Osteoarthritis in temporomandibular joint of Col2a1 mutant mice.

OBJECTIVE: Col2a1 gene mutations cause premature degeneration of knee articular cartilage in disproportionate micromelia (Dmm) and spondyloepiphesial dysplasia congenita (sedc) mice. The present study analyses the temporomandibular joint (TMJ) in Col2a1 mutant mice in order to provide an animal model of TMJ osteoarthritis (OA) that may offer better understanding of the progression of this disease in humans. DESIGN: Dmm/+ mice and controls were compared at two, six, nine and 12 months. Craniums were fixed, processed to paraffin sections, stained with Safranin-O/Fast Green, and analysed with light microscopy. OA was quantified using a Mankin scoring procedure. Unfolded protein response (UPR) assay was performed and immunohistochemistry (IHC) was used to assay for known OA biomarkers. RESULTS: Dmm/+ TMJs showed fissuring of condylar cartilage as early as 6 months of age. Chondrocytes were clustered, leaving acellular regions in the matrix. Significant staining of HtrA1, Ddr2 and Mmp-13 was observed in Dmm/+ mice (p<0.01). We detected upregulation of the UPR in knee but not TMJ. CONCLUSIONS: Dmm/+ mice are subject to early-onset OA in the TMJ. We observed upregulation of biomarkers and condylar cartilage degradation concomitant with OA. An upregulated UPR may exacerbate the onset of OA. The Dmm/+ mouse TMJ is a viable model for the study of the progression of OA in humans.

Cephalosporins can be given to penicillin-allergic patients who do not exhibit an anaphylactic response.

STUDY OBJECTIVES: To assess the risk of intraoperative allergic reactions to cephalosporins in patients who claim to be allergic to penicillin. DESIGN: Retrospective chart review. SETTING: University-affiliated hospital. MEASUREMENTS: 2,933 intraoperative anesthesia records of all adult orthopedic patients treated at our institution during a 14-month period (7/96-8/97) were reviewed for antibiotic use and allergic reactions. MAIN RESULTS: Most of the 2,933 orthopedic patients, including 413 patients who were allergic to penicillin, received a cephalosporin (usually cefazolin) during their procedure. Only one of the penicillin-allergic patients may have had an allergic reaction to the cephalosporin, because diphenhydramine and hydrocortisone were given at the beginning of the case. However, no mention was made on the chart about itching or a rash or hives. One of the non-penicillin-allergic patients did develop a rash while the cephalosporin was infusing, requiring discontinuation of the antibiotic. CONCLUSIONS: Given the low incidence of allergic reactions, it appears to be safe to administer cephalosporins to patients who claim to be allergic to penicillin. However, no conclusion can be made concerning patients who report severe or anaphylactic reactions to penicillin, because these patients probably were excluded from the study.

Acquired retinochoroiditis in hamsters inoculated with ME 49 strain Toxoplasma.

PURPOSE: These studies were undertaken to establish an animal model for use in studies of ocular toxoplasmosis. An animal model is needed to examine the development, progression, and resolution of ocular Toxoplasma infections and to study the effects on the disease of currently used and experimental therapies. METHODS: Cysts of the ME 49 strain of Toxoplasma gondii were injected intraperitoneally into each of 60 golden hamsters. The hamsters' eyes were examined before inoculation and at intervals after inoculation, and fundus photographs were taken. Histologic sections were analyzed and photographed to document the ocular effects of the infection. RESULTS: Retinochoroiditis was found in both eyes of all hamsters within 2 to 3 weeks of inoculation. The disease resolved spontaneously without treatment and was quiescent in most cases at 12 weeks after inoculation. The animals remained in good general health, and those tested had high antibody titers to Toxoplasma (1:256 to 1:32,000) at 6 months after the infection. The discovery of cysts and lesions in the retina confirmed the diagnosis. CONCLUSIONS: Although the lesions were not identical to those of human disease, this animal model of ocular toxoplasmosis offers several advantages: reproducibility, short incubation time, spontaneous resolution without treatment, consistent production of cysts, and ease of inoculation intraperitoneally without intraocular injection.

Cytochemical localization of glycogen in Chlamydia trachomatis inclusions.

