquine is still the drug of choice for the treatment of malaria attack in this area.


[Rapid diagnosis of malaria. The microwave furnace]. Thick blood is a well known method for malaria diagnosis, very faithful and sensitive, but it could not be used in emergency because it had to dry for many hours. Micro-wave over allows a quick drying (two minutes). Do it can now be used in emergency.


[Acanthamoeba keratitis. Morphologic and isoenzymatic study of a strain isolated from a medically treated case]. We report here the third case of Acanthamoeba keratitis discovered in France in a 30 year-old man wearing soft contact lenses. Amoebas of Acanthamoeba genus could be isolated twice from his corneal ulcer with simple ocular swabs. Owing to medical treatment alone, a successful healing of his eye was obtained. The isolated strain was studied morphologically and biochemically: the cysts observed microscopically after silver stain possessed less than six arms surrounded by a non reticulated ectocyst and belonged to group II defined by Pussard and Pons. But the isoenzymatic study of the ocular strain by isofocusing did not allow us to identify it with any one of the 18 reference strains used, although these represent 14 different described species.


[Complement to the epidemiologic study of the focus of Schistosoma mansoni bilharziasis in Kara (northern Togo)]. The present study on the focus of schistosomiasis mansoni of Kara (Northern Togo) complements a report already published in the Bulletin de la Société de Pathologie Exotique in 1988 (9). The following results were obtained through different experiments carried out with snails collected in Kara. According to the season, 3 to 15% of the Biomphalaria pfeifferi studied, were infected. The maximal release of cercariae was observed between 11 am and 1 pm. The survival of cercariae and their ability to infect mice were not superior to 16 hours. Data obtained with the chaetotaxy of miracidia of the Kara strain were compatible with those obtained with african strains by other authors. Moreover, B. pfeifferi from Kara was more susceptible to the Kara strain of Schistosoma mansoni than B. pfeifferi collected in the south of Togo or than B. glabrata from the new world. On the contrary, B. pfeifferi from Kara was susceptible to a strain of S. mansoni from Brazil and was able to produce infectious cercariae. In addition, the search for an animal reservoir was carried out; 3 of the 112 (2.7%) wild rodents captured around the river Kara, were infected. This relatively low number of infected rodents was compatible with the usual findings in Africa where the animal reservoir is considered from human origin and has no importance in the transmission of S. mansoni as it could have in South America and West Indies.


[Measure and analysis of urinary bilharziasis incidence levels in the village of Douloumi (Cameroon)]. A study on urinary schistosomiasis was carried out in the village of Douloumi to evaluate the changes in transmission over a one year period. It showed a stable prevalence rate between 1987 and 1988. However close scrutiny of the different population groups showed this apparent stability was the result of several trends: 1) a high incidence among the resident cohort with increase of prevalence among the younger age groups; 2) a high prevalence among individuals emigrating and 3) a low prevalence among immigrants. Incidence is considered as a better tool to evaluate the recent transmission of schistosomiasis, however incidence evaluation depends on the sensitivity of the case ascertainment method. In this study, a comparison of prevalences among a resident cohort was a good substitute to incidence.


[Alveolar echinococcosis in China: current data]. Very few information about alveolar echinococcosis in China is available outside the country. The aim of the authors is to give some precisions about the human cases and the infection in the natural animal hosts. It occurs in 3 distinct foci which comprise poor and remote rural areas. Approximately, 420 cases of human disease have been detected, and the most intense focus is Ningxia province in central China. In all areas, the adult tapeworm is frequently found in V. vulpes, V. corsac, and in the domestic dog. The intermediate hosts differ from an area to the others. Their infection rate is high in the central and the northern foci. More researches are needed for improving our knowledge about the epidemiology of the disease. But the actual major requirement is to apply control measures as health education and medical information.


[Ivermectin and pregnancy in mass treatment in Mali]. During july 1989, we have done a retrospective study, in Koba's valley in a savanna onchocerciasis area of Mali, after a mass treatment in rural community with ivermectin. 435 women aged from 15 to 45 years, among 461 with can be submitted to ivermectin risk during their pregnancy period, was seen and overhauled. In 1987, the frequency of women who received ivermectin during their pregnancy period (treated by error), was 17.7%, and 17.3% in 1988. We have seen any difference, between exposed and not exposed women to this error ivermectin treatment, comparing their in utero-mortality, new born mortality an the level of malformations. In spite of careful clinical monitoring in the field, the risk to give ivermectin to pregnancy women in rural community, is very high during mass treatment. Because traditionally (taboos), pregnancy is a forbidden subject. Sociological and anthropological studies, pregnancy field laboratory test and health education, are needed in african rural community before mass treatment with ivermectin.


[Therapeutic attitudes and malaria in Sanaga Basin (Cameroon)]. In order to evaluate malaria problems such as they are perceived by Sanaga Basin populations in Cameroon, authors appealed to a cluster sample survey on a population sample obtained from a 2 degree population poll in four representative sites. Malaria so defined represents the outstanding endemia by which the most usual therapeutic recourse makes self-medication interfere particularly by amino-4-quinolines. It is convenient to verify the quality of treatment so administered to adapt possibly training objectives of the interested population. In the present context of the chemoresistance extension of plasmodial strains to usual antimalarials, setting up a surveillance system of efficiency is necessary.


