A technique for immunoultrastructural identification of T6-positive Langerhans cells and indeterminate cells in mycosis fungoides.

An immunoelectron microscopic method was developed using T6 antiserum in an immunoperoxidase technique to label the dendritic cells of the epidermis in mycosis fungoides (MF). The technique allows simultaneous identification of intracellular Birbeck granules and T6 membrane positivity. Ultrastructural examination of the epidermal infiltrate of MF, using this method, confirmed the T6-positive nature of Langerhans cells and indeterminate cells. All Sézariform and mature lymphocytes were T6-negative and a small population of T6-negative histiocytic cells were also observed.

Ultrastructural identification of T lymphocytes in tissue sections of mycosis fungoides.

An immunoelectronomicroscopic method has been employed to demonstrate in situ the T lymphocyte nature of the dermal and epidermal infiltrates to mycosis fungoides. A specific antiserum to the human T lymphocyte surface antigen (HTLA) was used in an indirect reaction with a peroxidase labeled anti-immunoglobulin. After histochemically revealing the peroxidase activity, T cells were easily identified by the deposition of electron dense material on the cytoplasmic membrane. Counterstaining with uranyl acetate and lead citrate enabled us morphologically to observe the maturity of infiltrating T lymphocytes and to identify Sézary cells and other cells such as eosinophils in the infiltrate. Our results confirm the T cell nature of the dermal infiltrate in mycosis fungoides and show that the epidermal infiltrate of cells forming Pautrier micro-abscesses are predominantly T lymphocytes.

Evaluation of acid alpha-naphthyl acetate esterase activity as a marker of T lymphocytes in benign and neoplastic cutaneous infiltrates.

Activity of the enzyme acid alpha-naphthyl acetate esterase (ANAE) in T lymphocytes and histiocytes in cutaneous tissue sections of patients with histologically confirmed lichen planus, chronic discoid eczema and mycosis fungoides was compared with results obtained using an in situ immunohistochemical technique employing an antiserum against the human T lymphocyte surface antigen (HTLA). In lichen planus and chronic discoid eczema, most of the cells in the cutaneous infiltrate exhibited both positive ANAE activity and the presence of HTLA. In mycosis fungoides, cells identified as T lymphocytes by the presence of HTLA often showed no ANAE activity. ANAE activity appears to be a useful marker of T lymphocytes in benign cutaneous infiltrates but is less reliable in labelling abnormal T cells.

Regulation of two rat liver microsomal carboxylesterase isozymes: species differences, tissue distribution, and the effects of age, sex, and xenobiotic treatment of rats.

The preceding paper described the purification of two rat liver microsomal carboxylesterases, designated hydrolases A and B, that have high affinity (Km approximately 25 microM) and low affinity (Km approximately 400 microM) for para-nitrophenylacetate, respectively. The present study describes the preparation and purification of polyclonal antibodies against these purified enzymes. Each antibody was subjected to immunoabsorption chromatography to remove antibodies against epitopes common to both hydrolases A and B. The resulting isozyme-specific antibodies were used to study the regulation of hydrolases A and B by Western immunoblotting and Ouchterlony immunodiffusion. Liver microsomes from mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and humans contained one or more proteins that were immunochemically related and similar in size (M(r) approximately 60 kDa) to hydrolase A and/or hydrolase B. These proteins were preferentially recognized by the antibody against hydrolase A, except for cat liver microsomal esterase, which was preferentially recognized by antibody against hydrolase B. In rats, the levels of hydrolases A and B in liver microsomes were coregulated as a function of age, sex, and xenobiotic treatment of rats. The levels of both enzymes were very low in 1- and 2-week-old rats, but increased abruptly at 3 weeks of age in both male and female rats. Treatment of mature male rats with 11 known microsomal enzyme inducers caused little (< 35%) or no induction of hydrolase A or B, whereas treatment of rats with beta-naphthoflavone, pregnenolone- 16 alpha-carbonitrile or dexamethasone suppressed the levels of both enzymes. The kinetic analysis of para-nitrophenylacetate hydrolysis described in the preceding paper identified a high-affinity esterase (Km 20-35 microM) in rat liver, testis, lung, prostate, and pancreas and identified a low-affinity enzyme (Km 300-800 microM) in liver, kidney, small intestine, lung, brain, spleen, and heart. Immunoblot analysis established that hydrolase A was present in liver, testis, lung, and prostrate at concentrations that accounted for the high-affinity esterase activity in these tissues. Hydrolase A was not detected in the pancreas, even though this tissue contained low levels of a high-affinity esterase. Hydrolase B was detected in liver and kidney at concentrations that accounted for the low-affinity esterase activity in these tissues. Hydrolase B was not detected in the other tissues examined, some of which (e.g., small intestine) contained high levels of a low-affinity esterase. These results indicate that hydrolases A and B are independently expressed in a wide variety of extrahepatic tissues in rats.(ABSTRACT TRUNCATED AT 400 WORDS)

The cell population in the cutaneous infiltrate of mycosis fungoides: in situ using monoclonal antisera.

