Monoclonal antibodies against IREM-1: potential for targeted therapy of AML.

IREM-1 is an inhibitory cell surface receptor with an unknown function and is expressed on myeloid cell lineages, including cell lines derived from acute myeloid leukemia (AML) patients. We have generated a series of monoclonal antibodies (mAbs) against the extracellular domain of IREM-1 and further assessed its expression in normal and AML cells. IREM-1 was restricted to cells from myeloid origin and extensive expression analysis in primary cells obtained from AML patients showed IREM-1 expression in leukemic blasts of 72% (39/54) of samples. We therefore searched for specific IREM-1 mAbs with activity in functional complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Lead mAbs against IREM-1 showed specific cytotoxic activity against a variety of AML-derived cell lines and freshly isolated blasts from AML patients. Internalization of mAbs upon IREM-1 binding was also shown. In vivo anticancer activity of lead mAbs was observed in an established HL-60 xenograft model with a tumor growth delay of up to 40% and in a model using primary human AML cells, where treatment with anti-IREM-1 mAb resulted in a significant reduction of engrafted human cells. These results demonstrate IREM-1 as a potential novel target for immunotherapy of AML.

[Connexins and junctional channels. Roles in the spreading of cardiac electrical excitation and heart development]. / Connexines et canaux jonctionnels. Leurs rôles dans la propagation de l'activité électrique cardiaque et le développement du coeur.

The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The junctional channels that couple the cardiomyocytes are responsible for this propagation process. These channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Four Cxs - Cx30.2, -40, -43 and -45--have been demonstrated to be synthesized in the cardiomyocytes. In addition, each of these Cxs has a unique expression pattern in the myocardium. A fruitful approach of the role of these Cxs in the cardiac functions came with the development of transgenic mouse models. It has been shown that Cx43 was mainly involved in influx propagation in the ventricles and that inactivation in the cardiomyocytes of the gene of this Cx predisposed to development of cardiac abnormalities. Cx40 very significantly contributes to the propagation of electrical activity in the atria and the conduction system. Cx45 is essential to coordinate the synchronization of contractile activities of embryonic cardiomyocytes and for the normal progress of cardiogenesis. Finally, Cx30.2 contributes to the slowing of propagation of excitation in the atrioventricular node. These observations enable to better understand the relationships between alteration in Cx expression or gap junction remodelling and arrhythmias in the human heart.

Stereotactic vacuum biopsy of calcifications with a handheld portable biopsy system: a validation study.

To prospectively evaluate a compact portable 10-gauge handheld battery-operated biopsy system for stereotactic biopsy of microcalcifications. The ethics committee of the hospital approved this prospective multicentric study, and informed consent was obtained. Biopsy under stereotactic guidance was performed in 215 patients for 219 lesions consisting of microcalcifications without mass. The feasibility and the tolerance of the procedure were evaluated. The mean weight of the specimen was calculated. In patients with surgical diagnoses, the underestimation rate in biopsy diagnoses of atypical ductal hyperplasia and ductal carcinoma in situ were evaluated. The sampled specimens were separated according to the presence of calcifications on magnified specimen radiographs and to the probe the rotation number in order to evaluate the contribution of each rotation and the contribution of the specimen with and without calcifications on the radiographs. The macrobiopsy was feasible in 98.5% of the patients and was well tolerated in 82% of patients. It identified 4.6% invasive carcinomas, 18.5% ductal carcinomas in situ, 14.8% atypical ductal hyperplasias, 22.2% benign proliferative mastopathies and 39.8% benign non-proliferative mastopathies. The underestimation rate was 26.6% when an atypical ductal hyperplasia was diagnosed at biopsy, and 7.7% when a ductal carcinoma in situ was diagnosed. In the 77 patients with surgical correlation, the accurate diagnosis was obtained in specimens sampled during the first, second, and third in 69%, 9%, and 4% of the biopsies, respectively, and the analysis of specimens without microcalcification had an added value in 8% of patients. The compact portable battery-operated biopsy system can be used successfully for stereotactic biopsy of microcalcifications and constitutes a valid alternative to current systems.

