Immune modulatory effects of tulathromycin, gamithromycin, and oxytetracycline in cattle.

Certain classes of antibiotics, including tetracyclines and macrolides, are known to exert immune suppressive effects in other species but the immune modulatory effects of these antibiotics have not been previously studied in cattle. To address this question, we investigated the effects of oxytetracycline, gamithromycin, and tulathromycin on T cell and macrophage responses to activation, using in vitro assays. In addition, we assessed the impact of these antibiotics on T cell responses in vivo following treatment of healthy cattle with currently recommended doses of each of the three antibiotics. We found that all 3 antibiotics markedly suppressed T cell proliferation in vitro at relevant therapeutic drug concentrations and significantly suppressed macrophage activation responses to LPS. In cattle treated with a single dose of each antibiotic, we observed significant suppression of T cell proliferation and cytokine production beginning as early as 6 h after administration, with increasing immune suppression observed at 48 h. Taken together, these results indicate that commonly used antibiotics in cattle exert significant immune modulatory activity, in addition to their antimicrobial activity. These off-target effects should be considered when using antibiotics for prophylaxis or metaphylaxis in high-risk dairy or beef cattle (192 words).

GLUT4 localisation with the plasma membrane is unaffected by an increase in plasma free fatty acid availability.

BACKGROUND: Insulin-stimulated glucose uptake into skeletal muscle occurs via translocation of GLUT4 from intracellular storage vesicles to the plasma membrane. Elevated free fatty acid (FFA) availability via a lipid infusion reduces glucose disposal, but this occurs in the absence of impaired proximal insulin signalling. Whether GLUT4 localisation to the plasma membrane is subsequently affected by elevated FFA availability is not known. METHODS: Trained (n = 11) and sedentary (n = 10) individuals, matched for age, sex and body mass index, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic-euglycaemic clamp. Sequential muscle biopsies (0, 2 and 6 h) were analysed for GLUT4 membrane localisation and microvesicle size and distribution using immunofluorescence microscopy. RESULTS: At baseline, trained individuals had more small GLUT4 spots at the plasma membrane, whereas sedentary individuals had larger GLUT4 spots. GLUT4 localisation with the plasma membrane increased at 2 h (P = 0.04) of the hyperinsulinemic-euglycemic clamp, and remained elevated until 6 h, with no differences between groups or infusion type. The number of GLUT4 spots was unchanged at 2 h of infusion. However, from 2 to 6 h there was a decrease in the number of small GLUT4 spots at the plasma membrane (P = 0.047), with no differences between groups or infusion type. CONCLUSION: GLUT4 localisation with the plasma membrane increases during a hyperinsulinemic-euglycemic clamp, but this is not altered by elevated FFA availability. GLUT4 appears to disperse from small GLUT4 clusters located at the plasma membrane to support glucose uptake during a hyperinsulinaemic-euglycaemic clamp.

Iron burden and endocrine complications in transfusion-dependent thalassemia patients In Sarawak, Malaysia: a retrospective study.

INTRODUCTION: Thalassaemia is one of the major health problems in Malaysia. With safe blood transfusion regime, the lifespan of patients with transfusion-dependent thalassaemia (TDT) has improved but at the cost of a higher risk of developing endocrine disorders. It is crucial for us to monitor the iron overload to prevent end organ damage. This study aims to evaluate the iron burden and prevalence of endocrinopathies in patients with TDT in Sarawak. MATERIALS AND METHODS: This retrospective cohort study was conducted between January 2020 to June 2020 in six government hospitals in Sarawak. A total of 89 patients with TDT, aged 10 years and above, were recruited. RESULTS: Out of the 89 patients, there were 54 males (60.7%) and 35 females (39.3%) with a median age of 21 years (range 10.0-65.0). Sixty-seven (75.3%) patients had betathalassaemia major and 15 (16.9%) patients had haemoglobin E beta-thalassaemia (HbE beta-thalassaemia), remaining seven patients had other genotypes. Thirty-one (34.8%) patients had mean serum ferritin 2500ng/ml and above, and 44 (66.6%) had liver iron concentration (LIC) ≥7mg/g. The prevalence of endocrine disorders in our cohort was 69.7%. The most common endocrinopathies were short stature (n=46, 51.7%), followed by hypogonadism (n=24, 26.9%), delayed puberty (n=23, 25.8%), hypothyroidism (n=10, 11.2%), diabetes mellitus (n=9, 10.1%), impaired glucose tolerance (n=6, 6.7%) and hypoparathyroidism (n=3, 3.3%). Endocrinopathies were significantly associated with age (p=0.01), age at initiating regular blood transfusion (p<0.01) and duration of regular blood transfusion (p<0.01). CONCLUSION: Our data shows that the development of endocrinopathies in TDT can be time dependent. Early detection of endocrine-related complications and prompt treatment with iron chelation therapy are important to improve morbidity and mortality. A multidisciplinary approach with good patient-doctor collaboration is the key to improving patient care in our settings.

