A novel colorimetric assay of ß-D-glucans in basidiomycete strains by alcian blue dye in a 96-well microtiter plate.

Basidiomycete strains synthesize several types of ß-d-glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these ß-d-glucans in mushroom strains is of great biochemical importance. Because published assay methods for these ß-d-glucans present some disadvantages, a novel colorimetric assay method for ß-d-glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (∼14 nm) in UV-Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high-throughput colorimetric assay method on microtiter plates was used for quantification of ß-d-glucans in the range of 0-0.8 µg, with a slope of 44.15 × 10(-2) and a limit of detection of 0.017 µg/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for ß-1,3-d-glucan. The present assay method exhibited a 10-fold higher sensitivity and a 59-fold lower limit of detection compared with the published method with congo red. ß-d-glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify ß-d-glucans from other biological sources.

A procedure for Alcian blue staining of mucins on polyvinylidene difluoride membranes.

The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Recently, we developed a novel membrane electrophoretic method, supported molecular matrix electrophoresis (SMME), which separates mucins on a polyvinylidene difluoride (PVDF) membrane impregnated with a hydrophilic polymer. Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes; however, this method cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, but is incompatible with glycan analysis, which is indispensable for mucin characterizations. Here we describe a novel staining method, designated succinylation-Alcian blue staining, for visualizing mucins on a PVDF membrane. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2-fold higher than that of Pro-Q Emerald 488, a fluorescent periodate Schiff-base stain. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan analysis using porcine gastric mucin as a model mucin.

Identification and quantification of mucin expression.

The major phenotype of CF is the accumulation of mucus, a phenomenon whose relation to the dysfunctional CFTR is still not fully understood. This means that studies of mucus and its main component, the mucins, are important. Due to the large size and high glycosylation level, such questions need special considerations and methodology. We describe methods for the general quantification of heavily glycosylated proteins as the mucins using dot/slot blot. We also describe the separation of the mucins by gel electrophoresis and the identification with specific antibodies on Western blot and by proteomics.

Effects of infection with toxoplasma gondii oocysts on the intestinal wall and the myenteric plexus of chicken (Gallus gallus) / Efeitos da infecção por oocistos de toxoplasma gondii sobre a parede intestinal e o plexo mientérico de Gallus gallus

This paper aims to analyze the effects of the Toxoplasma gondii infection in the intestinal wall and myenteric plexus of chicken (Gallus gallus). Ten 36-day-old chickens were separated into two groups: control and experimental, orally inoculated with oocysts of the T. gondii strain M7741 genotype III. After 60 days the birds were submitted to euthanasia and had their duodenum removed. Part of the intestinal segments was submitted to histological routine, HE staining, PAS histochemical technique, and Alcian Blue. Qualitative analysis of the intestinal wall and comparative measurements among the groups with respect to total wall thickness, muscle tunic, mucosa, and tunica mucosa were carried out. Caliciform cells were quantified. The other part of the intestinal segments was fixed in formol acetic acid and dissected having the tunica mucosa and the tela submucosa removed. Neurons were stained with Giemsa, counted, and measured. Chickens from the experimental group presented diarrhea and inflammatory infiltrates in the tunica mucosa, thickness reduction of all the parameters assessed in the intestinal wall, and an increase of the number of caliciform cells. There was a ~70 percent reduction regarding the intensity of myenteric neurons; and the remaining cells presented a reduction of ~2.4 percent of the perikarion and ~40.5 percent of the nucleus (p<0.05). Chronic infection induced by T. gondii oocysts resulted in intestinal wall atrophy, mucin secretion increase, death and atrophy of chicken myenteric plexus neurons. Death and atrophy of myenteric plexus neurons may be related with the causes of diarrhea observed in chickens with toxoplasmosis.

O objetivo deste trabalho foi analisar os efeitos da infecção pelo Toxoplasma gondii sobre a parede intestinal e o plexo mientérico de Gallus gallus. Dez galinhas de 36 dias de idade separadas em dois grupos: controle e experimental inoculado com oocistos da cepa M7741 de T. gondii (genótipo III) pela via oral. Após 60 dias os animais foram submetidos à eutanásia e o duodeno coletado. Parte dos segmentos intestinais foi submetida à rotina histológica, coloração por HE e técnica histoquímica de PAS e Alcian Blue. Realizou-se uma avaliação qualitativa da parede intestinal e medidas comparativas entre os grupos da espessura da parede total, túnica muscular, muscular da mucosa e túnica mucosa. As células caliciformes foram quantificadas. Outra parte dos segmentos intestinais foi fixada em formol acético e dissecada retirando-se a túnica mucosa e a tela submucosa. Os neurônios foram corados pela técnica de Giemsa, contados e mensurados. Os animais do grupo experimental apresentaram diarréia e infiltrados inflamatórios na túnica mucosa, redução da espessura de todos os parâmetros avaliados da parede intestinal e aumento do número das células caliciformes. Houve uma redução de ~70 por cento da densidade dos neurônios mientéricos e as células remanescentes sofreram redução de ~2,4 por cento do pericário e ~40,5 por cento do núcleo (p<0,05). A infecção crônica induzida por oocistos de T. gondii levou a atrofia da parede intestinal, aumento da secreção de mucinas, morte e atrofia dos neurônios do plexo mientérico de galinhas. A morte e atrofia dos neurônios do plexo mientérico podem estar envolvidas na causa da diarréia observada em galinhas com toxoplasmose.

