Feulgen reaction conducted on unfixed smears]

After hydrolysis with 5N HCl at room temperature Feulgen's reaction either with Schiff's reagent or with some basic dyes instead of it can be carried out on the air-dried smears without any chemical fixation. Both reactions give quantitative results.     

Some aspects of residual formaldehyde testing when reusing haemodialysers

To assess the adequacy of quantifying residual formaldehyde concentrations when reusing, four semi quantitative methods of concentration estimation (Clinitest tablets, Schiff's reagent, Formalert Formotest) were compared. Two of these methods (Schiff's reagent and Clinitest) were inadequate in detecting low concentrations of formaldehyde and were associated with false positives from interference by chemicals contained in the dialysate. When using the two reagents specifically manufactured for this purpose, we also observed false positives with one (Formotest) while the other was capable of detecting formaldehyde concentrations as low as 4.5 mg/l. To minimise patients' exposure and the formation of anti-N like antibodies resulting from formaldehyde damage of red cells, it would be prudent when reusing, to select the most sensitive of these semi-quantitative techniques for routine use, to perform regular screening for anti-N antibodies and to periodically check formaldehyde levels by the use of the highly specific Hantzsch reaction.     

Histochemical detection of lipid peroxidation in the liver of bromobenzene-poisoned mice.

The possibility of detecting lipid peroxidation histochemically was investigated in liver tissue in vivo, in conditions in which the process has been demonstrated by biochemical methods. The technique was based on the detection of aldehyde functions with the use of the Schiff's reagent. The study was carried out on bromobenzene-intoxicated mice, which generally exhibit levels of lipid peroxidation considerably higher than those observed in the case of other hepatotoxins. Liver sections from control animals were unstainable by the reagent, while sections from bromobenzene-poisoned mice showed a purple stain of various intensity, unhomogeneously distributed, sometimes with a mediolobular localization. Microphotometric measurements were performed at 565 nm by means of a computer-controlled microscope photometer. The ratios of Schiff-positive area relative to total section area, as well as the total extinctions referred to 100 sq mu of the sections, showed a high correlation with the corresponding hepatic contents of malonic dialdehyde, chosen as biochemical index of lipid peroxidation. In vitro studies in which liver sections were incubated in the presence of NADPH-Fe2+, showed a Schiff positivity which increased with the incubation time, confirming the reliability of the histochemical method. Another procedure, based on the use of 2-OH-3-naphtoic acid hydrazide coupled with fast blue B, was also developed and proved to be possibly more sensitive than Schiff's reagent in the detection of lipid peroxidation in liver tissue.     

The use of histological techniques for the demonstration of ion exchange resins

AIM: To establish the staining characteristics of certain ion exchange resins in histological material, with a view to enabling confident differential identification. METHODS: Various histological staining procedures were applied to selected pathological material and prepared agar blocks containing the cation exchange resin calcium polystyrene sulphonate and the anion exchange resin cholestyramine. RESULTS: Calcium polystyrene sulphonate uniquely stained strongly by a direct Schiff's reagent procedure without any preoxidation and by the Ziehl-Neelsen method. Cholestyramine was negative by the former method but stained strongly with a standard Congo red technique. CONCLUSIONS: These staining results are consistent with the known structure and properties of polystyrene sulphonate and cholestyramine resins. Polystyrene sulphonate resins have the virtually pathognomonic feature of direct Schiff positivity, while morphology, location, and strong non-birefringent Congo red positivity facilitate the identification of cholestyramine. It is possible that the intrinsic staining characteristics of cholestyramine may be lost once it has bound to its target.     

