Favorable clinical response and drug retention of anti-IL-6 receptor inhibitor in rheumatoid arthritis with high CRP levels: the ANSWER cohort study.

OBJECTIVE: Currently, biological disease-modifying anti-rheumatic drugs (bDMARDs) with different modes of action [tumour necrosis factor inhibitor (TNFi), interleukin-6 receptor inhibitor (IL-6Ri), or cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig)] are used in clinical practice to treat rheumatoid arthritis (RA). However, it is unclear which type of bDMARD is the most efficacious for a specific clinical situation. C-reactive protein (CRP) is an acute-phase reactant driven by IL-6 signalling. Here, we aimed to establish whether therapeutic efficacy differs between IL-6Ri and other bDMARDs with alternative modes of action in RA patients according to their CRP level. METHOD: RA patients treated with bDMARDs were enrolled from an observational multicentre registry in Japan. Patients were classified into three groups according to baseline CRP tertiles. The overall 3 year retention rates of each bDMARD category were assessed. The Clinical Disease Activity Index (CDAI) was also assessed before and 3, 6, and 12 months after bDMARD initiation. RESULTS: A total of 1438 RA patients were included and classified into three groups according to tertiles of baseline CRP levels (CRP1, 0-0.3; CRP2, 0.3-1.8; CRP3, 1.8-18.4 mg/dL). In CRP3, the overall 3 year drug retention rates were significantly higher for IL-6Ri than for TNFi and CTLA4-Ig (77.5 vs 48.2 vs 67.3, respectively). No significant difference was evident in terms of CDAI 12 months after bDMARD initiation in CRP1-CRP3. CONCLUSION: IL-6Ri may be a favourable therapeutic option over TNFi and CTLA4-Ig in RA patients with high CRP levels.

The effect of oxygen concentration on arterial blood partial pressure of oxygen in dogs under general anesthesia.

Oxygen is used for medical treatment and general anesthesia. However, high concentrations of oxygen can have toxic effects on cells. In veterinary medicine, 100% oxygen is usually used during general anesthesia and it can be toxic to animals. However, there is little concern about its harmful effects in humans. The objective of this study was to demonstrate that using a high con- centration of oxygen increases the partial pressure of oxygen in arterial blood (PaO2) more so than a lower concentration, by comparing PaO2 at three different oxygen concentrations (100%, 60%, and 40%) in six dogs under general anesthesia for 3 hours. The mean PaO2 and standard error values at the 100%, 60%, and 40% oxygen concentrations were 535.8 ± 24.01, 374 ± 17.19, and 239 ± 8.78 mmHg, respectively (p⟨0.05). These results show that 100% and 60% oxygen concentrations could increase oxidative stress. Further studies are needed to examine the oxygen concentration that causes toxicity.

Monocyte CD64 expression as a novel biomarker for the disease activity of systemic lupus erythematosus.

OBJECTIVE: Interferon alpha (IFN-α) is a key cytokine associated with systemic lupus erythematosus (SLE). IFN-α induces the expression of CD64 on monocytes (mCD64). Although enhanced mCD64 expression has been reported in patients with SLE, it has never been assessed quantitatively. The aim of this study was to investigate whether or not mCD64 expression correlates with SLE disease activity. METHODS: The mCD64 expression levels were assessed quantitatively in 40 patients with active or inactive SLE by using flow cytometry. The mCD64 expression levels were subsequently compared with the SLE disease activity index (SLEDAI) and levels of existing SLE activity biomarkers, such as anti-DNA antibody, complements, and so on. RESULTS: The mCD64 expression was significantly higher in active disease than in inactive disease SLE (median molecules/cell, interquartile range: 34,648, 8174-24,932 and 20,865, 6357-21,503, respectively; p < 0.001). The levels of mCD64 expression strongly correlated with SLEDAI (r = 0.68, p < 0.001). CONCLUSION: The mCD64 expression is a simple and useful biomarker for evaluating disease activity in patients with SLE.

Hydrogen sensing properties of protective-layer-coated single-walled carbon nanotubes with palladium nanoparticle decoration.

Protective-layer-coated single-walled carbon nanotubes (SWNTs) with palladium nanoparticle decoration (Pd-SiO(2)-SWNTs) were fabricated and their sensing properties for hydrogen (H(2)) were investigated. SWNTs were coated with a 3-4 nm thick SiO(2) layer by pulsed laser deposition and subsequently decorated with Pd nanoparticles by electron beam evaporation. Even though the SWNTs were completely surrounded by a protective layer, Pd-SiO(2)-SWNTs responded to H(2) down to a concentration of 1 part per million. Compared with the Pd nanoparticle-decorated SWNTs without a protective layer (Pd-SWNTs), Pd-SiO(2)-SWNTs exhibited highly stable sensor responses with variations of less than 20%; Pd-SWNTs showed a variation of 80%. The density of the Pd-SWNTs significantly decreased after the sensing test, while that of the Pd-SiO(2)-SWNTs with the netlike structure remained unchanged. The hydrogen sensing mechanism of the Pd-SiO(2)-SWNTs was attributed to the chemical gating effect on the SWNTs due to dipole layer formation by hydrogen atoms trapped at the Pd-SiO(2) interface. Moreover, the relationship between H(2) concentration and sensor response can be described by the Langmuir isotherm for dissociative adsorption.

