Presacral myelolipoma. Report of a case with fine needle aspiration cytology and immunohistochemical and histochemical studies.

The cytomorphologic features of a case of the rare presacral myelolipoma, diagnosed by computed tomography (CT)-guided percutaneous fine needle aspiration (FNA) biopsy, are presented. It occurred in a 40-year-old man on prolonged steroid therapy for bronchial asthma. On the air-dried, Diff-Quik-stained smear, hematopoietic cells of all lineages and fragments of collapsed stroma were seen in a background of fat droplets. Megakaryocytic, myelocytic and eosinophilic lineages were further demonstrated by Factor VIII immunostain, chloroacetate esterase and LUNA-E histochemical stain, respectively. The reticulum stain of the resected tumor demonstrated that the hematopoietic cells and fat were supported by numerous reticulum fibers of varying thickness; that accounted for the collapsed stroma in the aspirated material. Presacral myelolipoma, occurring predominantly in female adults and presenting secondarily as a space-occupying lesion, is best regarded as encapsulated, heterotopic bone marrow supported by a network of reticulum fibers; it has a characteristic image on CT scan and can be diagnosed by FNA.     

Neurite outgrowth and proliferation of non-neuronal cells on cryostat sections of adult muscle

Rat spinal cord cells were cultured on cryostat sections of innervated and denervated muscle. Neurite outgrowth was greater on sections of denervated muscle, which therefore appeared to act on in vivo nerve regeneration. It seems that muscle sections were able to release into the culture medium factors that increase proliferation of fibroblasts. The muscle therefore appeared able to modulate its interaction with its environment by acting on different types of cells.     

Neurite outgrowth and proliferation of non-neuronal cells on cryostat sections of adult muscle

Rat spinal cord cells were cultured on cryostat sections of innervated and denervated muscle. Neurite outgrowth was greater on sections of denervated muscle, which therefore appeared to act on in vivo nerve regeneration. It seems that muscle sections were able to release into the culture medium factors that increase proliferation of fibroblasts. The muscle therefore appeared able to modulate its interaction with its environment by acting on different types of cells.     

Ultrastructural visualization of virus-induced surface antigens. Comparative studies of three immunohistochemical techniques.

Three immunohistochemical techniques used for ultrastructural localization of cell surface antigens were compared with respect to ease of reagent preparation and qualitative aspects of cell surface staining. The techniques compared were: (a) ferritin-conjugated antibody, (b) "hybrid" antibody and (c) a modification of the "bridging" technique which employs soluble immune complexes. Used in conjuction with strain MC29 avian oncornavirus-infected chick embryo cells and chicken antiviral immune serum, the results of all three techniques were comparable. In terms of ease of preparation, the "bridging" technique employing soluble complexes was found to be the most satisfactory.     

A solvent-free coating-procedure for the improved preparation of cryostat sections in light microscope histochemistry.

A new technique is presented for the external stabilization of cryostat sections by spraying the specimen surfaces with an aqueous solution of poly(vinyl alcohol) before each sectioning stroke. The spray freezes upon the surface and forms a tough coating which facilitates subsequent sectioning and handling especially of difficult material. The sections are affixed upon cold glass slides covered with an improved formulation of pressure-sensitive adhesive. During further processing of the affixed sections, the PVA-coating and any surrounding supporting medium dissolve without traces in the first aqueous incubation or staining solution.     

Fine needle aspiration cytology of perineurioma. Report of a case with histologic, immunohistochemical and ultrastructural studies

The fine needle aspiration (FNA) findings are presented for a case of perineurioma, a benign soft tissue tumor composed of perineurial cells, which are epithelial-like and ensheath the endoneurial connective tissue space of nerve fibers. A subcutaneous FNA performed on a posterior thigh mass of a 14-month-old boy produced a specimen consisting of numerous spindle-shaped tumor cells with bipolar cytoplasmic extensions and occasional pseudosignet-ring cells in a prominent myxoid background. Cytologically, a diagnosis of benign myxoid tumor was made. Histologic examination of the resected tumor revealed the characteristic features that have been described for perineurioma; the diagnosis was confirmed with immunocytochemical and ultrastructural studies. Perneurioma should be included in the differential diagnosis of benign myxoid neoplasms seen in FNA biopsy specimens.     

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections.

The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study show that the enzyme histochemical technique--except for some limitations--is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.     

Fine needle aspiration cytology of clear cell sarcoma. Report of a case with immunocytochemical, immunohistochemical and ultrastructural studies.

