Rheumatoid factor and immunoglobulin M mark hepatitis C-associated mixed cryoglobulinaemia: an 8-year prospective study.

OBJECTIVES: The prevalence and factors of hepatitis C virus (HCV) -associated mixed cryoglobulinaemia in Asia remain elusive, and we aimed to investigate these topics. METHODS: An 8-year prospective cohort study was conducted in 678 consecutive Taiwanese individuals with chronic HCV infection (438 completed an anti-HCV therapy course). RESULTS: Of 678 individuals, 437 (64.5%) had mixed cryoglobulinaemia and 20 (2.9%) had mixed cryoglobulinaemic syndrome. At baseline, IgM (cut-off >122 mg/dL), triglycerides and IgG levels, and HCV genotype 3 were independently associated with mixed cryoglobulinaemia. Rheumatoid factor (RF) levels were associated with mixed cryoglobulinaemic syndrome (cut-off >12.2 IU/mL). At 24 weeks post-therapy, the 362 individuals with a sustained virological response (SVR) had higher cured (106/362 (29.3%) versus 10/76 (13.2%), p = 0.003) and lower persistent (100/362 (27.6%) versus 33/76 (43.4%), p = 0.003) mixed cryoglobulinaemia rates than non-SVR patients. Among SVR patients, compared with baseline levels, RF, IgG and IgM levels decreased, except in individuals with new mixed cryoglobulinaemia. Pre-therapy IgM levels were associated with 24-week post-therapy new (95% CI of OR 1.002-1.023) and persistent (95% CI of OR 1.004-1.015) mixed cryoglobulinaemia in SVR patients. After up to 8 years, 24-week post-therapy IgM levels were associated with mixed cryoglobulinaemia in SVR patients (9/51; 17.64%; 95% CI of HR 1.004-1.011). Among 17 SVR patients with pre-therapy mixed cryoglobulinaemic syndrome, 5 (29.4%) had long-term mixed cryoglobulinaemia and 4 (23.5%) had mixed cryoglobulinaemic syndrome. CONCLUSIONS: Over 60% of chronic HCV-infected individuals had mixed cryoglobulinaemia, and 17.64% of SVR patients had mixed cryoglobulinaemia 8 years post-therapy. Pre-therapy RF and IgM levels marked HCV-associated mixed cryoglobulinaemic syndrome and mixed cryoglobulinaemia, respectively.

Nerve conduction block in diabetic rats using high-intensity focused ultrasound for analgesic applications.

BACKGROUND: Nerve conduction block using high-intensity focused ultrasound (HIFU) has been conducted with nerves of mixed fibres in normal animal models. This study tested the feasibility and safety of HIFU for sensory nerve conduction block in diabetic neuropathic nerves to determine its potential for pain relief. METHODS: Diabetes was induced in Sprague-Dawley rats using streptozotocin, and HIFU at 2.68 MHz was used for the block. This study consisted of two sections, in vitro and in vivo. For the in vitro experiments, the entire contiguous sciatic-sural nerves were obtained. Compound action potentials and sensory action potentials were recorded in the sciatic and sural nerves, respectively. For the in vivo experiments, compound muscle action potentials (CMAPs) were recorded from the gastrocnemius muscles. All data were expressed as median (range). RESULTS: The in vitro results showed that HIFU temporarily inhibited sensory action potentials of the control and diabetic rat nerves to 33.9 (8.2) and 14.0 (10.7)% of the baseline values, respectively, whereas the compound action potentials were suppressed to 53.6 (8.4) and 76.2 (7.5)% of baseline, respectively. The in vivo results showed that HIFU acutely blocked CMAPs to 32.9 (12.6) and 19.9 (10.9)% of baseline in control and diabetic rat nerves, respectively. Measurements of CMAPs and histological exanmination were used for indirect assessment of the safety of the HIFU technique. CONCLUSIONS: High-intensity focused ultrasound safely and reversibly suppressed nerve conduction in diabetic rat nerves when the stimulation parameters were appropriate. The results suggest that HIFU may have potential to block sensory nerves reversibly and provide peripheral pain relief.

Naproxen-induced Ca2+ movement and death in MDCK canine renal tubular cells.

Naproxen is an anti-inflammatory drug that affects cellular calcium ion (Ca(2+)) homeostasis and viability in different cells. This study explored the effect of naproxen on [Ca(2+)](i) and viability in Madin-Darby canine kidney cells (MDCK) canine renal tubular cells. At concentrations between 50 µM and 300 µM, naproxen induced [Ca(2+)](i) rises in a concentration-dependent manner. This Ca(2+) signal was reduced partly when extracellular Ca(2+) was removed. The Ca(2+) signal was inhibited by a Ca(2+) channel blocker nifedipine but not by store-operated Ca(2+) channel inhibitors (econazole and SKF96365), a protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, and a PKC inhibitor GF109203X. In Ca(2+)-free medium, pretreatment with 2,5-di-tert-butylhydroquinone or thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+) pumps, partly inhibited naproxen-induced Ca(2+) signal. Inhibition of phospholipase C with U73122 did not alter naproxen-evoked [Ca(2+)](i) rises. At concentrations between 15 µM and 30 µM, naproxen killed cells in a concentration-dependent manner, which was not reversed by prechelating cytosolic Ca(2+) with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl. Annexin V/propidium iodide staining data suggest that naproxen induced apoptosis. Together, in MDCK renal tubular cells, naproxen induced [Ca(2+)](i) rises by inducing Ca(2+) release from multiple stores that included the endoplasmic reticulum and Ca(2+) entry via nifedipine-sensitive Ca(2+) channels. Naproxen induced cell death that involved apoptosis.