Charge and Spin Order Dichotomy in NdNiO_{2} Driven by the Capping Layer.

Superconductivity in infinite-layer nickelates holds exciting analogies with that of cuprates, with similar structures and 3d-electron count. Using resonant inelastic x-ray scattering, we studied electronic and magnetic excitations and charge density correlations in Nd_{1-x}Sr_{x}NiO_{2} thin films with and without an SrTiO_{3} capping layer. We observe dispersing magnons only in the capped samples, progressively dampened at higher doping. We detect an elastic resonant scattering peak in the uncapped x=0 compound at wave vector (∼⅓,0), remindful of the charge order signal in hole doped cuprates. The peak weakens at x=0.05 and disappears in the superconducting x=0.20 film. The role of the capping on the electronic reconstruction far from the interface remains to be understood.

Phase Ib study of atezolizumab combined with cobimetinib in patients with solid tumors.

BACKGROUND: Preclinical evidence suggests that MEK inhibition promotes accumulation and survival of intratumoral tumor-specific T cells and can synergize with immune checkpoint inhibition. We investigated the safety and clinical activity of combining a MEK inhibitor, cobimetinib, and a programmed cell death 1 ligand 1 (PD-L1) inhibitor, atezolizumab, in patients with solid tumors. PATIENTS AND METHODS: This phase I/Ib study treated PD-L1/PD-1-naive patients with solid tumors in a dose-escalation stage and then in multiple, indication-specific dose-expansion cohorts. In most patients, cobimetinib was dosed once daily orally for 21 days on, 7 days off. Atezolizumab was dosed at 800 mg intravenously every 2 weeks. The primary objectives were safety and tolerability. Secondary end points included objective response rate, progression-free survival, and overall survival. RESULTS: Between 27 December 2013 and 9 May 2016, 152 patients were enrolled. As of 4 September 2017, 150 patients received ≥1 dose of atezolizumab, including 14 in the dose-escalation cohorts and 136 in the dose-expansion cohorts. Patients had metastatic colorectal cancer (mCRC; n = 84), melanoma (n = 22), non-small-cell lung cancer (NSCLC; n = 28), and other solid tumors (n = 16). The most common all-grade treatment-related adverse events (AEs) were diarrhea (67%), rash (48%), and fatigue (40%), similar to those with single-agent cobimetinib and atezolizumab. One (<1%) treatment-related grade 5 AE occurred (sepsis). Forty-five (30%) and 23 patients (15%) had AEs that led to discontinuation of cobimetinib and atezolizumab, respectively. Confirmed responses were observed in 7 of 84 patients (8%) with mCRC (6 responders were microsatellite low/stable, 1 was microsatellite instable), 9 of 22 patients (41%) with melanoma, and 5 of 28 patients (18%) with NSCLC. Clinical activity was independent of KRAS/BRAF status across diseases. CONCLUSIONS: Atezolizumab plus cobimetinib had manageable safety and clinical activity irrespective of KRAS/BRAF status. Although potential synergistic activity was seen in mCRC, this was not confirmed in a subsequent phase III study. CLINICALTRIALS.GOV IDENTIFIER: NCT01988896 (the investigators in the NCT01988896 study are listed in the supplementary Appendix, available at Annals of Oncology online).

