Crinophagic inclusions in gastric chief cells of mice with Chediak-Higashi syndrome

Immunostaining for pepsinogen with an immunoglobulin-peroxidase bridge procedure has been undertaken in conjunction with cytochemical staining for complex carbohydrate to investigate the composition and nature of large inclusions in gastric chief cells of beige mice with an analog of the human Chediak-Higashi syndrome (CHS). By these methods to stain chief cells and immunostaining for carbonic anhydrase to distinguish parietal cells, stomachs of the beige mouse were compared with those of normal black mice from which the genetic defect arose. The staining has confirmed that the chief cells are involved in CHS and has affirmed that one type of megabody in CHS chief cells contains both group I and group II pepsinogens and apparently arises in a process akin to crinophagy.     

An ultrastructural and cytochemical investigation of the development of inclusions in gastric chief cells and parietal cells of mice with the Chediak-Higashi syndrome

Gastric epithelium of the beige mouse, with a mutation thought to be analogous to that in Chediak-Higashi syndrome of man, has been examined by ultrastructural morphologic and cytochemical methods. The gastric chief cell in beige mice at 2 months of age or older disclosed two types of abnormal inclusion bodies each having distinctive morphologic and cytochemical features and a different distribution pattern and relationship to other organelles. On the basis of these findings, the first type of inclusion was thought to originate from zymogen granules, in a process of crinophagy, and the second type was interpreted as arising from the maturing face of the Golgi lamellae by the route for genesis of secondary lysosomes or lipofuscins. Each type of inclusion showed evidence both for participating in autophagic processes and for fusing with each other to produce giant inclusions. Additional observations in this study provided evidence for a role of Golgi endoplasmic reticulum lysosome in genesis of secretory granules and of the mature face of the Golgi complex in development of secondary lysosomes in chief cells. The findings also afforded evidence of migration of chief cells toward the bottom of the gland in the course of their maturation. The gastric parietal cell of control black mice disclosed secondary lysosomes, thought to arise from fusion between multivesicular bodies and mitochondria. These autophagic secondary lysosomes were enlarged in beige mice.     

The common heat shock protein receptor CD91 is up-regulated on monocytes of advanced melanoma slow progressors

Despite advances in our understanding of tumour immunology there is no therapy of proven survival benefit for advanced melanoma. Nevertheless, disease progression is slow in a small proportion of patients with metastatic melanoma, suggesting a contribution to outcome from host factors. Recent data have indicated the importance of the heat shock protein receptor CD91 in immune responses to, and progression of, infectious disease. Here we investigate the relationship between CD91 expression and outcome in malignancy. Rare melanoma patients were recruited with advanced disease that was progressing unusually slowly. CD91 expression on their monocytes was compared with control patients with more typical rapidly advancing metastatic disease. Th1 and Th2 cytokines, as well as innate and adaptive immune subsets, were also measured in the two groups. A significant increase in median CD91 expression levels was observed in slow progressors (P = 0.006). There were no differences in other immune subset markers or inflammatory cytokines. The ability of CD91 to internalize and cross-present tumour antigens through the major histocompatibility complex class I pathway may maintain CD8-positive cytotoxic T cell responses and contribute to slow progression of advanced melanoma.     

Use of Gen-Probe and Bactec for rapid isolation and identification of mycobacteria. Correlation of probe results with growth index

Gen-Probe culture confirmation tests (Gen-Probe, San Diego, CA) for Mycobacterium tuberculosis complex and Mycobacterium avium complex were performed on 276 mycobacterial isolates. All 138 M. tuberculosis complex isolates and 79 of 80 M. avium complex isolates were identified correctly. No falsely positive test results were obtained; 58 nontuberculous mycobacteria other than M. avium complex were negative by Gen-Probe. In a second phase of testing, Gen-Probe tests were performed using concentrates from 101 patient Bactec 12B cultures. Positive results by Gen-Probe tests were correlated with the growth index (GI) reading on the day of processing as well as the accumulated GI readings. For those 51 with high (greater than or equal to 999) final GIs, 40/40 (100%) M. tuberculosis complex isolates and 9/11 M. avium complex isolates were positive by Gen-Probe, and six other mycobacteria were negative. Of the 25 with moderate final readings (400 less than or equal to GI less than 999), 12/17 M. tuberculosis complex isolates and 1/1 M. avium complex isolates were correctly identified by Gen-Probe; seven other mycobacteria were negative. Of 25 with low readings (GI less than 400), 8/24 M. tuberculosis isolates were correctly identified by Gen-Probe, and no falsely positive test results were obtained with the other probes. All true negative tests on seven other mycobacteria (not M. tuberculosis complex or M. avium complex) had less than 2% hybridization. Of the 24 falsely negative tests on M. tuberculosis complex isolates or M. avium complex isolates, 22 had greater than 2% hybridization with their respective probes. Thus, percent hybridization greater than 2% may be a useful indicator of the need for retesting.     

