Blockage of amyloid induction by colchicine in an animal model.

Colchicine was found to have a strong inhibitory effect on amyloid induction in an animal model. When CBA/J mice were treated with colchicine concurrently with the amyloid induction regimen, the incidence of amyloidosis was, depending upon the dosage of colchicine, significantly decreased (0.005-0.010 mg colchicine per day) or completely blocked (more than 0.015 mg colchicine per day). The colchicine treatment was effective not only when colchicine was given for the entire course of the amyloid induction regimen but also when it was given only in the late pre-amyloid or the amyloid phase of the regimen or to the recipients after the transfer of amyloid. The data suggest the colchicine is effective in blocking amyloidogenesis at its final stage(s), while it may not affect significantly amyloid already deposited in the tissue.

Suppression of in vitro antibody response by a serum factor (SAA) in experimentally induced amyloidosis.

Serum from CBA/J mice made amyloidotic by chronic casein injections has been shown to suppress in vitro antibody response to SRBC. Similar suppression was also found with normal mouse serum but to a much lesser degree. This suppressive activity of both amyloidotic serum and normal serum was removed by absorption of the sera with antiserum to protein AA, the major constituent of casein-induced (secondary) amyloid fibrils. This antiserum to the amyloid fibril protein AA (mol wt 8,400 daltons) detects an immunologically cross-reacting serum alpha globulin (SAA) (mol wt approx. 100,000). It is postulated that the serum factor (SAA) is a regulator of antibody response and may be present in elevated amounts as the result of chronic antigenic stimulation.

High-resolution electron microscopic analysis of the amyloid fibril.

The ultrastructural organization of the fibrous component of amyloid has been analyzed by means of high resolution electron microscopy of negatively stained isolated amyloid fibrils and of positively stained amyloid fibrils in thin tissue sections. It was found that a number of subunits could be resolved according to their dimensions. The following structural organization is proposed. The amyloid fibril, the fibrous component of amyloid as seen in electron microscopy of thin tissue sections, consists of a number of filaments aggregated side-by-side. These amyloid filaments are approximately 75-80 A in diameter and consist of five (or less likely six) subunits (amyloid protofibrils) which are arranged parallel to each other, longitudinal or slightly oblique to the long axis of the filament. The filament has often seemed to disperse into several longitudinal rows. The amyloid protofibril is about 25-35 A wide and appears to consist of two or three subunit strands helically arranged with a 35-50-A repeat (or, less likely, is composed of globular subunits aggregated end-to-end). These amyloid subprotofibrillar strands measure approximately 10-15 A in diameter.

Combined cytochemical and electron microscopic demonstration of beta-glucuronidase activity in rat liver with the use of a simultaneous coupling azo dye technique.

Rat liver was fixed in formal-cacodylate-sucrose and frozen sections were incubated in a simultaneous-coupling medium containing naphthol AS-BI glucuronide as substrate and hexazonium pararosanilin as the diazo reagent. By light microscopy, the sections demonstrated beta-glucuronidase activity as red discrete granules in the pericanalicular cytoplasm and as a generalized cytoplasmic stain in the parenchymal cells. Brief treatment of sections in cold ethanol prior to incubation markedly enhanced the staining for the enzyme and made it possible to demonstrate sufficient amounts of the reaction product in sections embedded in epoxy resin following dehydration and propylene oxide treatment. Electron microscopy revealed that the reaction product was moderately electron opaque and deposited in greater amounts in the vacuolated dense bodies and occasionally in the dense bodies which did not show obvious vacuoles. In each dense body, the deposits occurred preferentially at the edge as well as in the area surrounding the vacuoles in the matrix. Control sections incubated in the presence of glucosaccharo-1:4-lactone were devoid of the reaction product. No deposits of the reaction product were found in the nucleus, mitochondria, or microbodies. The limitations of the present cytochemical technique for use in electron microscopy are briefly discussed.

Two weeks of permanence in negatively-charged air conditions causes alteration of natural killer cell function.

The effects of negatively-charged air conditions were analyzed as one of the approaches to improve health and quality of life. We previously reported that the use of a charcoal coating and application of an electric voltage yielded predominantly negatively-charged particles in an experimental room, and that 2.5 hours of living in these conditions caused a slight activation of the immune system (slight elevation of serum interleukin (IL)-2), regulated blood flow, and stabilized the autonomic nervous system when compared with control conditions (no dominance of negatively-charged particles). In this study, we expanded the previous study and placed 15 subjects in negatively-charged air conditions for two weeks during the night and analyzed various biological parameters. Although individual biological reactions differed from subject to subject, natural killer (NK) cell activity increased significantly following living in negatively-charged air conditions. Taken together, the results of the previous investigation and those of this study show that repeated elevation of IL-2 (although it immediately returned to the baseline level) causes chronic and recurrent stimulation to NK cells and results in the steady activation of NK cells. Negatively-charged air particles may be a good tool to improve health and quality of life.