Nivolumab versus docetaxel in previously treated advanced non-small-cell lung cancer (CheckMate 017 and CheckMate 057): 3-year update and outcomes in patients with liver metastases.

Background: Long-term data with immune checkpoint inhibitors in non-small-cell lung cancer (NSCLC) are limited. Two phase III trials demonstrated improved overall survival (OS) and a favorable safety profile with the anti-programmed death-1 antibody nivolumab versus docetaxel in patients with previously treated advanced squamous (CheckMate 017) and nonsquamous (CheckMate 057) NSCLC. We report results from ≥3 years' follow-up, including subgroup analyses of patients with liver metastases, who historically have poorer prognosis among patients with NSCLC. Patients and methods: Patients were randomized 1 : 1 to nivolumab (3 mg/kg every 2 weeks) or docetaxel (75 mg/m2 every 3 weeks) until progression or discontinuation. The primary end point of each study was OS. Patients with baseline liver metastases were pooled across studies by treatment for subgroup analyses. Results: After 40.3 months' minimum follow-up in CheckMate 017 and 057, nivolumab continued to show an OS benefit versus docetaxel: estimated 3-year OS rates were 17% [95% confidence interval (CI), 14% to 21%] versus 8% (95% CI, 6% to 11%) in the pooled population with squamous or nonsquamous NSCLC. Nivolumab was generally well tolerated, with no new safety concerns identified. Of 854 randomized patients across both studies, 193 had baseline liver metastases. Nivolumab resulted in improved OS compared with docetaxel in patients with liver metastases (hazard ratio, 0.68; 95% CI, 0.50-0.91), consistent with findings from the overall pooled study population (hazard ratio, 0.70; 95% CI, 0.61-0.81). Rates of treatment-related hepatic adverse events (primarily grade 1-2 liver enzyme elevations) were slightly higher in nivolumab-treated patients with liver metastases (10%) than in the overall pooled population (6%). Conclusions: After 3 years' minimum follow-up, nivolumab continued to demonstrate an OS benefit versus docetaxel in patients with advanced NSCLC. Similarly, nivolumab demonstrated an OS benefit versus docetaxel in patients with liver metastases, and remained well tolerated. Clinical trial registration: CheckMate 017: NCT01642004; CheckMate 057: NCT01673867.

Training alters the distribution of perilipin proteins in muscle following acute free fatty acid exposure.

KEY POINTS: The lipid droplet (LD)-associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, although whether the abundance and association of the PLIN proteins with LDs is related to the diverse lipid storage in muscle between trained and sedentary individuals is unknown. We show that lipid infusion augments IMTG content in type I fibres of both trained and sedentary individuals. Most importantly, despite there being no change in PLIN protein content, lipid infusion did increase the number of LDs connected with PLIN proteins in trained individuals only. We conclude that trained individuals are able to redistribute the pre-existing pool of PLIN proteins to an expanded LD pool during lipid infusion and, via this adaptation, may support the storage of fatty acids in IMTG. ABSTRACT: Because the lipid droplet (LD)-associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, we investigated the hypothesis that differential protein content of PLINs and their distribution with LDs may be linked to the diverse lipid storage in muscle between trained and sedentary individuals. Trained (n = 11) and sedentary (n = 10) subjects, matched for age, sex and body mass index, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic-euglycaemic clamp. Sequential muscle biopsies (0, 2 and 6 h) were analysed using confocal immunofluorescence microscopy for fibre type-specific IMTG content and PLIN associations with LDs. In both groups, lipid infusion increased IMTG content in type I fibres (trained: +62%, sedentary: +79%; P < 0.05) but did not affect PLIN protein content. At baseline, PLIN2 (+65%), PLIN3 (+105%) and PLIN5 (+53%; all P < 0.05) protein content was higher in trained compared to sedentary individuals. In trained individuals, lipid infusion increased the number of LDs associated with PLIN2 (+27%), PLIN3 (+73%) and PLIN5 (+40%; all P < 0.05) in type I fibres. By contrast, in sedentary individuals, lipid infusion only increased the number of LDs not associated with PLIN proteins. Acute free fatty acid elevation therefore induces a redistribution of PLIN proteins to an expanded LD pool in trained individuals only and this may be part of the mechanism that enables fatty acids to be stored in IMTG.

