Normal rabbit alveolar macrophages. I. The phagocytosis of tubular myelin.

Normal rabbit alveolar macrophages are engorged with large, dense inclusions which contain whorls of myelin figures, suggesting an exogenous source of polar lipids in their diet. One contributory source of such lipids is surfactant, since macrophages were seen ingesting tubular myelin and vacuoles containing remnants of it were found in the cytoplasm. Thus, as indicated previously in kinetic studies, it appears that alveolar macrophages participate in the turnover of surfactant. However, the relative importance of the macrophage in comparison to other pathways of surfactant removal remains to be determined. It is also noteworthy that although tubular myelin and myelin figures were abundant in the fixative used to wash out the lungs, bacteria were not found in it or in the macrophages. Thus, removal of obsolete surfactant may prove to be one of the mojor endocytic functions of alveolar macrophages.

Normal rabbit alveolar macrophages. II. Their primary and secondary lysosomes as revealed by electron microscopy and cytochemistry.

In this investigation, vacuoles containing tubular myelin proved to be digestive compartments with cytochemical reactivity for acid phosphatase and arylsulfatase. These cytochemical markers identify the secondary lysosomes, known to contain enzymes capable of hydrolyzing phospholipids like surfactant. Therefore, it appears that alveolar macrophages possess the enzymatic machinery for the degradation of the tubular myelin found in their digestive vacuoles. Although it thus appears evident that alveolar macrophages participate in the turnover of surfactant, the quantitative significance of this route of disposal is undetermined. This investigation has also established that acid hydrolases, so prominently displayed in the secondary lysosomes, are also localized in the rough endoplasmic reticulum and in Golgi-endoplasmic reticulum-lysosomes (GERL). Moreover, small vesicles which are produced from GERL serve as primary lysosomes in transporting digestive enzymes to the vacuoles.

Appearance of peroxidase reactivity within the rough endoplasmic reticulum of blood monocytes after surface adherence.

Rabbit blood monocytes, which contain no cytochemically demonstrable peroxidase, develop peroxidatic activity in the RER and perinuclear cisternae within 2 h after adherence to serum- or fibrin-coated surfaces. A similar reactivity appears in surface-adherent human and rat blood monocytes. In both localization and characteristics, this enzyme reactivity in monocytes resembles that normally seen in the resident peritoneal macrophages of the rabbit, as well as in several types of tissue macrophages in other species. Thus this observation supports the concept, presently based on the kinetic data of other investigators, that blood monocytes are the precursors of such cells. Moreover, the appearance of new enzyme activity after adherence may reflect alterations in cellular metabolism resulting from plasma membrane:surface interactions.

Differentiation of monocytes. Origin, nature, and fate of their azurophil granules.

The origin, content, and fate of azurophil granules of blood monocytes were investigated in several species (rabbit, guinea pig, human) by electron microscopy and cytochemistry. The life cycle of monocytes consists of maturation in bone marrow, transit in blood, and migration into tissues where they function as macrophages. Cells were examined from all three phases. It was found that: azurophil granules originate in the Golgi complex of the developing monocyte of bone marrow and blood, and ultimately fuse with phagosomes during phagocytosis upon arrival of monocytes in the tissues. They contain lysosomal enzymes in all species studied and peroxidase in the guinea pig and human. These enzymes are produced by the same pathway as other secretory products (i.e., they are segregated in the rough ER and packaged into granules in the Golgi complex). The findings demonstrate that the azurophil granules of monocytes are primary lysosomes or storage granules comparable to the azurophils of polymorphonuclear leukocytes and the specific granules of eosinophils. Macrophages from peritoneal exudates (72-96 hr after endotoxin injection) contain large quantities of lysosomal enzymes throughout the secretory apparatus (rough ER and Golgi complex), in digestive vacuoles, and in numerous coated vesicles; however, they lack forming or mature azurophil granules. Hence it appears that the monocyte produces two types of primary lysosomes during different phases of its life cycle-azurophil granules made by developing monocytes in bone marrow or blood, and coated vesicles made by macrophages in tissues and body cavities.

