Stress distribution of faceted particles in a silo after its partial discharge.

We present experimental and numerical results of the effect that a partial discharge has on the morphological and micro-mechanical properties of non-spherical, convex particles in a silo. The comparison of the particle orientation after filling the silo and its subsequent partial discharge reveals important shear-induced orientation, which affects stress propagation. For elongated particles, the flow induces an increase in the packing disorder which leads to a reduction of the vertical stress propagation developed during the deposit generated prior to the partial discharge. For square particles, the flow favors particle alignment with the lateral walls promoting a behavior opposite to the one of the elongated particles: vertical force transmission, parallel to gravity, is induced. Hence, for elongated particles the flow developed during the partial discharge of the silo leads to force saturation with depth whereas for squares the flow induces hindering of the force saturation observed during the silo filling.

Citrullinated antigens as C1q-binding and monoclonal rheumatoid factor (mRF)-binding peptides in synovial fluids from rheumatoid arthritis patients.

OBJECTIVE: Previous studies have demonstrated that immune complexes (ICs) may be involved in the pathogenesis of rheumatoid arthritis (RA). However, autoantigens contained in rheumatoid ICs remain to be elucidated. In the present study, we investigated whether the peptides captured by C1q and monoclonal rheumatoid factor (mRF), presumably associated with ICs, were citrullinated in synovial fluids from patients with RA. METHODS: Sixteen rheumatoid arthritis synovial fluids (RASFs), 7 osteoarthritis synovial fluids (OASFs), and 20 sera from RA patients were used for experiments. ICs were measured using commercially available kits based on the C1q-binding (C1q-IC) and mRF-binding (mRF-IC) assays. Citrullination of the peptides captured by C1q and mRF was detected by anti-modified citrulline antibody (Senshu Ab) after chemical modification. RESULTS: There was a significant correlation between levels of citrullination of C1q-binding peptides and those of mRF-binding peptides in RASFs (r=0.77), both of which were significantly higher than those in OASFs. No citrullinated Ags captured by C1q and mRF were detected in sera from patients with RA. CONCLUSIONS: We have demonstrated the presence of citrullinated Ags as C1q- and mRF-binding peptides in RASF. We suggest that citrullinated Ags may contribute to the pathogenesis of RA through IC formation in the joint.

Multiple intravascular papillary endothelial hyperplasia affecting skin and bone.

We report a 75-year-old man with multiple recurrent black papules involving his entire body. In the course of 3 years, 20 lesions were resected, which were histologically confirmed as intravascular papillary endothelial hyperplasia (IPEH). A similar vascular lesion was found on his tibia. The occurrence of multiple IPEH affecting skin and bone is extremely rare. The patient's medical history included hepatitis C, hepatoma and associated coagulopathy. We suggest that this patient's multiple lesions were induced by microthrombus formation due to liver dysfunction.

Graft-versus-adult T-cell leukemia/lymphoma effect following allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has proven effective in adult T-cell leukemia/lymphoma (ATL) patients. To study the graft-versus-ATL (Gv-ATL) effects after allo-HSCT, we analyzed 21 ATL patients who had been treated at our hospital. Of these, 18 had acute-, 2 had lymphoma- and 1 had chronic-type ATL; at allo-HSCT, seven patients were in CR, one was in PR, five had stable disease (SD) and eight had progressive disease (PD). Disease state after allo-HSCT was CR in 14, PR in 3, SD in 1 and PD in 3 patients. Among 15 patients who survived longer than 100 days, ATL relapsed in 10 patients, skin relapsed in 9 patients and 5 had relapsed on the skin alone. After we discontinued immunosuppressant therapy in these 10 patients, 8 manifested GVHD; ATL was ameliorated to CR in 6 patients. Donor lymphocytes were infused into two patients who did not show GVHD; one obtained CR. In five patients with skin relapse alone, four patients achieved CR following the discontinuation of the immunosuppressants. Our results demonstrate that relapse of ATL after allo-HSCT tends to develop on skin, and Gv-ATL effects played a critical role in the outcome of allo-HSCT for ATL.

9-Cis-retinoic acid exhibits antifibrotic activity via the induction of cyclooxygenase-2 expression and prostaglandin E2 production in scleroderma fibroblasts.