Characterization of zebrafish Cx43.4 connexin and its channels.

Connexins (Cx) form intercellular junctional channels which are responsible for metabolic and electrical coupling. We report here on the biochemical and immunohistochemical characterization of zebrafish connexin zfCx43.4, an orthologue of mammalian and avian Cx45, and the electrophysiological properties of junctional channels formed by this protein. The investigations were performed on transfected COS-7 cells or HeLa cells. Using site-directed antibodies, zfCx43.4 cDNA (GenBank accession no. X96712) was demonstrated to code for a protein with a M(r) of 45 000. In transfected cells, zfCx43.4 was localized in cell-cell contact areas as expected for a gap junction protein. zfCx43.4 channels were shown to transfer Lucifer Yellow. The multichannel currents were sensitive to the transjunctional voltage (V(j)). Their properties were consistent with a two-state model and yielded the following Boltzmann parameters for negative/positive V(j): V(j,0) = -38.4/41.9 mV; g(j,min) = 0.19/0.18; z = 2.6/2.3. These parameters deviate somewhat from those of zfCx43.4 channels expressed in Xenopus oocytes and from those of Cx45, an orthologue of zfCx43.4, expressed in mammalian cells or Xenopus oocytes. Conceivably, the subtle differences may reflect differences in experimental methods and/or in the expression system. The single channel currents yielded two prominent levels attributable to a main conductance state (gamma(j,main) = 33.2 +/- 1.5 pS) and a residual conductance state (gamma(j,residual) = 11.9 +/- 0.6 pS).

Gap junctional connexins in the developing mouse cardiac conduction system.

Gap junctional channels which couple myocytes mediate conduction phenomena in the heart. These channels are dodecamers of transmembrane proteins belonging to the connexin family (Cx). Three Cxs, Cx43, -40 and -45, have been found to be expressed in cardiomyocytes. Each of them has a distinct spatiotemporal pattern of expression, which is regulated during development. In the adult mouse heart, Cx43 is expressed in all the working myocytes and most of the conductive myocytes; Cx45 is weakly expressed in all conductive myocytes, including those of the nodal tissues; Cx40 expression is restricted to the atria and ventricular conduction system. Analysis of mice with deletions of Cx genes has provided evidence that Cx43, -40 and -45, and consequently the gap junctional channels they form, are involved in both heart function and development. For example, Cx40 deficiency results in sinoatrial conduction impairments, a significant decrease of the conduction velocity in the atria, and a delay of the propagation of impulse in the His bundle. Transgenic mouse lines with modified Cx40 genes are now being used to draw up a detailed map of the conduction system in the adult and developing heart, and to identify the regulatory elements involved in the transcriptional regulation of the Cx40 gene. Some preliminary results of these studies are described.

LacSwitch II regulation of connexin43 cDNA expression enables gap-junction single-channel analysis.

Metabolic and electrical coupling through gap junction channels is implicated in cell differentiation, tissue homeostasis, and electrotonic propagation of signals in excitable tissues. The characterization of gating properties of these channels requires electrophysiological recordings at both single- and multiple-channel levels. Hence, a system that is able to control connexin expression by external means would provide a useful tool. To regulate the expression of connexins in cells, plasmids encoding a transactivator and/or a lac-operon IPTG response-dependent Cx43 target gene were transfected into communication-deficient N2a neuroblastoma cells. Immunoblotting, dye coupling, and electrophysiological methods revealed that expression of Cx43 in selected clones could be tightly regulated. After 15-20 h of acute induction with IPTG, cell-to-cell communication reached its peak with junctional conductances of 15-30 nS. Chronic induction at specific doses of IPTG produced constant, controlled levels of Cx43 expression, which were reflected by predictable junctional coupling levels. These conditions allowed prolonged recordings from either lowly or highly coupled cells, making lac operon an ideal regulatory system for channel gating studies at a single-channel level.

Inhibition of endothelial wound repair by dominant negative connexin inhibitors.