Cytochemical localization of glucose-6-phosphatase in rabbit mononuclear phagocytes during differentiation.

Cytochemical and biochemical investigations have revealed glucose-6-phosphatase (G-6-Pase) activity in Kupffer cells of the liver. To determine whether other mononuclear phagocytes are also reactive for G-6-Pase, rabbit bone marrow, blood, and alveolar macrophages were tested for G-6-Pase by a modified Wachstein-Meisel method and prepared for electron microscopy. Some mononuclear phagocytes from all three tissues were intensely reactive; others were unreactive. In promonocytes, monocytes, and alveolar macrophages, reaction product for the enzyme was localized throughout all cisternae of the endoplasmic reticulum (ER) and the perinuclear cisternae, but it was absent from the Golgi complex, lysosomes, and occasional smooth tubular channels. These results indicate that mononuclear phagocytes at all stages of development contain cytochemically demonstrable G-6-Pase and that the distribution of the enzyme is not altered during their differentiation from immature cells in the bone marrow to mature macrophages in the lung.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Differences in peroxidase localization of rabbit peritoneal macrophages after surface adherence.

Unlike resident peritoneal macrophages, which contain peroxidase in the rough endoplasmic reticulum (RER) and perinuclear cisternae (PN), macrophages elicited into the rabbit peritoneal cavity by various stimulants lack the enzyme. Since we had previously found that such peroxidase reactivity rapidly appears in the RER and PN of blood monocytes after surface adherence in vitro, we wondered whether the enzyme could be similarly produced in elicited macrophages by adherence. Cells from peritoneal exudates (96 hours after endotoxin injection) were harvested, suspended in culture medium, and allowed to adhere to fibrin-coated or plastic surfaces. Following culture for various intervals, they were fixed, incubated for peroxidase, and examined by electron microscopy. We observed that these elicited cells, which initially contained no cytochemically detectable peroxidase, acquired peroxidatic activity in the RER and PN within 2 hours after adherence in culture. Thus macrophages, like blood monocytes, may rapidly acquire peroxidase reactivity as a consequence of plasma membrane: external surface interaction. In view of this finding, it would seem unwise to use peroxidase localization as the basis for advocating the existence of two separate lines of peritoneal macrophages, as has been proposed by previous investigators.

Ultrastructural localization of peroxidase in leukocytes of rat bone marrow and blood.

The differentiation of leukocytes in the bone marrow and blood of normal adult male rats was studied by electron microscopy and peroxidase cytochemistry. Tissue samples were fixed in glutaraldehyde, or paraformaldehyde-glutaraldehyde, and incubated in a peroxidase medium containing 3,3'-diaminobenzidine and H2O2 ad pH 7.6. Mature cells of blood were identified, and then the earlier stages of maturation in bone marrow were analyzed. In immature cells of four cell lines, neutrophils, monocytes, basophils, and eosinophils, peroxidase is synthesized and could be demonstrated in the rough endoplasmic reticulum (RER), Golgi complex, and in cytoplasmic granules. Later in maturation, reaction product for peroxidase could not be found in RER or Golgi complex, indicating that peroxidase synthesis had ceased. In two cell lines, neutrophils and monocytes, peroxidase-negative granules were formed, and the mature cells contained two populations of cytochemically distinct granules. All granules of mature eosinophils were peroxidase-positive. In mature basophils, some granules were clearly peroxidase-positive; others displayed variable density, making interpretation uncertain. Mast cells were never seen in blood, but were abundant in bone marrow; peroxidase was never found in their granules by either electron microscopic cytochemistry or a variety of light microscopic methods. Hence, these cells differ from basophils, not only in morphology but also in the enzyme content of their granules.

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.