[The supernatural, health and community action in Black Africa]. Based on the experience of community participation for tsetse control in the Congo, the author attempts to enlarge the reflexion on general health problems in Black Africa. He desires to draw the attention of the readers unfamiliar with anthropology, 1) to the current existence and social importance in the African communities of a particular approach, grounded on belief in the supernatural, radically different from Western thinking, 2) the intrinsic ambiguousness and richness of these ways of thinking, 3) to its great influence in community participation in sanitation programs. The ethical problems involved in the intervention of mediators using traditional or confessional ways of thinking are raised and included in a psychosocial problematic.


Iron disorders can mimic anything, so always test for them. Routinely measuring iron status is necessary because not only are about 6% of Americans in significant negative iron balance, but about 1% have iron overload. Serum ferritin is in equilibrium with body iron stores, and is the only blood test that measures them. Barring inflammation, each one ng (0.0179 pmol) ferritin/ml of serum indicates approximately 10 mg (0.179 mmol) of body iron stores. Very early Stage I positive balance is best recognized by measuring saturation of iron binding capacity. Conversely, serum ferritin best recognizes early (Stage I and II) negative balance. Deviations from normal are: 1. Both stages of iron depletion (i.e. low stores, no dysfunction). Negative iron balance Stage I is reduced iron absorption producing moderately depleted iron stores. Stage II is severely depleted stores, without dysfunction. These stages include over half of all cases of negative iron balance. Treated with iron, they never progress to dysfunction, i.e. to disease. 2. Both stages of iron deficiency. Deficiency is inadequate iron for normal function, i.e. dysfunction, disease. Negative balance Stage III is dysfunction without anemia; Stage IV is with anemia. 3. Positive iron balance: Stage I is a multi-year period without dysfunction. Supplements of iron and/or vitamin C promote progression to dysfunction (disease). Iron removal prevents progression. Stage II is iron overload disease, encompassing years of insidiously progressive damage to tissues and organs from iron overload. Iron removal arrests progression.


Detection and measurement of cytokines.

Detection and measurement of cytokines. Accurate and sensitive methods for the measurement and detection of cytokines are an obvious pre-requisite for the study of cytokine biology, biochemistry and the possible involvement of these molecules in pathology. In this review, the various methods available for cytokine measurement and detection (bioassays, immunoassays and other procedures) are described and compared. A critical appraisal of the potential advantages and limitations of the techniques is included.


Autocrine growth factors and leukaemic haemopoiesis. Studies on the structure of haemopoiesis in acute myeloblastic leukaemia (AML) has shown the presence of a small population of malignant cells with extensive proliferative and self-renewal properties which are features of stem cells. The requirements of these cells for proliferation have been studied both in clonogenic assays in semi-solid media and in liquid suspension culture. These have demonstrated that AML clonogenic cells from the majority of patients, can be stimulated to proliferate by colony-stimulating factors (GM-CSF, G-CSF and IL-3) as well as other cytokines including interleukin-1 and interleukin-6, all of which are known to stimulate normal haemopoietic progenitors. Unlike normal haemopoietic cells, leukaemic blasts from many patients with AML express transcripts for haemopoietic growth factors including GM-CSF, G-CSF and IL-1 but not IL-3, and secrete growth factor protein. When leukaemic cells are cultured at sufficiently high density to permit cell-cell interactions, autonomous growth of clonogenic cells can be seen. Autonomous growth is related to the autocrine secretion of haemopoietic growth factors including GM-CSF, G-CSF and IL-6. The degree of autonomous colony growth is variable but approximately 70% of AML samples exhibit either partial or totally autonomous growth; the remaining cells being absolutely dependent on exogenous CSF or fail to grow in the culture systems employed. Similar patterns of growth have been found in murine haemopoietic cells lines which have been transformed as the result of the retroviral insertion of genes for GM-CSF or IL-3.(ABSTRACT TRUNCATED AT 250 WORDS)


Monoclonal antibody (MoAb) therapy in non-Hodgkin's lymphomas. The advance of monoclonal antibody (MoAb) technology in recent years provides a further treatment modality for cancer therapy. In this review, we discussed the various strategies available for MoAb therapy. We also addressed the problems encountered so far in early clinical trials and suggested possible solutions. We finally reviewed some of the interesting clinical studies conducted so far on the use of MoAbs in patients with non-Hodgkin's lymphomas.


Mechanisms of action of, and modes of resistance to, alkylating agents used in the treatment of haematological malignancies. Although the alkylating agents were amongst the first non-hormonal compounds to be shown to be active against malignant cells they still rank as some of the most valuable cytotoxic drugs available for the treatment of patients with leukaemia and lymphoma. Melphalan, chlorambucil, busulfan, cyclophosphamide, ifosfamide and the nitrosoureas are all members of this class of drug, which are believed to exert their cytotoxic effects through the covalent linkage of alkyl groups to DNA. In the first report describing the use of alkylating agents in clinical practice the problem of drug resistance was recognised. In spite of this there is still comparatively little known about the mechanisms underlying the development of resistance as it occurs in patients. Studies using animal models and cell lines have suggested that both cellular and extracellular factors may be involved, but the precise relevance of these to the clinical setting is unclear. A greater understanding of the mode of action and mechanisms of resistance to alkylating agents should enable the development of modulators capable of the restoration of sensitivity to resistant cells, and the more effective use of these well established drugs.