Clinical outcomes of orthogonal plating to treat radial and ulnar fractures in toy-breed dogs.

OBJECTIVES: To describe the results of orthogonal plating (OP) as a treatment for fractures of the radius and ulna in toy-breed dogs. MATERIALS AND METHODS: The medical records (June 2011 to April 2019) of toy-breeds in which the OP technique using non-locking cuttable plates was employed to treat radial and ulnar fractures were reviewed. The inclusion criteria included a bodyweight of 3.5 kg or less, fracture of the diaphysis of the radius and ulna of one or both forelimbs, and the availability of follow-up radiographs. Revision surgeries were also included. RESULTS: Fifteen limbs that underwent initial fracture repair and five that underwent revision surgery met the inclusion criteria. The radial and ulnar fractures healed in 19 limbs at the final follow-up. Synostosis of the radius and ulna at the fracture sites was observed in one limb. Re-fracture after cranial plate removal was observed in one case. All dogs exhibited successful return of normal limb function at the final clinical and radiographic follow-up (mean, 104.7 ± 67.1 days; median, 79.5 days; range: 35 to 248 days). Long-term follow-up data obtained via telephone interviews to owners or referring veterinarians were available for 15 cases and confirmed maintenance of normal limb function in all dogs (mean, 32.5 ± 17.6 months; median, 26 months; range: 11 to 69 months). CLINICAL SIGNIFICANCE: OP allowed the successful open reduction of radial and ulnar fractures, facilitating bone union in both the radius and ulna and a return to normal limb function in a series of toy-breed dogs.

Increased mRNA expression of collagen V gene in pulmonary fibrosis of systemic sclerosis.

BACKGROUND: Collagen V shows promise as an inducer of interstitial lung fibrosis in experimental systemic sclerosis (SSc). MATERIALS AND METHODS: Remodelling of the pulmonary interstitium was evaluated based on the clinical data and open lung biopsies from 15 patients with SSc. Normal lung tissues obtained from eight individuals who died of traumatic injuries were used as control group. Immunofluorescence, immunohistochemistry, morphometry, tri-dimensional reconstruction and a real-time polymerase chain reaction were used to evaluate the quantity, structure and molecular chains of collagen V. The impact of these markers was tested on clinical data. RESULTS: The main difference in collagen V content between SSc patients and the control group was an increased, abnormal and distorted fibre deposition in the alveolar septa and the pre-acinar artery wall. The lungs from SSc patients presented [alpha1(V)] and [alpha2(V)] mRNA chain expression increased, but [alpha2(V)] was proportionally increased compared with the control group. High levels of collagen V were inversely associated with vital capacity (r = -0.72; P = 0.002), forced vital capacity (r = -0.76; P < 0.001), forced expiratory volume in 1-s (r = -0.89; P < 0.001) and diffusing capacity for carbon monoxide (r = -0.62; P = 0.04). CONCLUSIONS: Abnormal collagen V fibres are overproduced in lungs from SSc patients and may play an important role in the pathogenesis of the disease as this molecule regulates tissue collagen assembly. The aberrant histoarchitecture observed in SSc can be related to the overexpression of the [alpha2(V)] gene of unknown origin.

Thallphytic alleopathy: isolation and identification of laetisaric Acid.

Laetisaria arvalis, a soil-dwelling basidiomycete fungus, secretes an allelopathic agent that induces rapid hyphal lysis in several phytopathogenic fungi. The active compound was isolated from chloroform:methanol extracts of L. arvalis mycelia and characterized as a previously unknown hydroxy fatty acid, (Z,Z-9,12-8-hydroxyoctadecadienoic acid.

CikA, a bacteriophytochrome that resets the cyanobacterial circadian clock.

The circadian oscillator of the cyanobacterium Synechococcus elongatus, like those in eukaryotes, is entrained by environmental cues. Inactivation of the gene cikA (circadian input kinase) shortens the circadian period of gene expression rhythms in S. elongatus by approximately 2 hours, changes the phasing of a subset of rhythms, and nearly abolishes resetting of phase by a pulse of darkness. The CikA protein sequence reveals that it is a divergent bacteriophytochrome with characteristic histidine protein kinase motifs and a cryptic response regulator motif. CikA is likely a key component of a pathway that provides environmental input to the circadian oscillator in S. elongatus.

DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells.

Although autophagy enhances cell survival in nutrient-deprived cells by increasing adenosine triphosphate (ATP) production, it remains unclear if autophagy functions similarly in cells treated with cytotoxic chemotherapy agents. To address this issue, we measured both the ability of DNA damaging agents (Temozolomide, and Etoposide) to induce an autophagy-dependent production of ATP, and the effects of modulation of autophagy on drug-induced cell death. Both drugs induced an autophagy-associated increase in ATP production in multiple glioma cell lines. The drug-induced ATP surge could not be blocked by glucose starvation, but could be blocked by preincubation with the autophagy inhibitor 3-methyladenine (3-MA), an siRNA targeting beclin 1, or the mitochondrial inhibitor oligomycin. Inhibition of autophagy-induced ATP production increased non-apoptotic cell death associated with micronucleation, while restoration of the 3-MA-inhibited ATP surge by addition of pyruvate suppressed cell death. These results show that DNA damaging agents induce an autophagy-associated ATP surge that protects cells and may contribute to drug resistance.

Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow.

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family (HP1Hs alpha, HP1Hs beta and HP1Hs gamma), which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1Hs alpha, HP1Hs beta and HP1Hs gamma was determined by realtime RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow.

Tamoxifen inhibits transforming growth factor-alpha gene expression in human breast carcinoma samples treated with triiodothyronine.

OBJECTIVES: To examine the effects of triiodothyronine (T3), 17beta-estradiol (E2), and tamoxifen (TAM) on transforming growth factor (TGF)-alpha gene expression in primary breast cancer cell cultures and interactions between the different treatments. METHODS AND RESULTS: Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3- mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T3; dish 3: T3+TAM; dish 4: TAM; dish 5: E2; dish 6: E2+TAM. TGF-alpha mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T3 for 48 h significantly increased TGF-alpha mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-alpha mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities. CONCLUSION: We demonstrate that TGF-alpha mRNA expression is more efficiently upregulated by T3 than E2. Concomitant treatment with TAM had a mitigating effect on the T3 effect, while E2 induced TGF-alpha upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-alpha, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER alpha and beta; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E2.

Gadolinium-enhanced three-dimensional magnetization-prepared rapid gradient-echo (3D MP-RAGE) imaging is superior to spin-echo imaging in delineating brain metastases.

BACKGROUND: Precisely defining the number and location of brain metastases is very important for establishing a treatment strategy for malignancies. Although magnetic resonance imaging (MRI) is now considered the best modality, various improvements in sequences are still being made. PURPOSE: To prospectively compare the diagnostic ability of three-dimensional, magnetization-prepared rapid gradient-echo (3D MP-RAGE) imaging in detecting metastatic brain tumors, with that of two-dimensional spin-echo (2D SE) T1-weighted imaging. MATERIAL AND METHODS: A total of 123 examinations were included in this study, and 119 examinations from 88 patients with known malignancies were analyzed. All patients underwent T1- and T2-weighted 2D SE transverse imaging, followed by gadolinium-enhanced T1-weighted transverse and coronal 2D SE imaging and 3D MP-RAGE transverse imaging. Four radiologists interpreted the images to compare the accuracy and the time required for interpretation for each imaging. RESULTS: 3D MP-RAGE imaging was significantly better than 2D SE imaging for detecting metastatic brain lesions, regardless of the readers' experience. The sensitivities of the 3D MP-RAGE and 2D SE imaging for all observers were 0.81 vs. 0.80 (P>0.05), specificities were 0.93 vs. 0.87 (P<0.05), positive predictive values were 0.92 vs. 0.86 (P<0.05), negative predictive values were 0.78 vs. 0.75 (P>0.05), and accuracies were 0.84 vs. 0.78 (P<0.05), respectively. There was no significant difference in the time required for image interpretation between the two modalities (15.6+/-4.0 vs. 15.4+/-4.1 min). CONCLUSION: 3D MP-RAGE imaging proved superior to 2D SE imaging in the detection of brain metastases.

Modification of the hepatic mitochondrial proteome in response to ischemic preconditioning following ischemia-reperfusion injury of the rat liver.