Focal nodular and diffuse haematopoietic marrow hyperplasia in patients with underlying malignancies: a radiological mimic of malignancy in need of recognition.

AIM: To report the authors' experience of focal nodular haematopoietic marrow hyperplasia (FNHMH) and diffuse haematopoietic marrow hyperplasia (DHMH) clinically masquerading as skip, distant, or disseminated metastasis in seven patients with underlying malignant neoplasms. MATERIALS AND METHODS: Five patients with FNHMH and two with DHMH mistaken radiologically as skip and disseminated metastasis, respectively, were compared and contrasted with four patients with osteosarcomas and two with chondrosarcomas harbouring skip metastasis, noting the temporal relationship with their haematological profile. RESULTS: FNHMH and DHMH were undetectable by plain radiography and computed tomography (CT) except one showing subtle sclerosis on CT. They showed either isointense or hyperintense, but not hypointense, attenuation at T1-weighted imaging, and all showed hyperintense attenuation at T2-weighted MRI relative to skeletal muscle. Of the five patients who underwent bone scintigraphy, one showed mildly increased uptake, and one out of two showed markedly increased 2-[18F]-fluoro-2-deoxy-d-glucose (FDG)-positron-emission tomography (PET) uptake. The rates for sarcoma skip metastasis by plain radiography, CT, MRI, and bone scintigraphy were 40%, 66.7%, 100%, and 66.7%, respectively. At MRI, 60% showed hypointense and 40% isointense attenuation at T1-weighted, 80% hyperintense and 20% hypointense attenuation at T2-weighted imaging. Combined FDG-PET and CT, which was performed in only one patient, failed to show the skip metastasis. Not every patient with FNHMH or DHMH received granulocyte colony-stimulating factor (GCSF), but all had low or falling haemoglobin levels, which may thus be the prime cause for HMH. CONCLUSIONS: Due to overlapping radiological features, FNHMH and DHMH are great radiological mimics of malignancy. In some cases, needle biopsy is required for their definitive differentiation.

Biomarkers associated with severe hypoglycaemia and death in ACCORD.

AIMS AND HYPOTHESIS: In patients with Type 2 diabetes, intensive glycaemic control is associated with hypoglycaemia and possibly increased mortality. However, no blood biomarkers exist to predict these outcomes. Using participants from the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study, we hypothesized that insulin deficiency and islet autoantibodies in patients with clinically diagnosed Type 2 diabetes would be associated with severe hypoglycaemia and death. METHODS: A nested case-control study design was used. A case (n = 86) was a participant who died with at least one episode of severe hypoglycaemia, defined as hypoglycaemia requiring assistance, at any point during ACCORD follow-up. A control (n = 344) was a participant who did not die and did not have severe hypoglycaemia during follow-up. Each case was matched to four controls (glycaemic intervention arm, race, age and BMI). Baseline insulin deficiency (fasting C-peptide ≤ 0.15 nmol/l) and islet autoantibodies [glutamic acid decarboxylase (GAD), tyrosine phosphatase-related islet antigen 2 (IA2), insulin (IAA) and zinc transporter (ZnT8)] were measured. Conditional logistic regression with and without adjustment for age, BMI and diabetes duration was used. RESULTS: Death during ACCORD in those who experienced at least one episode of severe hypoglycaemia was associated with insulin deficiency [OR 4.8 (2.1, 11.1): P < 0.0001], GAD antibodies [OR 2.3 (1.1, 5.1): P = 0.04], the presence of IAA or baseline insulin use [OR 6.1 (3.5,10.7): P < 0.0001], which remained significant after adjusting for age, BMI, and diabetes duration. There was no significant association with IA2 or ZnT8 antibodies. CONCLUSIONS: In patients with Type 2 diabetes, C-peptide or GAD antibodies may serve as blood biomarkers predicting higher odds of subsequent severe hypoglycaemia and death. (Clinical Trial Registry No: NCT00000620, www.clinicaltrials.gov for original ACCORD study).