Kinetics of serum amyloid protein A in casein-induced murine amyloidosis.

Serum amyloid protein A (SAA), the precursor of secondary amyloid protein, is elevated in chronic diseases which are associated with an increased incidence of amyloid. However, SAA is also elevated in acute bacterial and viral infections and somes forms of cancer. The murine model of casein-induced amyloidosis was studied to determine the relationship between SAA production and amyloid deposition. SAA levels measured by radioimmunoassay were found to be as high as 200 times the normal level in CBA/J mice receiving daily parenteral casein. After a single injection of casein the SAA level was elevated by 3h and peaked by 12-18 h. Similar levels were found in casein-treated A/J mice, a strain less susceptible to the induction of amyloid. Parenterally administered bovine serum albumin, which has low potential for amyloid induction, gave SAA levels in CBA/J and A/J mice comparable to casein treatment. These data show that, while SAA levels are elevated during chronic antigenic stimulation, there are other factors involved in amyloid formation. These factors may include alterations in the degradation of SAA by the reticuloendothelial system caused by substances such as casein. Nude (athymic) mice were shown to attain high levels of SAA after receiving casein parenterally. Therefore, thymus-derived lymphocytes are not necessary for the synthesis of SAA.

Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex.

Pyruvate dehydrogenase complex was purified from rat heart. The complex showed four polypeptide bands on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, corresponding to lipoate acetyltransferase (mol.wt. 68 000), lipoamide dehydrogenase (mol.wt. 56 000), alpha-subunit (mol.wt. 41 000) and beta-subunit (mol.wt. 35 000) of pyruvate dehydrogenase. Rat heart pyruvate dehydrogenase complex was dissociated into three component enzymes and the antibodies against each component enzyme were prepared. Anti-pyruvate dehydrogenase and anti-lipoate acetyltransferase antibodies effectively precipitated pyruvate dehydrogenase complex, but an anti-lipoamide dehydrogenase antibody released lipoamide dehydrogenase from the complex and effectively precipitated lipoamide dehydrogenase. Lipoamide dehydrogenase was synthesized in a cell-free reticulocyte lysate system with total RNA from rat liver. Its translation product was detected as a putative precursor which is 3000 Da larger than the mature subunit. In cell-free translation programmed with free and membrane-bound polysomes, activity of mRNA coding for the precursor of the enzyme was much higher in free polysomes than in membrane-bound polysomes.

Cyclooxygenase-2 expression is up-regulated in transitional cell carcinoma and its preneoplastic lesions in the human urinary bladder.

Cyclooxygenase (COX) is a key enzyme in the synthesis of prostaglandins from arachidonic acid. Much evidence, including that from epidemiological and experimental studies, suggests that the inducible form of COX, COX-2, is increased in colon tumor tissues and is involved in colon cancer tumorigenesis. To determine the significance of COX-2 in tumorigenesis in the urinary bladder, the expression of COX-2 in transitional cell carcinoma and preneoplastic lesions of the bladder was examined. Tumor specificity of COX-2 immunoblotting was 100% in 12 of 35 (34%) tumors, but in 0 of the 10 normal urothelia samples. COX-2 expression was significantly correlated with tumor stage in 9 of 20 (45%) muscle-invasive (pT2-4) tumors and in 3 of 15 (20%) superficially invasive (pT1) tumors (P < 0.05). Immunohistochemical examination revealed that 13 of 14 (93%) samples of carcinoma in situ (CIS), which may be the precursor of muscle-invasive-type tumors, expressed COX-2, whereas 10 of 21 (48%) samples of dysplasia, which may be the precursor of both superficially invasive and muscle-invasive tumors, expressed COX-2. From the expression profile of COX-2 in these various urothelia, it is suggested that COX-2 is involved in the development of transitional cell carcinoma of the urinary bladder, especially that of muscle-invasive tumors via CIS. Furthermore, COX-2 may be a therapeutic target for CIS because of the high expression rate of COX-2 in CIS lesions.

Overexpression of cyclooxygenase-2 in squamous cell carcinoma of the urinary bladder.