BACKGROUND: The pathogenesis of scleroderma (SSc) is not fully understood, and there is no effective treatment for this chronic disease. Retinoic acid (RA) can modulate connective tissue metabolism, exhibit antifibrotic activity and improve the clinical symptoms of patients with SSc. However, the mechanisms by which RA elicits its antifibrotic actions remain to be determined. OBJECTIVE: To elucidate the underlying mechanisms by which retinoids exert beneficial effects on SSc. METHODS: Cultured skin fibroblasts from patients with SSc were treated with retinoids (9-cis-, 13-cis- and all-trans-retinoic acid) and their effect on the expression of cyclooxygenase (COX)-2, connective tissue growth factor (CTGF) and type I and III collagen and on the production of PGE(2) was examined. COX-2 expression was analysed by western immunoblotting, PGE(2) production by enzyme immunoassay and CTGF expression, and type I and III collagen expression by reverse transcriptase PCR and western immunoblotting. RESULTS: In cultured SSc fibroblasts, 9-cis-RA significantly increased COX-2 protein expression and PGE(2) production and inhibited the expression of CTGF and type I and III collagen. We further found that expression of CTGF and of type I and III collagen mRNA was inhibited by exogenous PGE(2) in SSc fibroblasts. CONCLUSION: In vitro, 9-cis-RA induced COX-2 expression and PGE(2) production in SSc fibroblasts and PGE(2) downregulated CTGF expression, leading to the inhibition of type I and III collagen synthesis. Our results indicate that the clinical effects of 9-cis-RA on SSc are, at least in part, attributable to the induction of PGE(2) and the subsequent suppression of CTGF expression that results in the blockade of collagenogenesis.

The molecular lesion in the alpha-N-acetylgalactosaminidase gene that causes angiokeratoma corporis diffusum with glycopeptiduria.

Angiokeratoma corporis diffusum with glycopeptiduria is a recently recognized inborn error of glycoprotein catabolism resulting from the deficient activity of human alpha-N-acetylgalactosaminidase (E.C. 3.2.1.49; alpha-GalNAc). The first patient with this autosomal recessive disorder, a 46-yr-old consanguineous Japanese woman, presented with diffuse angiokeratoma, mild intellectual impairment, and peripheral neuroaxonal degeneration. Deficient alpha-GalNAc activity also has been reported in consanguineous brothers with an infantile-onset form of neuroaxonal dystrophy resulting from a missense mutation (designated E325K) in the alpha-GalNAc gene. To identify the mutation causing the phenotypically distinct adult-onset disorder, Southern and Northern hybridization analyses of DNA and RNA from the affected homozygote were performed which revealed a grossly normal alpha-GalNAc gene structure and normal transcript size and abundancy. Reverse transcription, amplification, and sequencing of the alpha-GalNAc transcript identified a single C to T transition at nucleotide (nt) 985 that predicted an arginine to tryptophan substitution in residue 329 (designated R329W) of the alpha-GalNAc polypeptide. This base substitution was confirmed by hybridization of PCR-amplified genomic DNA from family members with allele-specific oligonucleotides. Transient expression of an alpha-GalNAc construct containing the R329W mutation resulted in the expression of an immunoreactive polypeptide which had no detectable alpha-GalNAc activity. Comparison of the biosynthesis and stabilities of the transiently expressed and radiolabeled normal, E325K (infantile-onset) and R329W (adult-onset) alpha-GalNAc polypeptides in COS-1 cells indicated that both the mutant precursors were processed to the mature form; however, the E325K mutant polypeptide was more rapidly degraded than the R329W subunit, thereby providing a basis for the distinctly different infantile- and adult-onset phenotypes.

Lysosomal alpha-N-acetylgalactosaminidase deficiency, the enzymatic defect in angiokeratoma corporis diffusum with glycopeptiduria.

