Claes Juhlin

Tissue Distribution of Human gp330/Megalin, a Putative Ca2+-sensing Protein

We used riboprobes and monoclonal antibodies to characterize tissue distribution of the human 550-kD homologue to gp330/megalin, primarily identified in the rat kidney. Human gp330/megalin mRNA and protein are readily identified in human parathyroid cells, placental cytotrophoblasts, kidney proximal tubule cells, and epididymal epithelial cells. The immunoreactivity is found on the surface of the cells and is heterogeneously downregulated in parathyroid hyperplasia and adenomas. Cells of the proximal kidney tubule and epididymis express the protein on their luminal aspect. Moreover, the protein is expressed in Type II pneumocytes, mammary epithelial and thyroid follicular cells, and the ciliary body of the eye. Sequence analysis of cDNA fragments, obtained by RT-PCR, revealed identical nucleotide sequences in parathyroid, kidney, placenta, epididymis, and lung. Immunohistochemistry for parathyroid hormone-related protein (PTHrP) revealed partial co-expression with human gp330/megalin in parathyroid, placenta, and mammary gland. The findings substantiate human gp330/megalin expression in a variety of human tissues expected to possess calcium-sensing functions. It may constitute a protein of utmost importance to adult and fetal calcium homeostasis, although other important functions may also be coupled to this exceptionally large protein with highly restricted tissue distribution. (J Histochem Cytochem 45:383–392, 1997)

Parathyroid-like regulation of parathyroid-hormone-related protein release and cytoplasmic calcium in cytotrophoblast cells of human placenta

Immunohistochemical staining of human placenta revealed intense reactivity for amino terminal and midregional parathyroid-hormone-related protein (PTHrp) in the cytotrophoblast cells and weaker staining in the syncytiotrophoblasts. The cytotrophoblasts also displayed conspicuous surface staining with the monoclonal antibodies E11 and G11, which recognize a Ca2+ receptor mechanism regulating hormone release of parathyroid cells. Cytotrophoblasts enriched on Percoll gradients or by linking surface-bound E11 to magnetic beads revealed biphasic elevation of cytoplasmic Ca2+ ([Ca2+]i) upon a stepwise rise of external Ca2+ from 0.5 to 3.0 mM, with a half-maximal effect at 1.75 mM. Individual cytotrophoblasts identified by their E11 reactivity disclosed a temporary increase of [Ca2+]i upon elevation of external Mg2+, while Mn2+ triggered both a [Ca2+]i transient and an influx of itself. These effects were efficiently blocked by the G11 antibody. Depolarization with K+ or addition of the voltage-dependent Ca2+ channel blocker verapamil had only marginal effects on [Ca2+]i. Raised extracellular calcium inhibited release of PTHrp from the cells, and this inhibition was blocked by the G11 antibody. The virtually parathyroid-identical Ca2+ regulation of [Ca2+]i may mediate feedback control of PTHrp release from the cytotrophoblasts and thereby participate in the regulation of placental Ca2+ transport.

Tumor-specific deposition of immunoglobulin G and complement in papillary thyroid carcinoma

Despite its predilection for multifocal growth and regional metastasis, papillary thyroid carcinoma (PTC) is a clinically indolent malignancy with an exceptionally favorable long-term prognosis. Together with the often striking inflammatory reaction present in PTC, its quiescent behavior has been suggested to reflect the activation of a tumor-induced immune response. To examine this possibility, we have studied the deposition of immunoglobulins and complement in PTC tissue. Samples from 70 cases of neoplastic and autoimmune thyroid diseases, including PTC (n = 41), follicular, anaplastic, and medullary carcinomas (n = 12), follicular adenoma (n = 6), Graves disease (n = 8), and Hashimotos thyroiditis (n = 3) were analyzed immunohistochemically. Cellular deposits of immunoglobulin G (IgG), particularly subclasses IgG1 and IgG4, and complement factors C3d, C4d, and C5 were shown in up to 80% of the PTC cases, whereas the other thyroid diseases studied showed little or no cellular deposition. Nonneoplastic tissue of PTC-containing thyroid glands (n = 22) lacked staining for IgG in 50% of the cases, and 82% were devoid of complement. The results suggest a tumor-specific immune response in PTC with activation of the classical complement cascade.

