Elevated serum levels of soluble CX3CL1 in patients with microscopic polyangiitis.

OBJECTIVES: To test the hypothesis that CX3CL1 contributes to the pathogenesis of microscopic polyangiitis. METHODS: Serum samples from 18 patients with microscopic polyangiitis (MPA), who fulfilled the revised criteria of the American College of Rheumatology (ACR), were collected during both the newly diagnosed, untreated active disease states and inactive disease states. Also serum was from patients with large vessel vasculitis (LVV), including giant cell arteritis (n=4) and Takayasu arteritis (n=3), and from 52 healthy individuals. Soluble (s)CX3CL1 levels in serum were measured using an enzyme-linked immunosorbent assay. Disease activity was assessed using Birmingham vasculitis activity scores (BVAS). Expression of CX3CR1 was examined by flow cytometry. RESULTS: Serum sCX3CL1 levels were significantly higher in MPA patients than in either LVV group or healthy individuals. The elevated sCX3CL1 levels seen in MPA patients correlated positively with BVAS, as well as with CRP levels and ESR, and similarly increased expression of cell-surface CX3CR1 was seen on peripheral blood CD4 and CD8 T cells from patients with MPA. Notably, sCX3CL1 levels and CX3CR1 expression were diminished during clinical remission following treatment. CONCLUSION: Our findings suggest that CX3CL1 may be involved in the pathogenesis of MPA, and may serve as a useful serologic marker of disease activity in systemic vasculitis.

Activated leucocytes express and secrete macrophage inflammatory protein-1alpha upon interaction with synovial fibroblasts of rheumatoid arthritis via a beta2-integrin/ICAM-1 mechanism.

OBJECTIVE: To examine the expression and regulation of chemotactic factor, macrophage inflammatory protein-1alpha (MIP-1alpha) by fibroblast-like synoviocytes (FLS), monocytes and polymorphonuclear neutrophils (PMN) isolated from the synovial fluid (SF) of rheumatoid arthritis (RA) patients. METHODS: Monocytes or PMN obtained from RA SF were co-cultured with unstimulated semiconfluent RA FLS. Culture supernatants were assayed for MIP-1alpha by enzyme-linked immunosorbent assay. The expression of MIP-1alpha mRNA and protein was also determined by Northern blot analyss and immunohistochemistry respectively. RESULTS: Interaction of activated leucocytes with FLS synergistically increased MIP-1alpha expression and secretion via a mechanism mediated by beta2-integrin/ intercellular adhesion molecule 1. CONCLUSION: MIP-1alpha expression within inflamed joints appears to be regulated not only by inflammatory cytokines but also by the physical interaction of activated leucocytes and FLS, and plays a crucial role in the progression and maintenance of RA synovitis.

Epigallocatechin gallate (EGCG) inhibits the sulfation of 1-naphthol in a human colon carcinoma cell line, Caco-2.

We have previously reported that epigallocatechin gallate (EGCG) strongly inhibits the in vitro phenol sulfotransferase (P-ST) activity of a human colon carcinoma cell line, Caco-2. In the present study, we examined the ability of EGCG to inhibit the sulfation of 1-naphthol in intact Caco-2 cells. Sulfation of 1-naphthol was detected in Caco-2 cells after 2 h of incubation, and was observed to continue for 24 h, resulting in an accumulation of sulfated 1-naphthol. Sulfation was strongly inhibited by the addition of EGCG to the culture medium. The IC50 of EGCG was calculated to be 20 microM; this value is similar to that obtained from in vitro assays (14 microM) [Ref. Tamura et al., Biol. Pharm. Bull., 23, 695, (2000)]. These results indicate that catechins are capable of inhibiting P-ST activity in intact cells as well as in vitro. We believe that the inhibitory activity of catechins might be the mechanism by which catechins (and green tea) exert anti-carcinogenic activity against procarcinogenic compounds that require P-ST activation in vivo.

Using contrast-enhanced sonography to assess the effectiveness of transcatheter arterial embolization for hepatocellular carcinoma.

