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Hepatic giant cell carcinoma. An ultrastructural and immunohistochemical study.

Hepatocellular carcinoma with osteoclast-like giant cells (hepatic giant cell carcinoma [HGCC]) is a rare entity, with only three cases reported. The tumor is histologically similar to giant cell tumor (GCT) of bone, and the origin of the multinucleated giant cells and mononuclear stromal cells has not been determined. The purpose of this report is to present a case of this rare tumor and compare its ultrastructural and immunohistochemical features with those of a conventional GCT of bone. Histologically, the HGCC consists of sheets of osteoclast-like giant cells with a background of mononuclear cells. The giant cells lack the pleomorphism seen in hepatocellular carcinomas with anaplastic giant cells. At the light microscopic level, most of this tumor was nearly identical to a GCT of bone, but several microscopic fields (less than 5% of the tumor) had the histologic appearance of a "usual" hepatocellular carcinoma. The hepatic tumor was negative for HAM 56, epithelial cytokeratins, muramidase, and alpha-1-antitrypsin, with only focal positivity for chymotrypsin in mononuclear and giant cells. The GCT was strongly positive for alpha-1-antitrypsin and chymotrypsin in both the mononuclear and giant cells and showed focal, weak staining for AE1 and AE3 in the mononuclear stromal cells. Ultrastructurally, both mononuclear and giant cells of the HGCC showed features typical of hepatocellular carcinoma. Although the patient presented in this report died, the pattern of growth was different from most hepatocellular carcinomas. The overall histologic features of this tumor are distinctive and appear to justify separating this variant from other types of hepatocellular carcinoma.

Adult↗

Ability of monomeric peptidoglycan fragments from Neisseria gonorrhoeae to damage human fallopian-tube mucosa.

Purified disaccharide peptide monomers obtained from Neisseria gonorrhoeae by enzymatic digestion of gonococcal peptidoglycan damaged the mucosa of human fallopian tubes in organ culture. Two peptidoglycan fragments were tested: a nonreducing, anhydromuramyl-containing monomer (the principal fragment shed by growing gonococci) and the analogous reducing, muramidase-derived monomer. The damage produced by either of these peptidoglycan monomers resulted in sloughing of ciliated cells from the mucosa and resembled the damage observed in active gonococcal infection and that produced by filter-sterilized toxic supernatant fluids from gonococcal-infected organ cultures. The minimal toxic dose of peptidoglycan monomers was 0.75 micrograms/ml. Neither lipopolysaccharide, sodium dodecyl sulfate, nor Triton X-100, possible contaminants from the monomer-purification procedures, was present in sufficient quantity to account for the damage. Both of the gonococcal peptidoglycan monomers may be present in vivo and thus may play a role in the pathogenesis of gonococcal infection.

Cilia↗

Rheumatoid nodule and subcutaneous granuloma annulare. A comparative histologic study.

We performed histologic and selected histochemical and immunohistochemical studies on rheumatoid nodules (RN) from 12 patients and lesions of subcutaneous granuloma annulare (SGA) from nine patients. The mean age for patients with RN was 56 years, while that for SGA patients was 19 years. In contrast to those with RN, none of the patients with SGA had arthritis. RN tended to show homogeneous, eosinophilic necrobiosis, giant cells within palisaded foci, and significant stromal fibrosis; while lesions of SGA showed pale, edematous necrobiosis, an absence of giant cells, and lesser degrees of fibrosis. The single most helpful histochemical method was alcian blue, which stained necrobiotic foci in all cases of SGA but in only one case of RN. Epithelioid cells in both disorders stained positively for muramidase. IgM was found in vessel walls in two cases of RN. While the mechanisms of these disorders are not entirely clear, it appears that RN can usually be reliably distinguished from SGA on histologic grounds alone.

Adult↗

Congenital angiomatoid malignant fibrous histiocytoma. A light-microscopic, immunopathologic, and electron-microscopic study.

We present a case of a congenital angiomatoid malignant fibrous histiocytoma. This rapidly growing lesion, which was located in the subcutis of the left upper arm, was excised at the age of 8 1/2 months. The patient, a girl, was well and free of disease 10 months after surgical removal of the tumor. The tumor appeared grossly encapsulated. The gray-tan tissue contained cystic spaces filled with recent and organizing hemorrhages. Microscopically, the tumor was composed of solid masses of histiocyte- and fibroblast-like cells, inflammatory infiltrate, and multifocal irregular blood-filled spaces, which were predominantly devoid of endothelial cells. The tumor was studied immunohistochemically with antibodies specific for FVIII-related antigen, S-100 protein, epithelial membrane antigen, vimentin, desmin, alpha-1-antitrypsin, muramidase, laminin, and collagen type IV. Ulex europaeus lectin-I was also utilized. These studies, along with our ultrastructural findings, suggest that: (a) the tumor is composed of a mixture of mesenchymal cells; (b) an imperfect angiogenesis may be taking place, resulting in a wide spectrum of vascular structures; and (c) the cell of origin may be a pluripotent mesenchymal cell.

