Angiotensin II increases cytosolic calcium and stimulates catecholamine release in cultured bovine adrenomedullary cells.

In bovine adrenomedullary cells in primary culture, angiotensin II (AII) elicited virtually immediate, dose-related increments in cytosolic calcium [( Ca++]i) measured by the Quin 2 technique and stimulated approximately proportional secretion of norepinephrine, epinephrine, and dopamine measured by liquid chromatography with electrochemical detection. Peak responses of [Ca++]i to AII were similar to peak responses to nicotine or KCl. Pre-treatment with verapamil or washing the cells in calcium-free medium attenuated the stimulatory effect of AII on [Ca++]i. Pre-treatment with nicotine, which temporarily inactivates cholinergic receptor-activated calcium channels, did not affect [Ca++]i responses to AII. The results indicate functional effects of AII on cultured chromaffin cells. The mechanism of cellular activation by AII appears to include increases in [Ca++]i due to opening of membrane calcium channels which may be unrelated to cholinergic receptor-operated calcium channels.

Cytosolic calcium in platelets of spontaneously hypertensive rats.

To test the hypothesis that an abnormality of the intracellular concentration of ionized calcium, [Ca2+]i, is associated with high blood pressure, we measured [Ca2+]i in the platelets of spontaneously hypertensive (SHR) and Wistar-Kyoto control (WKY) rats using the Quin 2 technique after separation of the platelets in calcium-free medium, during calcium repletion, and upon exposure to agonists which increase platelet [Ca2+]i (thrombin, adenosine diphosphate, serotonin and ionomycin). Despite clear-cut changes in [Ca2+]i during these manipulations, there were no differences between the SHR and WKY rats in baseline levels of [Ca2+]i or in the kinetics of changes in [Ca2+]i. These results do not support the hypothesis that high levels of [Ca2+]i at rest or abnormal kinetics of changes in [Ca2+]i play a pathophysiological role in the hypertension of SHR.

Effects of ouabain on cytosolic calcium in lymphocytes, platelets and adrenomedullary cells.

It has been suggested that a circulating inhibitor of Na/K ATPase can stimulate natriuresis and cause vasoconstriction in essential hypertension by stimulating transmembrane Na/Ca exchange to produce increased cytosolic concentrations of ionized calcium ([Ca++]i) in renal tubular and arteriolar smooth muscle cells. If this inhibitor affected [Ca++]i in all cell types, then clinical assays for its presence could be applied to easily accessible cells such as blood cells or platelets, and the inhibitor could exert a hormonal action on Ca++-dependent adrenomedullary secretion of catecholamines. We used the Quin 2 technique for measuring [Ca++]i in lymphocytes, platelets and adrenomedullary cells in response to ouabain. Inhibition of Na/K ATPase by ouabain (10(-7) to 10(-3) mol/l enhanced transient [Ca++]i responses during Ca repletion but had no effect on steady-state [Ca++]i in any of the cell lines. Although it is possible that a Na/Ca exchange mechanism may exaggerate transient increases in [Ca++]i during Na/K ATPase inhibition, other mechanisms appear to buffer these acute perturbations of [Ca++]i in lymphocytes, platelets and adrenomedullary cells.

Dead Sea bath salts for the treatment of rheumatoid arthritis.

Thirty patients with classical or definite rheumatoid arthritis were randomly divided into two groups of fifteen patients each of similar age, sex, duration and severity of disease, and medical treatment. All patients were treated once a day with bath salts heated to 35 degrees C for twenty minutes. Group I received Dead Sea bath salts and Group II, the control group, received sodium chloride (NaCl). The study was double-blind and of two weeks' duration. All patients were evaluated by one rheumatologist both before treatment, and two weeks later at the end of the treatment period. Follow-up evaluations were made one and three months after conclusion of the treatments. The clinical parameters evaluated included duration of morning stiffness, fifteen meter walk time, hand-grip strength, activities of daily living, circumference of proximal interphalangeal joints, number of active joints, Ritchie index and the patient's own assessment of disease activity. The laboratory parameters evaluated included erythrocyte sedimentation rate and serum levels of amyloid A, rheumatoid factor, sodium, potassium, calcium and magnesium. A statistically significant improvement (p less than 0.01 or p less than 0.05) was observed in Group I only, in most of the clinical parameters assessed. Maximal therapeutic effect was obtained at the end of the treatment and lasted up to one month.