A calcium prerinse required to form calcium fluoride in plaque from a sodium fluoride rinse.

The purpose of this study is to determine whether a calcium (Ca) prerinse used before a 228 µg/g (ppm) fluoride (F) rinse would induce the formation of 'calcium fluoride-like' (CaF2-like) deposits in human dental plaque. Sixty minutes after the use of the Ca prerinse/F rinse, plaque samples were collected from 10 volunteers, homogenized, and split into 2 aliquots. The plaque mass from one aliquot was then extracted with a 'plaque-like' solution that extracted all the CaF2-like deposits. The total F in both aliquots was then determined and compared. The results demonstrated that, as in previous studies, the Ca prerinse induced large increases in plaque fluid and total plaque F. However, unlike previous results without the Ca prerinse, 30% of the plaque F deposits were CaF2 or CaF2-like. Given that maintaining an elevated F concentration in the vicinity of a developing lesion may play an important role in the cariostatic effect of this ion, and the potential advantages of CaF2-like deposits as an F source, these results suggest that a Ca prerinse may increase the cariostatic effect of topical agents.

Combination neratinib (HKI-272) and paclitaxel therapy in patients with HER2-positive metastatic breast cancer.

INTRODUCTION: Neratinib is a potent irreversible pan-ErbB tyrosine kinase inhibitor that has demonstrated antitumour activity and an acceptable safety profile in patients with human epidermal growth factor receptor (HER)-2-positive breast cancer and other solid tumours. METHODS: This was a phase I/II, open-label, two-part study. Part 1 was a dose-escalation study to determine the maximum tolerated dose (MTD) of neratinib plus paclitaxel in patients with solid tumours. Part 2 evaluated the safety, efficacy, and pharmacokinetics of the combination at the MTD in patients with HER2-positive breast cancer. RESULTS: Eight patients were included in the dose-escalation study; no dose-limiting toxicities were observed, and an MTD of oral neratinib 240 mg once daily plus intravenous paclitaxel 80 mg m(-2) on days 1, 8, and 15 of each 28-day cycle was determined. A total of 102 patients with HER2-positive breast cancer were enrolled in part 2. The overall median treatment duration was 47.9 weeks (range: 0.1-147.3 weeks). Common treatment-emergent adverse events (all grades/grade ≥3) included diarrhoea (92%/29%; none grade 4), peripheral sensory neuropathy (51%/3%), neutropenia (50%/20%), alopecia (46%/0%), leukopenia (41%/18%), anaemia (37%/8%), and nausea (34%/1%). Three (3%) patients discontinued treatment due to an adverse event (mouth ulceration, left ventricular ejection fraction reduction, and acute renal failure). Among the 99 evaluable patients in part 2 of the study, the overall response rate (ORR) was 73% (95% confidence interval (CI): 62.9-81.2%), including 7 (7%) patients who achieved a complete response; an additional 9 (9%) patients achieved stable disease for at least 24 weeks. ORR was 71% among patients with 0/1 prior chemotherapy regimen for metastatic disease and no prior lapatinib, and 77% among those with 2/3 prior chemotherapy regimens for metastatic disease with prior lapatinib permitted. Kaplan-Meier median progression-free survival was 57.0 weeks (95% CI: 47.7-81.6 weeks). Pharmacokinetic analyses indicated no interaction between neratinib and paclitaxel. CONCLUSION: The combination of neratinib and paclitaxel was associated with higher toxicity than that of neratinib as a single agent, but was manageable with antidiarrhoeal agents and dose reductions in general. The combination therapy also demonstrated a high rate of response in patients with HER2-positive breast cancer. A phase III trial is ongoing to assess the benefit and risk of this combination in the first-line setting.

Randomized trial of preoperative docetaxel with or without capecitabine after 4 cycles of 5-fluorouracil­ epirubicin­cyclophosphamide (FEC) in early-stage breast cancer: exploratory analyses identify Ki67 as a predictive biomarker for response to neoadjuvant chemotherapy.