OBJECTIVE: We evaluated the usefulness of contrast-enhanced wideband harmonic gray-scale sonography in assessing the therapeutic effects of transcatheter arterial embolization for patients with hepatocellular carcinoma and compared the findings of this imaging modality with those of helical CT. SUBJECTS AND METHODS: Twenty-nine patients with 39 hepatocellular carcinoma lesions were examined. We scanned lesions before and after therapy using contrast-enhanced wideband harmonic gray-scale sonography after injection of a galactose-palmitic acid contrast agent. All patients held their breath for 20--50 sec after injection while the vascularity of the tumor was observed. We then monitored tumor enhancement between 60 and 180 sec after injection with patients breath-holding for a few seconds. Lesions were considered to exhibit viable tumor residue if hypervascular enhancement was observed in the tumor. We compared this enhancement with helical CT findings. RESULTS: After therapy, 36 of the 39 lesions showed viable tumor residue on contrast-enhanced wideband harmonic gray-scale sonography, with no artifacts from iodized oil. Helical CT revealed a high-attenuation area in 12 of the 36 lesions, whereas 24 of the 36 lesions could not be evaluated for tumor residue as a result of artifacts from iodized oil accumulation in the tumor. The remaining three lesions showed complete deposition of iodized oil and complete necrosis on contrast-enhanced wideband harmonic gray-scale sonography. CONCLUSION: Contrast-enhanced wideband harmonic gray-scale sonography is useful in evaluating the therapeutic effects of transcatheter arterial embolization for hepatocellular carcinoma.

Primary aldosteronism in pregnancy.

Aldosteronism is a rare complication of pregnancy. We report a case of a 26-year-old woman who became pregnant soon after a diagnosis of primary aldosteronism due to left adrenal adenoma was made. Only oral potassium supplementation was required in addition to routine prenatal care until 36 weeks' gestation. Subsequently, antihypertensive medication was needed to control elevated blood pressure. A healthy male infant was delivered by cesarean section because of abruptio placentae. The postoperative course was uneventful. Left adrenalectomy was conducted eight months after delivery under laparoscopic visualization. In this case report, we discuss management of aldosteronism in pregnancy and review the literature.

Medullary thyroid carcinoma with multiple hepatic metastases: treatment with transcatheter arterial embolization and percutaneous ethanol injection.

A 54-year-old man with medullary thyroid carcinoma in the thyroid gland was unable to undergo total thyroidectomy because the tumor had invaded the mediastinum. Radiation therapy and chemotherapy were given. Seven years later, intractable diarrhea and abdominal pain appeared, and computed tomography demonstrated hypervascular tumors in the thyroid gland and in the liver. The tumors were successfully treated with percutaneous ethanol injection to a lesion in the thyroid gland and transcatheter arterial embolization followed by percutaneous ethanol injection to tumors in the liver. Transcatheter arterial embolization and percutaneous ethanol injection may be valuable in treating medullary thyroid carcinoma.

Midtrimester urine human chorionic gonadotropin beta-subunit core fragment levels and the subsequent development of pre-eclampsia.

OBJECTIVE: Our purpose was to determine whether midtrimester maternal urine human chorionic gonadotropin beta-subunit core fragment predicts later pre-eclampsia. STUDY DESIGN: Urine beta-core fragment levels standardized to spot creatinine concentration and expressed as multiples of the median were prospectively determined in 347 midtrimester singleton pregnancies undergoing genetic amniocentesis. All women considered in the analysis were white and nonsmokers. Obstetric chart review was undertaken after delivery to identify cases in which pre-eclampsia developed. The risk of pre-eclampsia at different threshold levels of beta-core fragment of human chorionic gonadotropin was determined. RESULTS: The median maternal age was 36.0 years, with a median gestational age at urine collection of 16.0 weeks. The median level of the beta-core fragment of human chorionic gonadotropin was 1385.5 ng/mg of creatinine in those with pre-eclampsia, whereas that in those without pre-eclampsia was 1061.2 ng/mg. The difference was significant (Mann-Whitney U test, P = .03). A significant linear association was found between the beta-core fragment concentration and the risk of pre-eclampsia (Mantel-Haenszel test of linear association, P = .03). The relative risk and 95% confidence interval of subsequent pre-eclampsia increased from 2.07 (1.06 to 4.05) at beta-core fragment levels of human chorionic gonadotropin > or = 2.0 multiples of the median to 5.17 (1.95 to 13.7) at > or = 4.0 multiples of the median. CONCLUSION: Clinically normal patients with elevated midtrimester levels of urine beta-core fragment of human chorionic gonadotropin are at increased risk for the subsequent development of pre-eclampsia. The clinical value of this urine analyte as a marker for pre-eclampsia needs to be further investigated.

