Takashi Kazama

Sclerotic Lesions in the Glomeruli of Buffalo/Mna Rats

Spontaneously developing focal and segmental glomerulosclerosis (FGS) in Buffalo/Mna rats was studied. There were no differences in the occurrence of the disease between male and female rats. Plasma macromolecules were detected in the urine samples of 2-month-old rats using sodium dodecyl sulphate polyacrylamide gel electrophoresis. All 4-month-old animals had proteinuria in excess of 30 mg/24 h. At the age of 4 months, sclerotic glomeruli were rarely observed. At the age of 6 months, all animals had a few sclerotic glomeruli in every kidney section examined. Animals which were 22 months old had sclerotic lesions in 37.8-52.1% of the glomeruli. Ultrastructural examination revealed that alterations in epithelial cells, such as effacement of foot processes, vacuolization, and a podocytic membrane-like structure, were found at the age of 2 months. The animals examined were neither uremic nor hypertensive. Our present study showed that female as well as male Buffalo/Mna rats had an earlier onset of the disease than the other strains of rats and that alterations in glomerular epithelial cells were first detected in the early stage of the disease.

Two cases of vulval pigmented extramammary Paget's disease: histochemical and immunohistochemical studies.

We describe two Japanese female patients with pigmented extramammary Paget’s disease (EMPD); one patient had a dark brown plaque and the other had a reddish patch with a pigmented area, both affecting the vulval region. Histochemical and immunohistochemical examinations confirmed EMPD with melanocyte colonization; plump tumour cells with a large nucleus and pale cytoplasm that were positive for CAM 5.2 and CEA proliferated singly or in nests in the epidermis, and scattered among the tumour cells were many dendritic cells with a large amount of melanin that were positive for HMB‐45 and S‐100 protein. Fontana–Masson (FM) stain showed many positive cells with well‐developed dendritic processes within and around tumour nests. Histochemical and immunohistochemical studies of non‐pigmented EMPD cases on the same region showed that HMB‐45 positive cells were sparse or not detected at all, and that also FM staining‐positive cells were decreased or not detected, and their dendritic processes were poorly formed. The present study suggests that there might be heterogeneity in EMPD in terms of relationships between Paget’s cells and melanocytes.

In vitro characteristics of rat mesangial cells in comparison with aortic smooth muscle cells and dermal fibroblasts

SummaryRat glomerular mesangial cells were cultured and their antigens were compared with those of aortic vascular smooth muscle cells and dermal fibroblasts. Glomeruli, aortic, and dermal expiants were cultured for 3 weeks and subcultured in the same conditions. These cultured cells were evaluated by indirect immunofluorescence studies using antibodies against Thy-1 antigen, desmin, and chicken gizzard actin. Most of mesangial cells were positive for Thy-1, desmin, and actin. On the other hand, fibroblasts were negative for desmin, and smooth muscle cells stained Thy-1 scarcely, and were negative for desmin. In the latter two cells, actin-positive fibrils were thinner and fainter than mesangial cells. These results indicated that mesangial cells could be distinguished in vitro from vascular smooth cells and fibroblasts by immunofluorescence microscopy.

Two-dimensional electrophoretic analysis of human hair keratins, especially hair matrix proteins.

SummaryHuman hair keratins are composed of hair fibrous proteins (HFP) forming 10-nm filaments and nonfilamentous cysteine-rich hair matrix proteins (HMP); these proteins are highly cross-linked by disulfide bonds. In order to obtain high-resultional separation of HFP and HMP by two-dimensional polyacrylamide gel electrophoresis according to isoelectric point (IP) in the first dimension and molecular weight (MW) in the second dimension, these proteins were converted to S-carbamoylmethylated (SCam) derivatives with nonionizable iodoacetamide; this treatment hardly modified the electrophoretic mobility. SCam-HFP were separated into polypeptides with MW 41.5–59 kD (IP pH 5.1-6.8). SCam-HMP were subdivided into two groups; 14 polypeptides of acidic HMP with MW 15-28 kD (IP pH 5.0-7.0) and 12 polypeptides of basic HMP with MW 18.5-28 kD (IP pH 7.8-8.8). Variation in electrophoretic patterns among hair samples obtained from 15 persons in four Japanese families was found in acidic HMP, but not in HFP in basic HMP. The present method appears to be very suitable for the biochemical analysis of human hair keratins, especially HMP of nonfilamentous proteins.