This randomized, multicenter study compared the efficacy of docetaxel with or without capecitabine following fluorouracil/epirubicin/cyclophosphamide (FEC) therapy in operable breast cancer and investigated the role of Ki67 as a predictive biomarker. Patients were randomized to 4 cycles of docetaxel/capecitabine (docetaxel: 75 mg/m2 on day 1; capecitabine: 1,650 mg/m2 on days 1­14 every 3 weeks) or docetaxel alone (75 mg/m2 on day 1 every 3 weeks) after completion of 4 cycles of FEC (5-fluorouracil 500 mg/m2, epirubicin 100 mg/m2 and cyclophosphamide 500 mg/m2 on day 1 every 3 weeks). The primary endpoint was the pathological complete response (pCR) rate. Predictive factor analysis was conducted using clinicopathological markers, including hormone receptors and Ki67 labeling index (Ki67LI). A total of 477 patients were randomized; the overall response in the docetaxel/capecitabine and docetaxel groups was 88.3 and 87.4 %, respectively. There were no significant differences in the pCR rate (docetaxel/capecitabine: 23 %; docetaxel: 24 %; p = 0.748), disease-free survival, or overall survival. However, patients with mid-range Ki67LI (10­20 %) showed a trend towards improved pCR rate with docetaxel/capecitabine compared to docetaxel alone. Furthermore, multivariate logistic regression analysis showed pre-treatment Ki67LI (odds ratio 1.031; 95 % CI 1.014­1.048; p = 0.0004) to be a significant predictor of pCR in this neoadjuvant treatment setting. Docetaxel/capecitabine (after 4 cycles of FEC) did not generate significant improvement in pCR compared to docetaxel alone. However, exploratory analyses suggested that assessment of pre-treatment Ki67LI may be a useful tool in the identification of responders to preoperative docetaxel/capecitabine in early-stage breast cancer.

Predictions of the pathological response to neoadjuvant chemotherapy in patients with primary breast cancer using a data mining technique.

Nomogram, a standard technique that utilizes multiple characteristics to predict efficacy of treatment and likelihood of a specific status of an individual patient, has been used for prediction of response to neoadjuvant chemotherapy (NAC) in breast cancer patients. The aim of this study was to develop a novel computational technique to predict the pathological complete response (pCR) to NAC in primary breast cancer patients. A mathematical model using alternating decision trees, an epigone of decision tree, was developed using 28 clinicopathological variables that were retrospectively collected from patients treated with NAC (n = 150), and validated using an independent dataset from a randomized controlled trial (n = 173). The model selected 15 variables to predict the pCR with yielding area under the receiver operating characteristics curve (AUC) values of 0.766 [95 % confidence interval (CI)], 0.671-0.861, P value < 0.0001) in cross-validation using training dataset and 0.787 (95 % CI 0.716-0.858, P value < 0.0001) in the validation dataset. Among three subtypes of breast cancer, the luminal subgroup showed the best discrimination (AUC = 0.779, 95 % CI 0.641-0.917, P value = 0.0059). The developed model (AUC = 0.805, 95 % CI 0.716-0.894, P value < 0.0001) outperformed multivariate logistic regression (AUC = 0.754, 95 % CI 0.651-0.858, P value = 0.00019) of validation datasets without missing values (n = 127). Several analyses, e.g. bootstrap analysis, revealed that the developed model was insensitive to missing values and also tolerant to distribution bias among the datasets. Our model based on clinicopathological variables showed high predictive ability for pCR. This model might improve the prediction of the response to NAC in primary breast cancer patients.

Observation of perfect diamagnetism and interfacial effect on the electronic structures in infinite layer Nd0.8Sr0.2NiO2 superconductors.