[SMANCS-TAE combined with PEI in the treatment for hepatocellular carcinoma].

From January 1996 to August 1997, 24 patients with advanced hepatocellular carcinoma (HCC) equal to or more than 2 cm (mean +/- SD; 4.1 +/- 3.0 cm) in main tumor diameter were treated by SMANCS-TAE (20 cases) or SMANCS-TAI (4 cases) combined with PEI. Six cases had solitary lesion, 16 cases had multiple lesions, and 2 cases had massive lesions. After this combination therapy, 21 of 24 cases had complete tumor necrosis. During 3 to 19 months follow up period, 12 cases had cancer-free survival (SMANCS-TAI; 3 cases), and 9 cases had tumor recurrences (3 cases were local recurrences and 6 cases involved new lesions). Two cases died of hepatic infarction and cancer death, however, the remaining 22 cases were surviving. SMANCS-TAE combined with PEI is useful treatment for advanced large or multiple HCC lesions in patients who are poor surgical risks.

Elevated serum nicked and urinary beta-core fragment hCG in preeclamptic pregnancies.

OBJECTIVE: To determine whether different molecular forms of hCG in serum and urine are elevated in preeclamptic pregnancies. METHODS: Forty-three pregnant women were studied: 25 preeclamptic women and 18 normotensive women. Immediately after blood and urine samples were collected, the protease inhibitors leupeptin (0.35 mM) and phenanthroline (22 mM) were added. Various molecular forms of hCG in serum (complete hCG, nonnicked hCG, complete free beta hCG) and in urine (complete hCG, beta-core fragment hCG) were measured by matched immunoassays with a common enzyme-labeled tracer antibody. The nicked hCG assay used a coating of beta-subunit monoclonal antibody with the addition of scavenger antibody to remove nonnicked hCG. Mann-Whitney U test and chi 2 test were used for statistical analyses. RESULTS: Preeclamptic women had significantly higher median (range) levels of serum complete and nicked hCG than did normotensive women (3620 [850-12,000] versus 2420 [310-4840] ng/mL, P = .024; and 102 [45-275] versus 71 [11-143] ng/mL, P = .010, respectively). Both median (range) urinary complete hCG-creatinine and beta-core fragment-creatinine ratios were significantly higher in preeclamptic women than in normotensive women (37.6 [0.5-185] versus 11.3 [1.9-54], P = .013; and 11.8 [2-67] versus 5.3 [0.3-29], P = .009, respectively). CONCLUSIONS: Various molecular forms of hCG in serum and urine were significantly higher in preeclamptic than in normotensive pregnancies.

Comparison of 12 assays for detecting hCG and related molecules in urine samples from Down syndrome pregnancies.