Establishment and characterization of a clear-cell sarcoma (malignant melanoma of soft parts) cell line.

A clear cell sarcoma (CCS) cell line, designated as NCS-1, was established in monolayer culture from a xenograft line originating from a metastatic CCS. Marked karyotypic aberrations and tumorigenicity in nude mice revealed the malignant derivation of the NCS-1 cell line. These cells contained abundant glycogen and were amelanotic by light microscopy. By electron microscopy, however, melanosomes in various developmental stages were seen, and some of them were partially melanized. The electron microscopic dopa reaction revealed the presence of tyrosinase activity. Enzyme-linked immunoadsorbent assay revealed that NCS-1 cells expressed a 75-kDa glycoprotein which was identified as a marker of highly differentiated melanoma cells. From these results, NCS-1 cells were found to retain both cytochemical and morphological properties of CCS. Application of NCS-1 cells to a panel of monoclonal antibodies recognizing melanocytic differentiation antigens showed that they corresponded approximately to highly differentiated melanoma cells. In conclusion, the present study strongly supports the close relationship between CCS and malignant melanoma.

Purpura with Cold Urticaria in a Patient with Hepatitis C Virus Infection-Associated Mixed Cryoglobulinemia Type III: Successful Treatment with Interferon-β

We describe a 54‐year‐old man with hepatitis C virus (HCV) infection‐associated cryoglobulinemia type III. The patient had suffered from cold‐induced urticaria that left purpuric eruptions up to 1 cm in diameter, intermittent migratory joint pain for seven years and mild liver dysfunction for nine years. Hemophilia A was diagnosed when the patient was 26 years old, and he was then given infusions of factor VIII for a short time. In both skin biopsy samples from urticarial and purpuric eruptions, mild inflammatory infiltration by polymorphonuclear leukocytes with nuclear dust, extravasation of erythrocytes and deposition of IgM and C3 in the superficial blood vessels were observed. After antiviral treatment with interferon‐β, the clinical symptoms and the cryoglobulin and HCV‐RNA in the serum disappeared. There has been no recurrence in the subsequent nine years.

Selective loss of chondroitin 6-sulphate from basement membrane during progression from actinic keratosis to squamous cell carcinoma

Immunohistochemical alterations of type IV collagen (C-4) [2, 3], laminin [3, 9] heparan sulphate proteoglycan (HSPG) [4, 7] and chondroitin 6-sulphate (C6S) [1, 7] in squamous cell carcinomas (SCC) have previously been reported [7]. One of the most marked features of SCC is the absence of C6S from the basement membrane zone (BMZ) [7]. Actinic keratosis is a carcinoma in situ and a precursor of SCC [5]. During the progression from carcinoma in situ to invasive carcinoma, tumour cells penetrate the BMZ [6]. Therefore, a difference may exist in the distribution of BMZ proteoglycans between SCC and actinic keratosis. C-4 and laminin have been reported to be preserved as a distinct linear band in the BMZ of actinic keratosis [8]. However, the distributions of HSPG and C6S have not been investigated. In the present study, the changes in the composition of the BMZ components during progression from carcinoma in situ to invasive carcinoma were studied. A monoclonal antibody (MoAb) (HS47) to the core protein of HSPG and rabbit antisera to C-4 and laminin were produced as previously described [4]. Chondroitinase ABC (from Proteus vulgaris) and MoAb to C6S (3B3) were purchased from Seikagaku-Kogyo Co. Ltd., Tokyo. Rhodamine-conjugated anti-rabbit IgG was purchased from Tago Inc., Burlingame, Calif. Three cases of SCC arising from actinic keratosis, cryostat sections of which contained the border between invasive tumour and non-invasive tissue, were chosen from our tumour file and examined. The tissue specimens had been embedded in OCT compound and quickly frozen in liquid nitrogen at resection. Sections were cut at a thickness of 6 gm on a cryostat and dried at room temperature for 30 min. They were stained with a rabbit antiserum for the first staining and a monoclonal antibody for the second staining by the double immuno-

Immunohistochemical localization of heparan sulfate-containing proteoglycan in normal human skin with monoclonal antibodies: comparison with that of fibronectin

