Localization of the novel Xin protein to the adherens junction complex in cardiac and skeletal muscle during development.

Previously, we demonstrated that chick embryos treated with antisense oligonucleotides against a striated muscle-specific Xin exhibit abnormal cardiac morphogenesis (Wang et al. [1999] Development 126:1281-1294); therefore, we surmised a role for Xin in cardiac development. Herein, we examine the developmental expression of Xin through immunofluorescent staining of whole-mount mouse embryos and frozen heart sections. Xin expression is first observed within the heart tube of embryonic day 8.0 (E8.0) mice, exhibiting a peripheral localization within the cardiomyocytes. Colocalization of Xin with both beta-catenin and N-cadherin is observed throughout embryogenesis and into adulthood. Additionally, Xin is found associated with beta-catenin within the N-cadherin complex in embryonic chick hearts by coimmunoprecipitation. Xin is detected earlier than vinculin in the developing heart and colocalizes with vinculin at the intercalated disc but not at the sarcolemma within embryonic and postnatal hearts. At E10.0, Xin is also detected in the developing somites and later in the myotendon junction of skeletal muscle but not within the costameric regions of muscle. In cultured C2C12 myotubes, the Xin protein is found in many speckled and filamentous structures, coincident with tropomyosin in the stress fibers. Additionally, Xin is enriched in the regions of cell-cell contacts. These data demonstrate that Xin is one of the components at the adherens junction of cardiac muscle, and its counterpart in skeletal muscle, the myotendon junction. Furthermore, temporal and spatial expressions of Xin in relation to intercalated disc proteins and thin filament proteins suggest roles for Xin in the formation of cell-cell contacts and possibly in myofibrillogenesis.     

Molecular and pathogenic characterization of Borrelia burgdorferi sensu lato isolates from Spain

Fifteen Borrelia burgdorferi sensu lato isolates from questing ticks and skin biopsy specimens from erythema migrans patients in three different areas of Spain were characterized. Four different genospecies were found (nine Borrelia garinii, including the two human isolates, three B. burgdorferi sensu stricto, two B. valaisiana, and one B. lusitaniae), showing a diverse spectrum of B. burgdorferi sensu lato species. B. garinii isolates were highly variable in terms of pulsed-field gel electrophoresis pattern and OspA serotype, with four of the seven serotypes described. One of the human isolates was OspA serotype 5, the same found in four of seven tick isolates. The second human isolate was OspA serotype 3, which was not present in ticks from the same area. Seven B. garinii isolates were able to disseminate through the skin of C3H/HeN mice and to cause severe inflammation of joints. One of the two B. valaisiana isolates also caused disease in mice. Only one B. burgdorferi sensu stricto isolate was recovered from the urinary bladder. One isolate each of B. valaisiana and B. lusitaniae were not able to disseminate through the skin of mice or to infect internal organs. In summary, there is substantial diversity in the species and in the pathogenicity of B. burgdorferi sensu lato in areas in northern Spain where Lyme disease is endemic.     

Antibodies to Recombinant Clostridium difficile Toxins A and B Are an Effective Treatment and Prevent Relapse of C. difficile-Associated Disease in a Hamster Model of Infection

Clostridium difficile causes antibiotic-associated diarrhea and colitis in humans through the actions of toxin A and toxin B on the colonic mucosa. At present, broad-spectrum antibiotic drugs are used to treat this disease, and patients suffer from high relapse rates after termination of treatment. This study examined the role of both toxins in pathogenesis and the ability of orally administered avian antibodies against recombinant epitopes of toxin A and toxin B to treat C. difficile-associated disease (CDAD). DNA fragments representing the entire gene of each toxin were cloned, expressed, and affinity purified. Hens were immunized with these purified recombinant-protein fragments of toxin A and toxin B. Toxin-neutralizing antibodies fractionated from egg yolks were evaluated by a toxin neutralization assay in Syrian hamsters. The carboxy-terminal region of each toxin was most effective in generating toxin-neutralizing antibodies. With a hamster infection model, antibodies to both toxins A and B (CDAD antitoxin) were required to prevent morbidity and mortality from infection. In contrast to vancomycin, CDAD antitoxin prevented relapse and subsequent C. difficile reinfection in the hamsters. These results indicate that CDAD antitoxin may be effective in the treatment and management of CDAD in humans.     

Quantitative nucleic acid sequence-based assay as a new molecular tool for detection and quantification of Leishmania parasites in skin biopsy samples

Currently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify Leishmania parasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA), which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA is a highly sensitive and specific method that allows quantification of both Old and New World Leishmania parasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of CL patients.     

Development of the human elbow joint

Many studies have been published on the development of the human elbow joint, but authors disagree on its morphogenetic timetable. Most discrepancies center on the cavitation of the elbow joint (including the humeroradial, humeroulnar, and superior radioulnar joints), and the organization of the tunnel of the ulnar nerve. We summarize our observations on the development of the elbow joint in 49 serially sectioned human embryonic (n = 28) and fetal (n = 21) upper limbs. During week 12, ossification begins in the epiphyses of the elements comprising the elbow joint. At the end of the embryonic period, the shallow groove between the posterior aspect of the medial epicondyle and the olecranon process, begins to be visible. The elbow joint cavity appears in O'Rahilly stage 21 (51 days) at the level of the humeroulnar and humeroradial interzones. Formation of the cavity begins at the medialmost portion of the humeroradial interzone and the lateralmost portion of the humeroulnar interzone. The annular ligament begins to develop in O'Rahilly stage 21 (51 days), and the superior radioulnar joint cavity appears between this ligament and the lateral aspect of the head of the radius during O'Rahilly stage 23 (56 days). We established the morphogenetic timetable of the human elbow joint.     

