Usefulness of grading of neutrophil aggregate size by laser-light scattering technique for characterizing stimulatory and inhibitory effects of agents on aggregation

We attempted to apply the particle counting method that employs laser-light scattering technique to quantify the change in numbers of neutrophil homotypic aggregates of 3 graded-sizes (small, medium and large). Ex vivo activation of human neutrophils by a chemotactic peptide, fMLP, predominantly produced small-sized aggregates (< 15 cells), and also, transiently, medium-sized aggregates (16-130 cells). Co-treatment of neutrophils with fMLP and cytochalasin B mainly produced medium-sized aggregates, with very few large-sized aggregates (> 130 cells). Interestingly, when protein kinase C was activated with phorbol 12-myristate 13-acetate small-, medium- and even large-sized aggregates of neutrophils were formed. Presence of extracellular calcium was required to produce these neutrophil aggregations. Both prostaglandin E2 (PGE2) and wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI-3K), inhibited neutrophil aggregation, whereas dbcAMP, a cell permeable analog of cyclic AMP, did not, confirming that PGE2 causes neutrophil aggregation probably through PI-3K inhibition rather than activation of adenylate cyclase. These results suggest that the application of the light scattering technique to characterize human neutrophil aggregates by both size and numbers, has advantages over conventional optical turbulent aggregometry, in that it discriminates neutrophil aggregations produced by different mechanisms of intracellular signaling.     

Fatal toxoplasmosis in a southern muriqui (Brachyteles arachnoides) from São Paulo state,Brazil: Pathological,immunohistochemical, and molecular characterization

We report the pathological, immunohistochemical, and molecular features of fatal acute systemic toxoplasmosis in an adult, female, free‐living southern muriqui (Brachyteles arachnoides) from São Paulo state, Brazil. PCR‐RFLP genotyping analysis identified the #21 genotype of Toxoplasma gondii. This represents the first report of acute toxoplasmosis involving this genotype in humans and animals.     

Spontaneous pulmonary adenosquamous carcinoma in a free‐living black capuchin monkey (Sapajus nigritus)

Pulmonary neoplasia is rare among wild New World primates. We report the gross, microscopical, and immunohistochemical features of a primary multicentric pulmonary adenosquamous carcinoma in a free‐living black capuchin monkey (Sapajus nigritus). Herein, the spectrum of pulmonary neoplasms in non‐human primates is widened and briefly reviewed.     

Hepatocellular carcinoma in a free‐living marmoset (Callithrix sp.) with concomitant biliary trematodiasis

Hepatocellular carcinoma (HCC) is rare in New World primates. We report the gross, microscopical, and immunocytochemical features of a spontaneous HCC in a free‐living marmoset (Callithrix sp.). Hepatitis B and C virus and aflatoxin immunohistochemistry were negative; however, concomitant intra‐ and extrahepatic biliary trematodiasis could have played a role.     

High glucose-6-phosphatase activity in non-pigmented epithelial cells of rabbit ciliary body.

For study of the origin of glucose in the aqueous humor, glucose-6-phosphatase (G6Pase) and hexokinase activities, and glycogen, were cytochemically examined in the ciliary body (CB) of rabbit. G6Pase activity was also assayed biochemically. The staining reaction for G6Pase activity was strong in the non-pigmented epithelium (NPE) in the pars plana and tips of ciliary processes in the region containing large ciliary pockets within the pars plicata. NPE cells contained abundant reaction product for G6Pase activity in the endoplasmic reticulum (ER) and nuclear envelope. However, NPE in other regions of the CB and pigmented epithelium (PE) of CB, and other areas surrounding the anterior and (PE) of CB, and other areas surrounding the anterior and posterior chambers, showed weak or no G6Pase staining reaction. Biochemical G6Pase activity in the whole ciliary body was relatively high. Both NPE and PE in the pars plana and the tips showed strong staining reaction for hexokinase activity but no staining for glycogen. Furthermore, NPE cells in the tips bore large aggregates of smooth ER and many Golgi apparati. These suggest that the high G6Pase activity in NPE cells in the pars plana and the tips is related to glucose release into the aqueous humor.     