Increased Sensitivity of Lymphocytes from Atopic Individuals to Histamine-Induced Suppression

Histamine depressed lymphocyte reactivity to phytohemagglutinin and, to a lesser degree, concanavalin A, when administered simultaneously with mitogen to lymphocyte cultures. Addition of histamine at later times to the cultures appeared to have a slightly enhancing effect on the lymphocyte response. Stimulation of lymphocytes with pokeweed mitogen was in some cases enhanced, even by high concentrations of histamine. Lymphocytes from atopic individuals were more sensitive to the inhibitory effect of histamine than lymphocytes from nonatopic individuals. The sensitivity appeared age-dependent, but within each age group histamine evoked significantly more suppression on lymphocytes from atopic than from nonatopic individuals. The possibility that the altered reactivity of lymphocytes to histamine, which appears to be associated with atopic allergy, is of pathogenic importance, is discussed, and a hypothesis for the development of atopic disease is proposed.     

Immunocytochemical localization of lysozymes in respiratory and other tissues.

Immunostaining paraffin sections of appropriately fixed tissues with an antiserum to human urinary lysozyme as the primary step in an immunoglobulin-peroxidase bridge method has localized lysozyme in previously recognized sites such as Paneth cells, renal tubules, and lymph node macrophages in several species. In addition, lysozyme was demonstrated in the ciliary layer of the trachea, and type II pneumocytes, as well as cells of presumed mucoid nature in laryngotracheal glands. Large stellate cells in follicle centers in the lymph nodes and spleen and in the medulla of the thymus evidenced strong lysozyme reactivity. Granular pneumocytes disclosed immunoreactivity for lysozyme also at the ultrastructural level. Lysoplate assay demonstrated lysozyme in abundance in both the cellular pellet and acellular supernatant of rat alveolar wash fluid and in rat lung after repeated washing of alveoli. Hamster lung differed from the others in failing to immunostain for lysozyme and affording no evidence for content of lysozyme as determined by lysoplate assay. Sites stained with antiserum to human urinary lysozyme failed to stain with antiserum to egg white lysozyme. However, the pyloric glands, Golgi elements in intestinal epithelium, the surface of the colon, and the proximal straight renal tubule of the mouse stained exclusively with the antiserum to hen egg white lysozyme. Many sites staining with antiserum to urinary lysozyme in respiratory, renal, and lymphoid tissue lacked reactivity in control sections exposed to this antiserum after it was absorbed with purified urinary lysozyme. However, mucous acini in submandibular glands, although failing to stain with other control procedures, retained towared the absorbed antiserum, possibly through reacting with an antibody other than that for human urinary lysozyme. A number of cell types containing proteinaceous cytoplasmic granules stained in control sections exposed to normal serum in place of antilysozyme serum in the immunoglobulin-peroxidase bridge procedure and, thus, possessed selective, but nonimmunospecific affinity for immunoglobulin. Cell types that stained with antiserum to hen egg white lysozyme lost affinity for the antiserum after its absorption with egg white lysozyme but retained the affinity after absorption with urinary lysozyme.     

Changes in leucocyte numbers without diapedesis in rats given platelet-activating factor (PAF-acether)

Changes occurring in the blood and the peritoneal cavity following the intraperitoneal injection of platelet-activating factor (PAF-acether) into rats were compared with those when antigen was injected intraperitoneally into actively sensitised rats. A blood eosinophilia had been produced in the rats by an intravenous injection of Sephadex G200 6 days before either challenge. 5 min after PAF-acether, the total number of cells in the peritoneal washings had decreased and the concentration of extravasated dye-labelled plasma protein had increased with no change in histamine levels. On the other hand, antigen at this time produced nor only a decrease in cells and an increase in dye but also an increase in histamine concentration. Only antigen produced a cellular infiltration into the peritoneal cavity with an increase in numbers of neutrophils in the peritoneal washings at 4 h and of mononuclear cells and eosinophils at 24 h. In the blood at 4 h after either challenge, there was a neutrophilia and an eosinopenia. When PAF-acether and antigen were injected together into actively sensitised rats, leucocyte counts in the peritoneal washings increased by a similar amount, both at 4 and 24 h, as those in rats given antigen alone.     