Nickel-based complex oxides have served as a playground for decades in the quest for a copper-oxide analog of the high-temperature superconductivity. They may provide clues towards understanding the mechanism and an alternative route for high-temperature superconductors. The recent discovery of superconductivity in the infinite-layer nickelate thin films has fulfilled this pursuit. However, material synthesis remains challenging, direct demonstration of perfect diamagnetism is still missing, and understanding of the role of the interface and bulk to the superconducting properties is still lacking. Here, we show high-quality Nd0.8Sr0.2NiO2 thin films with different thicknesses and demonstrate the interface and strain effects on the electrical, magnetic and optical properties. Perfect diamagnetism is achieved, confirming the occurrence of superconductivity in the films. Unlike the thick films in which the normal-state Hall-coefficient changes signs as the temperature decreases, the Hall-coefficient of films thinner than 5.5 nm remains negative, suggesting a thickness-driven band structure modification. Moreover, X-ray absorption spectroscopy reveals the Ni-O hybridization nature in doped infinite-layer nickelates, and the hybridization is enhanced as the thickness decreases. Consistent with band structure calculations on the nickelate/SrTiO3 heterostructure, the interface and strain effect induce a dominating electron-like band in the ultrathin film, thus causing the sign-change of the Hall-coefficient.

Genetic landscape of patients with ALK-rearranged non-small-cell lung cancer (NSCLC) and response to ceritinib in ASCEND-1 study.

OBJECTIVES: To better understand genetic determinants of response to ceritinib, an exploratory analysis was conducted using tumor biopsies from anaplastic lymphoma kinase (ALK)-rearranged (ALK+) non-small-cell lung cancer (NSCLC) patients treated with ceritinib at doses of ≥ 300 mg in the ASCEND-1 study. METHODS: ASCEND-1 was an open-label, multicentre, phase 1, dose-escalation and expansion study of ceritinib (fasted) in ALK inhibitor (ALKi)-naïve or ALKi-pretreated patients with locally advanced or metastatic ALK + NSCLC. Biopsies were assayed by next-generation sequencing (NGS) using a Foundation Medicine panel targeting 295 genes. Somatic alterations were correlated with clinical outcome (cut-off 14-Apr-2014). A total of 285 ALK + NSCLC patients were treated with ceritinib at doses ≥ 300 mg. RESULTS: NGS data were generated for 85 pts (ALKi-pretreated [n = 54]; ALKi-naïve [n = 31]), 57 were collected from patients before exposure to any ALKi. NGS did not detect ALK rearrangement in 14 of 85 patients; several of these ALK NGS negative cases harbored alternative drivers, e.g. EGFR mutation. Of the 71 biopsies with NGS confirmed ALK rearrangement, the most frequently detected rearrangements were EML4-ALK variant 1 (V1) and EML4-ALK V3 (36.6% [26/71] and 32.4% [23/71] respectively). Eight (six crizotinib-pretreated and two pretreated with crizotinib followed by alectinib) of the 21 ALKi-pretreated patients carried a point mutation of the ALK TKD, and had the biopsy collected between 1 and 14 days before ceritinib; with the exception of one patient with a G1202R point mutation, all patients derived clinical benefit from ceritinib treatment. Of the 14 ALKi-naïve patients, ceritinib was effective in almost all patients, including a patient carrying a concomitant ERBB4 and HGF amplification. CONCLUSIONS: This exploratory analysis highlights the potential role of NGS in improving our understanding of response and resistance to ceritinib. It also illustrates that ceritinib is active against almost all ALK resistance mutations found in ALKi-pretreated patients. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01283516. Registered January 26, 2011, https://clinicaltrials.gov/ct2/show/NCT01283516.

Highly efficient multipotent differentiation of human periodontal ligament fibroblasts induced by combined BMP4 and hTERT gene transfer.

Because periodontal ligament (PDL) cells are reported to contain progenitor or stem cell populations, they are considered a beneficial cell source for clinical periodontal regeneration. Both bone morphogenetic protein 4 (BMP4) and human telomerase reverse transcriptase (hTERT) have essential roles in the modulation of stem cell properties. In this study we report for the first time that the combined ectopic expression of BMP4 and hTERT significantly enhanced the multipotent differentiation efficiency and capacity of human PDL fibroblasts (PFs), as shown by osteogenic, adipogenic and neurogenic differentiation in vitro, and cementum/PDL-like tissue regeneration in vivo. These findings may be attributed, at least in part, to the original upregulation of important stem cell markers, such as scleraxis, Stro-1 and CD146, and the extremely lowered threshold for BMP concentration to activate BMP signaling by enhanced basal phosphorylation levels of Smad 1/5/8. In addition, the significantly reduced expression levels of CD146 and CD90 with the presence of Noggin confirms the direct effect of BMP4 on the stem cell-like phenotype of genetically modified PF cells (BT-PFs). Furthermore, BT-PFs exhibited a high neural differentiation capacity (>75%). After transplantation into NOD/SCID mice, genetically modified-PFs generated cementum/PDL-like structures on the surface of the carrier. The multipotency of these modified cells potentially provides an attractive source of stem cells for therapeutic purposes and regenerative medicine.