Blood Vessels↗

Immunohistochemical localization of cathepsin G in human tissues.

An antiserum to human cathepsin G has been raised in sheep and its reactivity with human tissues has been tested. The indirect immunoperoxidase staining sequence was employed and was applied to routinely processed paraffin sections. Mature granulocytes, especially those of the neutrophil variety, were intensely and consistently stained. Activity was not observed in other cell or tissue types. Many of the cells of acute and chronic myeloid leukemia were strongly stained, in contrast to those of acute lymphoblastic or chronic lymphocytic leukemias. The results of the technique are compared with those described with staining for muramidase (lysozyme), alpha 1-antitrypsin, leukocyte elastase, and naphthol-AS-D-chloroacetate esterase, and with certain monoclonal antisera directed against granulocyte determinants.

Cathepsin G↗

Proliferative myositis. An immunohistochemical and ultrastructural study.

We studied four cases of proliferative myositis by the avidin-biotin-peroxidase complex technique, using a panel of 12 antibodies, and by electron microscopy. The aim was to clarify the nature of their constituent cells, specifically the giant ganglion-like cells and spindle cells, and to discuss the implications for histogenesis. In all cases, both cell types showed positive cytoplasmic staining with antibodies to vimentin, actin (C4), and alpha-smooth muscle actin-1, but in only one was there positive staining with desmin. No staining was obtained with factor XIIIa, muramidase, alpha-1-antitrypsin, myoglobin, S-100 protein, CAM 5.2, factor VIII-related antigen, or neuron-specific enolase. By electron microscopy, both types of cells were seen to contain numerous thin filaments, dense bodies, coated and pinocytotic vesicles, active and dilated rough endoplasmic reticulum, few microvilli, and incomplete desmosomal junctions. Our findings imply a myofibroblastic nature for the giant ganglion-like cells and spindle cells. Our observations also support the hypothesis that they are derived from a pericytic cell.

Actins↗

Evaluation of CD68 and other histiocytic antigens in angiomatoid malignant fibrous histiocytoma.

Immunohistochemistry for the histiocyte antigens CD68, L1, factor XIIIa, and S100 protein was performed on 19 cases of angiomatoid malignant fibrous histiocytoma (MFH) and for keratin, leukocyte common antigen, factor VIII-related antigen, muramidase, and desmin on six of these cases. Nine of 19 cases expressed the histiocyte differentiation antigen CD68, and in three cases expression was seen in greater than 90% of cells. Of the 19 cases studied, three contained rare S100-positive tumor cells, and two expressed factor XIIIa within tumor cells. In three of the six cases, rare desmin-positive tumor cells were identified. The neoplastic cells did not express the other antigens studied. CD68 was not detected in any of 12 cases of storiform-pleomorphic and myxoid MFH or in any of 24 cases of normal tissues or nonfibrohistiocytic mesenchymal tumors except for granular cell tumors. These results indicate that either the cells of angiomatoid MFH differentiate along lines of a tissue macrophage or alternatively, and perhaps more likely, are mesenchymal tumors with a phenotypic alteration, paralleling an enhanced capacity for phagocytosis and acquisition of lysosomes. In either event, the immunophenotype contrasts with conventional MFH and provides another independent set of observations in support of a separation of angiomatoid from conventional MFH.

Antibodies, Monoclonal↗

An immunohistochemical study of primary ovarian sarcoma. An evaluation of nine tumors.