Wounding of endothelial cells is associated with altered direct intercellular communication. To determine whether gap junctional communication participates to the wound repair process, we have compared connexin (Cx) expression, cell-to-cell coupling and kinetics of wound repair in monolayer cultures of PymT-transformed mouse endothelial cells (clone bEnd.3) and in bEnd.3 cells expressing different dominant negative Cx inhibitors. In parental bEnd.3 cells, mechanical wounding increased expression of Cx43 and decreased expression of Cx37 at the site of injury, whereas Cx40 expression was unaffected. These wound-induced changes in Cx expression were associated with functional changes in cell-to-cell coupling, as assessed with different fluorescent tracers. Stable transfection with cDNAs encoding for the chimeric connexin 3243H7 or the fusion protein Cx43-betaGal resulted in perturbed gap junctional communication between bEnd.3 cells under both basal and wounded conditions. The time required for complete repair of a defined wound within a confluent monolayer was increased by ~50% in cells expressing the dominant negative Cx inhibitors, whereas other cell properties, such as proliferation rate, migration of single cells, cyst formation and extracellular proteolytic activity, were unaltered. These findings demonstrate that proper Cx expression is required for coordinated migration during repair of an endothelial wound.

Rat gap junction connexin-30 inhibits proliferation of glioma cell lines.

Connexins, the structural components of gap junctions, control cell growth and differentiation and are believed to belong to a family of tumour suppressor genes. Studies on connexin localization in brain showed that several of these proteins were expressed in distinct compartments of the brain in a cell-type specific manner, indicating that different gap junctions play specific roles in the physiology of the mammalian brain. In this report, we first cloned rat connexin-30 cDNA from brain and showed that it was expressed in long-term primary culture of rat astrocytes. In order to examine the potential role of connexin-30 in tumour cell proliferation, we transfected the connexin-30 cDNA into two rat glioma cell lines (9L and C6) which have lost its expression. Transfected clones adequately expressed membrane-bound connexin-30 protein. Connexin-30-expressing clones showed slower growth, lower DNA synthesis and reduced proliferation in soft agar as compared with the parental and control cells. We concluded that connexin-30 may also probably be considered as a tumour suppressor in rat gliomas.

Decreased linear growth associated with intestinal bladder augmentation in children with bladder exstrophy.

PURPOSE: We determine if enterocystoplasty results in delayed linear growth using a case controlled study to observe the effects of intestinal bladder augmentation on growth in patients with bladder exstrophy. MATERIALS AND METHODS: A total of 50 patients who had undergone bladder augmentation for incontinence due to classic bladder exstrophy were selected from our patient database and matched for gender, age and type of exstrophy with 50 patients who had nonaugmented bladder exstrophy. Patients were then contacted and asked to permit the pediatricians to release growth charts. Once consent was obtained the charts were requested from the pediatricians, and evaluable data, defined as at least 1 height before and after augmentation, were obtained for 17 of 50 (34%) augmented and 15 of 50 (30%) nonaugmented cases. RESULTS: Mean age at surgery was 7.7 years. Delayed growth as defined by a postoperative decrease in percentile height occurred in 14 of 17 (82%) augmented cases (mean loss 15.6 percentile points). Delayed growth after age 7.7 years occurred in 5 of 15 (33%) controls but average growth for the entire group was 6.7 percentile points (p = 0. 014). Mean followup was 5.7 years (median 4.9) for the augmented group and 7.3 years (median 8.2) for the control group. CONCLUSIONS: Intestinal bladder augmentation is associated with a nearly universal decrease in percentile height. Close long-term followup of these patients and analysis of subtle metabolic alterations may provide information to help minimize or prevent growth impediment in the future.

Downregulation of connexin 45 gene products during mouse heart development.

The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase-polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels.

Connexin expression in cultured neonatal rat myocytes reflects the pattern of the intact ventricle.