Urine is a new medium for Down syndrome testing. In an effort to determine the best type of human chorionic gonadotropin (hCG)-related immunoassay for urine testing, we examined 14 Down syndrome and 91 unaffected pregnancy urine samples with 12 established assays. The assays included (a) those that detect hCG beta-core fragment only; (b) those that detect beta-core fragment with less than 18 per cent free beta-subunit cross-reactivity; (c) that which equally detects free beta-subunit and beta-core fragment; and (d) those that detect hCG, free beta-subunit, or combinations thereof. The seven type a and b assays had the highest sensitivity for Down syndrome. The median MOM for Down syndrome was 5.93 (range 4.73-7.53). At a 10 per cent false-positive rate, the median observed detection rate was 93 per cent (range 79-100 per cent) and the median predicted detection rate was 85 per cent (range 69-96 per cent). The assays that did not mainly detect beta-core fragment (types c and d) had poorer screening performance. The median MOM for Down syndrome was 2.70 (range 2.16-3.63 MOM). At a 10 per cent false-positive rate, the median observed detection rate was 50 per cent (range 36-64 per cent) and the median predicted detection rate was 37 per cent (range 21-62 per cent). We infer that the assays that only detect beta-core fragment, or beta-core fragment with minor free beta-subunit cross-reactivity (types a and b), are the better urine-based tests for Down syndrome screening.

Screening for Down syndrome pregnancy using beta-core fragment: prospective study.

Two recent publications by Cuckle et al., and one each by Canick et al. and Kellner et al., describe the use of urine beta-core fragment measurements as a screening test for Down syndrome pregnancies. Median levels of over 5.4 MOM were reported for cases of Down syndrome, with an over 72 per cent detection rate for a 5 per cent false-positive rate. Urine beta-core fragment was suggested as a superior screening test for Down syndrome pregnancies. These four studies were retrospectives, with samples from affected cases collected at different sites from those from normal cases. In the present study, prospective data were collected for 726 pregnancies over a 9-month period at a single medical centre. Fresh samples were assayed continuously, without knowledge of the karyotype. Urinary beta-core fragment levels in 709 unaffected samples continually declined from 12 to 24 weeks of pregnancy. A logarithmic fit was optimal for the median curve. The log standard deviation of unaffected samples was 0.368. All 13 Down syndrome cases had levels exceeding 1.0 MOM, with a median value of 4.1 MOM. Eight of 13 Down syndrome cases (62 per cent) had levels exceeding the 95th centile. Results have not been adjusted for maternal age, which may improve the detection rate. The results reported here, while less impressive than those reported previously, confirm the usefulness of urine beta-core fragment as a screening test for Down syndrome. Because of the prospective nature of this study, the 62 per cent sensitivity suggested here might be more representative of the true performance of urinary beta-core fragment in clinical practice than the higher rates observed in previous studies. Results for this single urine test are similar to those for triple screen and other serum combination tests. Single analyte urine beta-core fragment tests, or beta-core fragment combination protocols, may eventually replace serum analytes in screening for Down syndrome pregnancies.

Urine beta-core fragment, a potential screening test for ectopic pregnancy and spontaneous abortion.

The incidence of ectopic pregnancy in the United States has risen 6-fold in the last three decades. It now accounts for about 2% of reported pregnancies. Tests are now needed to identify ectopic pregnancy before it is clinically evident. We evaluated human chorionic gonadotropin beta-core fragment as a test to predict ectopic pregnancy and spontaneous abortion. Urine samples were collected from women with in vitro fertilized pregnancies, 2 1/2-5 weeks after embryo transfer. Fifty samples were collected from those later shown to have normal intrauterine pregnancies, samples from 13 women subsequently found (at 5-9.3 weeks) to have ectopic gestations, and 15 from those with impending spontaneous abortion. Beta-Core fragment levels were determined by immunoassay, and results normalized to creatinine concentration. Median beta-core fragment levels at 2 1/2-3, 3-4, and 4-5 weeks after embryo transfer, were 6.7, 91 and 737 microg/g for unaffected pregnancies, 1.0, 5.9 and 0.6 microg/g for impending ectopic pregnancies (0.15, 0.065 and 0.0008, multiples of the unaffected pregnancy median, MoM), and 0.75, 6.8 and 12 microg/g for impending spontaneous abortions (0.11, 0.07 and 0.016 MoM). A gestation-linked curve was modeled to discriminate unaffected pregnancy from impending ectopic gestation or spontaneous abortion. Plotted beta-core fragment levels were below this curve in 12 of 13 (92%) women with impending ectopic pregnancy, in 10 of 15 (67%) with spontaneous abortion outcome, and in 2 of 50 (4%) with intrauterine pregnancy and term outcome. Measurement of urine beta-core fragment at 2 1/2-5 weeks after embryo transfer (4 1/2-7 weeks after last menstrual period) might be useful for identifying failing pregnancies. Over three quarters (predictive value positive 76%) of those with low beta-core fragment levels have ectopic pregnancy or spontaneous abortion. On the contrary, 95% (predictive value negative) of those with normal range test values may be predicted to have a nonfailing term pregnancy. Diagnosis of ectopic pregnancy could be confirmed by transvaginal ultrasound, and ectopic pregnancy terminated early by nonsurgical methods, with minimal mortality or fertility loss. Major fetal defects that cause spontaneous abort pregnancies may also be recognized by transvaginal ultrasound. In such cases, chorionic villous sampling or possibly termination may be considered.