Origin and propagation of elastogenesis in the developing cardiovascular system

Ectomesenchyme derived from cardiac neural crest is critical to aorticopulmonary septation in the heart. However, any unique contribution of the cardiac ectomesenchyme to the extracellular matrix of the conotruncus has not been demonstrated previously. In this study the chronology and topography of soluble tropoelastin (STE) and the aldehyde-rich protein (ARP) of the elastic connective tissues have been examined in the chick embryo, stages 21-38, and in the quail-chick chimera, stages 24-35 (quail neural fold grafted onto a chick embryo). STE was located with immunofluorescence histochemistry, and ARP with Schiff's reagent. With these procedures prevenient sites of elastin synthesis are observed readily. The results show that the myocardium proper appears to have a role in the instigation of elastogenesis and in elastic fiber orientation; that the mesenchymal cells whose matrix contains elastic fibers are ectomesenchymal, of neural crest origin; and that elastin is deployed in an orderly proximal-distal sequence. It is hypothesized that elastogenesis is a critical event in aorticopulmonary septation.     

Intravascular lymphomatosis: a case presenting with encephalomyelitis and reactive haemophagocytic syndrome diagnosed by renal biopsy

 

Aims:

To report an unusual instance of intravascular lymphomatosis presented with encephalomyelitis and reactive haemophagocytic syndrome. There was no skin involvement. The diagnosis was made on a renal biopsy. Methods and results : The marrow smear was air dried and stained with Diff-Quik. The tissue sections were stained with haematoxylin and eosin, Masson trichrome, periodic acid–Schiff's reagent, Elastic van Gieson's stain, modified hexamine-silver technique and Martius scarlet blue. Immunohistochemistry for CD45, CD20, CD45RO, Factor VIII related antigen, CD31 and CD34 was performed on paraffin-processed tissue. The marrow smear showed active haemophagocytosis in the histiocytes. The renal biopsy showed intravascular lymphomatosis with tumour cells positive for CD45 and CD20. Conclusion : The possibility of intravascular lymphomatosis should be considered in patients with reactive haemophagocytic syndrome where the underlying cause cannot be found after thorough investigation.     

Blastomycosis in a South Indian patient after visiting an endemic area in USA.

We describe a case of blastomycosis in a diabetic patient from South India who had visited Milwaukee, Wisconsin, an endemic area for blastomycosis in the USA. After his return to Bangalore, India, the patient developed intermittent fever of moderate to high grade, cough, loss of weight and appetite, and abscesses in the left cubital fossa and thigh regions. Systemic examination at our hospital revealed that he had dullness to percussion over the chest region and decreased breath sounds. Direct examination of Gram-stained smears of the pus from an abscess showed many broad-based budding yeast cells and culture yielded a dimorphic fungus later identified as Blastomyces dermatitidis. Histologic examination of the curettage tissue slides stained with hematoxylin and eosin, periodic acid Schiff's reagent, and Gomori's methenamine silver stain procedures showed many broad-based budding cells characteristic of B. dermatitidis. The patient was successfully treated, initially with amphotericin B, followed by oral itraconazole for a period of 6 months. Blastomycosis cases in India are reviewed and the likely source of infection in this patient is discussed.     

Pathophysiological aspects of hodgkin's disease

Summary Hodgkin's disease is characterized by unique features setting it apart from other malignancies. Clinically, systemic symptoms are often prominent and spontaneous remissions may be seen. As major immunological aberrations, a reactive humoral response with hypergammaglobulinemia and a depression of cell-mediated immunity are observed. Pathologically, the diagnosis is based on the presence of pleomorphic morphology; this consists of an infiltrate of reactive inflammatory cells sometimes forming granulomatous lesions with or without varying numbers of the cells presumed to be malignant. The nature of these malignant cells remains undefined, with data supporting mainly B-cell, but also macrophage origin. Clear-cut evidence for monoclonality is available only in prognostically unfavourable histological forms.