The origin and distribution of glycogen in inclusions of Chlamydia trachomatis were demonstrated with silver proteinate stain for electron microscopy. Glycogen particles were detected in all developmental stages of C. trachomatis, as well as free in the inclusions. Intrachlamydial glycogen was most common in elementary bodies but was also detected in intermediate forms and reticulate bodies (RBs). Abnormal divisions and breakdown of cytoplasmic membranes were common in RBs. Cytoplasmic contents, including glycogen particles, were released into the inclusions after rupture of the outer membranes of abnormal RBs and intermediate forms. From these observations, we conclude that glycogen in inclusions of C. trachomatis originates in the organisms themselves.

Endocytosis at the micropore of Toxoplasma gondii.

Ultrastructural studies were undertaken to reexamine the structure and function of the micropore of Toxoplasma gondii. By incubating tachyzoites with the tracer horseradish peroxidase (HRP), we showed for the first time cytochemically that an extracellular tracer was internalized into vacuoles at the micropore. Our morphological observations also demonstrated that the base of the micropore in both tachyzoites and bradyzoites was sometimes covered by a clathrin-like bristle coat. A coated vesicle was observed in continuity with a bradyzoite micropore, and large (150-nm) coated vesicles were occasionally present just beneath the micropore. These results suggest that receptor-mediated endocytosis occurs at the micropore. In other micrographs, however, the micropore appeared uncoated. In some bradyzoites, the uncoated micropore was greatly dilated, and it contained vesicles like those found in the cyst matrix associated with debris from disintegrated parasites. We had previously observed such debris from fragmented organisms in cysts prepared in vivo. These results indicate that residues from dead bradyzoites may provide nutrients for younger, developing parasites in the same cysts. Our observations also suggest that either receptor-mediated or bulk endocytosis can occur at the micropore, perhaps depending upon the availability of specific ligands. Investigation of a receptor-mediated pathway may reveal a means for targeting therapy selectively to the parasites to benefit patients with disseminated toxoplasmosis.

Dynamic intraventricular obstruction during dobutamine stress echocardiography. A new observation.

BACKGROUND: The implications of hypotension occurring during dobutamine stress echocardiography have not been elucidated. We observed in some patients that hyperdynamic left ventricular function developed during dobutamine stress echocardiography and hypothesized that intracavitary obstruction was occurring and might account for hypotension in some patients. METHODS AND RESULTS: Fifty-seven consecutive patients undergoing dobutamine stress echocardiography underwent pulsed-wave and continuous-wave Doppler examination of the left ventricular cavity at rest and at peak dobutamine infusion. The development of an intraventricular gradient with dobutamine stress was defined as a late-peaking left ventricular Doppler velocity profile that exceeded basal velocity by at least 1 m/sec. During dobutamine stress testing, left ventricular outflow velocity or intracavitary velocity increased in all patients. Obstruction occurred in 12 patients (21%, group 1). Group 2 was the remaining 45 patients. Peak velocities in group 1 ranged from 2.0 to 5.0 m/sec (mean, 3.5 m/sec), and the mean increase from velocity at rest was 2.3 m/sec. The mean change in systolic blood pressure was significantly lower in patients in group 1 (-15 versus 4 mm Hg, p = 0.02). When the 18 patients with an ischemic response to stress testing (evidenced by new or worsening wall motion abnormalities) were excluded from analysis, systolic blood pressure response was still significantly different for the two groups (-19 versus 2 mm Hg, p = 0.03). CONCLUSIONS: Dynamic left ventricular obstruction is a new observation; it may develop frequently in patients undergoing dobutamine stress echocardiography. Obstruction rather than ischemia may explain a decrease in blood pressure during dobutamine stress echocardiography.

Toxoplasma gondii: differentiation and death of bradyzoites.

