Masamitsu Kanai

Immunocytochemical demonstration of retinol-binding protein in the lysosomes of the proximal tubules of the human kidney

SummaryThe localization of plasma retinol-binding protein (RBP) in the human kidney was determined by two immunocytochemical techniques, the PAP method and the protein A-gold technique. By using the affinity purified antibody against RBP obtained from the urine of the patients with cadmium poisoning (Itai-Itai disease), the immunoreactive substances were located by light microscopy in the proximal tubules of the human kidney. By immuno-electron microscopy, the stained organelles were identified as lysosomes in both S1 and S2 segments of the tubules. These data suggested that the reabsorption of low molecular weight plasma proteins like RBP can occur in the two segments. We inferred a similarity between the S1 and S2 segments concerning the reabsorption of RBP.

Purification and partial characterization of cellular retinoic acid-binding protein from human placenta

Cellular retinoic acid-binding protein (CRABP) has been purified to homogeneity from human placenta by a series of procedures, including acetone powder extraction, gel filtration on Sephadex G-50, and ion-exchange chromatography on DEAE-cellulose and on SP-Sephadex. Cellular retinol-binding protein (CRBP) was isolated concurrently. CRABP was purified 75,400-fold, based on total soluble acetone powder extract of placenta. The protein is a single polypeptide chain with a molecular mass of 14,600 Da, estimated by sodium dodecyl sulfate (SDS) gel electrophoresis or gel filtration, and has an isoelectric point of 4.78 (apo-CRABP, 4.82). On analysis of absorption and fluorescence spectra, the protein was seen to exhibit an absorption peak at 350 nm, fluorescence excitation maxima at 350 and 370 nm, and a fluorescence emission maximum at 475 nm. Human CRABP was immunologically distinct from human CRBP and serum retinol-binding protein.

Immunohistochemical localization of plasma retinolbinding protein and prealbumin in human pancreatic islets

SummaryThis study was undertaken, employing the immunoenzyme method, to confirm the presence of retinol-binding protein in human pancreatic islets, and to compare its distribution with that of prealbumin, insulin, glucagon, somatostatin and pancreatic polypeptide. It was found that most islet cells contained retinol-binding protein, although centrally located cells showed stronger reactivity than those in the peripheral region. The distribution of each of the five polypeptides differed from that of retinolbinding protein, indicating that these peptides did not cross-react with anti-retinol-binding protein antibody. Islet cells which contained prealbumin, on the other hand, were mostly classified as A cells. Further studies are necessary to confirm whether the islet cells produce retinol-binding protein or only store it.

HISTOCHEMICAL COMPARISON OF SPECIFICITY OF THREE BOWEL CARCINOMA‐REACTIVE LECTINS, GRIFFONIA SIMPLICIFOLIA AGGLUTININ‐II, PEANUT AGGLUTININ and ULEX EUROPAEUS AGGLUTININ‐I

A comparison of the histochemical affinities of three lectins reputedly specific to human large bowel carcinoma, namely Griffonia simplicifolia agglutinin‐II (GSA‐II), peanut agglutinin (PNA) and Ulex europaeus agglutinin‐I (UEA‐I), was done using 28 specimens in which normal mucosa, adenoma and carcinoma tissue were present and in contact with each other. In the normal mucosa, GSA‐II and PNA revealed only weak affinity to the Golgi region of epithelial cells, whereas UEA‐I showed binding to the apical surface of columnar cells and goblet cell mucins, especially in the right colon. Adenoma was characterized by relatively intense reactivity of the Golgi regions of epithelial cells for GSA‐II and PNA as well as reactivity of the apical surface of the columnar cells for UEA‐I. In carcinomas the apical surface of columnar cell‐type tumor cells was stained most intensely with UEA‐I, and then in descending order with GSA‐II and PNA. GSA‐II‐ and PNA‐reactive carcinoma cells occurred more frequently in invasive carcinoma than in intramucosal carcinoma. Goblet cell‐type tumor cells retained the properties of their normal counterparts. Staining with these lectins, especially GSA‐II‐horseradish peroxidase, might be helpful in the identification of carcinoma cells and for analysis of carcinoma‐associated antigens.