The practical management of haemophilia. Haemophilia is a rare and complex disorder and its successful management will depend upon the establishment of a network of 'comprehensive care' including the services of haematologists, orthopaedic surgeons, rheumatologists, dental surgeons, physiotherapists, specialised nurses and counsellors. One of the major lessons to be learned from the HIV epidemic in haemophilia is that it is critical to strive to obtain the safest and purest forms of blood products for these patients. The advent of clinically available recombinant factor VIII is expected soon; in the meantime there is a move towards treating all patients with high purity products.


Transligamentary anesthesia: a review.

Transligamentary anesthesia: a review. Intraligamentary anesthesia or, as we prefer to call it, transligamentary anesthesia (TLA) is one of the more recent developments in the field of pain blockage in dentistry. This review article organizes the vast amount of information, both current as well as past, found in the dental literature. After a general historical introduction, indications for TLA use are explained. This is followed by a technical examination of the anesthesia, including the various syringes in use, the preferred injection technique, and the types and volume of anesthesia solutions used. Other aspects of TLA, including the rate of success, onset time, duration, and animal/laboratory studies, are then examined. The various advantages and disadvantages of TLA are discussed along with adverse reactions, limitations, and patient acceptance. The article closes with a look at the diagnostic possibilities of the injection as well as a call for future research on this important development.


Effects of epinephrine and norepinephrine contained in 2% lidocaine on hemodynamics of the carotid and cerebral circulation in older and younger adults. The effects of vasoconstrictor-containing local anesthetics on the carotid and cerebral circulation were investigated using an ultrasonic quantitative blood flow measurement system. Nineteen healthy adult volunteers were divided into two groups according to age. The local anesthetics used were 2% lidocaine containing 1:80,000 epinephrine and 2% lidocaine containing 1:25,000 norepinephrine. One (1.8 mL) or two (3.6 mL) cartridges of each anesthetic were injected into unilateral or bilateral maxillary gingivae. Epinephrine-containing lidocaine had little effect on carotid and cerebral hemodynamics. Norepinephrine-containing lidocaine increased arterial blood pressure, decreased heart rate, and decreased carotid arterial blood flow and velocity, which caused increased cerebrovascular resistance and decreased cerebrovascular capacitance. The results suggest that norepinephrine may induce vasoconstriction of the cerebral blood vessels in both young and old adults in a dose-dependent manner. These changes appear to be greater in older people.


Anesthesia for temporomandibular arthroplasty in a quadriplegic patient: a case report. A 41-year-old male quadriplegic patient with bilateral TMJ ankylosis was admitted for surgery under general anesthesia. Preoperative tests showed abnormal lung function and a low total serum calcium level. Premedication consisted of diazepam 10 mg orally and glycopyrrolate 0.2 mg intramuscularly 90 minutes before the start of the operation. Because of an inability to open the mouth more than 0.5 cm, a nasal fiberoptic endoscopic intubation was done under sedation and local anesthesia. For maintenance of anesthesia the patient received nitrous oxide 50% in oxygen, halothane, and alfentanil. No muscle relaxant was administered. The 2-hour operation was completed uneventfully and the patient needed no ventilatory support afterward. Major anesthetic problems of spinal injury patients can be compared to those with cervical cord transection. The stage that the condition has reached determines the dominant anesthetic problems. The chronic stage (greater than 3 months postinjury, as in this patient) is usually characterized by sympathetic overactivity. Anesthetic problems during this stage may include risk of hyperkalemia from succinylcholine, cardiovascular instability and autonomic hyperreflexia, impaired thermoregulation, anemia, chronic infections, risk of hypercalcemia, and alveolar hypoventilation. None of these problems was encountered in this patient. A surprising finding was that the low serum calcium concentration did not influence the anesthetic outcome.


Experience with triazolam in preschool children. Triazolam (0.125 to 0.25 mg) was administered preoperatively to 17 unmanageable children aged 22 to 65 months. The children were monitored visually and by pulse oximeter. No child experienced any airway obstruction and none had oxygen saturation below 90%. Older children had lower SaO2 values than younger children. There was no relationship between the mg/kg dose and SaO2 values.


Effects of different lidocaine sprays for topical anesthesia on cardiovascular parameters and oxygen saturation. The effects of three lidocaine-containing solutions with and without frigen (freon-113) as a propellant on cardiovascular parameters and oxygen saturation, after different waiting periods and with different dosages applied, were investigated in 130 outpatients undergoing dental treatment in the maxilla under local anesthesia. They were divided randomly into five groups: (A) Xylocaine spray with frigen, two applications (20 mg lidocaine); (B) Xylestesin spray with frigen, two applications (14 mg lidocaine); (C) Xylestesin spray with frigen, three applications (21 mg lidocaine); (D) Xylestesin pump spray without frigen, two applications (14 mg lidocaine); and (E) no topical anesthesia. They were further divided into 12 subgroups to evaluate the influence of waiting periods between the application of the local anesthetic and the injection (1, 2, or 3 minutes). Without topical anesthesia and after the 1-minute waiting period, cardiovascular parameters increased immediately after the injection; after 2- and 3-minute waiting periods the increases were far less pronounced. The mean oxygen saturations did not change during the observed period. The higher lidocaine dosage administered (21 mg) did not intensify the positive impact on the cardiovascular parameters compared to the lower dosage (14 mg). The new pump spray without frigen proved to be as effective as the traditional, ecologically harmful aerosol sprays.