The living parasites in Toxoplasma cysts cannot be eradicated by current therapy and maintain latent infections for many years. Relatively little is known about encysted Toxoplasma. We therefore undertook studies using mice infected with the avirulent ME 49 strain of Toxoplasma. The bradyzoites in young (12- to 17-day-old) cysts contained the same organelles as did tachyzoites. The bradyzoites of older cysts (4 weeks postinoculation) had differentiated, losing certain organelles and acquiring others. Our major new finding was that in animals inoculated greater than or equal to 4 weeks previously, some bradyzoites were totally disrupted, spilling their contents (perhaps including lytic substances) into the cyst matrix. Many older bradyzoites in the same cysts lacked internal membranes and their viability was questionable, but there were also occasional parasites resembling viable tachyzoites and mature bradyzoites, organisms that might possibly initiate daughter cyst formation after cyst rupture. The life span of an individual bradyzoite may be shorter than formerly appreciated despite the prolonged course of latent infections.

Transesophageal echocardiography: a sonographer's perspective.

Clinical transesophageal echocardiography is increasingly being applied for the evaluation of numerous functional and anatomic cardiac abnormalities. This new technology has opened an area of invasive ultrasonography that has changed and expanded the role of the cardiac sonographer. The sonographer is essential for the implementation and performance of this recent advance in echocardiography.

Value and limitations of transesophageal echocardiography in assessment of mitral valve prostheses.

BACKGROUND: Transthoracic Doppler echocardiography examination has become an integral part of the investigations performed in patients with mitral valve prostheses. The limitations of the transthoracic approach are well documented. Transesophageal echocardiography provides a unique window for achieving a clear view of the mitral prosthesis. METHODS AND RESULTS: This study shows the usefulness of transesophageal echocardiography in clinical practice for assessment of patients with a mitral valve prosthesis. This technique demonstrated an abnormality in 48% of patients who had normal results on transthoracic examination. The overall sensitivity of transesophageal echocardiography was 96%. CONCLUSIONS: Transesophageal echocardiography constitutes an essential part of a comprehensive two-dimensional/Doppler echocardiographic examination in patients with suspected malfunction of mitral prostheses.

Invasion of the guinea pig conjunctiva by Toxoplasma gondii.

Although interactions of Toxoplasma gondii with host cells have been studied extensively in vitro, relatively little is known about the initial interactions of Toxoplasma with mucosal surfaces in vivo. We therefore studied the onset of a Toxoplasma infection in guinea pig conjunctiva. Toxoplasma were inoculated onto the conjunctival epithelium. The tissue was fixed 15 min to 48 hr after inoculation and examined by electron microscopy. Guinea pigs similarly inoculated were maintained in the laboratory for 2 to 8 weeks and tested for antibody by the Toxoplasma dye test. We found that parasites invaded both epithelial and goblet cells within minutes of inoculation. Replication occurred within 4 hr of inoculation and took place mainly in epithelial cells. Within 48 hr, the organisms were found beneath the basal lamina of the epithelium. The host developed an inflammatory response consisting first largely of polymorphonuclear leukocytes and later largely of macrophages. The parasites also replicated in macrophages, showing their ability to evade host defenses in nonimmune animals. Inoculated guinea pigs kept in the laboratory for 8 weeks survived and developed elevated antibody titers against Toxoplasma. The guinea pig conjunctiva is a suitable tissue for studying the pathogenesis of toxoplasmosis.

Transesophageal echocardiography: technique, anatomic correlations, implementation, and clinical applications.

The introduction of transesophageal echocardiography has provided a new acoustic window to the heart and mediastinum. High-quality images of certain cardiovascular structures [left atrial appendage, thoracic aorta, mitral valvular apparatus, and atrial septum] can be obtained readily (average examination, 15 to 20 minutes). In this article, we discuss the technique of image acquisition, image orientation, and anatomic validation. In addition, we describe our experience with the first 100 awake patients who underwent transesophageal echocardiography at our institution. The procedure was well accepted by the patients and associated with no major complications. The clinical indications for this procedure have included thoracic aortic dissection, prosthetic cardiac valve dysfunction, detection of an intracardiac source of embolism, endocarditis, cardiac and paracardiac masses, and mitral regurgitation. Transesophageal echocardiography also proved to be useful in assessment of critically ill patients in whom standard transthoracic echocardiographic images did not provide complete assessment. In these patients (who had extensive chest trauma, had undergone an operation, or were in an intensive-care unit), rapid assessment of the cardiovascular status at the bedside was possible with transesophageal echocardiography. On the basis of our initial experience, we conclude that transesophageal echocardiography complements standard two-dimensional Doppler and color flow examinations and will considerably improve the care of patients with cardiovascular disorders by providing high-quality unique images.