Monoclonal anti-parathyroid antibodies interfering with a Ca2+-sensor of human parathyroid cells

Previous findings indicate that binding of Ca2+ to an external receptor is part of the mechanism by which extracellular Ca2+ regulates cytoplasmic Ca2+ and hormone release of parathyroid cells. We now present evidence that two newly generated monoclonal anti-parathyroid antibodies react with structures involved in this sensing and/or gating of Ca2+. Microfluorimetric studies of fura 2-loaded human parathyroid cells thus revealed that the antibodies competed with Ca2+ and antagonized the rise in cytoplasmic Ca2+ normally obtained when parathyroid cells are exposed to increasing concentrations of extracellular Ca2+.

Modulation of the Ca2+-sensing function of parathyroid cells in vitro and in hyperparathyroidism.

When raising the extracellular Ca2+ concentration stepwise from 0.5 to 3.0 mM, bovine parathyroid cells reacted with initial transient and sustained elevations of the cytoplasmic Ca2+ concentration (Ca2+i), as well as more than 50% inhibition of parathyroid hormone (PTH) release. Human parathyroid adenoma cells and bovine cells cultured for 1 day or exposed to a low concentration of a monoclonal antiparathyroid antibody exhibited right-shifted dependencies of PTH release and Ca2+i on extracellular Ca2+ and reduced Ca2+i transients. The protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) further right-shifted the dose response relationship for Ca2+ regulated Ca2+i of the adenoma cells, whereas the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) tended to normalize it, without affecting Ca2+i of normal bovine cells. In cells from an oxyphil adenoma and a parathyroid carcinoma as well as in bovine cells cultured 4 days or exposed to a high concentration of the antiparathyroid antibody, there were no Ca2+i transients, very small increases in steady-state Ca2+i and nonsuppressible PTH release. The results suggest that reduced availability of a putative Ca2+-receptor and increased protein kinase C activity may be important factors in the decreased Ca2+ sensitivity of abnormal parathyroid cells.

THE USE OF PRE- OR POSTOPERATIVE ANTIBIOTICS IN SURGERY FOR APPENDICITIS: A SYSTEMATIC REVIEW

Background and Aim: The aim of this study was to review the literature regarding the use of pre- and/or postoperative antibiotics in the management of appendicitis, using data obtained from PubMed and the Cochrane Library. Material and Methods: A literature search was conducted using the terms “appendicitis” combined with “antibiotics.” Studies were selected based on relevance for the evidence on prophylactic and postoperative treatment with regard to the route and duration of drug administration and the findings of surgery. Results: Patients with acute appendicitis should receive preoperative, broad-spectrum antibiotics. The use of postoperative antibiotics is only recommended in cases of perforation, and treatment should then be given intravenously, for a minimum period of 3–5 days for adult patients, until clinical signs such as fever resolve and laboratory parameters such as C-reactive protein curve and white blood cell (WBC) start to decline. Conclusion: Preoperative antibiotic prophylaxis is recommended in all patients with acute appendicitis, whereas postoperative antibiotics only in cases of perforation.