Emergency treatment in Australia.

Emergency treatment in Australia. This short communication describes the key points of the emergency medical treatment situation for dentists in Australia. Dental school training and postgraduate educational opportunities in emergency medicine, emergency equipment and drugs found in a typical dental office, and use of the EMS by dentists are covered.


Physical evaluation of the patient.

Physical evaluation of the patient. The physical evaluation of the patient before and during anesthesia and sedation is assuming an ever greater importance with the advent of sophisticated and reliable monitoring devices. This paper reviews the possible methods of evaluation and describes the types of mechanical monitors most suited to the dental office.


Isolation of human tumor cells that produce cementum proteins in culture. We have cultured cells from explants of a human cementum tumor. The cells obtained were multipolar, they formed network-like structures and they were alkaline-phosphatase positive. Immunostaining and Western blots using specific antibodies revealed that these cells produced bone sialoprotein and collagen types I and V, and they also mineralized in vitro. Conditioned medium was mitogenic to fibroblasts and mitogens present were separated by heparin-affinity chromatography. Based on affinity to heparin and antibody-inhibition studies, the heparin fractions were shown to contain cementum-derived growth factor, platelet-derived growth factor and fibroblast growth factors. The cementum tumor cells, but not gingival fibroblasts, were stained positively by an antibody to cementum-derived attachment protein. The attachment protein was separated by immunoaffinity chromatography, and Western blots revealed that the preparation contained 56-kDa and 43-kDa proteins as major bands. Cells pulse-labeled with radioactive amino acids contained a 43-kDa protein as the major component; however, this protein was absent after a cold chase in the presence of cycloheximide, but 56-kDa, 39-kDa and 26-kDa species became prominent. These data indicated that the 56-kDa cementum attachment protein is derived from a 43-kDa precursor. Our data show that the cells cultured from the cementum tumor represent cementum cells capable of synthesizing and secreting cementum proteins in culture.


Chromogranin-A secretion from individual parathyroid cells: effects of 1,25-(OH)2 vitamin D3 and calcium. Chromogranin-A (CgA) is a 50-kDa protein located in and secreted by most endocrine and neuroendocrine cells along with native hormone. In the parathyroid gland, CgA is cosecreted with parathyroid hormone (PTH). Although these peptides are secreted together, recent evidence has suggested that they are processed differently in response to stimuli, such as 1,25-(OH)2 vitamin D3 and calcium. We have validated a reverse hemolytic plaque assay for studying CgA release from parathyroid cells. This assay allows for the detection of quantitative changes in hormone secretion from individual parathyroid cells. Bovine parathyroid cells were mixed with protein-A-linked ovine erythrocytes (oRBC) and plated in a monolayer in the presence of CgA antiserum. After incubation, complement was added to the cells to induce cell lysis. Lysis of oRBC around a parathyroid cell indicated the release of CgA from a cell. Results showed that plaque formation was dependent on assay reagents and that serial dilution of the antibody reduced plaque formation. CgA secretion was inhibited by increasing concentrations of calcium and stimulated by increasing concentrations of 1,25-(OH)2vitamin D3.


Effects on cultured neonatal mouse calvaria of 1 alpha,25-dihydroxyvitamin D3, 26,26,26,27,27,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 and 26,26,26,27,27,27-hexafluoro-1 alpha,23S,25-trihydroxyvitamin D3. The potency of 26,26,26,27,27,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 (26,27-F6-1,25(OH)2D3) to enhance bone calcium (Ca) mobilization in vitro was higher than that of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In a 48-h organ culture system using fetal rat limb bones, effects of 26,27-F6-1,25(OH)2D3 on bone resorption were similar to those of 1,25(OH)2D3. Thus, we attempted to clarify whether or not the potency of 26,27-F6-1,25(OH)2D3 in bone resorption in vitro would be higher than that of 1,25(OH)2D3. Calvarial bones from neonatal mice were used as explants and the culture period was extended to 144 from 48 h. In cultures of 0 to 48 h, both drugs increased the release of prelabeled 45Ca from cultured calvariae, in a dose-dependent manner. In cultures of 48 to 144 h, 26,27-F6-1,25(OH)2D3 was much more effective than 1,25(OH)2D3 regarding indices of bone resorption. 26,26,26,27,27,27-F6-1 alpha,23S,25-trihydroxyvitamin D3 (26,27-F6-1,23,25(OH)3D3), a main metabolite of 26,27-F6-1,25(OH)2D3 in rats, stimulated 45Ca release equipotently to 1,25(OH)2D3. In mouse bone marrow cells cultured for 7 days, the potency of 26,27-F6-1,25(OH)2D3 on osteoclast formation was much higher than 1,25(OH)2D3. These results suggest that the higher potency of 26,27-F6-1,25(OH)2D3 in bone resorption may be due at least in part to both the higher potency of 26,27-F6-1,25(OH)2D3 in osteoclast formation and the larger retention of 26,27-F6-1,23,25(OH)3D3 in calvariae.