Epidemiological studies indicate that the development of squamous cell carcinoma of the urinary bladder is closely associated with chronic inflammation of the urinary tract, but the underlying mechanism is unknown. Cyclooxygenase (COX)-2 is involved in tumorigenesis in many tumors. The purpose of this study was to investigate the role of COX-2 in squamous cell carcinoma of the urinary bladder by immunoblot and immunohistochemical analyses. COX-2 protein was undetectable in normal bladder samples, but was expressed in 29 of 29 (100%) squamous cell carcinomas and in 8 of 8 (100%) squamous metaplasias. The expression of COX-2 showed intense, homogenous cytoplasmic immunostaining in squamous cell carcinomas. In contrast, COX-2 was heterogeneously expressed in 6 of 12 (50%) cases of transitional cell carcinoma of the bladder combined with squamous cell carcinoma, consistent with previous findings. We provide the first evidence that COX-2 is expressed in squamous cell carcinomas of the urinary bladder and in the precursor lesions, indicating its involvement in the development of this type of malignancy.

In vitro and in vivo induction of apoptosis by sphingosine and N, N-dimethylsphingosine in human epidermoid carcinoma KB-3-1 and its multidrug-resistant cells.

Sphingolipid breakdown products, including ceramide and sphingosine, regulate cell growth, cell differentiation, and apoptosis. We examined the effect of various agents, including sphingolipids, on apoptosis induction in human epidermoid carcinoma KB-3-1 and its multidrug-resistant (MDR) subclone KB-C2 cells which express P-glycoprotein. Adriamycin (ADM) induced apoptosis in KB-3-1 cells but not in KB-C2 MDR cells at the concentration of 50 microg/ml. On the other hand, 15 microM sphingosine or its methylated derivative N, N-dimethylsphingosine (DMS) induced apoptosis in both cell types in vitro. These results suggested that KB-C2 MDR cells were resistant to apoptosis induction by ADM but sensitive to that by sphingosine and DMS. Ceramide and sphingosine-1-phosphate, the initial metabolites of sphingosine, failed to induce apoptosis under the same experimental condition as sphingosine/DMS. The protein kinase C (PKC) inhibitors H7 and staurosporine did not induce apoptosis in either cell line, suggesting that PKC-independent signaling is involved in apoptosis induced by sphingosine and DMS, although both sphingosine and DMS have been shown to down-regulate PKC. Furthermore, DMS significantly inhibited the growth of KB-3-1 as well as KB-C2 MDR tumors in vivo, with evidence of increased apoptosis. The intracellular level of exogenously added [3H]sphingosine or [14C]DMS did not differ between the KB-3-1 parent cell line and its MDR subclone KB-C2, whereas that of [14C]ADM was reduced in KB-C2 MDR cells compared to KB-3-1 cells. These results suggest that P-glycoprotein acts as a transporter for ADM but not for sphingosine or DMS. Furthermore, DMS at the concentrations which induce apoptosis in KB-C2 cells did not affect the level of [14C]ADM. Because sphingosine and DMS induce apoptosis regardless of P-glycoprotein expression, they may provide a new strategy and a promising approach to the treatment of anticancer drug-resistant cancer.

Utility of subcutaneous fat aspiration for the diagnosis of systemic amyloidosis (immunoglobulin light chain).

To our knowledge, this is the first blind and controlled analysis of subcutaneous fat aspiration for the diagnosis of primary systemic amyloidosis. The procedure was performed on 82 patients with biopsy-proved systemic amyloidosis and 72 normal adult volunteers. Slides from 71 of the 72 controls were read as negative. Slides from 59 (72%) of the 82 patients with amyloidosis were read as positive or weakly positive after staining with alkaline Congo red. Subcutaneous fat aspiration was as sensitive as rectal biopsy and substantially more sensitive than bone marrow biopsy in diagnosing amyloidosis. In six instances fat aspiration would have obviated the need for a more invasive diagnostic biopsy. Subcutaneous fat aspiration is sensitive (72%) and specific (99%) for amyloidosis. It is technically simpler and less expensive than rectal biopsy and permits immediate assessment of specimen adequacy. The concordance rate for two independent pathologists was 95%. Equivocally positive stains should be interpreted with caution because weak nonspecific staining may be seen.

Proteoglycans in amyloidogenesis.

The potentially important role of proteoglycans and glycosaminoglycans in the modern concept of general amyloidogenesis has been discussed.

Alpha 1-antichymotrypsin is associated solely with amyloid deposits containing the beta-protein. Amyloid and cell localization of alpha 1-antichymotrypsin.