Cytoskeleton of Toxoplasma gondii.

The cytoskeleton of Toxoplasma gondii was studied by electron microscopy using whole mounts of detergent-extracted parasites and thin sections of routine preparations, tannic acid-stained organisms, and detergent-extracted parasites. In whole mounts, the spiral arrangement of the 22 pellicular microtubules closely corresponded to the pattern of surface ridges seen previously by scanning electron microscopy and reflected the torsion of the parasite body during locomotion. The microtubules had free posterior ends and were anchored anteriorly in the polar ring, presumed to be a microtubule organizing center (MTOC). The insertions of the microtubules were supported by blunt projections of the polar ring, forming a cogwheel pattern in transverse view. The internal microtubules had 13 protofilaments and were twice the length of the conoid. They extended through the conoid and ended at the anterior preconoidal ring, presumably a second MTOC. The subunits of the conoid were arranged in a counterclockwise spiral when traced from base to tip, as were the pellicular microtubules. We postulate that as the conoid moves, the polar ring complex moves along the spiral pathway of the conoid subunits. Retraction of the conoid would then rotate the polar ring, producing the torsion of the body we observed by SEM.

Unusual structure of rat conjunctival epithelium. Light and electron microscopy.

The conjunctiva of the adult Sprague-Dawley rat was studied by light microscopy of 3 micron glycol methacrylate sections of whole eyes with lids and by electron microscopy of conjunctiva from the lower fornix. Rat conjunctiva is unique among species studied. All the superficial epithelial cells are squamous cells rather than polyhedral or columnar cells. Furthermore, the goblet cells are aggregated into clusters rather than distributed randomly throughout the epithelium. These clusters are not found at the lid margin or limbus, but are present in the palpebral and bulbar conjunctivae and achieve maximal size and number near the fornix. The stratified squamous epithelium is typical, composed of a layer of basal cells, an intermediate zone of wing cells, and an upper zone of several layers of squamous cells. Dividing cells are seen only in the basal layer. Occasional mononuclear leukocytes are found in the basal and intermediate layers. The goblet cell clusters are largely composed of columnar cells. Goblet cells predominate, but there are also occasional tuft cells, characterized by thick microvilli at their apices. Basal cells form only an incomplete layer beneath the columnar cells, which in places span the entire epithelium. The conjunctiva of the adult rat has few cells with potential for immunological activity. It does not contain appreciable numbers of plasma cells, and lymphoid follicles are absent.

New view of the surface projections of Chlamydia trachomatis.

Two kinds of surface specializations of chlamydiae have been described: hemispheric projections and spikelike rods. We undertook the present studies to demonstrate chlamydial ultrastructure in greater detail in conventional thin-sectioned specimens. Chlamydia trachomatis (LGV strain L2/434/Bu), cultured for 40 h in L929 mouse fibroblasts, was fixed in glutaraldehyde-acrolein, p-formaldehyde-glutaraldehyde, or glutaraldehyde-osmium tetroxide mixtures, postfixed in osmium tetroxide, stained in uranyl acetate, dehydrated in ethanols, and embedded in Epon. By the use of fixatives that penetrate and fix rapidly, chlamydial outer and plasma membranes were clearly revealed. Our results indicate that the hemispheric projections are specializations of the plasma membrane of elementary bodies. The spikelike projections are found in intermediate forms, originate beneath depressions of the plasma membrane, and extend through the periplasmic space and outer membrane to end with pointed tips. Improved preservation of chlamydiae provides a new, informative view of their complex structure. Significant interactions between chlamydiae and host cells might be influenced by the surface structures shown in this study.

Demonstration of the mucous layer of the tear film by electron microscopy.