Screening for Down syndrome using urine hCG free beta-subunit in the second trimester of pregnancy.

Human chorionic gonadotropin (hCG) free beta-subunit levels were determined in 709 control and 13 Down syndrome urine samples from the second trimester of pregnancy. Results were normalized to urine creatinine concentration and converted to multiples of the unaffected pregnancy medium (MOM). The concentration of free beta-subunit in Down syndrome cases was 3.9 MOM. Seven of 13 Down syndrome pregnancies (54 per cent) had free beta-subunit levels at or above the 95th centile of unaffected pregnancies. Urine free beta-subunit may potentially be useful as a screening test for Down syndrome.

Combining beta-core fragment and total oestriol measurements to test for Down syndrome pregnancies.

Recent articles by Cuckle et al., Canick et al., and Isozaki et al. have evaluated urine beta-core fragment as a screening test for Down syndrome in second-trimester pregnancies. They found over four-fold elevation of beta-core fragment levels in Down syndrome pregnancies, and between 62 and 88 per cent detection of this trisomy at a 5 per cent false-positive rate. Urine beta-core fragment may be a superior screening test for Down syndrome pregnancies. In the present study, urinary total oestriol has been evaluated as a marker to use in combination with beta-core fragment in screening for Down syndrome pregnancies. The two markers were evaluated separately in relation to the urine creatinine concentration. To amplify screening performance, we evaluated the ratio of beta-core fragment to total oestriol levels (creatinine-independent). beta-core fragment and total oestriol levels were determined (normalized to creatinine, ng/mg creatinine) in urine samples from 480 unaffected and 12 Down syndrome pregnancies, collected consecutively at a single prenatal diagnosis centre. The median beta-core fragment level in Down syndrome cases was 4.5 MOM. Fifty-eight per cent of Down syndrome cases had beta-core fragment levels exceeding the 95th centile of unaffected pregnancies. The median total oestriol level in Down syndrome cases was 0.33 MOM. Forty-two per cent of Down syndrome cases had total oestriol levels exceeding the 95th centile of unaffected pregnancies. We investigated the ratio of the two determinants (beta-core fragment, ng/ml divided by total oestriol, ng/ml) in our sample set. The median beta-core fragment:total oestriol ratio in Down syndrome cases was 13 MOM. Seventy-five per cent of Down syndrome cases had a ratio exceeding the 95th and the 99.5th centile of unaffected pregnancies. Total oestriol complements beta-core fragment in urine screening for Down syndrome pregnancies. A test measuring the ratio of the two urine determinants may be a significant improvement over current serum methods for detecting Down syndrome.

IL-6 antisense-mediated growth inhibition of a choriocarcinoma cell line: an intracellular autocrine growth mechanism.