Relationship between the serum levels of calcitonin, calcium, phosphorus and red blood cell Ca-ATPase during calcium infusion to healthy volunteers.

The relationship between the serum levels of calcitonin (CT), Ca and P, and red blood cell Ca-ATPase (RBC Ca-ATPase) was investigated during Ca infusion given to 6 healthy volunteers. The following results were obtained: Ca infusion, during a 3-hour period, raised serum levels of CT, P and RBC Ca-ATPase with significant correlations between serum CT and RBC Ca-ATPase. The increase in RBC Ca-ATPase activity was accompanied by an appropriate decrease in K-Na-ATPase activity. This study supports the in vitro observations concerning the regulation of intracellular Ca level by CT. Activation of Ca-ATPase during Ca loading is important for red cell survival.

Importance of serum amyloid A (SAA) level in monitoring disease activity and response to therapy in patients with prostate cancer.

Serum amyloid A (SAA) and acid phosphatase (AcP) levels were determined in serial serum samples of 35 patients in different stages of dissemination and correlated with activity of carcinoma of the prostate. Up to 500-fold increases in SAA level were detected during active periods of cancer with a decrease towards the normal range in remission, in comparison with a 10-fold increase of AcP. The correlation between these two parameters was highly significant (P less than 0.001), but while SAA shows 100% sensitivity during the active stage, AcP shows only 85% sensitivity. It is suggested that although SAA is not a specific marker for any particular illness, due to its characteristic pattern of change in malignant diseases and its high sensitivity, it represents a useful biochemical parameter for the assessment of the activity of the disease to monitor response to therapy during follow-up.

Inhibitory effect of the calcium antagonist, verapamil, on measles and vaccinia replication in cell culture.

Kidney cell lines MA104 and BGM were infected with vaccinia and measles viruses respectively in the presence of 45Ca. Increased 45Ca level was detected in the virally infected cells as compared with control cells. An enhancement of 28 +/- 6% and 37 +/- 13% was shown in vaccinia and measles respectively following 5 h of infection. The effect of the calcium antagonist verapamil was studied in both vaccinia- and measles-infected cells. In one-step growth experiments, the mean inhibitory effect of 90 microM verapamil on viral yield after 13 h was 97 +/- 1% in the case of vaccinia. In the measles virus after 47 h a mean of 76 +/- 5% inhibition was detected. The suitability of verapamil as a potential antiviral agent is suggested and requires further investigation.

Acute phase protein, serum amyloid A, inhibits IL-1- and TNF-induced fever and hypothalamic PGE2 in mice.

The effect of serum amyloid A (SAA) on fever induced by recombinant interleukin-1 beta (rIL-1 beta) or recombinant tumour necrosis factor alpha (rTNF alpha) was studied in mice. Serum amyloid A is an acute phase protein whose rise in pathological events is induced by the cytokines IL-1, IL-6 and TNF. Administration of human serum amyloid A to mice inhibited fever induced by rIL-1 beta or rTNF alpha in vivo, while the addition of human serum amyloid A to mice hypothalamic slices inhibited IL-1 beta- or TNF alpha-induced prostaglandin E2 (PGE2) production in vitro. Since serum amyloid A did not affect body temperature or hypothalamic PGE2 levels when administered alone, it may represent a specific servo-mechanism for fever regulation in acute events, and it suggests, for the first time, a possible feedback relationship between serum amyloid A and the immunoregulatory cytokines.

Cytokeratin 20: a new marker for early detection of bladder cell carcinoma?

