Enhanced polymer one-step staining (EPOS) for proliferating cell nuclear antigen (PCNA) and Ki-67 antigen: Application to intra-operative frozen diagnosis

Enhanced polymer one-step staining (EPOS) is a novel, highly sensitive one-step immunostaining method. This simple and rapid technlque was applied to intra-operattve frozen diagnosis. The markers of choice were proliferating cell nuclear anmen (PCNA) and Ki-67 antigen. These cell prollferation markers were both identifiable in fresh frozen see tions of the human tonsil In approximately 7 min. The suitable staining sequences are as follows. Frozen sections prepared using 3-aminopropyitimethoxysilane-cpated glass slides are immediately fixed, without air drying, for 15s in a mixture of 50% formalin and 50% methanol for PCNA, and in 10% formalln for Ki-67 antigen. After a brief rinse in phosphate-buffered saline (PSS), sections are incubated with the EPOS antibody for 3 min, followed by PBS rinse for 1 min. The peroxidase activity is visualized in diaminobenzidine-H₂O₂ solution containing 10mmol/L imidazole for 2 min. After a light rinse in tap water, the nuclei are briefly counterstained with 5% methyl green. When necessary, endogenous peroxi-dase blockage in 1% periodic acid solution for 1 min is added before the EPOS antibody incubation. This procedure is applicable to frozen sections of gastric cancers, malignant lymphomas, and brain, liver and peritoneal lesions in which differential diagnosis between benignancy and malignancy was required.     

ISOLATED ADRENOCORTICOTROPIC HORMONE (ACTH) DEFICIENCY

The case to be reported is that of a 72-year-old woman with isolated adrenocorticotropic hormone (ACTH) deficiency, who complained of anorexia and generalized malaise. The secretions of human growth hormone(HGH), prolactin (PRL), luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH) were all within normal limit. In spite of the extremely low level of Cortisol, the plasma level of AGTH would not rise sufficiently, but a marked response of Cortisol to AGTH stimulation was recognizaed. The postmortem examination revealed a decrease In basophilic or PAS-positive cells of the anterior pituitary gland which also showed a selective loss of AGTH-secreting cells over immunohistochemical study. Electron microscope could easily visualize somatotroph, mammotroph, thyrotroph, FSH- and LH-gonadtroph, but corticotroph was difficult to be discerned. Adrenocortical cells demonstrated atrophy and degeneration, for which the zona fasciculata and zona reticularis were narrowed. The zona glomerulosa was slightly enlarged In width.     

CLINICOPATHOLOGICAL STUDY ON SMALL RENAL CELL CARCINOMAS WITH METASTASES

Seven metastasizing small renal cell carcinomas smaller than 30 mm in the greatest diameter were clinicopathologically studied for a better understanding of their characteristic features as compared to those of small tumors without metastases. Grayish-white infiltrating tumors in gross appearance and alveolar or solid microscopic structure consisting of granular or spindle cells and of atypical nuclei were suggestive of having metastases. Two tumors which had positive reactions to the lower nephron markers such as SB A, PNA, and/or DBA were considered to be of lower nephron origin and displayed poor prognosis. ACTA PATHOL. JPN. 37: 947–954, 1987.     

FINE STRUCTURE OF NEUROBLASTOMA—A CASE REPORT—

Ultrastructural observation was carried out on neuroblastoma cells in a 4-year-old girl. The nuclei of these neoplastic cells were irregular with occasional cytoplasmic invaginations, and proportionally large with diffusely dispersed chromatin substance. Rare mitotic figures were also present. The cell-to-cell interfaces were relatively simple with only finely wavy membranes but without interdigitations. A few desmosomes were, however, apparent. The cytoplasmic pseudopodes were frequently observed in a limited area of the cell surface and nearly free of organelles. The main findings of the intracytoplasmic organelles consisted of some mitochondrial changes, poor development of Golgi bodies, occasional formation of focal cytoplasmic degradation, and intracytoplasmic Abrillar aggregates. Numerous large round to oval dense bodies encircled by a double membrane were highly suggestive of lipid bodies. The central area of rosette-like cell aggregates was noted to be composed of cytoplasmic processes of neoplastic cells.     

Directed differentiation of telencephalic precursors from embryonic stem cells

We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells ( approximately 90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation ( approximately 35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6(+) cells yield up to 75% of Bf1(+) cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1(+) and/or Islet1/2(+) cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.     

The combination of IFN-alpha2 and IFN-alpha8 exhibits synergistic antiproliferative activity on renal cell carcinoma (RCC) cell lines through increased binding affinity for IFNAR-2.

Although there are at least 13 interferon-alpha (IFN-alpha) subtypes in humans, interactions between the subtypes remain unknown. To understand IFN-alpha interactions, we examined the antiproliferative activities and the receptor binding affinities of different combinations of IFN-alpha2 and IFN-alpha8 using six renal cell carcinoma (RCC) cell lines. Although IFN-alpha8 was the more potent subtype, synergistic and antagonistic antiproliferative effects were also observed in certain combinations of IFN-alpha2 and IFN-alpha8. To analyze the interactions between IFN-alpha2 and IFN-alpha8, the receptor-binding kinetics of different combinations of IFN-alpha2 and IFN- alpha8 to the IFN-alpha receptors, IFNAR-1 or IFNAR-2, were measured using a surface plasmon resonance-based biosensor. Unexpectedly, the receptor binding kinetics to IFNAR-2 but not to IFNAR-1 were mutually related to antiproliferative activity and increase in the binding speed (K(a)) for IFNAR-2. Moreover, we observed the increased fluorescence intensity (FI) of biotin-labeled IFN-alpha8 to IFNAR-2 by receptor binding inhibition assay with unlabeled IFN-alpha2 but not the other combinations. These findings indicate that the binding manner of IFN-alpha8 for IFNAR-2 is different from that of IFN-alpha2, suggesting that binding of IFN-alpha8 rather than binding of IFN-alpha2 to IFNAR-2 leads to activation and subsequent antiproliferative activity despite the same antiviral activity in RCC.     