Cytologic findings of clear cell sarcoma obtained by fine needle aspiration (FNA) of a tumor are described. The tumor probably originated in the retroperitoneal tissue, and the diagnosis was confirmed histologically by open biopsy. Percutaneous needle aspirates of the intraabdominal tumor and touch preparations obtained from the open biopsy specimen revealed numerous atypical cells with an extremely hyperchromatic nucleus, prominent nucleoli and clear cytoplasm. The cytoplasm was rich in glycogen. The immunocytochemical technique demonstrated S-100 protein and neuron-specific enolase in the cytoplasm, both of which were exhibited also in the histologic specimen. Clear cell sarcoma is a rare tumor of soft tissue, and to our knowledge, detailed cytologic appearances of this tumor obtained by FNA have not been reported. In addition, the present tumor was unique in location. It is possible to diagnose clear cell sarcoma accurately on an FNA cytologic specimen if the periodic acid-Schiff stain and immunocytochemical technique are utilized in addition to the routine Papanicolaou method.     

A cytophotometric and electron-microscopical study on catalase activity in serial cryostat sections of rat liver

Summary The validity of the histochemical procedure for demonstrating catalase activity in cryostat sections of rat liver at the light-and electron-microscopical level was studied cytophotometrically. Incubations in the presence of 5 mm diaminobenzidine, 44 mm hydrogen peroxide and 2% polyvinyl alcohol performed on fixed cryostat sections resulted in the highest amounts of final reaction product precipitated in a fine granular form which was specific for catalase activity. Serial sections processed for electron microscopy indicated that the osmiophilic final reaction product was exclusively localized in the matrix and core of peroxisomes. The relationship between incubation time and the amounts of final reaction product generated by catalase activity as measured at 460 nm in mid-zonal areas of liver lobules showed non-linearity for the test-minus-control reaction because first-order inactivation of the enzyme occurred during incubation. Linearity of the test-minus-control reaction and section thickness was observed up to 8 m. Catalase in rat liver showed a K_m value of 2.0 mm for its substrate hydrogen peroxide when the diaminobenzidine concentration was 5 mm. It is concluded that the procedure for demonstrating catalase activity in serial cryostat sections of rat liver at the light- and electron-microscopical level is specific and can be applied to quantitative purposes. This approach may be useful in pathology, when only small biopsies are available, when the tissue is heterogeneous, and when other histochemical markers also need to be studied in the same material.     

The effects of storage on the retention of enzyme activity in cryostat sections. A quantitative histochemical study on rat liver

Summary The effect of storage of unfixed cryostat sections from rat liver for 4 h, 24 h, 3 days and 7 days at -25°C was studied on the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine oxidoreductase, glutamate dehydrogenase, succinate dehydrogenase (all demonstrated with tetrazolium salt procedures), glucose-6-phosphatase (cerium-diaminobenzidine method), 5-nucleotidase (lead salt method), dipeptidyl peptidase II, acid phosphatase (both simultaneous azo coupling methods), d-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure) and catalase (diaminobenzidine method). The effect of drying of the cryostat sections at room temperature for 5 and 60 min was investigated as well. The enzyme activities were quantified by cytophotometric measurements of test and control reactions. The test minus control reaction was taken as a measure for specific enzyme activity. It was found that the activities of all the enzymes investigated, with one exception, were affected neither by storage of the cryostat sections at -25°C for up to 7 days, nor by drying of the sections at room temperature for up to 60 min. The exception was xanthine oxidoreductase, whose activity was reduced by 20% after 5 min drying of sections or after 4 h storage. Therefore, only incubations for xanthine oxidoreductase activity have to be performed immediately after cutting cryostat sections, whereas for the other enzymes a considerable margin appears to exist.     

Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein

Trehalose, a disaccharide present in many non-mammalian species, protects cells against various environmental stresses. Whereas some of the protective effects may be explained by its chemical chaperone properties, its actions are largely unknown. Here we report a novel function of trehalose as an mTOR-independent autophagy activator. Trehalose-induced autophagy enhanced the clearance of autophagy substrates like mutant huntingtin and the A30P and A53T mutants of alpha-synuclein, associated with Huntington disease (HD) and Parkinson disease (PD), respectively. Furthermore, trehalose and mTOR inhibition by rapamycin together exerted an additive effect on the clearance of these aggregate-prone proteins because of increased autophagic activity. By inducing autophagy, we showed that trehalose also protects cells against subsequent pro-apoptotic insults via the mitochondrial pathway. The dual protective properties of trehalose (as an inducer of autophagy and chemical chaperone) and the combinatorial strategy with rapamycin may be relevant to the treatment of HD and related diseases, where the mutant proteins are autophagy substrates.     