PURPOSE: Cytokeratins are constituents of the intermediate filaments of epithelial cells in which they are expressed in various combinations depending on epithelial type and degree of differentiation. Of the 20 known cytokeratins, cytokeratin 19 is expressed in normal urothelium cells, whereas the recently identified cytokeratin 20 (CK-20) is expressed in urothelial carcinoma but not normal urothelium cells. We examine whether CK-20 expression can be used as a bladder tumor marker for transitional cell carcinoma in cells isolated from urine. MATERIALS AND METHODS: The reverse transcriptase polymerase chain reaction method was used to determine expression of CK-20 in cells separated from urine of patients with bladder carcinoma. Cells were obtained from urine of 192 patients stratified into 3 groups of 21 healthy young volunteers without a history of transitional cell carcinoma, 27 with a negative bladder biopsy for transitional cell carcinoma and 144 with bladder transitional cell carcinoma. The parameters were tumor stage and grade, tumor size, number of tumors, urinary cytology and CK-20. RESULTS: CK-20 amplification band (370 base pairs) was obtained with messenger ribonucleic acid extracted from transitional cell carcinoma cells of bladder tumor. CK-20 in the urine samples of the control group was negative (no false-positive results, specificity 100%). Among the 27 patients with pTo disease CK-20 was negative in 20 (specificity 74.1%). In the 7 patients with positive CK-20 histology showed chronic inflammation in 2, atypical hyperplasia in 3 and metaplasia in 1. In 1 patient who had a known history of transitional cell carcinoma the urothelium was normal. Among 144 patients with bladder transitional cell carcinoma CK-20 was positive in 131. Sensitivity of the method was much higher than urinary cytology (91 versus 56.3%, p <0.0001). We demonstrated no correlation between CK-20 and tumor grade. CONCLUSIONS: Our results indicate that CK-20 is a potential marker for bladder cancer. The noninvasive detection method assesses urothelial cells from the voided urine specimen using reverse transcriptase-polymerase chain reaction. The CK-20 marker was significantly more sensitive than urinary cytology.

Modulation of prostaglandin I2 production from bovine aortic endothelial cells by serum amyloid A and its N-terminal tetradecapeptide.

The study was aimed to explore the possible involvement of the acute phase HDL apolipoprotein, serum amyloid A (SAA) in the regulation of PGI2 production by endothelial cells. This, in view of the recent detection of SAA mRNA in endothelial cells of human atherosclerotic lesions. Human SAA induces PGI2 formation in bovine aortic endothelial cells culture in a concentration relevant to moderate acute phase events. 50 micrograms/ml of purified human SAA increases PGI2 production from a mean basal level of 2,490 +/- 330 pg/ml by 1.80 +/- 0.1 fold (n = 10; p < 0.01). The PGI2 inducing activity resides apparently in the N-terminal, i.e. amino acid residues 1-14, of the SAA molecule, 50 micrograms/ml of the peptide induces 2.9 +/- 0.5 fold increase of PGI2 production (n = 4; p < 0.03). TNF and LPS each induce PGI2 production in a concentration and time dependent manner. TNF in concentration of 10 ng/ml induces, in the presence of calf serum, an increase of 24.9 +/- 2.3 fold (n = 4; p < 0.001) and LPS in concentration of 1 microgram/ml causes a 18.3 +/- 1.3 fold increase, (n = 4; p < 0.01). In serum-free cultures, only a 2.5 +/- 0.3 fold increase was detected by 10 ng/ml TNF (n = 4), and a 5.9 +/- 0.4 by 1 microgram/ml of LPS. Thus, serum has a strong effect on PGI2 induction. When 50 micrograms/ml SAA is coadministered with 1 ng/ml TNF it reduces the TNF-induction of PGI2 from 7.7 +/- 2.8 to 3.3 +/- 1.2 fold (n = 4; p < 0.01). SAA attenuates, as well, LPS-mediated activity, although in a less pronounced manner. Our finding suggest a potential physiological function for SAA in regulation of basal and cytokine-induced PGI2 production by vascular endothelium. The capacity of SAA to markedly moderate PGI2 induction by TNF and LPS suggest that it may play a role in defending against vessel damage, in cases of atherosclerosis, bacterial infection or septic shock. The induction of PGI2 by SAA through its N-terminal domain, which also exhibits an anti-platelet aggregation activity, suggests a potential therapeutical use for this peptide as an anti-hypertensive and an anti-aggregatory agent.

Serum amyloid A (SAA) in viral infection: rubella, measles and subacute sclerosing panencephalitis (SSPE).