Histochemical method for demonstrating quail mast cell types simultaneously.

The development and application of selective staining methods for routine detection of mast cells are of considerable interest, because these cells play an important role in health and disease. The composition of cytoplasmic mast cell granules depends on the species and type of mast cell. The study reported here was conducted to investigate the combined use of aldehyde fuchsin (AF) and the Alcian blue-critical electrolyte concentration (AB-CEC) (pH 5.8, 0.3 M MgCl(2)) techniques for differentiating avian mast cell subtypes. Tissue samples from skin, intestines, and lungs of six healthy adult quail and two control rats were fixed in Carnoy's solution and 10% formolin for routine histological processing. To determine the staining properties of sulfated glycosaminoglycans (GAGs), a three-step staining technique was applied using berberine sulfate, AF, and AB-CEC. In quail, AF positivity following application of the AB-CEC technique was found only in the lungs, mostly in cells that gave a berberine sulfate-positive reaction, and this positivity was determined to be localized particularly in the nucleus and perinuclear cytoplasm. In other regions, the pale AF staining of cells that did not emit fluorescence when stained with berberine sulfate was determined to be replaced by a blue color after application of AB-CEC. The AF/AB-CEC (pH 5.8, 0.3 M MgCl(2)) technique demonstrated that rat and quail mast cells varied in both GAG types and their distribution within the cell. Especially in avian species, this technique can be applied to distinguish mast cells according to their GAG content. It can be used as an alternative to the AB/safranin O staining procedure for differentiating mast cells that contain and lack heparin.

The flow path of alcian blue from the acupoint BL23 to the surface of abdominal organs.

Two hours after Alcian Blue (AB) dye was injected at the rat acupoint BL23, the abdominal cavity was examined and AB-stained threadlike structures were observed on the right abdominal cavity. Those threadlike structures were mainly distributed on the surfaces of the duodenum, colon and cecum. These threadlike structures were thin (about 50 microm) and moved freely, and were connected to corpuscles that were about 500 x 200 microm wide and also stained with AB. On analyzing the histology of the threadlike structures, rod-shaped nuclei, bundles of collagen fibers, reticulofibers, and squamous-like epithelial cells were observed. Immune cells and some sinuses were inside the threadlike structures. These characteristics describe those of Bonghan ducts. The flow paths from the acupoint to internal organs can possibly be used as paths for drug delivery.

One-dimensional cellulose acetate plate electrophoresis--a feasible method for analysis of dermatan sulfate and other glycosaminoglycan impurities in pharmaceutical heparin.

A cellulose acetate plate electrophoresis method for analysis of pharmaceutical heparin and its potential glycosaminoglycan impurities, e.g. dermatan sulfate, chondroitin sulfate and oversulfated chondroitin sulfate, is presented. Heparin is chemically degraded by application of nitrous acid and residual glycosaminoglycans are electrophoretically separated thereafter. After staining using Alcian blue 8GS, these glycosaminoglycan impurities can be quantified by means of comparison to a dermatan sulfate standard. Results of a validation study of this analytical method are shown, demonstrating its feasibility for routine use in analytical quality control labs under GMP conditions.

Morphological and immunocytochemical investigations on mast cells in porcine ureter.

Morphological, morphometric, histochemical and immunocytochemical investigations on mast cells, located in the wall of ureter of 8 months aged pigs were performed. Mast cells were found in all three layers of ureteral wall, but their distribution was irregular and the number unequal. It was established that alcian blue (AB)-positive mast cells were significantly more than toluidine blue (TB)-positive mast cells. A statistically significant smaller number of both AB and TB-stained mast cells were observed in the tunica mucosa. The largest number of mast cells was found in the tunica muscularis. In the adventitia, mast cells were higher in number in the main connective tissue than in the connective tissue near the blood vessels. Mast cells stained with TB showed variably expressed gamma-metachromasia, which was best visible in those situated in the lamina propria of the mucosa. The prevailing parts of mast cells, however, were AB-positive after AB-safranin staining. This was mostly found in mast cells of the tunica muscularis and in mast cells of perivascular location in the tunica adventitia. Immunocytochemically, mast cells were found to be positive for histamine and vasoactive intestinal polypeptide in the muscle coat, and to histamine in the adventitia, as well. On the basis of obtained results it was presumed that the mast cells in porcine ureter most probably took part not only in keeping of local homeostasis, but played also an important role of mobility of smooth muscle cells in the middle layer of ureter on one hand, and, on the other, in the adventitial blood vessels.