Hydrocephalus induced by immunological blockage of the subcommissural organ–Reissner's fiber (RF) complex by maternal transfer of anti-RF antibodies

Stenosis of the cerebral aqueduct seems to be a key event for the development of congenital hydrocephalus. The causes of such a stenosis are not well known. Overholser et al. in 1954 (Anat Rec 120:917-933) proposed the hypothesis that a dysfunction of the subcommissural organ (SCO) leads to aqueductal stenosis and congenital hydrocephalus. The SCO is a brain gland, located at the entrance of the cerebral aqueduct, that secretes glycoproteins into the cerebrospinal fluid that, upon release, assemble into a fibrous structure known as Reissner's fiber (RF). By the permanent addition of new molecules to its rostral end, RF grows and extends along the aqueduct, fourth ventricle, and central canal of the spinal cord. The immunological blockage of the SCO-RF complex has been used to test Overholser's hypothesis. The following was the sequence of events occurring in pregnant rats that had been immunized with RF glycoproteins: the mother produced anti-RF antibodies and transferred them to the fetus through the placenta and to the pup through the milk, and the antibodies reached the brain of the fetus and pup and blocked the SCO-RF complex. This resulted in a permanent absence of RF that was followed by stenosis of the cerebral aqueduct, and then by the appearance of hydrocephalus. The latter was patent until the end of the 6-month observation period. The chronic hydrocephalic state appeared, in turn, to induce new alterations of the SCO. It is concluded that a selective immunological knock out of the SCO-RF complex leads to hydrocephalus.     

Histological study of the proximal and distal segments of the embryonic outflow tract and great arteries

The normal development of the ventricular outlets and proximal region of the great arteries is a controversial subject. It is known that the conus, truncus arteriosus (truncus), and aortic sac participate; however, there are some doubts as to the actual prospective fate of the truncus. Some authors propose that it gives origin to the proximal region of the great arteries and that the myocardial cells of its wall become smooth muscle. Nevertheless, others think that the truncus only forms the arterial valve apparatus and that therefore the myocardial cells transform into fibroblasts. As a first approach to beginning to elucidate which process occurs, the aim of this article was to study the histological changes in the wall of these components of the developing heart in chick embryos whose hearts had been labeled at the truncoconal boundary at stage 22HH, tracing the changes up to stage 36HH. Also, the histological constitution of the wall of the pulmonary arterial trunk and its valve apparatus were studied in the posthatching and adult hearts of chickens and rats. The conus and truncus walls were always encircled by a myocardial sleeve from the outset of their development. Between stages 26HH to 28HH, the truncal myocardial cells adjacent to the mesenchymal tissue of the ridges began to lose cell-to-cell contacts and invaded the extracellular matrix. At stage 24HH, the aortic sac began to project into the pericardial cavity and became divided into two channels by the aortic-pulmonary septum at stage 26HH. The wall of the aortic sac is mostly constituted by a compact mesenchymal tissue. Initially, it does not have smooth muscle but this starts to appear at stage 30HH. The insertion ring of the valves, a broad structure, was formed by mesenchymal tissue. Both structures were always covered by a myocardial sleeve. The leaflets developed from the truncal ridges, the segment immediately proximal to the aortic sac. Our results indicate that the proximal region of the pulmonary and aortic arteries do not originate from the truncus arteriosus; rather, we found that they take origin from the aortic sac. Thus, our findings agree with the proposal that the myocardial cells of the external sleeve of the truncus become fibroblastic and suggest that the insertion ring of the arterial valves has a dual origin: fibroblasts produced by truncal myocardial transdiferentiation and the mesenchymal tissue of the proximal region of the truncal ridges, while the leaflets have their origin from the truncal ridges. We discuss the fact that, because the truncus arteriosus does not give origin to the trunks of the aortic and pulmonary arteries, it may be necessary to modify terminology. Based on our results, together with the new findings obtained by in vivo labeling, immunostaining, a chimeric approach, and ultrastructural studies, we propose a developmental model that correlates the fate of the conus, truncus, and aortic sac with the normal morphogenesis of the ventricular outlet tracts and the trunks of the great arteries. (c) 2005 Wiley-Liss, Inc.     

Improvement of leukocyte functions in young prematurely aging mice after a 5-week ingestion of a diet supplemented with biscuits enriched in antioxidants

In our previous studies, diet supplementation with therapeutic thiolic antioxidants improved the function of peritoneal leukocytes from mice, especially in adult prematurely aging mice (PAM). In the present work, we have studied the effects of ingestion during 5 weeks of a diet supplemented with 20% (wt/wt) of biscuits enriched with antioxidants (vitamin C, vitamin E, beta-carotene, zinc, and selenium) on several immune functions of peritoneal leukocytes from young PAM. The results show that, in macrophages, chemotaxis and phagocytosis as well as the intracellular free radical levels, which are depressed in PAM in comparison with the control nonprematurely aging mice (NPAM), increase after supplementation, especially in the PAM. An increase also occurs in lymphocyte chemotaxis, proliferative response to the mitogen concanavalin A, and interleukin-2 release, as well as in natural killer cell activity. However, the release of tumor necrosis factor-alpha, which increases with aging, decreases after 5 weeks of supplementation. As a well preserved function of the immune system is an excellent marker of health and longevity, the improvement of leukocyte functions after ingestion of the present diet suggests that this antioxidant supplementation may be useful for the preservation of health and functional longevity in aging populations.