The mucous layer on the ocular surface maintains the stability, spread, and coherence of the tear film and is essential for normal vision. In spite of its importance, the precise thickness and localization of mucus on the surface of the eye are not known because it is not preserved in conventional electron-microscopic preparations. The authors used two different methods to show mucus on the guinea pig cornea and conjunctiva. First, the authors precipitated mucous glycoproteins by adding a quaternary ammonium compound, either cetylpyridinium chloride or hexadecyltrimethylammonium bromide, to aldehyde fixatives. This procedure stabilized the mucus over the goblet cells and adjacent epithelium, although the mucous layer was not preserved uniformly in other areas. Tannic acid intensely stained mucus precipitated by these methods and showed it to be 0.8 micron thick on the cornea and 1.4 micron thick on the conjunctiva. To confirm these results, the authors also prepared specimens of cornea and conjunctiva by freeze substitution. This technique preserved the mucus in a smooth, uninterrupted layer. The thickness of the mucus was somewhat variable; it measured 1.0 micron over the cornea and varied from 2.0 to 7.0 micron over the conjunctiva because of the greater irregularity of the tissue. The authors' results show that mucus constitutes a considerable part of the precorneal tear film and is thicker than was recognized formerly.

Scanning electron microscopy of Toxoplasma gondii: parasite torsion and host-cell responses during invasion.

Scanning electron microscopy confirmed our previous finding that toxoplasmas actively invade mouse peritoneal cells that are inhibited from phagocytosis. The parasites entered cells with the conoid end first and sometimes showed a counter-clockwise torsion of the body during invasion. Counter-clockwise torsion was also noted in free toxoplasmas. Host-cell responses to active invasion varied with experimental conditions and with the type of host cell. Under adverse culture conditions for phagocytosis, normal macrophages formed rudimentary filopodia or lamellipodia around the tips of invading toxoplasmas; macrophages subjected to hyperthermia before similar incubation with toxoplasmas showed little or no response to invasion. Normal and heat-treated lymphocytes showed little surface reaction to invasion, but occasionally a flocculent collar was seen around the tip of an invading toxoplasma. Scanning electron microscopy provides clues to possible mechanisms of toxoplasma locomotion and host-cell invasion.

Glucose-6-phosphatase as a cytochemical marker of endoplasmic reticulum in human leukocytes and platelets.

Leukocytes and platelets, freshly isolated from normal human blood, were tested cytochemically for glucose-6-phosphatase (G-6-Pase) by a modified Wachstein-Meisel method. The enzyme was present in the endoplasmic reticulum (ER) and perinuclear cisternae of all five types of leukocytes and in the ER of platelets. The reaction product from the cytochemical test distinguished the ER from other intracellular membrane-limited cisternae (i.e., the smooth pinocytic tubules of monocytes and the surface-connected canalicular system of platelets) and thus is a valuable marker of the ER. The cytochemical test also showed that the ER of polymorphonuclear leukocytes (PMN), usually obscured by abundant granules in cells prepared for morphological examination, is more extensive than formerly appreciated. This is the first demonstration of G-6-Pase in human leukocytes. Its precise role in leukocyte metabolism can now be investigated.

Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion.

To determine whether the rhoptries of Toxoplasma gondii play a role in the invasion of host cells by this parasite, we inoculated toxoplasmas into the peritoneal cavities of normal mice and into macrophage cultures, fixed the specimens at various intervals thereafter, and analyzed them by electron microscopy. We found that during host-cell invasion, the rhoptry membrane fused with the anterior limiting membrane of the toxoplasma, producing an opening to the exterior. Since such openings were formed when the host-cell membrane was disrupted, it appears that the rhoptries may secrete a lytic product that facilitates invasion through the host-cell membrane. Such a "penetration-enhancing factor" was previously isolated from lysed toxoplasmas (Lycke and Norrby, 1966). Occasionally, when secretion was incomplete, masses of tubules were found in the rhoptries, sometimes as soon as 15 sec after the toxoplasms had been injected into mice. Similar tubules were found in the parasitophorous vacuole that was formed 10-15 min later, and such tubules are typical of vacuoles containing replicating parasites. Because these tubules are in continuity with the vacuolar membrane, it appears to be a hybrid membrane, composed in part of toxoplasma products. We speculate that the hybrid nature of the vacuolar membrane prevents it from fusing with the lysosomes of phagocytes and thereby contributes to the intracellular survival of the parasites.