Our recent studies demonstrated that alpha 1-antichymotrypsin (ACT), a serine protease inhibitor, was associated with the beta-protein in the brain amyloid deposits of Alzheimer's disease, aged human controls and aged monkeys, suggesting a role for the inhibitor in the amyloid deposition. In the present study we used immunohistochemistry to test for the presence of ACT in the amyloid deposits which contain, as their major component, a protein different from the beta-protein. ACT was not found in the amyloid deposits in primary or secondary amyloidosis, familial and amyloidotic polyneuropathy or Creutzfeldt-Jakob disease (non-beta-protein amyloidoses), but was found (together with beta-protein) in Alzheimer's disease, Down's syndrome, normal aging, and hereditary cerebral hemorrhage with amyloidosis of Dutch origin. These results suggest a specific association of ACT with beta-protein amyloid. We next examined the distribution of the inhibitor in normal human brain and in various human neuropathological states in order to identify cells that express this protein during brain degeneration. In addition to its association with amyloid, ACT immunoreactivity was also located in astrocytes near areas of neuronal or tissue loss, in a few neurons and pericytes and in the epithelium of the choroid plexus.

Suppression of bcl-2 gene expression by sphingosine in the apoptosis of human leukemic HL-60 cells during phorbol ester-induced terminal differentiation.

Our recent studies have shown that intracellular levels of sphingosine, an endogenous PKC inhibitor, increase during apoptosis resulting from phorbol ester (PMA)-induced terminal differentiation of human myeloid leukemic HL-60 cells, and have suggested that sphingosine may function as an endogenous mediator of apoptosis in these cells [Ohta, et al. (1995) Cancer Res. 55, 691-697]. We report here that apoptosis induced by PMA, sphingosine, and N,N-dimethylsphingosine (DMS) was accompanied by a concomitant decrease of bcl-2 expression in both RNA and protein levels in HL-60 cells, while expression of bcl-XL and bax mRNA did not change, and neither sphingosine nor DMS induced differentiation of HL-60 cells. In contrast, in apoptotic cells induced by pharmaceutical PKC inhibitors H7 or staurosporine, expression of bcl-2 did not change nor did the intracellular sphingosine concentration. These results suggest that sphingosine may function as an endogenous mediator of apoptotic signaling in PMA-induced terminal differentiation of HL-60 cells through bcl-2 down-regulation, probably independent from PKC inhibition.

Sphingosine induces apoptosis in androgen-independent human prostatic carcinoma DU-145 cells by suppression of bcl-X(L) gene expression.

Our recent studies have suggested that sphingosine, an endogenous protein kinase C (PKC) inhibitor, may mediate apoptosis induced by a phorbol ester (PMA) in human promyelocytic leukemia HL-60 cells [Ohta et al. Cancer Res. 1995;55:691-697], and that the apoptotic induction by both PMA and sphingosine is accompanied by down-regulation of bcl-2, a gene which acts to prevent apoptotic cell death [Sakakura et al. FEBS Lett. 1996;397:177-180]. In this study, we examined the sphingosine-induced apoptosis of the androgen-independent human prostatic carcinoma cell line DU-145, which expresses bcl-X(L) and Bax but not bcl-2, and found that treatment of DU-145 cells with sphingosine suppressed bcl-X(L) in both mRNA and protein levels but did not change bax expression at all. In contrast, in apoptotic cells treated with a PKC inhibitor, staurosporine, no effect on bcl-X(L) or bax expression was observed. The initial metabolites of sphingosine in the cells, ceramide and sphingosine 1-phosphate, failed to induce apoptosis. These results indicate that, in DU-145 cells, sphingosine, but not its metabolites, induces apoptosis through down-regulation of bcl-X(L), independently of PKC inhibition. Our present results, together with previous observations, strongly suggest that apoptosis regulatory genes differ according to cell type and apoptosis induction through sphingosine is accompanied by inhibition of either bcl-2 or bcl-X(L) activity in these cells.

Diagnosis of amyloidosis by abdominal fat aspiration. Analysis of four years' experience.

Abdominal fat aspiration samples from 443 consecutive patients were examined for amyloid after Congo red and hematoxylin staining. Of the aspirates from 83 patients known to have systemic amyloid disease prior to the biopsy, 70 (84 percent) were found to yield positive results. The results for four aspirates from patients with localized amyloid disease were negative. Of the aspirates from 356 patients of unknown clinical status referred for analysis by outside physicians, 26 (7 percent) yielded positive results for amyloid. On review of the clinical records of these 26 patients, 11 had proved systemic amyloidosis demonstrated on biopsy of another site; all had a clinical course consistent with amyloid disease. In no case was amyloid found in a fat aspiration sample from a patient without clinical evidence suggestive of systemic amyloid disease. This study supports the proposal that abdominal fat aspiration is the diagnostic procedure of choice in the evaluation of amyloidosis since it requires no specialty consultation or technical expertise, causes minimal patient discomfort, and is accompanied by virtually no risk of morbid complication. A positive result has a high predictive value of amyloid disease in patients of unknown clinical status.