Recently a novel case of angiokeratoma corporis diffusum with glycoaminoaciduria was described in a 46-yr-old Japanese woman. Known causes of the cutaneous manifestation were eliminated by enzyme analyses, and further characterization of the accumulated urinary O-linked sialopeptides revealed identity to those excreted by patients with an infantile neuroaxonal dystrophy due to lysosomal alpha-N-acetylgalactosaminidase deficiency. Investigation of the alpha-N-acetylgalactosaminidase activity and protein in the proband revealed less than 2% of normal activity and the absence of detectable immunoreactive enzyme protein, findings comparable to those in the patients with infantile neuroaxonal dystrophy and alpha-N-acetylgalactosaminidase deficiency. In addition, the proband's unaffected offspring had half-normal levels of alpha-N-acetylgalactosaminidase activity, consistent with this enzymatic deficiency being the primary metabolic defect in this autosomal recessive trait. Ultrastructural examination of skin and blood cells from the adult proband revealed the presence of prominent lysosomal inclusions containing diffuse amorphous and filamentous material. In contrast, these morphologic findings were not observed in the nonneural tissues from patients with infantile neuroaxonal dystrophy and alpha-N-acetylgalactosaminidase deficiency. These studies document the occurrence of two forms of alpha-N-acetylgalactosaminidase deficiency and sialopeptiduria, a severe infantile-onset form of neuroaxonal dystrophy without angiokeratoma or visceral lysosomal inclusions and an adult-onset form characterized by angiokeratoma, extensive lysosomal accumulation of sialoglycopeptides and the absence of detectable neurologic involvement.

Induction of urogenital anomalies and some tumors in the progeny of mice receiving diethylstilbestrol during pregnancy.

Pregnant mice were given a single dose (10 mug/g body weight) of diethylstilbestrol (DES) on Days 7 to 19, which correspond to the first to fifth lunar months in humans, after the authors, using a 14C-labeled compound, confirmed easy placental penetration by DES. Treatment with DES on Days 15 to 19 resulted in the induction of persistent urogenital sinus (15.8 to 92.5%) and hypertrophy of the portio vaginalis (11.8 to 73.3%) in female offspring, and treatment on Days 17 and 19 resulted in the induction of undescended testes and their hypogenesis (70.4 to 73.3%) in male offspring, although treatment with DES at other stages of pregnancy and after birth did not cause these alterations. The incidence of various tumors (lung adenoma, granulosa cell tumor, etc.) increased significantly (31.0 to 37.9%) when DES was given on Days 15 and 17, which correspond to the stage sensitive to other carcinogens. However, adenosis and adenocarcinoma of the vagina were not observed in the offspring.

Biological behavior of cloned cells of human malignant fibrous histiocytoma in vivo and in vitro.

A human malignant fibrous histiocytoma cell line was established in vitro. Cells showed a wide variety of morphologies, although the karyotype study showed that the tumor was monoclonal in origin because of the presence of unique marker chromosomes in 100% of the cells examined (50 of 50). Cells were cloned according to their characteristic morphologies and biological behavior in culture. The cloned cells were sparse spindle, packed spindle, epithelioid, and lymphoid. In colonies, sparse spindle cells grew separately from each other without cell to cell contact but produced a cartwheel pattern at confluency. Packed spindle cells grew in a tightly packed fashion and produced a storiform pattern at confluency. Epithelioid cells were spindle shaped as individuals but became epithelioid when in contact with each other and produced many multinucleated giant cells at confluency. Lymphoid cells were spindle shaped as individuals but became spherical at confluency. When tumors were grown in nude mice after transplantation of these cloned cells, the histology was shown to be unrelated to morphology in culture and was epithelioid (histiocytic), as was the original tumor. These results show that (a) a single cell derived from malignant fibrous histiocytoma cells exhibits a wide range of phenotypical expression in vitro, (b) cells have their own morphological and biological characteristics in vitro, which (c) however, are easily influenced by environmental factors and (d) which are unstable and even interchangeable. These characteristics may contribute to the endless variety of cellular forms and growth patterns of malignant fibrous histiocytomas in humans.

Morphological, biological, and biochemical characteristics of a benign human trichilemmoma cell line in vivo and in vitro.