Preferential osteopenia in women with osteoporotic fractures. One-hundred thirty-six women with spontaneous vertebral crush fractures had bone density measurements by photon absorptiometry. Their absolute and relative extent of osteopenia were expressed as Z scores with respect to values from normal white women. Absolute Z scores were used to express the mean bone mass of women with crush fractures vis-a-vis normal women at peak bone mass; relative Z scores were used to express the same means vis-a-vis age-matched controls. The skeletal sites measured included the distal radius, lumbar spine, and three sites on the femur (neck, Ward's triangle, and trochanter). The absolute deficit for the five sites ranged from -1.66 to -2.60. The relative deficit was close to 1 SD for all sites. A discriminant analysis revealed that the spine and femur were predictive for vertebral crush fractures but the radius was not. Comparison with a previously reported hip-fracture population (n = 31) showed that the low bone density of the spine separated the two fracture groups best. We propose the following: (a) vertebral-fracture patients have extensive generalized osteoporosis including the hip with a preferential deficit in the spine and (b) hip-fracture patients have generalized osteoporosis with preferential osteopenia of the hip and do not have a greater mineral deficit in the spine or radius than age-matched normal women. It may be that if a single site is to be selected for screening for spine and hip fractures, the greatest value would be in measurement of the femur, although ideally the skeletal site of interest would be measured. Prospective studies are needed to confirm the utility of measurements at the spine and hip in prediction of future fractures.


Lack of deleterious effect of slow-release sodium fluoride treatment on cortical bone histology and quality in osteoporotic patients. We evaluated the effects of intermittent slow-release sodium fluoride (SRNaF) and continuous calcium citrate therapy on cortical bone histology, reflection ultrasound velocity (material strength) and back-scattered electron image analysis (BEI) in 26 osteoporotic patients before and following therapy. All measurements were made on transiliac crest bone biopsies obtained before and following 2 years of therapy in each patient. For all 26 patients there were no significant changes in cortical bone histomorphometric parameters. In 15 patients in whom bone material quality was assessed by reflection ultrasound, there was no change in velocity (4000 +/- 227 SD to 4013 +/- 240 m/s). BEI disclosed no mineralization defects or the presence of woven bone. Mean atomic number (density) of bone increased slightly, but significantly (9.261 +/- 0.311 to 9.457 +/- 0.223, P = 0.031). While these changes are less marked than those observed for cancellous bone, they indicate that this form of therapy does not adversely affect cortical bone remodelling.


Comparison of single-photon and dual-energy x-ray absorptiometry of the radius. We compared dual-energy x-ray absorptiometry (DXA) for measurement of the radius with the conventional single-photon absorptiometry (SPA) method. To evaluate reproducibility, 34 healthy male and female subjects were measured twice each by both methods. While the instruments measured bone mineral content (BMC) similarly, projected area was consistently lower by SPA and therefore bone mineral density (BMD) was higher by an average of 10%. We report similar coefficients of variation for the two methods, which are 0.8% (SPA) and 0.7% (DXA) for BMC (P = 0.71) and 0.8% (SPA) and 1.4% (DXA) for BMD (P = 0.02). We also evaluated the relationship between the methods with data from 196 clinic patients who were measured once each on both instruments. The BMD measurements from the two instruments were highly correlated (r = 0.97) in these patients, which permits the conversion of databases from SPA to DXA-equivalents. We conclude that DXA can replace SPA for radial bone densitometry.


Trans-differentiation of hypertrophic chondrocytes into cells capable of producing a mineralized bone matrix. Trans-differentiation of hypertrophic chondrocytes into bone-forming cells was observed when femurs from 14-day-old chick embryos were cut through the region of hypertrophic cartilage and the separated pieces were cultured for 2-18 days. Inside many chondrocytic lacunae a new matrix was present which had the staining characteristics of bone matrix including birefringence and the capacity to mineralize. The cells within the lacunae had the characteristics of osteoblasts, such as alkaline phosphatase activity and positive immunocytochemical staining for osteocalcin, osteonectin, osteopontin and type I collagen. Chondrocyte necrosis and empty lacunae were only observed immediately at the cut edge, and in that region no bone-forming cells were present inside the lacunae. Where bone-matrix was present, the lacunae had remained intact, the cells were viable and no evidence of cell migration was observed. This suggested that the bone-forming cells had originated from the hypertrophic chondrocytes. The temporal sequence of events was followed closely. Two days following the cut only a few chondrocytes showed a positive reaction for osteocalcin, osteonectin, osteopontin and the type I collagen. At that time no such reaction product was observed in the chondrocytes of uncut femurs. Many hypertrophic chondrocytes divided, as shown by tritiated thymidine incorporation. The rate of cell division increased between 2-6 days, when several smaller basophilic cells were present inside the lacuna instead of the single hypertrophic chondrocyte. These cells expressed alkaline phosphatase activity, were positive for fibronectin, the above non-collagenous bone proteins and type I collagen. The bone matrix that was observed after 6-18 days was initially confined to the inside of the chondrocytic lacunae, but later spread beyond the lacunar confines. The bone proteins were still associated with the bone-forming cells, but fibronectin was absent when matrix formation was evident. Mineralization of the intra-lacunar osteoid took place after 12-18 days. It is speculated that the trans-differentiation was initiated by disruptions of the normal cell-cell associations.