Serum amyloid A (SAA) levels were determined in the serial serum samples of eight rubella, 10 measles and seven subacute sclerosing panencephalitis (SSPE) patients. An early rise in SAA levels was detected in the acute phase in rubella and measles, followed by a prompt decrease in the convalescent phase. In a number of measles and rubella patients from whom early serum samples were available, the rise of SAA levels could be demonstrated before specific viral antibodies could be detected by complement fixation (CF) (measles) and haemagglutination inhibition (HI) (rubella). In only one rubella and one measles patient was no rise of SAA level detected. In SSPE only a moderate increase in SAA levels was noted except in one patient during a temporary deterioration, at which time the SAA level was very high; it returned to close to normal shortly thereafter. The possibility that SAA levels might be of value in monitoring the severity of infections, the recovery process and effects of anti-viral agents is discussed.

Serum amyloid A, an acute phase protein, inhibits platelet activation.

The effect of serum amyloid A, an acute phase protein, on platelet function was studied. Serum amyloid A was isolated and purified from sera of patients with recent trauma. Serum amyloid A inhibited thrombin-induced gel-filtered platelet aggregation. However, it did not inhibit aggregation induced by collagen or adenosine diphosphate, nor did it influence the aggregation of platelet-rich plasma activated with thrombin. Further studies of its effect on thrombin-induced activities showed that serum amyloid A, at concentrations of 25 to 100 micrograms/ml (which are found in mild acute events), suppressed the increase of cytosolic [Ca2+], thromboxane generation, and carbon 14-labeled serotonin release in a dose-dependent fashion. Serum amyloid A did not interfere with the clotting or amidolytic activities of thrombin. Therefore, the data suggest a protective role for serum amyloid A in thromboembolic disease by specific interaction with thrombin-induced platelet activation. Amyloid A protein also markedly inhibited thrombin-induced platelet aggregation. Because amyloid A is homologous to the N-terminal portion of serum amyloid A, the observed activity probably resides in that part of the molecule. This finding may be of importance in localization of the active site on serum amyloid A.

Expression of cytokeratin-20 in endometrial carcinoma.

OBJECTIVE: Cytokeratins are constituents of the intermediate filaments of epithelial cells which are expressed in various combinations depending on the epithelial type and the degree of differentiation. The recently identified cytokeratin-20 (CK-20) was found with immunohistochemical methods to be expressed in gastrointestinal epithelium, uroepithelial cells, and Merkel cells. Clues were also found for low expression of CK-20 in endometrial carcinoma cells. The aim of this study was to examine whether CK-20 expression can be measured in endometrial carcinoma with the more sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) methods and therefore used as a potential diagnostic tumor biomarker for endometrial carcinoma. METHODS: In the present study we have used RT-PCR methods to determine expression of CK-20 in endometrial epithelial cells. Endometrial specimens were collected from 18 patients with endometrial carcinoma and 22 patients that underwent hysterectomy due to benign diseases. The specimens were prepared for both RT-PCR and immunohistochemical analysis. RNA, of the various cell pellets, was extracted and RT-PCR was performed with CK-20 and CK-19 primers (CK-19 was used as a marker for normal epithelial cells). Immunohistochemistry was carried out with the avidin-biotin-peroxidase complex method on formalin-fixed paraffin sections using CK-20 antibody. RESULTS: CK-20 amplification band (370 bp) was obtained with mRNA extracted from endometrial carcinoma cells of 17 of the patients with endometrial carcinoma (sensitivity, 94.4%). CK-20 was negative in 21 patients with benign endometrial disease (specificity, 91.3%). No positive results were obtained with the immunohistochemical methods. CONCLUSION: These results indicate that RT-PCR of CK-20, because of its high sensitivity, is a potential biomarker for detecting endometrial carcinoma.

Sulphur bath and mud pack treatment for rheumatoid arthritis at the Dead Sea area.

Forty patients with classical or definite rheumatoid arthritis in a stage of active disease were treated for two weeks at a spa hotel. The patients were divided into four groups of 10. Group I was treated with daily mud packs, group II with daily hot sulphur baths, group III with a combination of mud packs and hot sulphur baths, and group IV served as a control group. The patients were assessed by a rheumatologist who was blinded to the treatment modalities. Statistically significant improvement for a period of up to three months was observed in the three treatment groups in most of the clinical indices. Improvement in the control group was minor in comparison and not statistically significant. No significant improvement was observed in any of the laboratory variables measured. Except for three mild cases of thermal reaction there were no side effects.