Nine cases of primary ovarian sarcoma were studied with a panel of 10 antibodies by using the avidin-biotin-complex immunoperoxidase technique. Original diagnoses included five mixed müllerian tumors (MMT) homologous type, two MMT heterologous type, and two pure sarcomas (PS). Cytokeratin immunoreactivity was observed in epithelial components of all MMTs, but was not observed in PS. Two tumors originally classified as MMT also showed cytokeratin positivity in spindle-cell components. All the PSs and the spindle-cell components in six of seven MMTs showed positive staining for vimentin. Positive staining for desmin and myoglobin was found in one MMT in which cells with cytoplasmic cross-striations were identified on hematoxylin and eosin sections. However, less differentiated rhabdomyoblastic cells also stained for desmin and myoglobin, facilitating their recognition. Immunoreactivity for epithelial membrane antigen was noted in well-differentiated epithelial components of six MMT cases. One case stained with antibodies to carcinoembryonic antigen, four with S-100 protein, five with alpha-1-antitrypsin, and four with muramidase. No staining of stromal hyaline droplets was observed with any antibodies. Because of the immunohistochemical staining pattern, two of the original diagnoses were changed. Application of immunohistochemistry to spindle tumors of ovary is discussed.

Female↗

Inflammatory meningeal masses of unexplained origin. An ultrastructural and immunological study.

Two patients with inflammatory meningeal masses were studied. Lesions in both patients showed varying proportions of meningothelial and inflammatory components. The non-neoplastic nature of the inflammation was confirmed in one case by lymphocyte surface marker study, which showed T and B cells in one to four ratio, and by immunohistochemistry, which revealed polyclonal plasma cells. The abundant histiocytes contained muramidase and often enclosed intact lymphocytes or plasma cells within their cytoplasm, i.e., emperipolesis. Their surfaces bore slender interdigitating pseudopodia, intercellular junctions, and subplasmalemmal linear densities. The derivation of these histiocytes is uncertain: mononuclear phagocytes, meningothelial cells, and multipotential meningeal cells are all possible progenitors. A comparison with eleven similar reported cases reveals a tendency for inflammatory meningeal masses to occur in the young, as well as a predilection for posterior fossa involvement. They resemble the extranodal lesions of sinus histiocytosis with massive lymphadenopathy, as well as plasma cell granulomas or inflammatory pseudotumors of lung and other tissues. However, it is possible that these lesions represent a variant of meningioma in which an unusual immunological response has been evoked.

Adult↗

Purification and properties of autolytic endo-beta-N-acetylglucosaminidase and the N-acetylmuramyl-L-alanine amidase from Bacillus subtilis strain 168.

beta-N-Acetylglucosaminidase has been purified from the walls of Bacillus subtilis 168 and compared with the other known autolysin, N-acetylmuramyl-L-alanine amidase (amidase). The beta-N-acetylglucosaminidase was a dimer in LiCl buffers with a sub-unit molecular weight of 90000 and a pH optimum of about 5.0. It was very sensitive to proteolytic enzymes and was critically activated by 0.1 to 0.2 M-LiCl. It was insoluble in concentrations of LiCl lower than 0.05 to 0.1 M. It was less strongly bound to walls than was the amidase, which was a monomer of molecular weight 30000 to 40000 in LiCl buffers. The beta-N-acetylglucosaminidase is an endo-enzyme and showed no exo-activity. Lysozyme-like enzyme (muramidase) activity was undetectable in the wall extracts examined.

Acetylglucosaminidase↗

Turnover of the cell wall peptidoglycan during growth of Neisseria gonorrhoeae and Escherichia coli. Relative stability of newly synthesized material.

The peptidoglycan of a number of strains of Neisseria gonorrhoeae and Escherichia coli turned over during exponential growth as monitored by the loss of radioactivity (supplied as [14C]glucosamine) from SDS-insoluble material. However, no turnover of the peptide side chains of E. coli peptidoglycan was observed (monitored by diamino[3H]pimelic acid) even though turnover of glycan material was occurring. Turnover rates of 9 to 15% per generation were recorded for all the N. gonorrhoeae strains studied except for the autolytic variant RD5 which showed a higher rate of turnover (20 to 26% per generation). In contrast to previous interpretations, these rates of turnover were not affected by benzylpenicillin, unless sufficient antibiotic was present to affect culture turbidity, when lysis occurred. Examination of the fragments (monomer, dimer and their O-acetylated counterparts, and oligomers) produced by Chalaropsis B muramidase treatment of prelabelled peptidoglycan revealed that no fraction of the peptidoglycan was immune from turnover. However, peptidoglycan pulse-labelled for only 10 min did not show immediate turnover. The lapse of time before turnover commenced was strain dependent, with a maximum value of 1.5 generations. This work confirms that the peptidoglycan of N. gonorrhoeae undergoes a period of maturation and suggests that only mature peptidoglycan turns over.

Bacterial Proteins↗

O-acetylation of peptidoglycan in Neisseria gonorrhoeae. Investigation of lipid-linked intermediates and glycan chains newly incorporated into the cell wall.