Familial amyloid polyneuropathy. Demonstration of prealbumin in a kinship of German/English ancestry with onset in the seventh decade.

A family with autosomal dominant transmitted familial amyloid polyneuropathy residing in Texas is described. Clinically, the prominent sensory and severe autonomic nervous system involvement resembles the Andrade (Portuguese) type I familial amyloid polyneuropathy but is unique in that the age of onset is in the seventh decade in all family members affected to date. Using an immunoperoxidase technique, prealbumin was demonstrated in the amyloid deposits. This finding suggests that this family shares biochemical as well as clinical characteristics consistent with similar kinships with type I familial amyloid polyneuropathy of diverse geographic origin.

Hemodialysis-associated amyloidosis and beta-2 microglobulin. Clinical and immunohistochemical study.

The beta-2 microglobulin type of amyloidosis was identified in articular and para-articular tissues of 14 patients with non-amyloid nephropathies undergoing long-term hemodialysis. Ten patients had carpal tunnel syndrome, 13 had juxta-articular radiolucent cysts (complicated by spontaneous fractures of the femoral neck in three), and six had destructive arthropathies of the large joints of the limbs. Massive amyloid deposits were found in the synovium, capsule, ligaments, articular cartilage, and/or bone. They were characterized by Congo red-induced green birefringence that was sensitive to potassium permanganate treatment. They reacted with anti-beta-2 microglobulin antiserum, whereas they did not react with antibodies directed against AA protein, prealbumin, or immunoglobulins. These data suggest that the potentially disabling arthropathy of hemodialysis is due to amyloid lesions. The persistently elevated plasma beta-2 microglobulin levels may play a role in the pathogenesis of this recently recognized complication, and if so, this complication should be preventable.

Inhibition of MAP kinase by sphingosine and its methylated derivative, N,N-dimethylsphingosine.

Endogenous sphingolipid metabolites such as ceramides and sphingosines have been increasingly recognized as lipid mediators of cell growth, differentiation and apoptosis. We have previously studied the ability of sphingosine (Sph) and N,N-dimethylsphingosine (DMS) to induce apoptosis in a variety of solid tumor cell lines. Here we report that in tumor cell lines displaying high mitogen-activated protein kinase activity (MAPK), treatment with 5 mu M of these sphingolipids significantly inhibited MAPK activity within 2-5 min (p < 0.005-0.01 as compared to controls) and induced apoptosis within hours. In contrast, untransformed cells and those tumor cell lines with low MAPK activity showed no significant change in activity and no apoptosis. High concentrations of C2-ceramide (50-100 mM), which induced apoptosis in the solid tumor cells, did not show significant effect on MAPK activity. MAPK activity was not directly inhibited in vitro, but tyrosine phosphatase activity was increased 2-4 fold in solid tumor cells by Sph or DMS (p < 0.01-0.05), suggesting that a phosphatase may play an important role in sphingolipid-directed MAPK regulation. Sph/DMS-induced apoptosis, but not MAPK inhibition, was blocked by protease inhibitors, indicating that MAPK inhibition is an earlier step of Sph/DMS-induced apoptosis than proteolysis. Furthermore, in human breast carcinoma MDA468 cells and human epidermal carcinoma A431 cells, both of which overexpress the epidermal growth factor (EGF) receptor, 20-200 nM EGF inhibited MAPK (p < 0.005-0.01) and induced apoptosis. These observations suggest that inhibition of the MAPK cascade may be involved in apoptotic signaling by Sph/DMS in some solid tumor cells, or by EGF in some cancer cells which overexpress the EGF receptor. Finally, the PKC-specific inhibitor, calphostin C, under conditions in which PKC is completely suppressed, inhibited MAPK activity and induced apoptosis only weakly in these solid tumor cells, whereas the non-specific PKC inhibitor staurosporine induced both apoptosis and MAPK inhibition significantly, suggesting that MAPK inhibition and apoptosis by Sph/DMS occurs independently of PKC in these cell lines, although these pathways may act cooperatively in other cell types. This study provides insight into possible mechanisms involved in sphingolipid-induced apoptosis in solid cancer tumor cell lines.