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Abbreviations E-rosettes spontaneous rosette formation with sheep erythrocytes - EB Epstein Barr - FcIgG (receptor for) Fcpart of IgG - Fc IgM (receptor for) Fc-part of IgM - Ig immunoglobulin - PHA phytohaemagglutinin - PAS periodic acid Schiff's reagent - POX peroxidase - SmIg surface membrane Ig - T-antigen specific membrane antigen of T-lymphocytes     

Changes in cell surface proteins and glycoproteins during the encystation of Entamoeba invadens

Changes in cell surface components of axenically grown trophozoites of Entamoeba invadens which occur during encystation were followed. Protein patterns of trophozoites, immature and mature cyst forms, were analyzed by sodium dodecyl sulphate gel electrophoresis. Total protein profiles of trophozoites and cyst forms stained by Coomassie blue gave similar patterns. In contrast, a number of different bands were observed in gels stained with the carbohydrate-specific Schiff's reagent as well as when nitrocellulose blottings were treated with 125I-radiolabelled wheat germ or soybean agglutinins. The most notable differences were bands at 250 and 95-105 kDa present in the cyst forms and absent in the trophozoites, and two bands at 70 and 75 kDa present in the latter and missing in the cysts. Labelling of trophozoites and cyst cell surfaces by iodination with lactoperoxidase revealed a number of protein bands which were exposed on the trophozoite surface and missing in the cysts. Moreover, gel electrophoresis patterns of non-reduced or reduced samples also differed considerably, indicating that a number of proteins are linked by disulphide bonds. This study shows that specific glycoproteins are produced during cyst formation.     

Histochemical detection of anionic components in the cephalopod brain

Brains obtained from three species of mediterranean cephalopods (Loligo, Sepia, Octopus) were fixed in Bouin's solution. Paraffin sections were cut sequentially at the frontal plane and used for Alcian blue staining (critical electrolyte concentration method), colloidal iron hydroxide staining procedure, the periodic acid Schiff's reagent method, and the lead tetra-acetate-Schiff method. The stained sections were evaluated at 2 regions of different histological composition: the palliovisceral ganglion and the optic lobe. A high concentration of anionic components was found in synaptic regions of the neuropil whereas neuronal cell bodies showed a relatively weak staining of these constituents. There was a significant reaction of the perineuronal glia nets in the cellular rind of the palliovisceral ganglion. From the comparison of staining patterns obtained with the 4 methods in this study and literature data it can be concluded that the detected anionic sites are mainly carboxyl groups of acidic proteins and/or glycoproteins. Sulphate groups may be present in lower concentrations. Their distribution reveals that the role of anionic components other than sialic acids in the invertebrate brain might be discussed in the context of synaptic transmission similar to that in vertebrates. The possible involvement of the glia cell population has to be taken into consideration.     

Purification and characterization of an immunosuppressive factor from ovarian cancer ascites fluid

A nonspecific immunosuppressive factor present in malignant (ovarian carcinoma) ascites fluid has been purified by acid extraction from a high molecular weight (greater than 20000) complex followed by preparative isoelectric focusing on Bio-lyte media. It is an acidic protein (pI = 3.6) of mol. wt. 50000 to 52000 as estimated by gel filtration and composed of subunits of 25000 to 26000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing conditions. It inhibits the phytohemagglutinin-dependent mitogenic response of normal peripheral blood lymphocytes by 50% at 2 microgram/ml concentrations in vitro and suppresses 80% of the plaque-forming cell response to sheep erythrocytes at 100 microgram per mouse in vivo. Its chemical identity with any of the known plasma proteins has not been established. Its failure to stain with periodic acid Schiff's reagent indicates its minimal content of carbohydrates. It is susceptible to tryptic and pronase digestion but insensitive to deoxyribonuclease and ribonuclease digestion. A smaller suppressive factor identified in the same fluid appears to be a lymphotoxin; it differs from the acid-extracted nonspecific suppressive factor in its lack of susceptibility to trypsin.     

A Mycoplasma strain F38 growth-inhibiting monoclonal antibody (WM-25) identifies an epitope on a surface-exposed polysaccharide antigen.