The anabolic action of 17 beta-estradiol (E2) on rat trabecular bone is suppressed by (3-amino-1-hydroxypropylidene)-1-bisphosphonate (AHPrBP). We have previously found that high doses of 17 beta-estradiol (E2), similar to those seen in late pregnancy, stimulate bone formation in adult rats. In this communication we tested the effects of a combination of E2 and (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) on bone formation and bone volume in rat bone. E2 (4 mg/kg/day subcutaneously for 17 days) stimulated the bone formation rate to 6 times that of control rats. This was reduced by a single administration of AHPrBP (0.3 mg/kg subcutaneously) to 3 times control levels, and by similar daily injections of AHPrBP to levels not significantly different from those of untreated rats. Suppression of bone formation was effected predominantly through a reduction in the percentage of double-labelled surfaces, consistent with reduced osteoblast recruitment. We found only relatively minor effects of AHPrBP on the mineral apposition rate, suggesting that AHPrBP affected osteoblast function less than osteoblast recruitment. Suppression of histodynamic parameters of bone formation by AHPrBP was associated with suppression of the increase in bone volume otherwise induced by E2. The suppression by AHPrBP of the effect of E2 on bone formation contrasted with its lack of effect on other target tissues for E2, since AHPrBP did not affect the E2-induced changes in longitudinal bone growth or uterine weight. These results suggest that AHPrBP inhibits the anabolic effect of estrogen on rat trabecular bone.


Calcium ion transport kinetics during dentinogenesis: effects of disrupting odontoblast cellular transport systems. Due to strongly discrepant results in the literature, controversy exists about the timing of the transport of Ca2+ ions to the mineralization front during dentinogenesis and the role of the odontoblasts in this transport. The present study gives evidence, by means of autoradiography as well as by a radiochemical technique, that the transport time for Ca2+ ions into the dentin mineral phase is about 10-15 min in the rat incisor. The results also show that technical factors, such as mode of tracer injection and the use of perfusion fixation, may influence the results more or less strongly. Finally, by disturbing odontoblast microtubules, involved in intracellular transport processes, and by blocking odontoblast calcium uptake channels by nifedipine and neomycin, the Ca2+ ion transport into dentin mineral was found to be strongly impaired. This may be taken as an indication that transcellular calcium transport mechanisms have a role during dentinogenesis.


Prostaglandin E2 prevents ovariectomy-induced cancellous bone loss in rats. The object of this study was to determine whether prostaglandin E2 (PGE2) can prevent ovariectomy-induced cancellous bone loss. Thirty-five 3-month-old female Sprague-Dawley rats were divided into two groups. The rats in the first group were ovariectomized (OVX) while the others received sham operation (sham-OVX). The OVX group was further divided into three treatment groups. The daily doses for the three groups were 0, 1 and 6 mg PGE2/kg for 90 days. Bone histomorphometric analyses were performed on double-fluorescent-labeled undecalcified proximal tibial metaphysis (PTM). We confirmed that OVX induces massive cancellous bone loss (-80%) and a higher bone turnover (+143%). The new findings from the present study demonstrate that bone loss due to ovarian hormone deficiency can be prevented by a low-dose (1 mg) daily administration of PGE2. Furthermore, a higher-dose (6 mg) daily administration of PGE2 not only prevents bone loss but also adds extra bone to the proximal tibial metaphyses. PGE2 at the 1-mg dose level significantly increased trabecular bone area, trabecular width, trabecular node density, density of node to node, ratio of node to free end, and thus significantly decreased trabecular separation from OVX controls. At this dose level, these same parameters did not differ significantly from sham-OVX controls. However, at the 6-mg dose level PGE2, there were significant increases in trabecular bone area, trabecular width, trabecular node density, density of node to node, and ratio of node to free end, while there was significant decrease in trabecular separation from both OVX and sham-operated controls. The changes in indices of trabecular bone microanatomical structure indicated that PGE2 prevented bone loss as well as the disconnection of existing trabeculae. In summary, PGE2 administration to OVX rats decreased bone turnover and increased bone formation parameters resulting in a positive bone balance that prevented bone loss (in both lower and higher doses) and added extra bone to metaphyses of OVX rats (in higher dose). These findings support the strategy of the use of bone stimulation agents in the prevention of estrogen depletion bone loss (postmenopausal osteoporosis).