T cell antigens were studied in cutaneous sections from five patients with mycosis fungoides (MF). The method allowed cell counting to be undertaken for each monoclonal antiserum. OKT3 (pan T cell) antiserum confirmed the predominantly T lymphocytic nature of the infiltrate, labelling the majority of infiltrating cells. OKT4 (helper/inducer) antiserum positively labelled 90% of the lymphocytes identified as OKT3 +. OKT8 (suppressor) antiserum marked only single or small groups of dermal lymphocytes, which comprised 24% of the cells identified as T lymphocytes. OKT6 (anti-Langerhans) showed positive labelling of dendritic cells in the epidermis and dermis. Fewer positively labelled epidermal dendritic cells were observed in sections from patients receiving PUVA, but no difference was found in the number of OKT6 positive dermal cells. The ratio of helper to suppressor cells in the dermal infiltrate significantly exceeded the normal circulating ratio.

The chloroacetate esterase reaction for mast cells in dermatopathology: a comparison with metachromatic staining methods.

This paper compares standard metachromatic methods with Leder's chloroacetate esterase reaction on mast cells in paraffin wax-embedded tissue of urticaria pigmentosa and a variety of inflammatory and benign neoplastic cutaneous conditions. Our conclusions are that Leder's method allows easier identification of mast cells and can be a useful adjunct to conventional metachromatic methods. The technique could be conveniently adopted in routine dermatopathology for mast cell identification.

Demonstration of histiocytes in the epidermal infiltrate of mycosis fungoides.

The presence of histiocytes in the epidermal and dermal infiltrate of mycosis fungoides was observed histochemically by revealing a diffuse pattern of acid alpha-naphthyl acetate esterase (ANAE) activity in the cytoplasm, and immunohistochemically by demonstrating the presence of histiocyte-specific 'enzymes', lysozyme, alpha-I-antitrypsin and alpha-I-antichymotrypsin. Histiocytes in the infiltrate of mycosis fungoides may be involved in antigen processing and interaction with T lymphocytes.

Purification and characterization of two rat liver microsomal carboxylesterases (hydrolase A and B).

The enzymatic hydrolysis of para-nitrophenylacetate by rat liver microsomes is predominantly catalyzed by two esterases: one with high affinity (Km approximately 25 microM) and one with low affinity (Km approximately 400 microM) for the substrate. Two kinetically distinct esterases were similarly detected in liver microsomes from mouse, hamster, guinea pig, rabbit, cat, cynomolgus monkey, and human, but only the high-affinity enzyme was detectable in dog liver microsomes. The tissue distribution of these kinetically distinct esterases was examined in rats. High-affinity (Km 20-35 microM esterase activity toward para-nitrophenylacetate was detected in testis, lung, prostate, and pancreas. The activity in testicular microsomes was comparable to that in liver microsomes. Low-affinity (Km 200-700 microM) esterase activity was detected in kidney, small intestine, lung, spleen, heart, and brain. The activity in kidney microsomes was comparable to that in liver microsomes. The high-affinity esterase in testicular and liver microsomes was highly sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF), whereas the low-affinity esterase in kidney and liver microsomes was relatively resistant. These results suggested that rat liver microsomes contain two esterases with high activity toward para-nitrophenylacetate, a PMSF-sensitive esterase with high substrate affinity, and a PMSF-insensitive esterase with low substrate affinity. In support of the hypothesis, we have purified and characterized two esterases, designated hydrolases A and B, which appear be the only abundant enzymes in rat liver microsome that rapidly hydrolyze para-nitrophenylacetate. Hydrolase A hydrolyzed para-nitrophenylacetate with high affinity (Km approximately 25 microM), and was inhibited by extremely low concentrations of PMSF (IC50 approximately 100 nM). In contrast, hydrolase B hydrolyzed para-nitrophenylacetate with low affinity (Km approximately 400 microM) and was inhibited only by relatively high concentrations of PMSF (IC50 approximately 100 microM Paraoxon, the active metabolite of parathion, and cresylbenzodioxaphosphorin oxide, the active metabolite tri-ortho-tolylphosphate, completely inhibited the hydrolysis of pra-nitrophenylacetate by rat liver microsomes and by hydrolases A and B, whereas the sulfhydryl agent, para-chloromercurobenzoate, was not inhibition. These results suggest that hydrolases A and B are both serine esterases. The N-terminal amino acid sequence of hydrolases A and B were similar but distinct (23 the first 30 amino acid residues were identical), indicating that these two esterases are isozymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute inhalation toxicity of neutralized chemical agent identification sets (CAIS) containing agent in chloroform.

An acute head-only inhalation study was conducted in rats exposed for 1 h to product solution (wastestream) resultant from the chemical neutralization of Chemical Agent Identification Sets (CAIS) containing agent (sulfur mustard (HD), nitrogen mustard (HN-1) or lewisite (L)) in chloroform. Groups of Sprague-Dawley rats were exposed to varying concentrations (24000, 18000, 12000 or 6000 ppm) of CAIS wastestream. An additional group was exposed to the vehicle (chloroform/t-butanol) only, at a concentration equivalent to the concentration of vehicle at the highest exposure level. Animals were evaluated for toxic effects, including assessment of toxicant-induced alterations to the ocular and respiratory systems. Mortality on exposure to 24000 ppm of test article or to vehicle alone was high. Mortality in the other exposure groups was roughly proportional to the concentration of test article (wastestream). Toxic signs were consistent with exposure to solvent system components (chloroform/t-butanol) and to agent decomposition products/by-products. Incidence and severity of ocular effects were similar in vehicle control and treatment groups. The salient respiratory effect observed was a decreased minute volume, which was also noted in vehicle and treatment groups.