Radioactive labelling of the amino sugars in gonococcal peptidoglycan was followed by treatment with Chalaropsis muramidase and TLC separation of the products. Even after very brief periods of labelling (0.5 min) the peptidoglycan was already cross-linked to some 80% of the final value and little change occurred within 2 min. The remaining cross-linking was achieved only over a period of about one generation time. Streptomycete endopeptidase was used to show the extent to which new chains were cross-linked to old. Even at the earliest times many cross-linked units contained new material in both moieties and by 3 min there was little distinction in relative labelling, indicating that in Neisseria gonorrhoeae most newly synthesized glycan chains are cross-linked to other new chains rather than to pre-existing peptidoglycan. A model is proposed in which newly polymerized monomer units are predestined either towards dimer formation with other new chains, which are then rapidly O-acetylated and not further cross-linked, or towards the formation of trimers and higher oligomers, the latter being a slower process. Although significant O-acetylation of peptidoglycan was detectable even at the earliest times, efforts to detect O-acetylated lipid intermediates were unsuccessful. The chief lipid intermediate found was apparently the disaccharide-peptide unit linked to undecaprenol.

Acetylation↗

Cross-linking and O-acetylation of newly synthesized peptidoglycan in Staphylococcus aureus H.

Staphylococcus aureus H growing exponentially was labelled with N-acetyl[14C]glucosamine, which became incorporated into the peptidoglycan. The portion of peptidoglycan not linked to teichoic acid (60-75% of the whole) was degraded with Chalaropsis muramidase to yield disaccharide-peptide monomers and dimers, trimers and oligomers formed by biosynthetic cross-linking of the monomers. The degree of O-acetylation of these fragments was also examined. Pulse-chase experiments showed that the proportion of label initially in the monomer fraction immediately after the 1 min pulse declined rapidly during a 3 min chase, while the oligomer fraction (fragments greater than trimer) gained the radioactivity proportionately. The radioactivity of the dimer and trimer fractions remained virtually unchanged. At 4 min after the commencement of labelling (i.e. approx. one-tenth of a generation time) final values had been reached. The O-acetylation of all fragments had achieved final values even at 1 min, except for the monomer fraction, which showed an increase from 40% to 60% during the first 3 min of chase. Although O-acetylation was clearly a very rapid process, no O-acetylated peptidoglycan lipid-intermediates could be detected.

Acetylation↗

Cross-linking and O-acetylation of peptidoglycan in Staphylococcus aureus (strains H and MR-1) grown in the presence of sub-growth-inhibitory concentrations of beta-lactam antibiotics.

Staphylococcus aureus H was grown for 4 generation times with various sub-growth-inhibitory concentrations of beta-lactam antibiotics specific for particular penicillin-binding proteins (PBPs) - PBP2, clavulanic acid; PBP3, methicillin; PBP4, cefoxitin - and also with the non-specific benzylpenicillin. Isolated cell walls were digested with Chalaropsis muramidase and the resulting peptidoglycan fragments were fractionated by HPLC into disaccharide-peptide monomers and cross-linked dimers, trimers, tetramers and greater oligomers. The pattern of relative fragment concentrations with increasing amounts of drug was roughly the same regardless of the antibiotic used, monomers and dimers increasing while trimers and tetramers changed little and oligomers decreased rapidly. The patterns resembled closely those predicted by the 'random addition' model for multiple cross-link formation and not at all those predicted by the 'monomer addition' model. The O-acetylation of the peptidoglycan remained essentially unaffected under all these conditions. S. aureus MR-1, a constitutive producer of PBP2', gave similar results when treated with methicillin.

Acetylation↗

pGIL01, a linear tectiviral plasmid prophage originating from Bacillus thuringiensis serovar israelensis.

Bacillus thuringiensis serovar israelensis harbours, in addition to several circular plasmids, a small linear molecule of about 15 kb. Sequence analysis of this molecule, named pGIL01, showed the presence of at least 30 ORFs, five of which displayed similarity with proteins involved in phage systems: a B-type family DNA polymerase, a LexA-like repressor, two potential muramidases and a DNA-packaging protein (distantly related to the P9 protein of the tectiviral phage PRD1). Experimental evidence confirmed that pGIL01 indeed corresponds to the linear prophage of a temperate phage. This bacteriophage, named GIL01, produces small turbid plaques and is sensitive to organic solvents, which suggests the presence of lipid components in its capsid. Experiments using proteases and exonucleases also revealed that proteins are linked to the genomes of both pGIL01 prophage and GIL01 phage at their 5' extremities. Altogether, these features are reminiscent of those of phages found in the Tectiviridae family, and more specifically of those of PRD1, a broad-host-range phage of Gram-negative bacteria. Dot-blot hybridization, PFGE, PCR and RFLP analyses also showed the presence of pGIL01 variants in the Bacillus cereus group.