Differential chemotactic responses of different populations of fetal rat calvaria cells to platelet-derived growth factor and transforming growth factor beta. We tested the chemotactic response to platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta) of cells released enzymatically from fetal rat calvaria (RC). Both factors were chemotactic for RC cells, but the magnitude of the chemotactic response differed markedly between different populations and varied with time in culture of the cell populations. Cells released earlier from the calvaria showed a greater response than osteoblast-enriched populations released later. The optimal concentration of PDGF was the same for both alkaline phosphatase (AP)-positive and AP-negative cells within the populations. However AP-positive cells showed two peaks of response to TGF beta; one peak coincided with the TGF beta concentration also maximally affecting AP-negative cells, while the other occurred at a concentration 50-100 times higher. The results indicate that PDGF and TGF beta are chemotactic for both AP-positive and AP-negative cells in populations of cells derived from fetal calvariae, that chemotactic response declined with longer periods of time in culture, and that AP-positive osteoblast-like cells respond to a concentration of TGF beta that does not affect the AP-negative cells in the population.


The dose-dependency of alcohol-induced hypoparathyroidism, hypercalciuria, and hypermagnesuria. Ingestion of alcohol evokes hypoparathyroidism, hypercalciuria, and hypermagnesuria. The dose-dependency of these changes has not been assessed before. We measured the serum concentrations of intact parathyroid hormone (PTH), and serum and urine calcium and magnesium in six normal men before and at intervals up to 6 h after the ingestion of fruit juice (control) and 0.5, 1.0 and 1.3 g of alcohol per kg of body weight. As compared with the control experiment the maximum reductions in the mean PTH concentration were 31% (P = 0.19), 31% (P = 0.20) and 45% (P = 0.01) with the three alcohol doses, respectively. After stopping drinking, the urinary excretion of calcium was 85%, 142% and 207% higher during the three alcohol experiments than during the control session (P < 0.05, < 0.01 and < 0.01, respectively), also urinary magnesium increased up to threefold. We conclude that in nonalcoholic subjects acute alcohol intake induces hypoparathyroidism, hypercalciuria, and hypermagnesuria, the latter two being dose-dependent. The direct renal effects of alcohol are the major mechanisms for hypercalciuria and hypermagnesuria, but hypoparathyroidism contributes to hypercalciuria at high levels of alcohol intoxication.


Bone mineral density during puberty in western Canadian children. To assess the influence of puberty and its associated changes in body weight and height on bone mineral density (BMD), lumbar spine (L2-L4) and femoral neck BMD were measured in 74 healthy, active children (9-16 years) using dual-photon absorptiometry. Competitive swimmers were recruited to minimize the potential effect variability in mechanical loading regime may have on bone density of the lumbar spine. Tanner staging was used to assess stage of puberty. Current dietary calcium intake was assessed by analysis of 6-day dietary records. Significant differences in spinal and femoral neck BMD occurred between early (Tanner 1 and 2) and late stages of puberty (Tanner 4 and 5), P < 0.05. A significant correlation was found between bone density and dietary calcium intake. However stepwise regression analyses demonstrated stage of puberty or body weight were the only factors which significantly affected spinal BMD, accounting for 77% and 68% of the variability respectively; while at the femoral neck, body weight accounted for 52% of the variability. These results demonstrate that when potential interacting factors are controlled for through regression analyses, differences in BMD occur mainly as a function of puberty and the associated gains in body weight.


Hormone responses of in vitro bone nodule cells: studies on changes of intracellular calcium and membrane potential in response to parathyroid hormone and calcitonin. We used two techniques to study the responses of individual in vitro bone nodule cells to parathyroid hormone (PTH) and calcitonin (CT). These techniques are laser scanning confocal imaging with a fluorescent indicator to measure intracellular free [Ca2+], and microelectrode impalement to measure the electrical potential difference across the cell membrane. We applied these measurement techniques to cells in the top cellular layer of nodules that form in vitro in cultures of cells obtained from fetal rat calvaria. Our measurements showed a transient increase in intracellular free [Ca2+] following application of PTH or CT. The duration of the increase in fluorescent intensity following PTH application varied from about 100 to more than 300 s, and the duration following CT application was from 30 to 80 s. In some measurements we applied both hormones in sequence, and observed that some cells showed an intracellular [Ca2+] response to both hormones, while other cells apparently responded to only one or the other of the hormones, or to neither. We also observed membrane potential changes in response to PTH and to CT. The membrane potential response to CT was quite small. The time courses of these membrane potential changes consisted of a depolarizing phase lasting about 100 s (with both hormones) followed by a hyperpolarizing phase (with PTH). Control measurements using only the vehicle solutions were carried out with both techniques, producing negligible responses.


The effect of rat parathyroid hormone (1-34) infusion on urinary 3-hydroxypyridinium cross-link excretion in the rat. The utility of measurement of the urinary excretion of the 3-hydroxypyridinium cross-links, pyridinoline (Pyr) and deoxypyridinoline (Dpyr) as indices of bone resorption in rats was investigated. Total Pyr and Dpyr excretion were measured in young rats treated by s.c. infusion with rat parathyroid hormone (1-34) (PTH) at 22-30 micrograms/kg/day or with diluent (controls) for 14 days. During infusion, average urinary excretion of both cross-links was significantly higher in PTH rats (Pyr: 11.77 +/- 0.44 nmol/day), Dpyr: 15.81 +/- 0.95 nmol/day) than in controls (Pyr: 10.17 +/- 0.35 nmol/day, Dpyr: 12.03 +/- 0.67 nmol/day). These results were consistent with the magnitude of the expected increase in bone resorption rate with this dose of PTH. The method appears to provide a sensitive measure of bone resorption for in vivo bone studies in rats.