BACKGROUND/AIM: Ischemic preconditioning (IPC) may reduce hepatic ischemia-reperfusion (IR) injury, but efficacy of IPC on mitochondrial proteome is not demonstrated. We investigated how IPC modifies the mitochondrial proteome after IR injury. METHODS: Rats were subjected to 25 min of portal triad crossclamping (IR group, n = 8). In the IPC group (n = 8), 10 min of temporal portal triad clamping was performed before 25 min of portal clamping. Samples were obtained after 24 h. The mitochondrial inner-membrane potential was measured by the uptake of a lipophilic cationic carbocyanine probe and mitochondrial proteome was also investigated using 2-dimensional differential in-gel electrophoresis and liquid chromatography-tandem mass spectrometry. RESULTS: Mitochondrial inner-membrane potential and glutathione were lower and serum transaminase was higher in the IPC group than in the IR group. The mitochondrial precursor of aldehyde dehydrogenase 2 and alpha-methylacyl-CoA-racemase were upregulated in the IPC group in comparison to the IR group. In contrast, protein disulfide-isomerase A3 precursor, 60S acid ribosomal protein P0, carbonic anhydrase 3 and superoxide dismutase were significantly more downregulated in the IPC group than in the IR group. CONCLUSIONS: A hepatoprotective effect by IPC was not shown; however, IPC caused significant up- or downregulation of several mitochondrial proteins.

Measurement of radical-scavenging ability in small bowel ischemia/reperfusion injury in rats using an in vivo and ex vivo electron paramagnetic resonance technique.

Reperfusion of ischemic tissues results in the formation of toxic reactive oxygen species (ROS), such as superoxide anion, hydroxyl radicals, hydroperoxide, and peroxynitrite. ROS are potent oxidizing agents, fully capable of damaging cellular membranes by lipid peroxidation. In this study, we applied for the first time the in vivo electron paramagnetic resonance (EPR)/spin probe and ex vivo EPR technique to provide direct evidence of ROS following experimentally induced small bowel ischemia/reperfusion (I/R) injury. The decay rate (spin clearance rate) was determined over the first 3 minutes at 6 hours after reperfusion. Decay rates in rats subjected to I/R injury were lower than those in the sham group. Superoxide scavenging activity (SSA) in rats subjected to I/R injury was significantly lower than that in the sham group. In conclusion, this study demonstrated that it is possible to detect the accumulation of ROS following experimentally induced small intestine I/R injury using an in vivo and an ex vivo EPR technique with a spin probe.

Real-time monitoring of nitric oxide (NO) and pO2 levels under ischemic conditions associated with small bowel ischemia/reperfusion injury using selective electrodes for NO and oxygen molecules.

The present study demonstrated the feasibility of monitoring nitric oxide (NO) and pO2 levels under ischemic conditions associated with small bowel ischemia/reperfusion (I/R) injury through the use of selective electrodes for NO and oxygen molecules. NO levels gradually increased during ischemia. When reperfusion was started, the NO level decreased suddenly and returned to pre-ischemia values within 10 minutes. After clamping, pO2 decreased rapidly. When reperfusion was started, pO2 increased suddenly, returning to pre-ischemia values within 10 minutes. We concluded that it is feasible to monitor NO and pO2 levels under ischemic conditions of small bowel I/R injury through the use of electrodes selective for NO and oxygen molecules.

High-yield synthesis of conductive carbon nanotube tips for multiprobe scanning tunneling microscope.

We have established a fabrication process for conductive carbon nanotube (CNT) tips for multiprobe scanning tunneling microscope (STM) with high yield. This was achieved, first, by attaching a CNT at the apex of a supporting W tip by a dielectrophoresis method, second, by reinforcing the adhesion between the CNT and the W tip by electron beam deposition of hydrocarbon and subsequent heating, and finally by wholly coating it with a thin metal layer by pulsed laser deposition. More than 90% of the CNT tips survived after long-distance transportation in air, indicating the practical durability of the CNT tips. The shape of the CNT tip did not change even after making contact with another metal tip more than 100 times repeatedly, which evidenced its mechanical robustness. We exploited the CNT tips for the electronic transport measurement by a four-terminal method in a multiprobe STM, in which the PtIr-coated CNT portion of the tip exhibited diffusive transport with a low resistivity of 1.8 kOmega/microm. The contact resistance at the junction between the CNT and the supporting W tip was estimated to be less than 0.7 kOmega. We confirmed that the PtIr thin layer remained at the CNT-W junction portion after excess current passed through, although the PtIr layer was peeled off on the CNT to aggregate into particles, which was likely due to electromigration or a thermally activated diffusion process. These results indicate that the CNT tips fabricated by our recipe possess high reliability and reproducibility sufficient for multiprobe STM measurements.

Hydrogen sulfide donor micelles protect cardiomyocytes from ischemic cell death.

Hydrogen sulfide, an important gaseous signaling molecule in the human body, is known to protect cardiomyocytes from ischemia, a condition characterized by insufficient oxygen supply to the cells. Here we show that a nanosized H2S donor micelle releases H2S intracellularly and prevents cardiomyocyte apoptosis in an in vitro ischemia model.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer.

OBJECTIVES: Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. METHODS: Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 µg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. RESULTS: Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. CONCLUSIONS: Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Gene expression profiling of clinical stages II and III breast cancer.

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.