Detection of tumor circulating cells by cytokeratin 20 in the blood of patients with endometrial carcinoma.

BACKGROUND: Cytokeratins are constituents of the intermediate filaments of epithelial cells which are expressed in various combinations depending on the epithelial type and the degree of differentiation. Using the reverse transcriptase-polymerase chain reaction technique (RT-PCR) we recently demonstrated that: (1) Cytokertin 20-the most recent discovered cytokeratin-is expressed in endometrial carcinoma tumors but not in the endometrium of patients with benign diseases, and (2) CK-20 is not expressed in blood cells. The aim of this study is to examine whether CK-20 expression in blood can be used as a biomarker for the detection of the dissemination of malignant cells in patients treated for endometrial carcinoma. METHODS: In the present study, we have used RT-PCR to determine the expression of CK-20 in the peripheral blood of the following groups: (1) preop new diagnosed patients (n = 20), (2) patients with no clinical evidence of disease following completion of definitive treatment (n = 33; 17 at low risk; 16 at high risk), (3) patients with recurrent disease (n = 6), and (4) a control group of healthy subjects (n = 16). RNA was extracted from cell pellets and analysed by RT-PCR using primers for CK-20. RESULTS: Of the 20 patients of the first group 7 (35%) were CK-20 positive. Of the 33 patients of the second group 17 (51%) were CK-20 positive. Subdivision of this group showed that 9 of 17 (53%) were positive in the low-risk subgroup, and 8 of 16 (50%) were positive in the high-risk subgroup. All 6 patients with recurrent disease were positive, and all subjects in the control group were negative. CONCLUSION: These results indicate that RT-PCR of CK-20, because of its high sensitivity, is a potential biomarker for detecting metastasis in blood samples of patients with endometrial carcinoma.

Degradation of amyloid A and serum amyloid A by red blood cell haemolysate in patients with familial mediterranean fever.

Enzymatic activity for the degradation of serum amyloid A (SAA) and amyloid A (AA) was detected in erythrolysates of normal subjects and patients with familial mediterranean fever. A significant difference between the activity of normal subjects and patients was not found. Serum inhibited the SAA (but not the AA) haemolysate proteolytic activity. Interindividual variation in the susceptibility of SAA to degradation by RBC haemolysates was shown. The original digestible fraction of SAA became gradually resistant to proteolytic cleavage over a 9 month period while the susceptibility of AA to degradation remained unchanged in this time period. These findings suggest that enzymatic degradation of SAA depends on the source of SAA, as well as inhibitory activity in serum.

Detection of micrometastasis by cytokeratin-20 (reverse transcription polymerase chain reaction) in lymph nodes of patients with endometrial cancer.

BACKGROUND: Cytokeratins are constituents of the intermediate filaments (IFs) of epithelial cells which are expressed in various combinations, depending on the type of epithelium and degree of differentiation. We have reported (R. Zemer, A. Fishman, J. Bernheim, S. Zimlichman, O. Markowitz, M. Altaras, and A. Klein, Gynecol Oncol 70:410-413, 1998) on the determination of cytokeratin-20 (CK-20) by reverse transcription polymerase chain reaction (RT-PCR) in the detection of endometrial cancer cells as a potential biomarker. In that study, we also found that by using immunocytochemistry, most carcinomas were found to be negative for CK-20. The sensitivity and specificity rates obtained by using the RT-PCR method were 94.4 and 91%, respectively. OBJECTIVE: The aim of this study is to investigate the feasibility and potential of the specific mRNA marker, CK-20, to detect endometrial cancer cells-micrometastases (MMs)-by RT-PCR in lymph node (LN) samplings of patients undergoing hysterectomy for endometrial carcinoma. METHOD: We used the RT-PCR method to determine the expression of CK-20 in the LNs of 20 patients [study group (SG)] who were being surgically staged and treated for endometrial carcinoma. The specificity of the mRNA CK-20 marker was examined in LNs obtained from five healthy patients [control group (CG)] who underwent abdominal hysterectomy and bilateral salpingo-oopherectomy for benign gynecologic conditions. The LNs obtained from the SG and CG patients were prepared together before mRNA extraction. RNA of the various cell pellets was extracted and RT-PCR was performed with CK-20 primers. RT-PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining against PCR size markers. Specificity of the RT-PCR products was examined by Southern blotting. RESULTS: Histopathologic examinations demonstrated the presence of metastases in two (10%) SG patients. These patients were also CK-20 positive. Of the remaining 18 patients with negative histopathologic results, 6 (33%) were CK-20 positive and 12 (67%) were negative. All the CG patients were CK-20 negative (specificity, 100%). CONCLUSIONS: The results obtained in this study suggest that RT-PCR of CK-20 is more sensitive than traditional histopathologic methods in the diagnosis of MMs in LNs of patients with endometrial cancer. Thus, due to the aforementioned characteristics of CK-20, it may be considered a powerful biomarker in the detection of MMs in LNs of patients with endometrial cancer.