A cell line of a benign tumor, trichilemmoma, was established in vitro and has been maintained in culture for 1.5 years with more than 30 passages. Plating efficiency was less than 0.1%, and population doubling time was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) e was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) e was 10 days. Saturation density was 10(5) cells/sq cm at the time of a monolayer with 98% cell viability. Ultrastructurally, tissue-cultured trichilemmoma cells showed desmosome-tonofilament complexes at cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 microgram/10(6) cells) as observed in vivo. Gas chromatographic analysis revealed that extracted glycogen was composed of glucose alone. Chromosome analyses with trypsin-Giemsa banding showed an abnormal karyotype with hypodiploid modal numbers of 44 and 45. There were four marker chromosomes observed in 100% of cells in 100 metaphase cells examined. Cells did not grow on fibroblast monolayers or in soft agar in vitro but did induce tumors in athymic nude mice (12 of 15) after the s.c. injection of tissue-cultured cells (2.5 x 10(6) to 4.5 x 10(7) cells/mouse). The histological characteristics of the tumors in nude mice were similar to those of the original tumor. This is the first time, to our knowledge, that a benign human tumor cell line has been established in vitro which can induce tumors in nude mice.

Lack of the latent transforming growth factor beta binding protein in malignant, but not benign prostatic tissue.

Transforming growth factor beta (TGF-beta) is a family of proteins which act as a potent growth inhibitor for most cell types including epithelial cells. TGF-beta is synthesized as latent high molecular weight complexes, composed of TGF-beta, the NH2-terminal part of the TGF-beta precursor and the third molecule, the latent TGF-beta binding protein (LTBP). We here ascertain that TGF-beta is expressed in human prostatic cancer tissue as well as in cystectomized prostatic tissue and in materials from transurethral resections with benign prostatic hyperplasia, analyzed by immunohistochemistry. TGF-beta is observed in both epithelial cells and stromal cells. No significant correlation was obtained between TGF-beta expression in tumor cells and their degree of differentiation. However, analysis by immunohistochemistry using antibodies against LTBP revealed that specimens from histopathologically verified human prostatic cancer are mostly negative for this molecule, although it is expressed in cystectomized prostatic and benign prostatic hyperplasia tissues. These results indicate that in cystectomized prostatic and benign prostatic hyperplasia tissues, TGF-beta may be produced in a complex associated with LTBP; whereas in prostatic carcinoma, TGF-beta is produced without associating with LTBP. The biological significance of the production of TGF-beta in relation to LTBP and the possible association with prognosis are discussed.

Antiteratogenic effects of tumor inhibitors, caffeine, antipain, and retinoic acid in mice.

To learn the effects of tumor inhibitors on chemically induced malformations, caffeine, antipain, and 13-trans-retinoic acid were given to pregnant ICR/Jcl mice after a single dose of urethan, N-hydroxyurethan, N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea, or 4-nitroquinoline 1-oxide, which induces about 50% of the malformed fetuses. When caffeine was given immediately after carcinogen treatment on Day 10, urethan- and N-ethyl-N-nitrosourea-induced malformations were significantly suppressed by caffeine posttreatment, while N-hydroxyurethan- and N-methyl-N-nitrosourea-induced malformations were not suppressed by caffeine. 4-Nitroquinoline 1-oxide-initiated teratogenesis was also suppressed, but not significantly so (p not equal to 0.07). The results were very similar to those of the effects of caffeine on tumors induced by these carcinogens. Malformations of genetic origin (cleft palates and cleft lips) in CL/Fr mice were also suppressed significantly by caffeine treatment on Days 8 to 11, although the level of inhibition was less than that in chemically induced malformations. A protease inhibitor (antipromotor), antipain, also suppressed urethan-induced malformations. The antiteratogenic effects of antipain were most effective when it was given during the period of 24 to 48 hr after urethan treatment, while those of caffeine were most effective when it was given immediately after urethan. The promoting process might be involved in chemically induced teratogenesis, as it was in carcinogenesis. A natural retinoid (13-trans-retinoic acid) also suppressed urethan-induced malformations. Thus, tumors and malformations induced by chemical carcinogens were suppressed by tumor inhibitors, suggesting the similarity of both processes in the subcellular level, in spite of their morphological differences.

The effect of a thymidine phosphorylase inhibitor on angiogenesis and apoptosis in tumors.