Cellular physiology and pathophysiology of the parathyroid glands

This report provides insight into parathyroid gland physiology and the pathophysiology of hyperparathyroidism (HPT). Increases in the extracellular calcium concentration constitute the primary physiological signal for inhibition of parathyroid hormone (PTH) release. Transduction of the external signal into a cellular response involves activation of a cation receptor mechanism on the plasma membrane with rapid rise in the cytoplasmic calcium concentration of the cells. This recently discovered parathyroid calcium receptor has been characterized as a glycoprotein of unusually high molecular weight, which may play a key role in calcium homeostasis since it is also expressed in the kidney and placenta. Binding of external calcium to the receptor is associated with mobilization of intracellular calcium as well as calcium influx into the cells and phosphoinositol hydrolysis. These events rapidly interfere with the release process through essentially unknown mechanisms and probably also at sustained stimulation inhibit PTH gene transcription. The relative calcium insensitivity of the PTH release in HPT is associated with a deranged regulation of cytoplasmic calcium within pathological parathyroid cells. The molecular basis for this disturbance comprises down regulation of the cation receptor, whereby external calcium is translated into abnormally low levels of cytoplasmic calcium and insufficient inhibition of PTH release. Studies on expression of the functionally important cation sensing glycoprotein and its associated cellular signal systems may provide novel means for interference with the pathophysiological derangements of HPT.RésuméCet article traite le physiologie normale de la parathyroÏde et la physiopathologie de lhyperparathyroÏdie (HPT). La libération de la parathormone (PTH) est inhibée par une augmentation de la concentration extra-cellulaire en calcium. La transduction de ce signal externe en une réponse cellulaire comporte lactivation dun récepteur de cation au niveau de la membrane cellulaire couplée avec la hausse rapide de la concentration intracellulaire en calcium. Ce recepteur de calcium, de connaissance relativement récente, est une glycoprotéine dun poids moléculaire élevé, qui pourrait jouer un rÔle essentiel dans lhoméostasie du calcium puisquil est également retrouvé au niveau des cellules rénales et placentaires. La liaison entre le calcium extra-cellulaire au récepteur fait appel à une mobilisation du calcium intra-cellulaire et une rentrée cellulaire du calcium ainsi quune hydrolyse de phosphoinositol. Ces évènements interviennent rapidement sur linhibition de la libération de la PTH par des mécanismes encore mal élucidés. Il se peut également que la transcription du gène PTH soit inhibée. La diminution de la libération de la PTH dans lHPT est associée avec une perturbation du taux intra-cellulaire de calcium. Au plan moléculaire, les choses se passent comme si le calcium extra-cellulaire se transposait vers la cellule en quantité anormalement basse. Létude de cette glycoprotéine et son système de transmission de signal pourrait fournir dintéressants moyens pour inverser les perturbations physiopathologiqes dans lHPT.ResumenEl presente reporte provee información sobre la fisiología de las glándulas paratiroides y la patofisiología del hiperparatiroidismo (HPT). El aumento en la concentración del calcio extracelular constituye la señal fisiológica primaria para inhibir la liberación de hormona paratiroidea (PTH). La transducción de la señal externa a una respuesta celular involucra la activación de un mecanismo receptor catiónico de la membrana con aumento rápido de la concentración citoplásmica de calcio. Este receptor paratiroideo de calcio, recientemente descubierto, ha sido caracterizado como una glicoproteína de un poco cumÚn elevado peso molecular, el cual puede jugar un papel clave en la homeostasis general del calcio, por cuanto también se expresa en el riñón y en la placenta. La ligadura del calcio extracelular al receptor se asocia con movilización del calcio intracelular y con influjo de calcio a la célula e hidrólisis de fosfoinositol. Estos fenómenos interfieren en forma rápida con el proceso de liberación mediante mecanismos todavía no identificados y pueden también influir, a niveles de estimulación sostenida, sobre la transcripción genética del PTH. La relativa insensibilidad del calcio a la liberación de PTH en el HPT se asocia con una alterada regulación del calcio citoplásmico dentro de las células paratiroideas anormales. Las bases moleculares para esta alteración incluyen una inhibición del receptor de calcio, por lo cual el calcio extracelular es traducido a niveles anormalmente reducidos de calcio citoplásmico e insuficiente inhibición de la liberación del PTH. Estudios sobra la expresión de la funcionalmente importante glicoproteína sensora catiónica y su asociado sistema de señales puede proveer nuevas maneras de corregir las alteraciones patofisológicas del HPT.