Effect of serum amyloid A, HDL-apolipoprotein, on endothelial cell proliferation. Implication of an enigmatic protein to atherosclerosis.

The possible contribution of apo-HDL serum amyloid A (SAA) to the protective effect of HDL against atherosclerosis was studied by evaluating its effect on bovine aortic endothelial cell (BAEC) proliferation. Our results suggest that human SAA, both purified and recombinant, in concentrations relevant to mild acute phase events, significantly inhibit endothelial cell proliferation in a dose-dependent manner (e.g., 50 micrograms/ml causes approximately 88% inhibition; p < 0.001). This inhibition was attenuated by addition of fibroblast growth factor (FGF), which antagonized the SAA-mediated effect. As levels of TNF may be highly elevated during the acute phase response, its effect on BAEC proliferation was evaluated and found, at concentrations of > 1 pg/ml, to be substantially inhibitory Co-incubation of cells with both SAA and TNF was inhibitory, albeit neither additive nor synergistic. FGF antagonized the effect of both proteins. Amyloidic deposit (AA, i.e. SAA 1-76), derived from pathological proteolysis of SAA, practically retains the inhibitory activity (e.g. 50 micrograms/ml causes approximately 66% inhibition; p < 0.001) but apparently lacks the regulatory site towards FGF. In contrast to the above inhibitory effect, synthetic SAA-related peptide corresponding to the sequence 29-33 of SAA enhances BAEC proliferation (50 micrograms/ml causes approximately 64% increase; p < 0.001). The present data, coupled with our previous observations in which SAA was found to induce endothelial PGI2 formation and to inhibit overproduction of PGI2 by TNF and LPS as well as platelet aggregation, may suggest that SAA contributes to the protective effect of HDL against atherosclerosis. This, by means of its modulatory effect on endothelial cell and platelet activation, primarily in the presence of other regulatory proteins. SAA-derived peptides may, potentially, be used as therapeutic agents in the treatment of atherosclerosis and cardiovascular diseases.

Serum and synovial fluid levels of serum amyloid A protein and C-reactive protein in inflammatory and noninflammatory arthritis.

Serum and synovial fluid (SF) levels of serum amyloid A (SAA) and C-reactive protein (CRP) were measured in 46 cases of various inflammatory arthritis (Group 1), and in 40 cases of noninflammatory arthritis: 18 cases of osteoarthritis (Group 2) and 22 cases of traumatic arthritis (Group 3). Serum and SF SAA levels were markedly elevated in Group 1: 126.4 micrograms/ml +/- 19.2 SEM and 46.4 micrograms/ml +/- 10.5 SEM, respectively; moderately elevated in Group 2: 10.1 micrograms/ml +/- 2.9 SEM, 4.0 micrograms/ml +/- 1.1 SEM and moderately elevated in Group 3: 10.4 micrograms/ml +/- 1.2 SEM, 4.0 micrograms/ml +/- 1.2 SEM, respectively. Serum/SF SAA ratios were similar in all 3 groups and ranged between 2.52-2.72. In comparison to SAA, the increment of serum and SF CRP above normal levels was moderate. A positive strong correlation was found between serum SAA and serum CRP: r = 0.64 (p less than 0.001) and between SF SAA and SF CRP: r = 0.59 (p less than 0.0001). SF SAA did not correlate with the number of SF white blood cells but did correlate with the percent of SF polymorphonuclear cells: r = 0.23 (p less than 0.05).