OBJECTIVE: Primary cultures of neonatal rat ventricular myocytes have become a widely used model to examine a variety of functional, physiological and biochemical cardiac properties. In the adult rat, connexin43 (Cx43) is the major gap junction protein present in the working myocardium. In situ hybridization studies on developing rats, however, showed that Cx40 mRNA displays a dynamic and heterogeneous pattern of expression in the ventricular myocardium around birth. The present studies were performed to examine the expression pattern of the Cx40 protein in neonatal rat heart, and to examine the connexins present in cultures of ventricular myocytes obtained from those hearts. METHODS: Cryosections were made of hearts of 1-day-old Wistar rats. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain > 75, approximately 50%, and < 25% confluency) and cultured for 24, 48 or 96 h. Cx40 and Cx43 were detected by immunofluorescence and immunoblotting. RESULTS: Immunohistochemical stainings confirmed that gap junctions in the atrium and His-Purkinje system were composed of at least Cx43 and Cx40. From the subendocardium towards the subepicardium Cx40 expression gradually decreased, resulting in the sole expression of Cx43 in the subepicardial part of the ventricular wall. In ventricular myocytes cultured at high density (> 75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolabeling which showed a homogeneous distribution pattern, Cx40 staining was heterogeneous, i.e. in some clusters of cells abundant labeling was present whereas in others no Cx40 staining could be detected. The pattern of Cx43 immunoreactivity was not altered by the culture density. In contrast, in isolated ventricular myocytes cultured at low density (< 25% confluency) the relative number of cell-cell interfaces that were Cx40-immunopositive decreased as compared to high density cultures (35 vs. 70%). Western blots did not reveal significant differences in the level of Cx40 and Cx43 expression at different culture densities. CONCLUSIONS: These results show that cultured ventricular myocytes retained typical features of the native neonatal rat ventricular myocardium with regard to their composition of gap junctions. This implicates that these cultures may serve as a good model for studying short-term and long-term regulation of cardiac gap junction channel expression and function.

Heart defects in connexin43-deficient mice.

Cardiac malformation in connexin43 (CX43)-disrupted mice is restricted to the junction between right ventricle and outflow tract, even though CX43 is also expressed abundantly elsewhere. We analyzed cardiac morphogenesis in immunohistochemically and hybridohistochemically stained and three-dimensionally reconstructed serial sections of CX43-deficient embryos between embryonic day (ED) 10 and birth. The establishment of the D configuration in the ascending loop of CX43-deficient hearts is markedly retarded, so that the right ventricle retains a craniomedial position and is connected with the outflow tract by a more acute bend in ED10 and ED11 embryos. Because of the subsequent growth of the right ventricle, this condition usually evolves into a D loop, but when it persists, a "crisscross" configuration develops, with the atrioventricular cushions rotated 90 degrees, a horizontal muscular ventricular septum, and a parallel course of the endocardial ridges of the outflow tract. After ED12, large intertrabecular pouches develop at the ventricular side of both shelflike myocardial structures that support the endocardial ridges of the outflow tract, ie, at the location that was earlier characterized by the acute bend between the right ventricle and the outflow tract and that subsequently develops into the anterosuperior leaflet of the tricuspid valve. Retarded development of the D configuration in the ascending loop of the embryonic heart predisposes the myocardium at the junction of the right ventricle and outflow tract to excessive development of intertrabecular pouches during subsequent development.

Senology-the urgent need for a specialty.

Expert physicians from many European and Latin American countries are engaged in a systematic effort to persuade influential colleagues and government officials to recognize senology as a well-defined scientific discipline and establish it as an independent specialty. The task is complex, as the establishment of a new specialty always is. Senology is not a superfluous specialty; it is similar in characteristics and indications to many other single organ-targeted specialties such as cardiology, hematology, nephrology, neurology, and others. The quest for its recognition may be a long effort that will require vigor, intelligence, firmness, persistence, and the establishment of a dialogue between interested physicians and government representatives, leaving personal interests aside. To take the care of patients with breast diseases to its optimal stage, it will be imperative to create specialized breast centers governed by principles of unity of place and action. Gathering under the same roof all specialists skilled and interested in the diagnosis and management of benign and malignant diseases of the breast and all the diagnostic and therapeutic equipment to deliver care to these patients will bring us much closer to our goal.