Routine whole body CT of high energy trauma patients leads to excessive radiation exposure

BackgroundWhole body computed tomography (WBCT) is an important adjunct in trauma care, which is often part of standard protocol in initial management of trauma patients. However, WBCT exposes patients to a significant dose of radiation. The use of WBCT was assessed in a modern trauma cohort in Sweden.MethodsA two-center retrospective cohort study was performed. All consecutive trauma alert patients at a university hospital (July-December 2008), and a rural county hospital (January 2009- December 2010) were included. Patients were stratified into three groups (high, intermediate and low risk) based on documented suspected injuries at primary survey at the site of accident or at the emergency department. Injury severity score (ISS) was calculated. Case records were reviewed for clinical and radiological findings at the time of trauma, and during a ≥36xa0months of follow-up period to identify possible missed injuries.ResultsA total of 523 patients were included in the study (university hospital nu2009=u2009273; rural county hospital nu2009=u2009250), out of which 475 patients (91.0 %) underwent radiological examinations, 290 patients (55.4 %) underwent WBCT, which identified trauma related findings in 125 patients (43.1 % of those examined). The high-risk group (nu2009=u200962) had a mean age of 38.5xa0years (21.1 SD). Mean ISS was 16.48 (18.14 SD). In this group, WBCT resulted in a positive finding in 38 (74.5 %) patients. In the intermediate-risk group (nu2009=u2009322; mean age 37.66, 20.24 SD) ISS was 4.42 (6.30 SD). A positive finding on WBCT was found in 87 of the intermediate group patients (44.8 %). The low-risk group (nu2009=u2009139; mean age 32.5xa0years; 21.4 SD) had a mean ISS of 0.84 (1.57 SD) with no positive findings on WBCT and no missed injuries in medical records at ≥36xa0months.DiscussionThe risk of developing radiation induced cancer is significant for young people if exposed to relatively high dose radiation as is the case in WBCT. WBCT in high-energy trauma is important for planning of treatment in severely injured patients while it can be questioned in the seemingly not injured where it is used mainly to permit early discharge from the ED.ConclusionsRisk stratification criteria could in this retrospective study identify high energy trauma patients not in need of radiological imaging. WBCT in high-energy trauma does not affect patient care if the patient is mentally alert, not intoxicated nor shows signs of other than minor injuries when evaluated by a trauma-team. The risk of missing important traumatic findings in these patients is very low. Observation of the patient with reexamination instead of imaging may be considered in this group of often young patients where radiation dose is an issue.

Monoclonal antiparathyroid antibodies revealing defect expression of a calcium receptor mechanism in hyperparathyroidism