Decreased heterotopic osteogenesis in vitamin-D-deficient, but normocalcemic guinea pigs. The effect of vitamin D deficiency unhampered by hypocalcemia on de novo bone formation was studied in guinea pigs. Heterotopic induction of osteogenesis was evaluated 4 weeks after intramuscular transplantation of allogenic urinary bladder transitional epithelium from vitamin-D-repleted (+D) donors into +D and -D recipients. In -D recipients the frequency of osteogenesis and the amount of induced bone were significantly diminished; induced bone was less mature, scantly cellular woven bone poorly repopulated with bone marrow. No effect of vitamin D deficiency on orthotopic bone growth and on mineralization of orthotopic and heterotopically induced bone was observed. It is proposed that in addition to inducing factors (BMPs, growth factors) which may be responsible for transformation of mesenchymal cells to osteoprogenitor cells, normal concentrations of 1,25-(OH)2D3 may be required for proliferation and further differentiation of these cells into osteoblasts and for expression of genes engaged in extracellular matrix formation and maturation.


Familial idiopathic hyperphosphatasia (FIH): response to long-term treatment with pamidronate (APD). A 5-year-old child suffering from familial idiopathic hyperphosphatasia (FIH) was treated by: (1) intravenous infusion of pamidronate (APD) (3 h) (0.75 mg/kg/day) for 5 days; and (2) oral administration of APD (8 mg/kg/day) for 1 year, in association with calcium (1 g/day) as calcium gluconate. A decrease of both serum calcium and phosphate, and a slight PTH increase were observed immediately after the IV treatment; serum alkaline phosphatase did not change, but a marked and rapid decline in the hydroxyprolinuria was observed: basal 659 +/- 207 during IV treatment 169 +/- 59 (mean +/- SD mg/24 h, P < 0.005). At the end of one year of oral APD treatment clinical and radiological findings showed a remarkable improvement. Serum calcium, phosphate and PTH returned to the initial values. Plasma alkaline phosphatase levels showed a 70% decrease: basal 1370 IU/l, 1 year 410 IU/l whereas the hydroxyprolinuria values were similar to those determined at the end of the intravenous treatment (212 +/- 13 mg/24 h), but still significantly lower than the basal levels (P < 0.01). No side-effects were observed. APD appears to be a promising treatment for patients with FIH.


Osteoporosis and calcification of the aorta. In an age-stratified random sample of 200 Rochester, Minnesota women, the prevalence of aortic calcification rose with aging, as did the prevalence of vertebral fractures, while bone mass fell. The statistically significant positive association of aortic calcification with vertebral fractures and the negative associations with bone mass at six skeletal sites were mainly accounted for by age. After age-adjustment, the only association remaining was a negative one between calcified aortic plaques and bone mineral density (BMD) of the lumbar spine (P < 0.05). Aortic calcification was not associated with any measures of calcium metabolism, after adjusting for age, except for a slight negative association between linear aortic calcifications and 25(OH) vitamin D levels (P < 0.05). BMD values of the lumbar spine were somewhat greater than predicted for age in women with severe aortic calcification, but similar findings were seen at other skeletal sites and none of the differences was statistically significant. While overestimation of bone mass was generally minimal, severe aortic calcification may distort lumbar spine assessments in a minority of postmenopausal women.


Cytological and ultrastructural investigation on osteoblastic and preosteoclastic cells grown in vitro in the presence of ipriflavone: preliminary results. The effects of ipriflavone on bone cells were studied in vitro on pre-osteoclastic (FLG 29.1) and osteoblast-like (Saos-2) cells grown for 48 h either separately or in co-cultures, with or without the addition of PTH. Histological, ultrastructural and histochemical (TRAP-activity demonstration) methods were used. The main results show that ipriflavone reduces replication of FLG 29.1 cells and inhibits TRAP production by these cells both in controls and in co-cultures treated with PTH. Moreover, it has a moderate stimulatory effect on proliferation of osteoblast-like cells and reduces the PTH-induced degenerative changes of Saos-2 cells. These results suggest that the inhibitory effect of ipriflavone on FLG 29.1 cells might be indirect and might be mediated by the osteoblast-like cells.


Effects of ipriflavone on bone remodeling in primary hyperparathyroidism. The aim of this study was to evaluate the effects of ipriflavone treatment on bone remodeling in primary hyperparathyroidism. Nine patients, 6 females and 3 males (mean +/- SD age 56 +/- 12.5 years) were treated with 1200 mg/day of ipriflavone by oral administration divided in 3 daily doses. All patients were treated for 21 days; in 5 patients treatment was prolonged to 42 days. In all patients the main serum and urinary parameters of bone remodeling were evaluated. The results suggest that ipriflavone affects bone remodeling by inhibiting bone resorption without affecting bone formation. Ipriflavone is, therefore, indicated in the treatment of metabolic bone diseases characterized by a high bone turnover.