Microphotometric analysis of cytochrome P-450 in periportal, midzonal, and perivenular hepatocytes of mice treated with phenobarbital

To obtain detailed information on the increase of cytochrome P-450 (P-450) content in periportal, midzonal, and perivenular hepatocytes after phenobarbital (PB) administration, and to study the mechanism of increased P-450 in the endoplasmic reticulum (ER), we estimated microphotometrically the P-450 content and morphometrically the area of ER in hepatocytes of three zones from mice injected with 35, 50, 100, or 150 mg/kg of PB for 3 days. The amount of P-450 per unit cytoplasmic volume and the number of P-450 molecules per unit ER area (P-450 number) were increased by injection of 50, 100, or 150 mg/kg, and the ER area per unit cytoplasmic volume was increased by injection of 100 or 150 mg/kg, in hepatocytes from all three zones. Thus, the amount of P-450 in hepatocytes appeared in general to increase multiplicatively by simultaneous increases in both the P-450 number and the ER area. Furthermore, we could recognize two general types of relationship in the P-450 number and ER area between the patterns of change and the increasing doses: (a) increase in the P-450 number without ER proliferation (active type) in periportal and perivenular hepatocytes after injection of low doses; and (b) increase in ER proliferation without increase in the P-450 number (passive type) in hepatocytes of all three zones after injection of high doses.     

A new microphotometric method for measurement of cytochrome P-450 in sections of liver

We developed a new microphotometric method for measuring the amounts of cytochrome P-450 (P-450) in fresh frozen sections of liver. Four serial frozen sections cut from the liver were separately incubated in 50 mM Tris-HCl buffer (pH 8.0) alone, in buffer containing sodium dithionite, in buffer saturated with carbon monoxide (CO), and in buffer saturated with CO and containing sodium dithionite. The difference between absorbance at 450 nm and that at 490 nm was measured in these sections with a simple microphotometer system. This method yielded precise amounts of P-450 in sections by measuring the true extinction of P-450 and by minimizing the effect of contaminating hemoproteins. Livers of adult rats contained large amounts of P-450, which was greater in perivenular hepatocytes than in periportal hepatocytes. In livers of newborn rats, however, small amounts of the enzyme were distributed evenly throughout the lobule.     

Immunocytochemistry applied to aspiration biopsy cytology. Diagnostic contribution in 100 cases of previously stained, routine specimens

OBJECTIVE: To assess the contribution of immunocytochemistry (ICC) to aspiration biopsy cytology (ABC), in a diagnostic context, on routine, previously stained cytologic specimens. STUDY DESIGN: Among 5,221 consecutive cases of ABC, 5.3% were subjected to ICC in the clinical-morphologic context. One hundred of these cases, with a final clinical and histopathologic diagnosis, were studied to determine the contribution of this ancillary technique to the final cytologic diagnosis. All cases had histopathologic study and prospective ICC, performed on usual smears, alcohol fixed and previously stained by the Papanicolaou technique, and were subjected to an avidinbiotin-peroxidase complex method. RESULTS: ICC was contributory in 82% of cases. The contribution of ICC to ABC of lymphoid tissue, thyroid and related organs, soft tissue and miscellaneous cases was, respectively, 84% (39 cases), 88% (26), 72% (18) and 76% (17). ICC was noncontributory in 18 cases, due mainly to misleading interpretation (6%), uncharacteristic profile (5%) and inconclusive immunostain (7%). CONCLUSION: ICC could be successfully applied in routine ABC specimens since the usually investigated antigenic determinants are preserved, allowing previous morphologic study and screening of the smears. The principal contribution of ICC applied to lymph nodes, thyroid and soft tissue aspirates was, respectively, confirmation of metastatic neoplasms, differential of follicular versus C-cell proliferation and assessment of mesenchymal lineages.