Monoclonal antibodies were generated with a hybridoma technique after immunization of mice with intact human parathyroid cells. Three antibodies of the IgG type reacted in immunohistochemistry and immunofluorescense with parathyroid epithelial cells and proximal tubule cells of the kidney but not with a large number of other human tissues. Adenomatous and hyperplastic parathyroid glands of patients with hyperparathyroidism (HPT) demonstrated a reduced immunohistochemical reactivity with the antibodies as compared to the intense staining of normal human parathyroid tissue. Experiments with dispersed parathyroid cells from the normal and pathological glands showed that 2 of the antibodies blocked the effects of ambient calcium on cytoplasmic calcium and parathyroid hormone release. This indicates that the antibodies interfere with a newly recognized receptor mechanism of parathyroid cells involved in the sensing and gating of calcium and thereby also in the regulation of parathyroid hormone release. A reduced expression of the putative calcium receptor mechanism of abnormal parathyroid cells may, thus, be principally responsible for the hypercalcemia of HPT by causing a decreased sensitivity to extracellular calcium of the parathyroid hormone release from pathological parathyroid glands. The antibodies should improve the parathyroid histological diagnosis by their ability to identify and to distinguish between normal and abnormal parathyroid tissue.RésuméDes anticorps monoclonaux ont été fabriqués avec la technique hybridoma après immunisation des souris avec des cellules parathyroïdes humaines intactes. Trois anticorps du type IgG ont réagi en immunohistochimie et en immunofluorescence avec les cellules épithéliales et tubulaires proximales du rein, mais pas avec la plupart des autres tissus humains. Les glandes parathyroïdes adénomateuses et hyperplasiques des patients hyperparathyroïdiens ont présenté une réactivité immunohistoclinique réduite vis-à-vis des anticorps en comparaison avec la coloration intense du tissu parathyroïde humain normal. En utilisant expérimentalement des cellules parathyroïdiennes dispersées provenant des glandes normales et pathologiques, 2 des anticorps bloquaient les effets du calcium ambiant sur le largage du calcium cytoplasmique et de la parathormone. Cela indique que les anticorps interfèrent avec le mécanisme récepteur, récemment reconnu, des cellules parathyroïdiennes, qui détermine la présence du calcium et qui en contrôle les mouvements de lion, et par conséquent joue un rôle dans le relargage de la parathormone. La réduction de lexpression du mécanisme suppose des récepteurs de calcium des cellules parathyroïdiennes anormales pourrait donc être responsable de lhypercalcémie dans lhyperparathyroïdisme. Le mécanisme serait une baisse de la sensibilité au calcium extracellulaire provoquant une baisse de la sécrétion de parathormone par les glandes parathyroïdiennes pathologiques. Ces anticorps pourraient améliorer le diagnostic histologique par leur capacité didentifier et de distinguer entre les tissus parathyroïdes normal et pathologique.ResumenAnticuerpos monoclonales fueron generados mediante una técnica de hibridoma después de immunizar a ratones con células paratiroideas humanas intactas. Tres anticuerpos del tipo IgG reaccionaron en immunohistoquímica e immunofluorescencia con células paratiroideas epiteliales y con células del túbulo proximal del rinón, pero nó con un gran número de otros tejidos humanos. Las glándulas paratiroides adenomatosas e hiperplásicas de pacientes con hiperparatiroidismo (HPT) mostraron una reducida reactividad immunohistoquímica con los anticuerpos en comparación con la intensa coloración del tejido paratiroideo humano normal. Experimentos con células paratiroideas dispersas provenientes de glándulas normales y patológicas mostraron que 2 de los anticuerpos bloqueaban los efectos del calcio ambiental sobre el calcio citoplásmico y la liberación de hormona paratiroidea. Esto indica que los anticuerpos interfieren con un recientemente descubierto mecanismo receptor de las células paratiroideas involucradas en la percepción del calcio y la regulation de la liberación de hormona paratiroidea. Una expresión reducida del mecanismo receptor putativo de calcio de las células paratiroideas anormales puede ser, por consiguiente, el responsable principal de la hipercalcemia del HPT al causar una disminuida sensibilidad al calcio extracelular en el proceso de la liberación de la hormona por las células paratiroideas patológicas. Estos anticuerpos deben contribuir a mejorar el diagnóstico histológico paratiroideo por su capacidad de identificar y diferenciar el tejido normal del patológico.

Interaction of monoclonal antiparathyroid antibody with Ca2+ agonistic actions of Mn2+ in normal human parathyroid cells

Effects of the monoclonal antiparathyroid antibodies G11 and E11 on Mn2+ interaction with individual normal human parathyroid cells were studied. At 0.5mM Ca2+, 3mM Mn2+ induced a rapid transient increase in cytoplasmic Ca2+ [Ca2+i] followed by quenching of the fluorescence from the Ca2+ indicator fura-2 as Mn2+ entered into the cells. Whereas the antibody E11 had no effects, treatment with G11 abolished the Ca2+i transient and considerably delayed the entry of Mn2+. The results support the presence of a cation-sensitive receptor mechanism on parathyroid cells and indicate that the antibody G11 not only blocks the interaction between Ca2+ and this receptor mechanism but also that of Mn2+.