Monoclonal antibody (MAb) WM-25 differentiates by in vitro growth inhibition Mycoplasma capricolum subsp. capripneumoniae (Mycoplasma strain F38), which causes contagious carpine pleuropneumonia, from other Mycoplasma spp. (F. R. Rurangirwa, T. C. McGuire, A. J. Musoke, and A. Kibor, Infect. Immun. 55:3219-3220, 1987). The antigen identified by MAb WM-25 was isolated from solubilized Mycoplasma strain F38 organisms by MAb WM-25 affinity chromatography and was stained with Schiff's reagent, but not with Coomassie blue, after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of purified F38 polysaccharide with periodate abolished binding with MAb WM-25, and MAb WM-25 binding was blocked with laminarin, a complex oligosaccharide with beta(1-->3) sugar linkages. Purified F38 polysaccharide blocked both growth inhibition and agglutination of live F38 organisms caused by MAb WM-25 and rabbit antiserum to F38 organisms. The results in this paper demonstrate that MAb WM-25 binds a periodate-sensitive epitope on the F38 polysaccharide which is also exposed on the surface of Mycoplasma strain F38. Because MAb WM-25 also causes in vitro growth inhibition of F38, the reactive polysaccharide epitope may induce protective immune responses.     

An insulin delivery system from oxidized cellulose

Cellulose oxidized with periodate (OC) has been shown to be a biocompatible, biodegradable matrix suitable for immobilization and sustained release of drugs. In the present work, insulin was immobilized on the OC-matrix and the release profile in vivo and in vitro was examined. The hormone is bound to the matrix by Schiff's bond formation and is released by hydrolysis of this bond as well by degradation of the OC-matrix. The release can be sustained over longer periods by reducing the Schiff's base with NaBH4. The insulin depots could be conveniently injected as suspensions and are able to maintain glucose levels in diabetized rabbits at normal values over several days.     

SDS-PAGE analysis of M. leprae protein antigens reacting with antibodies from sera from lepromatous patients and infected armadillos.

Studies have been conducted to characterize M. leprae bacilli derived from infected armadillos. First, the proteins of the mycobacterial extracts were fractionated by SDS-PAGE. Subsequently, the proteins in the gel were electrophoretically transferred on a strip of nitrocellulose paper by the technique of 'electrophoretic blotting'. The separated bacterial protein bands, thus immobilized on the nitrocellulose paper were made to react immunologically with sera from the lepromatous patients, infected armadillo sera and other experimental mycobacterial antisera. It was observed that a majority of M. leprae proteins contained antigenic determinants also present on proteins of BCG. In addition, only two specific antigen bands of 33KD and 12KD were conspicuously detected by the patients' sera and the infected armadillo sera. These substances were further identified as polysaccharides or glycoproteins since they could only be stained by Schiff's reagent or alcian blue. Only 12KD glycoprotein band reacted with concanavalin A, whereas wheat germ agglutinin (WGA) did not show any reaction with them. These 33KD and 12KD glycoprotein antigens were found to lose their antigenicity after pepsin treatment and can be considered as glycoproteins. Further, radiolabelling experiments showed that 12KD antigen underwent radioiodination under usual conditions, but 33KD glycoprotein failed to be similarly radiolabelled. It is suggested that these protein antigens have M. leprae specific determinants on a cross-reacting component.     