Thymidine phosphorylase (TP) is an enzyme involved in the reversible conversion of thymidine to thymine and is identical to an angiogenic factor, platelet-derived endothelial cell growth factor. TP is expressed at higher levels in a wide variety of solid tumors than in the adjacent nonneoplastic tissues. Patients with TP-positive colon and esophageal tumors have a poorer prognosis than those with TP-negative tumors. We have recently synthesized a new TP inhibitor (TPI), 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil hydrochloride. We investigated the effect of TPI on angiogenesis in KB cells transfected with platelet-derived endothelial cell growth factor cDNA, KB/TP, and a mock transfectant, KB/CV, using the mouse dorsal air sac assay model. We found that KB/TP cells had a higher angiogenic ability than KB/CV cells and that TPI completely suppressed angiogenesis by KB/TP. Furthermore, at a dose of 50 mg/kg/day, TPI considerably decreased the growth rate of KB/TP cells xenografted into nude mice. Microvessel density in KB/TP tumors was higher than that in KB/CV tumors, and TPI did not significantly change the density in either of the tumors. The apoptotic index in KB/TP tumors was significantly lower than that in KB/CV tumors, and TPI significantly increased the apoptotic index in KB/TP tumors but not in KB/CV tumors. These findings, taken together with previous reports, suggest that the expression of TP plays an important role in tumor growth and that TPI suppresses tumor growth by increasing the proportion of apoptotic cells and probably inhibiting angiogenesis.

Inhibition of BAD phosphorylation either at serine 112 via extracellular signal-regulated protein kinase cascade or at serine 136 via Akt cascade sensitizes human ovarian cancer cells to cisplatin.

We studied the roles of the phosphatidylinositol 3-kinase (PI-3K)-protein kinase B/Akt-BAD cascade in both cisplatin-resistant Caov-3 and -sensitive A2780 human ovarian cancer cell lines. Treatment of both Caov-3 and A2780 cells with cisplatin but not with the trans-diaminodichloroplatinum (transplatin) isomer stimulated the activation of Akt, and the PI-3K inhibitor wortmannin blocked the cisplatin-induced activation of Akt. Treatment of both Caov-3 and A2780 cells with cisplatin but not with the trans-diaminodichloroplatinum isomer also stimulated the phosphorylation of BAD at both the Ser-112 and Ser-136 sites. Whereas the phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin, its phosphorylation at Ser-112 was blocked by a MAP/ERK kinase inhibitor, PD98059. Exogenous expression of a dominant-negative Akt in both Caov-3 and A2780 cells decreased the cell viability after treatment with cisplatin. In contrast, no sensitization to cisplatin was observed in cells expressing wild-type Akt. We further examined the role of BAD in the viability after cisplatin treatment using BAD mutants. Exogenous expression of each of the singly substituted BADS112A or BADS136A in both Caov-3 and A2780 cells decreased the viability after treatment with cisplatin to a degree intermediate between that caused by exogenous expression of wild-type BAD and doubly substituted BAD2SA. Cisplatin did not stimulate the phosphorylation of BAD Ser-136, but did stimulate the phosphorylation of BAD Ser-112 in cells expressing a dominant-negative Akt, suggesting that BAD Ser-136 but not Ser-112 was phosphorylated by Akt. Our findings suggest that cisplatin-induced DNA damage causes the phosphorylation of both BAD Ser-112 via an extracellular signal-regulated protein kinase (ERK) cascade and BAD Ser-136 via a PI-3K-protein kinase B/Akt cascade and that inhibition of either of these cascades sensitizes ovarian cancer cells to cisplatin.

Fatty acid specificity of acyl-CoA synthetase in rat glomeruli.

The fatty acid specificity of acyl-CoA synthetase in rat glomeruli for physiologically and pathologically important long-chain fatty acids was studied. The apparent Michaelis constants (Km) for substrate fatty acids increased in the order, linolenic less than linoleic less than eicosapentaenoic less than arachidonic less than oleic less than palmitic acid. The maximum velocities with these fatty acids decreased in the order, oleic greater than linoleic greater than palmitic (approximately equal to) linolenic greater than arachidonic greater than eicosapentaenoic acid. The syntheses of radioactive arachidonyl-CoA and palmitoyl-CoA from radioactive arachidonic and palmitic acid, respectively, were both inhibited by all fatty acids mentioned above including the substrate fatty acids, their inhibitory effects being inversely correlated with their apparent Km values. These results suggest that the enzyme in glomeruli has a unique specificity for fatty acids and that there is no arachidonic acid-specific acyl-CoA synthetase in glomeruli. The possible contribution of the glomerular enzyme with this specificity to the abnormal fatty acid levels in diabetic animals is discussed.