An ultrastructural study of human eosinophil granules: Maturational stages and pyroantimonate reactive cation

For ultrastructural study of the formation and maturation of human eosinophil granules, bone marrow and buffy coat specimens were fixed with an aqueous solution of potassium pyroantimonate and osmium tetroxide and by conventional methods. The antimonate-osmium tetroxide method of fixation, which is thought to permit ultrastructural localization of sodium or other cations, offered an advantage over routine methods in that it permitted recognition of four rather than two varieties of cytoplasmic granules in human eosinophils at various stages of cell development. These four granule varieties, designated A, B, C, and D, differed primarily in distribution and content of crystalloids and antimonate deposits: A granules lacked antimonate deposits and cystalloids; B granules contained a rim of deposits but lacked crystalloids; C granules possessed deposits that were present in the peripheral matrices but not in the central crystalloids; and D granules lacked deposits but contained crystalloids. Evidence is provided that these four varieties of granules represent progressive stages in the maturation of a single granule type and that granules without crystalloids are transformed into granules with crystalloids. The results also provide evidence for the presence of an as yet unidentified pyroantimonate precipitable cation in human eosinophil granules.     

The effects of drugs on leucocyte changes following the injection of antigen into the peritoneal cavities of actively sensitised rats

The injection of antigen into the peritoneal cavities of actively sensitised rats produced an immediate reaction characterised by an increase in concentrations in the peritoneal fluids, collected 5 min later, of extravasated dye labelled plasma proteins, histamine and slow reacting substance of anaphylaxis (SRS-A). Changes were also produced in the numbers of leucocytes in the blood and peritoneal cavity. 5 min after antigen challenge there was a reduction in the number of cells that could be washed from the peritoneal cavity. 4 h after antigen there was an increase in numbers of neutrophils both in the blood and peritoneal washings and these fell to the levels in control rats at 24 h. 24 h after antigen, and continuing for 72 h, there was an increase in numbers of eosinophils and mononuclear cells in the peritoneal washings.The rats were injected intravenously with sephadex particles to produce a blood eosinophilia at the time of antigen challenge, this increased the numbers of eosinophils migrating into the peritoneal cavity but had no effect on antibody levels, the numbers of other leucocytes or on the immediate reaction.An inhibitor of lipoxygenase and cyclo-oxygenase metabolism of arachidonic acid, phenidone, at 100 mg/kg p.o., inhibited SRS-A release to control levels, in the immediate reaction, but had not effect on the leucocyte changes. The glucocorticosteroid, dexamethasone, at doses of 0.1 and 1 mg/kg p.o., produced little inhibition of SRS-A release but significantly inhibited neutrophil, eosinophil and mononuclear cell infiltration into the peritoneal cavity. These results suggest that arachidonic acid metabolites released in the immediate reaction are not the prime mediators of the cellular changes. Isoprenaline, at 0.05 and 0.2 mg/kg s.c., inhibited extravasation in the immediate reaction with no effect on histamine release but only the higher dose inhibited neutrophil and eosinophil infiltration into the peritoneal cavity. Aminophylline, at 50 mg/kg p.o., had no effect on the immediate reaction but inhibited the neutrophil infiltration. Disodium cromoglycate (DSCG) at 20 and 100 mg/kg s.c. inhibited the immediate reaction but this had no effect upon the cellular changes taking place after 5 min. Cyproheptadine at 1 mg/kg s.c. inhibited extravasation but had no effect on the cellular changes. It appears therefore that factors other than those derived from the mast cell were responsible for the cellular changes in this system. DSCG at 100 mg/kg s.c. and aminophylline at 25 and 50 mg/kg p.o. prevented the reduction in the number of cells that could be washed from the peritoneal acvity 5 min after antigen challenge.