Celecoxib-induced increase in cytosolic Ca(2+) levels and apoptosis in HA59T human hepatoma cells.

Celecoxib has been shown to have antitumor effect in previous studies but the mechanisms are unclear. The effect of celecoxib on cytosolic Ca(2+) concentrations ([Ca(2+)]i) and viability in HA59T human hepatoma cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)]i. Celecoxib at concentrations of 10-50 µM induced a [Ca(2+)]i rise in a concentration-dependent manner. The response was reduced by 80% by removing Ca(2+). Celecoxib induced Mn(2+) influx, leading to quenching of fura-2 fluorescence. Celecoxib-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365, and protein kinase C modulators. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin nearly abolished celecoxib-induced [Ca(2+)]i rise. Incubation with celecoxib abolished thapsigargin-induced [Ca(2+)]i rise. Inhibition of phospholipase C with U73122 abolished celecoxib-induced [Ca(2+)]i rise. At 1-50 µM, celecoxib inhibited cell viability by less than 20%, which was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid/acetoxy methyl (BAPTA/AM). Celecoxib (10-50 µM) also induced apoptosis. In sum, in HA59T hepatoma cells, celecoxib induced a [Ca(2+)]i rise by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive store-operated Ca(2+) channels. Celecoxib also caused cell death via apoptosis.

Effect of diindolylmethane on Ca(2+) homeostasis and viability in MDCK renal tubular cells.

The effect of the natural product diindolylmethane (DIM) on cytosolic Ca(2+) concentrations ([Ca(2+)]i) and viability in MDCK renal tubular cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)]i. DIM at concentrations 1-50 µM induced a [Ca(2+)]i rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). DIM induced Mn(2+) influx leading to quenching of fura-2 fluorescence. DIM-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) greatly inhibited DIM-induced [Ca(2+)]i rise. Incubation with DIM abolished TG or BHQ-induced [Ca(2+)]i rise. Inhibition of phospholipase C with U73122 reduced DIM-induced [Ca(2+)]i rise by 50%. At 1, 10, 40 and 50 µM, DIM slightly enhanced cell proliferation. The effect of 50 µM DIM was reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In sum, in MDCK cells, DIM induced a [Ca(2+)]i rise by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive store-operated Ca(2+) channels. DIM did not induce cell death.

Fendiline-evoked [Ca2+]i rises and non-Ca2+-triggered cell death in human oral cancer cells.

The effect of fendiline on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) and proliferation has not been explored in human oral cancer cells. This study examined whether fendiline altered Ca(2+) levels and caused cell death in OC2 human oral cancer cells. [Ca(2+)](i) and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Fendiline at concentrations above 10 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). The fendiline-induced Ca(2+) influx was sensitive to blockade of L-type Ca(2+) channel blockers. In Ca(2+)-free medium, after pretreatment with 50 microM fendiline, 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor)-induced [Ca(2+)](i) rises were inhibited; and conversely, thapsigargin pretreatment nearly abolished fendiline-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with 2 microM U73122 did not change fendiline-induced [Ca(2+)](i) rises. At concentrations between 5 and 25 microM, fendiline killed cells in a concentration-dependent manner. The cytotoxic effect of 15 microM fendiline was not reversed by prechelating cytosolic Ca(2+) with BAPTA/AM. Collectively, in OC2 cells, fendiline induced [Ca(2+)](i) rises by causing Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx from L-type Ca(2+) channels. Furthermore, fendiline-caused cytotoxicity was not via a preceding [Ca(2+)](i) rise.

Gossypol, a component in cottonseed, induced increases in cytosolic Ca2+ levels in Chang liver cells.

The effect of gossypol, a compound found in cottonseed, on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. Gossypol (0.2-5microM) increased [Ca2+](i) in a concentration-dependent manner with an EC(50) value of 1.5microM. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase within 5min after drug application. Removal of extracellular Ca2+ markedly reduced the [Ca2+](i) signals by 80+/-2%. Preincubation with 0.1mM La3+ or 10microM nimodipine abolished the Ca2+ influx. Gossypol (5microM)-induced release of intracellular Ca2+ was reduced by 75% by pretreatment with 1microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+. Conversely, pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. After pretreatment with 5microM gossypol in Ca2+-free medium for several min, addition of 3mM Ca2+ induced a [Ca2+](i) increase of a magnitude nine-fold greater than control. Gossypol (5microM)-induced Ca2+ release was not affected by inhibiting phospholipase C with 2microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Together, this study shows that gossypol induced significant [Ca2+](i) increases in Chang liver cells by releasing Ca2+ from intracellular pools in a phospholipase C-dissociated fashion and by causing La3+- and nimodipine-sensitive Ca2+ influx.