The receptor for advanced glycation end products mediates the chemotaxis of rabbit smooth muscle cells.

Long-term incubation of proteins with glucose leads to advanced glycation end products (AGEs) with fluorescence and a brown color. We recently demonstrated immunologically the intracellular AGE accumulation in smooth muscle cell (SMC)-derived foam cells in advanced atherosclerotic lesions. To understand the mechanism of AGE accumulation in these foam cells, we have now characterized the interaction of AGE proteins with rabbit-cultured arterial SMCs. In experiments at 4 degrees C, 125I-labeled AGE-bovine serum albumin (AGE-BSA) showed a dose-dependent saturable binding to SMCs with an apparent dissociation constant (Kd) of 4.0 microg/ml. In experiments at 37 degrees C, AGE-BSA underwent receptor-mediated endocytosis and subsequent lysosomal degradation. The endocytic uptake of 125I-AGE-BSA was effectively inhibited by unlabeled AGE proteins such as AGE-BSA and AGE-hemoglobin, but not by acetylated LDL and oxidized LDL, well-known ligands for the macrophage scavenger receptor (MSR). Moreover, the binding of 125I-AGE-BSA to SMCs was affected neither by amphoterin, a ligand for one type of the AGE receptor, named RAGE, nor by 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole-hexanoic acid-BSA, a ligand for the other AGE receptors, p60 and p90. This indicates that the endocytic uptake of AGE proteins by SMCs is mediated by an AGE receptor distinct from MSR, RAGE, p60, and p90. To examine the functional role of this AGE receptor, the migratory effects of AGE-BSA on these SMCs were tested. Incubation with 1-50 microg/ml of AGE-BSA for 14 h resulted in significant dose-dependent cell migration. The AGE-BSA-induced SMC migration was chemotactic in nature and was significantly inhibited (approximately 80%) by an antibody against transforming growth factor-beta (TGF-beta), and the amount of TGF-beta secreted into the culture medium from SMC by AGE-BSA was sevenfold higher than that of control, indicating that TGF-beta is involved in the AGE-induced SMC chemotaxis. These data suggest that AGE may play a role in SMC migration in advanced atherosclerotic lesions.

Sulfhydryl and disulfide stainings in amyloids of skin-limited and systemic amyloidoses.

Disulfide (S-S) bonds and sulfhydryl (-SH) groups in skin-limited and systemic amyloidoses in frozen and paraffin-embedded sections were examined with a thiol reagent, N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide (DACM). In frozen sections, dermal amyloids of skin-limited amyloidoses contained a large number of S-S bonds but no -SH groups [macular amyloidosis (9 cases), lichen amyloidosis (4), and skin tumor-associated (seborrheic keratosis) amyloidosis (1)]. In contrast, amyloids of systemic amyloidoses contained no S-S bonds or -SH groups [primary and myeloma-associated amyloidoses (1 each)]. The identical results were obtained from paraffin-embedded sections in skin-limited amyloidoses [macular (31), biphasic (4), lichenoid (9) and skin tumor-associated Bowen's disease (3), seborrheic keratosis (2), solar keratosis (2), porokeratosis Mibelli (1), and basal cell epithelioma (1) amyloidoses], systemic amyloidoses [primary (3), myeloma-associated (2), and secondary (2) amyloidoses] and tumefactive amyloidoses of the tongue (2). Furthermore, amyloid-like deposits confirmed by various histochemical stainings were found in the epidermis in 27/67 cases of skin-limited amyloidoses in both frozen and paraffin sections. These intraepidermal amyloid-like deposits contained S-S bonds in all cases (27/27) and -SH groups in 10 of 27 cases. In contrast, an intraepidermal amyloid-like deposit was not observed in any systemic amyloidoses (0/9) or tumefactive amyloidoses of the tongue (2). These results showed that skin-limited amyloidoses could be differentiated from systemic amyloidoses by DACM methods (this appears to depend upon the differences of amino acid composition between skin-limited and systemic amyloidoses) and that paraffin-embedded sections were usable for DACM methods. Present study further suggests that amyloids ion skin-limited amyloidoses are, at least in part, derived from epidermal keratinocytes.