IL-12p40 and IL-18 modulate inflammatory and immune responses to respiratory syncytial virus infection.

Respiratory syncytial virus-induced bronchiolitis has been linked to the development of allergy and atopic asthma. IL-12 and possibly IL-18 are central mediators orchestrating Th1 and/or Th2 immune responses to infection. To determine a possible role for IL-12 in regulating the immune response to acute respiratory syncytial virus infection, IL-12p40 gene-targeted (IL-12p40-/-) and wild-type mice were intratracheally infected with respiratory syncytial virus, and lung inflammatory and immune responses were assessed. Lung inflammation and mucus production were increased in the airways of IL-12p40-/- mice as compared with those of wild-type mice, concurrent with increased levels of the Th2 effector cytokines IL-5 and IL-13. Respiratory syncytial virus clearance and levels of Th1 effector cytokine IFN-gamma were not altered. Interestingly, IL-18, another mediator of IFN-gamma production, was significantly increased in the lungs of IL-12p40-/- mice early during the course of infection. Abrogation of IL-18-mediated signaling in IL-12p40-/- mice further enhanced Th2 immune response and mucus production in the airways during respiratory syncytial virus infection but failed to modulate IFN-gamma production or viral clearance. These findings implicate a role for IL-12 and IL-18 in modulating respiratory syncytial virus-induced airway inflammation distinct from that of viral clearance.

Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride.

Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.

Novel findings in inhibition of mast cell-dependent immediate-type cutaneous reactions by Gahmi-Shini-San.

This report describes an inhibitory effect of Gahmi-Shini-San (GSS) on mast cell-mediated immediate-type allergic reactions. GSS is an Oriental herbal medication, which has been successfully used in Korea for the treatment of allergic disorders, mainly skin anaphylactic diseases. GSS inhibited the ear swelling response induced by intradermal injection of compound 48/80 in a mouse model on a concentration-dependent basis. The mast cells in mouse ear tissue were stained by alcian blue/nuclear fast red. GSS significantly inhibited the compound 48/80-induced degranulation from mast cells in ear tissue. GSS dose-dependently inhibited the histamine release from the rat peritoneal mast cells by compound 48/80. We also studied the effect of GSS on mast cell-dependent passive cutaneous anaphylaxis activated by dinitrophenyl IgE antibody. GSS showed inhibition of passive cutaneous anaphylaxis following oral administration. These results indicated that GSS has inhibitory effect on mast cell-dependent immediate type cutaneous reactions.

Patterns of hyaluronan staining are modified by fixation techniques.

The apparent intensity of hyaluronan (HA) staining in tissue sections can vary as a function of fixation techniques. We examined the histochemical distribution of HA in normal human skin using an HA-specific binding peptide derived from bovine nasal cartilage. The HA, particularly in the dermis, was best preserved in sections fixed in 10% acid-formalin with 70% ethanol. In contrast, sections fixed in the routine 10% neutral-buffered formalin had a much weaker intensity of HA staining. Furthermore, acid-formalin/ethanol-fixed sections retained much of their apparent HA after incubation with saline, in contrast to the neutral formalin-fixed sections, in which most of the stainable HA was lost. Such marked differences in staining intensity were not observed in slides stained with Alcian blue, a procedure presumed to stain HA as well as other glycosaminoglycans. Staining using the HA binding peptide was entirely absent when sections were first preincubated in hyaluronidase, whereas similar Alcian blue-stained sections retained most of their staining intensity. Caution should be exercised in evaluating the distribution of HA in tissues using the HA binding peptide, particularly when different fixation techniques among several laboratories are being compared. In addition, the ability to evaluate the HA content of tissues using Alcian blue staining should be reconsidered. The sulfated glycosaminolglycans of the "ground substance" appear to be the predominant substrates for Alcian blue.

Cytokeratins, CEA, and mucin histochemistry in the diagnosis and characterization of extramammary Paget's disease.