A case of the esophageal candidiasis supposedly caused by rhinenchysis steroid chronic administration before sleep]

In general, steroid is mainly used as anti-inflammatory action in case of allergic diseases. As one of the side effects of inhalation steroid, a report is given below regarding buccal capsule/esophageal candidiasis. The patient came to the hospital with the chief complaint regarding passage dysphagia in the time of deglutition; pharyngitis and esophageal candidiasis were found by endoscopy of upper gastrointestinal tract.The interview after the endoscopy revealed that the patient, a 69-year-old female was diagnosed as chronic perennial allergic rhinitis a few years ago, and had been inhaling rhinenchysis Beclometasone dipropionate (BDP) before sleep every day for the past two years because using this collunarium seemed to mitigate the nasal obstruction and mucus during sleep. The patient did not report this fact before the endocsopy because she did not associate it with her subjective symptom. In this case, it was assumed that nebulized rhinenchysis BDP was accidentally swallowed to the pharynx and esophagus during sleep. As a treatment, rhinenchysis BDP was canceled and instead Azunol mouth washing (gargling/nasal douche) was used. No antifungal agent was used. In two weeks, the patient reported some improvement, and this was confirmed by reexamination of the upper gastrointestinal tract using endoscope in one month and a half. Pharyngitis was improved, and in the digital endoscopic assessment of esophageal candidiasis complicating inhaled steroid therapy the esophageal candidiasis became Grade I (mild grade). As for the later progress, the patient did not report any subjective symptoms such as nasal obstruction and dysphagia. In addition, the inflammation caused by candidiasis and found in the early examination was improved. The patient in this case was under treatment for thrombosis in the vein of lower extremity, but no complications such as diabetes mellitus or immune deficiency syndrome were observed. DISCUSSION: Esophageal candidiasis by chronic administration of inhalation of steroid before sleep for asthmatic patients has been reported. However, there has not been a report of esophageal candidiasis by chronic administration of rhinenchysis steroid before sleep for patients with allergic rhinitis. Similarly, in the case of the use of steroid in the form of collunarium before sleep, steroid stayed in the esophagus via the transendothelial nasal cavity, and that seemed to cause, in the long run, to develop esophageal candidiasis. CONCLUSIONS: One of the implications of the above case is that collunarium can go down, even when it is nebulized in the nasal cavity, to the esophagus via the nasal cavity to buccal capsule. This suggests the necessity for preventative measures in the case of chronic administration of steroid as follows. A. Blowing of the nose just after the use of collunarium B. Daily rinsing (gargling and nasal douche).     

Peptide analysis,stability studies,and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2-A(g7)

The MHC class II molecule H2-A(g7) is the chief genetic determinant in insulin-dependent diabetes mellitus of the non-obese diabetic (NOD) mice. Poor peptide binding ability, as well as presentation of a unique subset of peptides by this molecule was suggested to promote autoimmunity in this strain. However, several laboratories have presented results in favor of an H2-A(g7) molecule that can avidly bind many different peptides. The crystal structures of H2-A(g7) in complex with two different peptides did not completely resolve this issue. To analyze the peptide binding capacity and the motif requirements of H2-A(g7), we eluted natural ligands from purified H2-A(g7) molecules isolated from the H2-A(g7)-transfected M12-C3 cells. A low peptide yield dominated by a few peptide ligands was found. Pool sequencing and alignment of individual ligands on the basis of molecular modeling revealed a peptide-binding motif with basic/aliphatic/small hydrophilic amino acids at relative position 1 (p1), aliphatic amino acids at p4, Ala at p6, and acidic amino acids and Ser/Gly at p9, as well as acidic residues at p10/11. Though weak, the binding of individual ligands, as well as the importance of an acidic C-terminal residue was confirmed by peptide binding studies to isolated H2-A(g7) molecules. Furthermore, the H2-A(g7) molecule incompletely dissociated into its constituent chains in SDS-electrophoresis under nonreducing conditions. This provides additional evidence of its weak affinity for peptides, which probably arises from the combination of beta56His/beta57Ser/beta78Ala and other unique H2-A(g7) residues in contact with the antigenic peptide. These results allow a better understanding of the role of this molecule in the development of autoimmunity and the identification of epitopes relevant to diabetes.     

Synthesis and properties of thermotropic liquid-crystalline polymers containing transition metals

New thermotropic, liquid-crystalline polyesters ( 5a, b, 6a, b ) with mesogenic bipyridinediyl units were synthesized. They form complexes with Fe(II) and Cu(II) salts. Homopolyesters 5a, b , containing no metal or less than a certain amount of metal, are smectic. A nematic mesophase was observed in the case of copolyester 6a, b . The structural properties of a polyester complex 5a with Cu, as crystal, liquid-crystal and isotropic liquid, were examined by ESR.