HLA-DR and melanoma-associated antigen (p97) expression during the cell cycle in human melanoma cell lines, and the effects of recombinant gamma-interferon: two-color flow cytometric analysis.

Using monoclonal antibodies, recombinant human gamma-interferon, and fluorescence-activated cell sorter, 2 human melanoma cell lines (KHm-1/4 and A101D) were examined quantitatively for HLA-DR and 97-kD melanoma-associated antigen (p97) expression throughout the cell cycle. Two-color flow cytometric analysis showed that the mean cell volume increased (KHm-1/4, 2.6 times; A101D, 3.6 times) during the progression of the cell cycle, and that fluorescence intensity of HLA-DR and p97 correlated well with cell volume, i.e., both antigens were maximally detected during the G2-M phase. The density of HLA-DR and p97 on the cell surface remained relatively constant throughout the cell cycle with the exception that cells in S phase showed a slightly lower density compared with those in G0/G1 and G2-M phases. gamma-Interferon treatment (500 IU/ml, 72 h) increased HLA-DR+ cells (KHm-1/4, 65% to 89%; A101D, 34% to 84%) and p97+ cells (KHm-1/4, 8% to 12%; A101D, 19% to 35%). Increased antigen densities were also relatively constant throughout the cell cycle as in nontreated cells. Cells treated with gamma-interferon tended to accumulate at G0/G1 phase (KHm-1/4, 21% to 37%; A101D, 17% to 53%), and had a reduced cell volume (0.82-0.95 times) throughout cell cycle. This study revealed that both melanoma cell lines showed heterogeneity in the expression of HLA-DR and p97, and that this heterogeneity was influenced, at least in part, by cell cycle and immunologic events such as gamma-interferon treatment.

A novel point mutation affecting the tyrosine kinase domain of the TRKA gene in a family with congenital insensitivity to pain with anhidrosis.

A nerve growth factor receptor encoded by the TRKA gene plays an important part in the formation of autonomic neurons and small sensory neurons in dorsal root ganglia and in signal transduction through its intracytoplasmic tyrosine kinase domain. Recently, three mutations in the tyrosine kinase domain of TRKA have been reported in patients with congenital insensitivity to pain with anhidrosis, which is an autosomal recessive disorder characterized by recurrent fever due to absence of sweating, no reaction to noxious stimuli, self-mutilating behavior, and mental retardation. We examined the TRKA gene in five generations of a large Japanese family with many consanguineous marriages who live in a small remote island of the southern part of Japan. We found a novel point mutation at nucleotide 1825 (A-->G transition) resulting in Met-581-Val in the tyrosine kinase domain. Two of the three affected patients were homozygous for this mutation; however, the third affected patient was heterozygous. Further analysis revealed that the third patient was a compound heterozygote with the Met-581-Val mutation in one allele and with a single base C deletion mutation at nucleotide 1726 in exon 14 in the other allele, resulting in a frameshift and premature termination codon.

Monoclonal antikeratin antibody: production, characterization, and immunohistochemical application.

A monoclonal antikeratin antibody, EKH4, was produced from a hybridoma cell line which was established by fusing P3X63SAg8 mouse myeloma cells with spleen cells of mice immunized with human trichilemmoma cells. Immunoblot analysis showed that EKH4 antibody reacts predominantly with 50 kilodalton keratin polypeptide in normal epidermis. By indirect immunofluorescence and immunoperoxidase techniques, EKH4 antibody reacted with the lower 2-3 cell layers of the epidermis as well as most cells of pilosebaceous follicle of human and animal skin. Tumor cells of human basal cell epitheliomas and squamous cell carcinomas were also stained with this antibody. The staining was much more regular and intense compared with an available monoclonal antikeratin antibody, AE1. In the lesion of epidermal proliferative disorders, such as psoriasis and actinic keratosis, the entire epidermis instead of the lower layers was stained with EKH4 antibody. Normal skin overlying or adjacent to epithelial tumors also showed positive staining in the entire epidermis. By using indirect immunoperoxidase technique, EKH4 also stained alcohol-fixed, paraffin-embedded tissue sections.