Ion chromatographic determination of metals in biocompatibility testing

The increasing numbers of materials used in surgical implants require a wide spectrum of tests to evaluate their biocompatibility. The release of metals is an important additional feature for determining the suitability for clinical applications of biomaterials such as ceramics and metals. Due to the difficulty of performing time-consuming experiments in vivo, one approach for rapidly determining the suitability of biomaterials and their interactions with the tissues with which they will come in contact is to perform in vitro tests, based on cell culture analysis, using solutions that reproduce the cleavage and binding capabilities of body fluids and tissues. The present work reports on the application of ion chromatography to the simultaneous determination of some metal ions in a few biologic media. Agar-Agar solution, "199 medium, " and betadine and Schiff's reagents were selected as some of the most representative biologic simulating solutions. A method is recommended for the pretreatment of the sample with minimal reagent addition since it is very rich in organic compounds and cannot be analyzed without pretreatment. The proposed procedure requires the sample to be subjected to oxidative UV photolysis for about 60 min in an UV digester at 85 degrees +/- 5 degrees C, followed by the ion chromatographic determination. Lead (II), cadmium (II), iron (III), copper (II), nickel (II), zinc (II), and cobalt (II) simultaneously were determined at microg/L levels.     

Micromethods for the analysis of tear proteins in pharmacological studies.

Although it is well established that ocular mucins and other proteins are essential for tear film stability, whether certain drugs, like non steroidal antiinflammatory drugs (NSAIDs), could cause ocular dryness by inhibiting their secretion is not known. To perform these and other studies of pharmacological interest, we evaluated several micromethods for the analysis of tear samples. The major proteins of the tear fluid collected in capillaries, i.e. IgA, lactoferrin, tear specific prealbumin and lysozyme, were analyzed by SDS-PAGE and gel permeation HPLC, using 2.5-5 microliters of sample. Gastric mucin (PGM), examined as a standard, was analyzed by solid phase assays based on previously described histochemical staining methods: dot blot assays were performed using small disks of polyvinylidene difluoride or nylon membranes, prepared by an ordinary paper punch, which were coated with PGM and stained by Alcian blue or the periodic acid Schiff's reagent. The densitometric analysis was carried out using an ordinary flat scanner controlled by a personal computer equipped with an inexpensive software. The sensitivity of these simple assays was low (100-500 micrograms) but considered sufficient for certain studies. A more sensitive assay (5-20 micrograms) was developed by immobilizing PGM in small agarose gels (100 microliters), prepared in the wells of 96-well microplates, which could by stained by stains-all and analyzed by an automatic plate reader at 595 nm.     

A sensitive automated procedure for the determination of glucuronic acid.

A newly automated method for the detection of uronic acids, particularly glucuronic acid is described. Data showing the limitations of this method are also presented. The advantages of the method are: (1) greatly increased sensitivity compared to the carbazole methods and increased range; (2) lowered interference from other sugars, proteins and salts; (3) variability to allow small sample volumes and/or continuous flow; (4) a more stable color reagent solution.     

A Sensitive Automated Procedure For the Determination of Glucuronic Acid

A newly automated method for the detection of uronic acids, particularly glucuronic acid is described. Data showing the limitations of this method are also presented. The advantages of the method are: (1) greatly increased sensitivity compared to the carbazole methods and increased range; (2) lowered interference from other sugars, proteins and salts; (3) variability to allow small sample volumes and/or continuous flow; (4) a more stable color reagent solution.     

New thermo-crosslinking reactions of copolymers of phenyl methacrylates by use of polyfunctional epoxy compounds

Successful new thermo-crosslinking reactions of copolymers of various phenyl methacrylates by use of polyfunctional epoxy compounds were carried out in the film state at 100–150°C in presence of quaternary ammonium salts, quaternary phosphonium salts, tert-amines, or the crown ether dicyclohexyl-18-crown-6/potassium salt systems as a catalyst. Addition reactions of 4-nitrophenyl, 4-chlorophenyl or phenyl ester groups in the copolymers with ethylene glycol diglycidyl ether (EGGE) result in gel compounds without other side reactions. The rate of gel production of the copolymer having electron-attracting groups such as the 4-nitro group on phenoxide is faster than that of the other copolymers. It was also found that the rate of gel production of the copolymer is affected by the amount of phenyl methacrylate component in the copolymer, the glass transition temperature (T_g) of the copolymer, the structure of polyfunctional epoxy compounds as a crosslinking reagent, the length of alkyl chain in the catalyst, and the kind of counter anion of the catalysts, respectively.