Determination of cellular retinol‐binding protein in human hepatocellular and colorectal carcinomas by radioimmunoassay

A study was conducted to determine the tissue levels of cellular retinol‐binding protein (CRBP), serum retinol‐binding protein (RBP) and retinoids in hepatocellular carcinoma (HCC) and in colorectal adenocarcinoma. CRBP, which has a molecular weight of 14 900 daltons and an isoelectric point of 4.9, was purified from human liver. An antihuman CRBP antibody was raised in the turkey and further purified by immunosorbent affinity chromatography on CRBP‐coupled Sepharose column. A radioimmunoassay for CRBP using this antibody was established. RBP was measured by an enzyme immunoassay and retinoids were measured by high‐performance liquid chromatography analysis. Retinol levels in liver tumours were significantly decreased compared with those in respective non‐cancerous adjacent tissues. Retinyl ester levels in liver tumours were also significantly decreased compared with those in the adjacent tissues. CRBP levels in liver tumours were significantly decreased compared with those in the adjacent tissues, whereas no significant difference was observed in the CRBP level between the colon tumours and adjacent colon tissues. RBP levels in liver tumours were also significantly decreased compared with those in the adjacent tissues. The decreased CRBP levels in liver tumours may, at least in part, account for the local deficiency of retinol in HCC, whereas colon tumours may grow independently, regardless of retinol status.

Cellular FITC-linked immunospecific assay (Cell-FLISA) for detection of monoclonal antibodies against cell-surface antigens

A cellular fluorescein isothiocyanate (FITC)-linked immunospecific assay (Cell-FLISA) has been established using the recently developed fluorophotometer for microplates. In the Cell-FLISA system, monoclonal antibodies specific for the surface antigens of live cells are detected by measuring the fluorescence intensity of an FITC-labeled second antibody: goat anti-mouse immunoglobulin antibody. It takes only 2 min to count 96 samples in microplate wells using the fluorophotometer for microplates. Moreover, by this system, the analysis is finished within 2 hr. Thus, the Cell-FLISA system has advantages in screening a large number of samples, such as hybridoma cell lines secreting monoclonal antibodies against cell-surface antigens.

Significance of IgM antibody to hepatitis B core antigen for the differential diagnosis of acute and chronic hepatitis B virus infection and for the evaluation of the inflammatory activity of type B chronic liver diseases

SummaryIgM antibody to hepatitis B core antigen (anti-HBc) was assayed using a commercial kit in acute and chronic hepatitis B virus (HBV) infection and evaluated for its diagnostic and clinical significance. IgM anti-HBc was positive in all of 21 cases with type B acute hepatitis in the acute phase, and was also detected in 5 of 20 cases with type B chronic persistent hepatitis, in 4 of 20 patients with type B chronic active hepatitis and in one of 10 with type B liver cirrhosis. The absence of this marker was noted in all of 20 asymptomatic hepatitis B surface antigen (HBsAg) carriers and in 50 with HBsAg-negative patients with liver disease and in 200 healthy blood donors. The cut-off index of IgM anti-HBc was greater than 2.0 in all serum samples obtained in the acute phase of type B acute hepatitis, but was below 2.0 in type B chronic liver disease. A close relationship was found between the presence of IgM anti-HBc and the degree of inflammatory activity in patients with HBsAg-positive chronic liver disease.These data show that examination of IgM anti-HBc is useful in distingushing type B acute hepatitis from type B chronic liver disease, and also in evaluating the severity of disease in type B chronic liver disease.