To identify a sensitive marker for extramammary Paget's disease and to identify histochemical and immunohistochemical features that suggest occult pelvic cancer in patients with extramammary Paget's disease, we retrieved all cases between 1983 and 1992 with a Standardized Nomenclature of Medicine code of extramammary Paget's disease in the Vanderbilt University Medical Center (Nashville, Tenn) surgical pathology archives. All were stained for alcian blue/dPAS (periodic acid-Schiff), mucicarmine, AE1/AE3, cytokeratin (CAM 5.2), cytokeratin (CK) 7, CK 20, carcinoembryonic antigen (CEA), orthokeratin, prostate-specific antigen, and S-100. Sixteen cases (2 men, 14 women) were retrieved. Two had pelvic malignancies: one rectal adenocarcinoma and one transitional carcinoma. Only CK7 marked all cases. Mucins were sensitive but focal, a potential problem in small biopsy specimens. The transitional tumor had a unique staining profile (CEA- and mucin-negative). CK20 strongly marked Paget cells associated with rectal cancer; its presence suggests a large bowel lesion but is not specific. No case expressed prostate-specific antigen; its presence in a man suggests prostatic carcinoma.

Alcian blue and epithelial membrane antigen are useful markers in differentiating benign from malignant papillae in thyroid lesions.

Immunohistochemistry for epithelial membrane antigen (EMA) and histochemistry for alcianophilic substances were performed in 17 cases of papillary thyroid carcinoma (PTC) and 11 cases of benign thyroid lesions showing papillary changes (7 diffuse hyperplastic goitres-Graves' disease; 4 colloid cystic goitres). In all PTCs the glycocalix of the cells lining the papillary structures was strongly positive with anti-EMA antiserum. Alcian blue pH 2.5 stain (AB 2.5) was also positive in 15 of these cases. In contrast, no cases of benign thyroid lesions showed AB 2.5 positivity in the cells lining the papillary structures and the positivity with anti-EMA antiserum, present in only 5 out the 11 cases, was focal and very weak. These results indicate that the presence and distribution of EMA and alcianophilic substances may be useful in distinguishing benign from malignant thyroid lesions containing papillae.

Clear cell metaplasia of the breast: a lesion showing eccrine differentiation.

Clear cell metaplasia of the human breast is known to be a benign metaplastic change which has no pre-malignant connotation. Despite its proposed relationship to focal lactational change and to lactating breast, morphological and immunocytochemical features failed to demonstrate a clear relationship between these. Mucin secretion showed a characteristic pattern of granularity, and endocrine differentiation was not present. The mucin and immunocytochemical features suggest a relationship with eccrine sweat glands and a better name would perhaps be 'eccrine metaplasia' to underline the special relationship breast metaplasias have to sweat gland epithelium.

Hydrophobic interaction of alcian blue with soluble and erythrocyte membrane proteins.

Alcian Blue (AB), a cationic dye widely employed for monitoring negative surface charge variations on red blood cell (RBC), platelet and glomerular membranes of patients with nephrotic syndromes, was found in fact to aggregate with itself and precipitate in the pH range 7.0-7.8, i.e., at the physiological pH values used for performing the binding assay between the dye and cell surfaces. This aggregation appears to be essentially hydrophobic as it is insensitive to urea but fully prevented in presence of 2% zwitterionic detergent. In addition, AB binds to most RBC membrane proteins solubilized by urea-detergent extraction, again suggesting hydrophobic interaction. AB also interacts with freely soluble proteins such as haemoglobin and myoglobin; such binding is disrupted by ethylurea and/or 2% zwitterionic detergent, typical inhibitors of hydrophobic liaisons. AB also strongly binds to myoglobin with all the negative charges blocked by esterification of the carboxyl groups, again ruling out direct interaction via surface negative charges. It is concluded that AB binding to the RBC surface can hardly monitor variations in surface charge due to sialic acid residues but, at best, variations in surface hydrophobicity.

Value of immunocytochemistry in the study of malignant effusions.

Recognition of malignant effusion relies heavily on cytologic examination despite the difficulty of distinguishing atypical mesothelial hyperplasia from metastatic carcinoma. The combination of CEA, EMA, vimentin, keratin, high-molecular-weight cytokeratin (HMWK), low-molecular-weight cytokeratin (LMWK), and Alcian blue was tested in 51 cytologic specimens of pleural, peritoneal, and pericardial effusions. These showed metastatic carcinoma in 38 cases (ovary, 14; lung, 8; breast, 7; GI, 4; endometrium, 4; bladder, 1) and mesothelial processes in 13 (hyperplasia, 9; mesothelioma, 4). Strong positivity for EMA (92%), CEA (90%), and Alcian blue (71%) was noted in metastatic carcinoma but not in the mesothelial processes. Keratin was positive in all cases of mesothelioma but occurred also in mesothelial hyperplasias (44%) and metastatic carcinomas (47%). In mesothelial cells, HMWK was consistently stronger than LMWK, whereas in adenocarcinoma the reverse was true. There was no difference in the degree or distribution of positivity of any of the markers among the various primary sites of the neoplasms. Our findings are consistent with the view that immunocytochemistry with a battery of antibodies is useful in the recognition of malignant effusions but cannot, as yet, determine the site of origin of metastatic neoplasms.