Expression pattern of connexin gene products at the early developmental stages of the mouse cardiovascular system.

The synchronized contraction of myocytes in cardiac muscle requires the structural and functional integrity of the gap junctions present between these cells. Gap junctions are clusters of intercellular channels formed by transmembrane proteins of the connexin (Cx) family. Products of several Cx genes have been identified in the mammalian heart (eg, Cx45, Cx43, Cx40, and Cx37), and their expression was shown to be regulated during the development of the myocardium. Cx43, Cx40, and Cx45 are components of myocyte gap junctions, and it has also been demonstrated that Cx40 was expressed in the endothelial cells of the blood vessels. The aim of the present work was to investigate the expression and regulation of Cx40, Cx43, and Cx37 during the early stages of mouse heart maturation, between 8.5 days post coitum (dpc), when the first rhythmic contractions appear, and 14.5 dpc, when the four-chambered heart is almost completed. At 8.5 dpc, only the reverse-transcriptase polymerase chain reaction technique has allowed identification of Cx43, Cx40, and Cx37 gene transcripts in mouse heart, suggesting a very low activity level of these genes. From 9.5 dpc, all three transcripts became detectable in whole-mount in situ-hybridized embryos, and the most obvious result was the labeling of the vascular system with Cx40 and Cx37 anti-sense riboprobes. Cx40 and Cx37 gene products (transcript and/or protein) were demonstrated to be expressed in the vascular endothelial cells at all stages examined. By contrast, only Cx37 gene products were found in the endothelial cells of the endocardium. In heart, Cx37 was expressed exclusively in these cells, which rules out any direct involvement of this Cx in the propagation of electrical activity between myocytes and the synchronization of contractions. Between 9.5 and 11.5 dpc, Cx40 gene activation in myocytes was demonstrated to proceed according to a caudorostral gradient involving first the primitive atrium and the common ventricular chamber (9.5 dpc) and then the right ventricle (11.5 dpc). During this period of heart morphogenesis, there is clearly a temporary and asymmetrical regionalization of the Cx40 gene expression that is superimposed on the functional regionalization. In addition, comparison of Cx40 and Cx43 distribution at the above developmental stages has shown that these Cxs have overlapping (left ventricle) or complementary (atrial tissue and right ventricle) expression patterns.

Urolithiasis in the exstrophy-epispadias complex.

PURPOSE: We sought to determine the incidence of urolithiasis in patients with the exstrophy-epispadias complex, associated risk factors and guidelines for the proper clinical management of this problem. MATERIALS AND METHODS: We retrospectively reviewed the chart and x-rays of 530 patients with the exstrophy-epispadias complex treated at our institution. RESULTS: Stones formed in 77 of the 530 patients (15%), including 16% of those with classic exstrophy, 25% of those with cloacal exstrophy and 3% of those with epispadias. White and male individuals were most commonly affected. Most calculi formed in the bladder, whether native or augmented by enterocystoplasty. Standard techniques were successful for stone removal. Most calculi consisted of calcium apatite, calcium oxalate monohydrate or magnesium ammonium phosphate, usually in combination with at least 1 other element. The risk of stone formation was associated with augmentation cystoplasty (p < 0.001) and a bladder neck procedure to increase outlet resistance (p < 0.001). Other risk factors included urinary tract infection, foreign bodies, vesicoureteral reflux and urinary stasis but not acidosis or immobilization. Stones recurred in 30 patients (39%), including equal numbers of those treated with open and closed techniques, and recurrence was associated with urinary tract infection or struvite composition (p < 0.05). Serum calcium abnormalities were not present in any patient, including those with metabolic acidosis or prolonged immobilization. Urinary chemistry studies were incomplete or unavailable. CONCLUSIONS: These data suggest that urolithiasis in the exstrophy-epispadias complex is related to risk factors associated with surgical reconstruction of this condition. The role of metabolic abnormalities that may predispose to urolithiasis is unknown but under investigation. Standard treatment is effective but stone recurrence remains a significant problem. Urine chemistry data may provide information to help minimize stone development in this patient population.