Electron microscopic localization of 5'-nucleotidase in rat salivary glands. Comparative enzyme- and immunohistochemical studies

The localization of 5'-nucleotidase in rat parotid and submandibular glands was investigated at the electron microscope level by an immunohistochemical technique using a highly specific antibody, and the results were compared with those obtained using the newley developed cerium method for enzyme histochemistry. Both methods demonstrated that 5'-nucleotidase is located on the external surface of the luminal plasma membranes of acinar cells as well as on intercalated and striated ductal cells. In the basolateral membranes of these cells, the portions adjacent to myoepithelial cells exhibited intense reaction products, but the other areas of plasma membranes contained only trace amounts of the reaction products. Both cerium-based enzyme histochemistry and immunohistochemistry showed that myoepithelial cells retain the enzyme on their plasma membranes. Neither method produced reaction products in the intracytoplasmic structure of constitutive cells of the salivary glands. We discuss the usefulness of the cerium-ion method for the demonstration of 5'-nucleotidase activity and compare it with the traditional lead-ion method.     

X-ray diffraction studies of the corneal stroma.

A low-angle diffraction pattern has been obtained from corneal stroma. This pattern arises both from the arrangement of the collagen fibrils and from the packing of the tropocollagen molecules along the axes of the fibrils. The spacing arising from the packing of the fibrils increases homogeneously on swelling although the tissue as a whole swells only radially referred to the intact eye. The necessary rearrangement of the fibrils for this type of swelling to occur might result in the formation of regions devoid of collagen fibrils and the water not in the lattice of collagen fibrils could be synonymous with the lakes postulated by Benedek (1971) to explain the loss of transparency on swelling.The spacings due to the packing of the tropocollagen molecules are unusual in that, although they index as the third and fifth orders of the well-known 66 nm repeat, the first order of this spacing is absent. Calculation of the Patterson function for corneal collagen leads to peaks in electron density separated by distances of 0.38 and 0.24 of the repeat distance.     

Histofluorescence studies of central monoamines. Comparison of Falck, cryostat and SPG methods.

The aim of the presented paper was to compare the Falck-Hillarp histofluoresce method for monoamines with its cryostat modification and the glyoxylic acid SPG method. The sensitivity and specificity of these methods and also the occurence of unspecific fluorescence is discussed.     

Opposing effects of cryostat sections of preneoplastic and neoplastic mouse mammary lesions on in vitro migration of peritoneal exudate cells.

Cryostat sections of normal mouse tissues and of preneoplastic HAN and neoplastic mammary tumors were used as "antigens" in MMI tests. Nonspecific inhibition of normal and sensitized PEC migration was induced by HAN and some normal tissues, including normal mammary gland. This inhibition did not require the presence of lymphocytes, was not species specific, and could be blocked by sera from HAN-bearing mice. Cryostat sections of mammary tumors did not inhibit, indeed occasionally enhanced PEC migration. Further, the presence of tumor cryostats and eluates interfered with inhibition induced by HAN cryostats and by PPD with PEC from donors sensitized to that antigen. Histologic examination of HAN and of mammary tumor tissue revealed inflammatory cells to be distributed diffusely in the former and localized peripherally around the latter type of lesion.     

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections

Summary The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study whow that the enzyme histochemical technique — except for some limitations — is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.     

Conjunctival impression cytology with transfer in the assessment of vitamin A status in Nigerian children.

OBJECTIVE: To determine vitamin A status by conjunctival impression cytology with transfer (CIC-T) and assess its ability to predict low and deficient serum retinol concentrations. STUDY DESIGN: CIC-T was performed on 128 healthy, well-nourished and 230 malnourished children aged under 6 years by a 3-5-second application of cellulose acetate paper to each bulbar conjunctiva followed by transfer of the adhered cells onto glass slides. The slides were stained with Alcian green 2GX, and smears were classified as normal, borderline normal, borderline abnormal and deficient. Corresponding serum retinol levels were determined in each subject. RESULTS: The results showed that CIC-T is a simple procedure with a failure rate of 7.3% caused by tearing and agitation. The power of CIC-T to predict vitamin A status varied with both the CIC-T smear classification used and serum retinol concentration threshold. CIC-T smear classification as abnormal and normal appears to be the most robust and predictive of serum retinol, < 10 and > 10 < 20 micrograms/dL, respectively. CONCLUSION: The simplicity, sensitivity and specificity of CIC-T suggest that this procedure is a good screening tool for epidemiologic survey of vitamin A status.     

Improved localization of thiamine pyrophosphatase activity in mounted cryostat sections using gel fixation and gel incubation media.

The presence of 1% agar in the fixation and substrate solutions for the histochemical demonstration of thiamine pyrophosphatase (4.4 mM TPP; 3.6 mM Pb2+; 0.025 Tris-maleate buffer, pH 7.2) clearly facilitates the localization of the enzyme in Golgi apparatus in cold microtome sections prepared from unfixed specimens.