The growth of tumor cells can be regulated by a variety of cytokines. To investigate the pathogenesis of choriocarcinoma and explore a new therapeutic approach for the carcinoma, we examined the role of IL-6 in the growth of a human choriocarcinoma cell line (JEG-3). IL-6 was identified in the supernatant of the cell culture medium by enzyme-linked immunosorbent assay, indicating that this cell secreted IL-6. The mRNAs of IL-6, IL-6 receptor and gP130, the IL-6 signal transducer, in this cell were shown to be present by reverse transcriptase polymerase chain reaction assay and confirmed by Southern blot hybridization and direct sequencing. The addition of hrIL-6 to the cell culture failed to stimulate cell growth. Monoclonal antibodies against IL-6, IL-6 receptor, and gP130 were also unable to inhibit the proliferation of this cell line. The antisense oligonucleotides targeting IL-6 mRNA, however, inhibited both cell growth and IL-6 production. Taken together, these findings indicate that endogenous IL-6 plays an important role in the growth of the JEG-3 cell line, and it exerts its action by an intracellular autocrine growth mechanism. The results also suggest that the therapeutic trials with monoclonal antibodies designed to neutralize IL-6 or block its receptor will likely fail, whereas the antisense oligonucleotides targeted to IL-6 mRNA may have some value for the treatment of choriocarcinoma and other cancers with intracellular autocrine growth fashion mediated by IL-6.

The behaviour of red cells in narrow tubes in vitro as a model of the microcirculation.

To investigate the behaviour of red cells in the microcirculation, we established a new capillary method using narrow fluorinated ethylenepropylene copolymer tubes with internal diameters of 12.5 and 25.0 microns. Red cell flow in the tubes under a given range of pressure was analysed through a video system connected to a microscope. The experimental condition was adjusted so that the velocity of the control normocytes would be compatible with that in corresponding vessels in vivo, 0.5-1.5 mm/s. In the 12.5 microns tube, normocytes obtained from 12 young normal volunteers ran in an axisymmetric edge-on orientation with a folded shape at higher pressures, but rolled along freely without deformation at lower pressures. Deformation during the passage of the microcytes obtained from four patients with polycythaemia vera complicated with iron-deficient microcytosis and 10 patients with iron deficiency anaemia was relatively mild, whereas that of the macrocytes obtained from eight patients with refractory anaemia was marked. Even after the screening effect at the tube entrance was taken into consideration, the velocities of both microcytes and macrocytes were found to be significantly lower than the control normocytes. Therefore this method may be a new way to investigate the flow properties of red cells in the microcirculation.

Active urea transport in the rat inner medullary collecting duct: functional characterization and initial expression cloning.

Active transport of urea has been proposed to exist in the inner medullary collecting duct (IMCD) of low-protein fed mammals for over 30 years. We perfused IMCD subsegments from rats fed a standard (18%) or a low (8%) protein diet and tested for the presence of active urea transport. We found no active urea transport in terminal IMCDs, regardless of diet. In initial IMCDs from rats fed 18% protein or fed 8% protein for one to two weeks, we again found no active urea transport. However, in rats fed 8% protein for three to four weeks, we found significant net urea reabsorption. This active urea reabsorption was inhibited when Na+, K(+)-ATPase activity was inhibited by adding 1 mM ouabain or removing bath potassium, suggesting a secondary active transport process. Removing sodium from the perfusate completely inhibited net urea reabsorption, demonstrating that this active urea transport is dependent upon the presence of sodium in the tubule lumen. Unlike the facilitated urea transporter, the active urea transporter was not inhibited by phloretin nor stimulated by vasopressin, suggesting that it is a distinct transport protein. To test this hypothesis, we size-separated poly(A)(+)-RNA prepared from inner medullae of rats fed 8% protein for three weeks and injected it into Xenopus laevis oocytes. RNA from a 4.4 to 8.4 kb size fraction increased urea permeability fourfold compared to water-injected oocytes or injecting RNA from other size-fractions. We conclude that feeding rats a low-protein diet for three weeks induces the expression of an unique, secondary active, sodium-dependent urea transporter whose cDNA is between 4.4 and 8.4 kb in size. In addition, our results suggest that it will be possible to clone the cDNA for this sodium-urea cotransporter by expression in Xenopus laevis oocytes.