Amino Acid Sequence↗

Crystal structures of three complexes between chito-oligosaccharides and lysozyme from the rainbow trout. How distorted is the NAG sugar in site D?

Like all c-type lysozymes, those from rainbow trout act as 1,4-beta-acetyl-muramidases to destroy bacteria by cleaving the polysaccharide chains of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) units in the cell walls. Lysozymes also hydrolyse chitin, the analogous N-acetylglucosamine polymer. The rainbow trout enzymes have been shown to be particularly effective in bacterial defence. We have determined the crystal structures of three complexes between rainbow trout lysozyme (RBTL) and the chito-oligosaccharides (NAG)(2), (NAG)(3) and (NAG)(4) to resolutions of 1.8, 2.0 and 1.6 A, respectively. Crystals of these complexes were obtained by co-crystallization, and intensity data were collected on a FAST area detector system. Refinement and model building gave final R values of 16.6, 15.9 and 16.5% for the di-, tri- and tetrasaccharide complexes, respectively. The results show that the chito-oligosaccharides bind to sites A, B and C as previously observed for complexes between the hen egg-white lysozyme (HEWL) and a variety of saccharides. The NAG ring in site D is not bound so deeply and is only slightly distorted towards a half-chair conformation as observed for the equivalent NAM residue in HEWL. From our results, there is reason to question the position and the degree of strain of the D saccharide and the mode of binding and importance of two saccharides in sites E and F for correct orientation of sugar D and effective hydrolysis of a productive substrate-lysozyme complex. Simple model building study from our structures implies a 'left-sided' binding mode of (NAG)(6) in the lower part of the active site of RBTL.

Journal Article↗

Structure of a complex between bulgecin, a bacterial metabolite, and lysozyme from the rainbow trout.

Bulgecin, a sulfonated glycopeptide produced by Pseudomonas acidophila and Pseudomonas mesoacidophila, induces bulge formation and enhances lysis of bacterial cell walls when used in combination with beta-lactam antibiotics. The compound does not itself exhibit any antibacterial activity, but has been shown to inhibit a soluble lytic transglycosylase (SLT70) from Escherichia coli which has a lysozyme-like domain. Recently, the crystal structure of an SLT-bulgecin complex has been determined to 3.5 A resolution. We report here the crystal structure of a complex between lysozyme from the rainbow trout (RBTL) and bulgecin A at 2.0 A resolution. As for the SLT-bulgecin complex, bulgecin is bound with the glycosaminyl moiety in subsite C and the proline residue in site D of the active-site cleft of RBTL, where it makes hydrogen-bonding interactions with the catalytic residues. The taurine moiety is bound to the left side of subsites E and F in the lower part of the active-site cleft. From the observed position of the bulgecin molecule, it seems reasonable that it is an inhibitor of rainbow trout lysozyme. The lysozymes may, in general, be a target for the design of a novel type of antibiotics distinct from the beta-lactams which are insensitive to the muramidases.

Journal Article↗

Ultrastructural and histochemical identification of sclerosing liposarcoma.

A paratesticular tumour by light microscopy showed large amounts of partly hyalinized collagenous stroma, spindle shaped fibroblast-like cells, pleomorphic cells with foamy cytoplasm, multinucleated tumour type giant cells and lipid inclusions. By light microscopy, there was a problem as to whether the tumour should be classified with the liposarcomas or with the fibrous histiocytic group. Ultrastructurally, spindle shaped cells resembled fibroblasts and, in a few cells, intracellular collagen fibres were seen. Histochemical studies revealed the presence of mannose-rich glycoprotein which is characteristic for collagen producing cells, but frequently these cells also showed numerous lipid inclusion. The foamy cytoplasmic areas of pleomorphic cells were shown to consist of dilated ergastoplasmic sacs and large vacuoles lined by smooth endoplasmic reticulum negating the possibility that these cells were of histiocytic origin. This is supported further by the dearth of lysosomes and absence of lysozyme (muramidase) in the tumour cells. The presence of numerous lipid inclusions in many of the tumour cells, which are otherwise ultrastructurally similar to fibroblast, suggests that these cells represent precursors of lipoblasts. The above findings taken together allowed the tumour to be categorized as a sclerosing liposarcoma.

Aged↗