Distribution of retinol-binding protein in the human digestive tract

SummaryBy employing polyclonal antibodies for retinol-binding protein (RBP), its distribution in the human pancreas and digestive tract mucosa was compared with those of transthyretin (TTR) and various peptide hormones. The materials used included surgically removed pancreas, esophagus, stomach, small and large intestines. Paraffin sections were stained by the indirect immunoenzyme method. The results indicate that RBP-containing cells are found in the pancreas and the gastro-intestinal mucosa, but most frequently in the gastric antrum and duodenum. In the pancreas, RBP-containing cells are found in the islets and among acinar and ductal epithelial cells, and consistently stain for chromogranin A. RBP-containing cells in the gastrointestinal mucosa showed typical features of endocrine cells and also stained for chromogranin A. The distribution of TTR in these tissue sites resembled that of RBP, but the immunoreactive intensities of both peptides altered independently. Comparison of the distribution of RBP, TTR, and various gastrointestinal peptide hormones revealed that the distribution of RBP coincided with none of the other peptides, although some of the RBP-containing cells stained for most of the peptides examined and vice versa. These results suggest that RBP may be a consistent component of gastrointestinal endocrine cells.

In Vitro Activity of Cefotaxime,Ceftizoxime,Cefmenoxime,and Latamoxef in Comparison with Other β-Lactam Antibiotics against Recent Clinical Isolates of Haemophilus influenzae and Haemophilus parainfluenzae

The importance of Haemophilus injluenzae as an etiological agent of meningitis, septicemia and respiratory tract infection has been well documented for many years. Moreover, a number of infections due to H. parainjluenzae, such as septicemia, meningitis and endocarditis, have been reported (I, 2, 4, II, 12, 30). Ampicillin has long been considered the drug of choice for Haemophilus infections ( 18, 28). However, the emergence of strains resistant to ampicillin has recently been noted ( 5, 18, 23, 25, 26, 28), stimulating some investigators to study the in vitro efficacy of other antimicrobial agents against this species (3, 7, 9, 13-15, 21, 22, 29, 31). In the present study, we tested the in vitro activity of the newly developed semisynthetic third-generation cephalosporins, cefotaxime, ceftizoxime, cefmenoxime, and latamoxef, together with ampicillin, sulbenicillin, cefazolin, cefotiam, and cefoxitin, against recent clinical isolates of H. injluenzae and H. parainjluenzae. The 32 strains of H. injluenzae tested included 14 from pharyngeal swabs, 9 from sputa, 4 from ears, 2 from bronchial brushings, 2 from cerebrospinal fluid, and I from blood. All of the 68 H. parainjiuenzae strains examined were from pharyngeal swabs. The isolates were identified as H. injiuenzae or H. parainjiuenzae by testing for their requirement of X and V factors (16), for fermentation of glucose, sucrose, lactose, maltose, xylose, and mannitol (17), for indo! production (16), for urease activity ( 1 7), for ornithine decarboxylation ( 1 7), and for hemagglutination ( 16), and by the porphyrin test ( 19). All the strains were classifiable by biotype according to the proposal of Kilian (20). In addition, H. injiuenzae strains were serotyped with antisera types a to f (Difco) by using fresh agar cultures grown on chocolate agar plates containing 7.5% horse blood as the antigens. The presence of ,B-lactamase was assayed by the phenol red test as described by Escamilla (8). Minimal inhibitory concentrations (MICs) were determined by the agar plate dilution method. Chocolate agar plates containing the drug in twofold serial concentrations (from 100 to 0.00038 p,gfml) were prepared by adding 7.5% defib-

Serum auto‐antibodies in patients with hepatocellular carcinoma: The clinical significance of antinuclear antibody

Auto‐antibodies including antinuclear antibody (ANA), antismooth muscle antibody, antimitochondrial antibody, rheumatoid factor, lupus erythematosus factor, antimicrosomal antibody and antithyroglobulin antibody were assayed in sera from 84 patients with hepatocellular carcinoma, 70 with liver cirrhosis, 50 with chronic hepatitis, 30 with cancer of the alimentary tract and 100 normal subjects. A significantly higher incidence and higher titre of ANA was found in patients with hepatocellular carcinoma, and the patterns of nuclear fluorescence detected by the indirect immunofluorescent test using cultured baby hamster kidney cells were predominantly speckled and nucleolar. In 16 patients with this disease, serial assays of ANA were done in sera obtained before and after development of hepatoma and/or after resection of the hepatic tumour. Antinuclear antibodies evolved in six patients in conjunction with the progression from cirrhosis to hepatoma, and two of three ANA‐positive patients who had the hepatic tumour resected lost ANA from their sera after resection.