Heparan sulfate-containing proteoglycan (HSPG) has been immunohistochemically demonstrated in the basement membrane zones (BMZs) in the human skin [1, 6, 8, 11]. We reported on the isolation of an unique HSPG, the core protein of which has an affinity for fibronectin (FN), from human placenta and the preparation ofmonoclonal antibodies (MoAbs), HS42 and HS47, which recognize the core protein [8]. In the present paper we studied the detailed localization of the antigen reacting with these MoAbs in the human skin and compared it with that of FN. Normal skin specimens were embedded in OCT compound (Miles Laboratories Inc., Naperville, IL, USA) and quickly frozen in liquid nitrogen with and without preincubation in phosphate-buffered saline (PBS) containing 10% glycerol for 1 h at 4~ for immunoperoxidase and immunofluorescent stainings, respectively. For immunofluorescent microscopy, the cryostat sections were stained with rabbit antiserum to FN as described previously [12] and rhodamine conjugated goat rabbit-specific IgG (Tago Inc., Burlingame, CA, USA). After rinsing, the same sections were then stained with the MoAb and FITC conjugated rabbitmouse-specific IgG (DAKOPATTS a/s, Glostrup, Denmark). The control sections were incubated with PBS instead of the MoAbs. Immunofluorescences of both FITC and rhodamine on the same area of each section were photographed.

Monoclonal antibodies to human glomerular antigens

Using cultured human fetal kidney cortical cells as antigen, two monoclonal antibodies (moAbs) against human glomeruli were produced. One of these moAbs, H-4, recognized the cell surface of glomerular epithelial cells, and the other, H-13, recognized the extracellular matrix present in the mesangial area. Both also reacted with liver, H-4 recognizing antigen present on the hepatocyte, and H-13 recognizing antigen distributed along the sinusoid. Species specificity for these moAbs was examined using mouse, rat, guinea pig and rabbit glomeruli, which revealed that H-4 reacted with rat glomerular epithelial cells and H-13 stained guinea pig glomerular mesangium. In the human fetal kidney, H-13 reacted with the mesangium, glomerular and tubular basement membrane and Bowmans capsule, and H-4 with the glomerular and tubular epithelial cells. Dot immunobinding assay of fibronectin purified from glomerular culture supernatant and plasma revealed that H-13 recognized both plasma and cellular fibronectin. Immunoblot analysis of 2.0 M guanidine HCl extract after dissociation in sodium dodecyl sulfate and electrophoresis demonstrated binding of H-4 to a 125 kd polypeptide. Immunoblot analysis of thermolysin-digested fibronectin exhibited binding of H-13 to 145 kd and 110 kd fragments, but not to 38 kd − 29 kd fragments. In renal biopsy specimens from patients with membranous nephropathy, H-13 stained the glomerular basement membrane (GBM), but not the mesangium, whereas anti-fibronectin antisera stained both the GBM and the mesangium. In those from patients with minimal change nephrotic syndrome (MCNS), IgA glomerulonephritis (IgAGN) and membranoproliferative glomerunephritis (MPGN), the staining pattern with H-13 was similar to that with polyclonal anti-fibronectin antisera. These results indicate that H-4 recognizes a 125 kd polypeptide constituent of the glomerular epithelial cell membrane and that H-13 recognizes the cell binding domain of fibronectin as well as revealing structural alterations in the mesangium and GBM.

Heterogeneity in Basement Membranes of Skin—Electrostatic Properties of Basal Lamina‐associated Anionic Sites

In order to evaluate the charge‐selective filter function of basement membranes (BMs), electrostatic properties of anionic sites on the BMs of dermo‐epidermal junction were compared with those of the nuclear (the thick capillary wall) and the attenuated (the thin capillary wall) portions of capillary and other small blood vessels by determining salt concentrations required to remove ruthenium red from these BMs of the rat skin previously perfused with the cationic dye. The concentration of NaCl which was required to remove ruthenium red was more than 1.4M for the attenuated portion of capillary and only 1.2M for the other BMs, indicating that the former is more highly negatively charged than the latter. It is suggested that the BM of the attenuated portion of the capillary expedites the charge‐selective exchange of macromolecules more effectively than the other BMs.