The axis of interleukin 12 and gamma interferon regulates acute vascular xenogeneic rejection.

Recent advances using transgenic animals or exogenous complement inhibitors have demonstrated prevention of hyperacute rejection of vascularized organs, but not graft loss due to acute vascular rejection. Using various wild-type and cytokine-deficient mice strains, we have examined the mechanisms of acute vascular rejection. C57BL/6 mice deficient in interleukin12 or gamma interferon showed faster acute vascular rejection than did wild-type mice. Furthermore, mice defective in B-cell development showed no acute vascular rejection. These results demonstrate that the axis of interleukin 12 and gamma interferon provides a survival advantage in vascularized xenografts by delaying or preventing acute vascular rejection caused by a B cell-dependent mechanism.

Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn.

Recent observations suggest that the src-related tyrosine protein kinase p59fyn may be involved in antigen-induced T lymphocyte activation. As a result of alternative splicing, p59fyn exists as two isoforms that differ exclusively within a short sequence spanning the end of the Src Homology 2 (SH2) region and the beginning of the tyrosine protein kinase domain. While one p59fyn isoform (fynB) is highly expressed in brain, the alternative product (fynT) is principally found in T lymphocytes. To further understand the role of p59fyn in T cell activation and to test the hypothesis that p59fynT serves a tissue-specific function in T lymphocytes, we have examined the effects of expression of activated versions (tyrosine 528 to phenylalanine 528 mutants) of either form of p59fyn on the physiology of an antigen-specific mouse T cell hybridoma. Our results demonstrated that the two forms of fyn, expressed in equivalent amounts, efficiently enhanced antibody-induced T cell receptor (TCR)-mediated signals. In contrast, only p59fynT increased interleukin 2 production in response to antigen stimulation. This finding implies that the distinct p59fyn isoform expressed in T lymphocytes regulates the coupling of TCR stimulation by antigen/major histocompatibility complex to lymphokine production.

Proteomic identification of membrane proteins regulating antimicrobial peptide resistance in Vibrio parahaemolyticus.

AIMS: To identify proteins regulating antimicrobial peptide (AMP) resistance in Vibrio parahaemolyticus using membrane subproteome analysis. METHODS AND RESULTS: Three synthetic AMPs (Q4, Q6 and H1) and a natural one from fish (pleurocidin) were used for selection of AMP-resistant strains. Differential expression patterns of the outer and inner membrane proteins (OMPs and IMPs) among wild-type and the resistant strains were obtained using two-dimensional gel electrophoresis. Two OMPs (TolC and flagellin) and five IMPs [transcription termination factor (NusA), long-chain fatty acid transport protein (FadL), elongation factor Tu (EF-Tu), ATP synthase F1, alpha subunit (F1-ATPa) and dihydrolipoamide dehydrogenase (DLD)] were identified using LC-ESI-Q-TOF MS/MS and Mascot program. Real-time quantitative polymerase chain reaction was also performed to determine the mRNA expression level of the target genes. All seven membrane proteins except FadL were upregulated in the AMP-resistant clones, both in the translational and transcriptional levels. CONCLUSIONS: Our results suggested that V. parahaemolyticus may obtain their resistance against AMPs through upregulation of the multidrug efflux transporter, effective repair of damaged membranes and prevention of cellular penetration of AMPs. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report describing bacterial AMP resistance mechanism using proteomic methodologies. Elucidating the mechanism could help in the development of more sustainable antimicrobial agents.