A subset of histamine receptor ligands improve thermotolerance of the yeast Saccharomyces cerevisiae.

AIMS: Histamine interacts with the stress response in eukaryotes. This study investigated the effects of antihistamines on the heat shock (HS) response in yeast, thereby exploring their functions in a well-established histamine receptor (H(x) R)-free model. METHODS AND RESULTS: Stress response was evaluated by determining growth and viability of postlogarithmic phase grown yeast cultures after HS at 53°C for 30 min. The effects of H(x) R ligands were investigated following short- and long-term administration. The H(1) R antagonist dimethindene exerted dose-related antifungal actions, whereas the H(2) R antagonist ranitidine failed to elicit any effect. In contrast, the H(3/4) R and H(4) R ligands, thioperamide and JNJ7777120, respectively, induced the thermotolerant phenotype. The circumvention of thermotolerance by cycloheximide and the induction of Hsp70 and Hsp104 expression indicated the contribution of de novo protein synthesis in the adaptive process, likely directed towards alterations in Hsp expression. CONCLUSIONS: The data provide evidence for the differential function of H(x) R ligands in thermotolerance induction in yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: First demonstration of the action of antihistamines in the HS response in yeast. The work supports the potential H(x) R-independent functions of histaminergic compounds in fungal adaptation and stimulates research on the prospect of their exploitation in eukaryotic (patho)physiology.

DNA aptamers against the MUC1 tumour marker: design of aptamer-antibody sandwich ELISA for the early diagnosis of epithelial tumours.

Aptamers are functional molecules able to bind tightly and selectively to disease markers, offering great potential for applications in disease diagnosis and therapy. MUC1 is a well-known tumour marker present in epithelial malignancies and is used in immunotherapeutic and diagnostic approaches. We report the selection of DNA aptamers that bind with high affinity and selectivity an MUC1 recombinant protein containing five repeats of the variable tandem repeat region. Aptamers were selected using the SELEX methodology from an initial library containing a 25-base-long variable region for their ability to bind to the unglycosylated form of the MUC1 protein. After ten rounds of in vitro selection and amplification, more than 90% of the pool of sequences consisted of target-binding molecules, which were cloned, sequenced and found to share no sequence consensus. The binding properties of these aptamers were quantified using ELISA and surface plasmon resonance. The lead aptamer sequence was subsequently used in the design of an aptamer-antibody hybrid sandwich ELISA for the identification and quantification of MUC1 in buffered solutions. Following optimisation of the operating conditions, the resulting enzyme immunoassay displayed an EC50 value of 25 microg/ml, a detection limit of 1 microg/ml and a linear range between 8 and 100 microg/ml for the MUC1 five tandem repeat analyte. In addition, recovery studies performed in buffer conditions resulted in averaged recoveries between 98.2 and 101.7% for all spiked samples, demonstrating the usability of the aptamer as a receptor in microtitre-based assays. Our results aim towards the formation of new diagnostic assays against this tumour marker for the early diagnosis of primary or metastatic disease in breast, bladder and other epithelial tumours.

Infliximab trough concentrations during maintenance therapy are associated with endoscopic and histologic healing in ulcerative colitis.

BACKGROUND: Endoscopic and histologic healing are emerging as new therapeutic goals in ulcerative colitis (UC), as these endpoints are associated with less relapse, hospitalization and colectomy. AIM: To investigate the association of serum infliximab trough concentrations during maintenance therapy with endoscopic or histologic healing in UC. METHODS: In this multi-center retrospective cohort study, we included consecutive patients with moderate-to-severe UC on infliximab maintenance therapy who had an endoscopic evaluation and underwent therapeutic drug monitoring within three months of the colonoscopy, between February 2008 and March 2016. Per event analysis was performed. Endoscopic healing was defined as Mayo endoscopic sub-score of ≤1. Histologic healing was defined as no or only focal mild active inflammation. RESULTS: Seventy colonoscopies from 56 patients were evaluated. Infliximab trough concentrations (median [interquartile range]) were significantly higher in patients with endoscopic (11.3 [7.6-14.5] vs 6.3 [0-9.8] µg/mL, P < .001) or histologic (11.1 [6.7-14.5] vs 6.7 [0-9.9] µg/mL, P = .002) healing, respectively, compared to patients without healing. Receiver-operating characteristic analyses identified infliximab trough concentration thresholds of 7.5 (area under the curve [AUC]: 0.758) and 10.5 (AUC: 0.721) µg/mL to be associated with endoscopic and histologic healing, respectively. Multiple logistic regression analysis identified infliximab trough concentration ≥7.5 (P = .013; odds ratio [OR]: 4.3; 95% confidence intervals [CI]: 1.4-13.3) and ≥10.5 µg/mL (P = .013; OR: 3.8; 95% CI: 1.3-11) as independent factors associated with endoscopic and histologic healing, respectively. CONCLUSIONS: This study demonstrated that infliximab trough concentrations during maintenance therapy are associated with endoscopic and histologic healing in patients with UC.

Effect of the Hsp90 modulators on the heat-shock response in eukaryotic cells.

The possible role of the heat-shock protein 90 (Hsp90) complex on the heat-shock (HS) response in yeast using the Hsp90 inhibitors geldanamycin (GA) and 17-allylamino-17-demethoxygeldanamycin (AAG), and prednisolone and 17beta-estradiol as modulators was investigated. Following long- or short-term administration of the drugs, either alone or in combination, the response was determined as cell viability and growth after exposure to HS. Upon short-term preconditioning, both Hsp90 inhibitors conferred cycloheximide-dependent thermal resistance to the yeast cultures, while upon long-term treatment the induction of thermotolerance was confined only to AAG. Co-administration of prednisolone or 17beta-estradiol failed to significantly alter the response to Hsp90 inhibitors. However, since short-term incubation with prednisolone alone induced thermotolerance, increased the budding cell fraction and tended to reduce the adaptive response to GA, its effect on GA-induced thermotolerance is not yet explained. Generally, GA and AAG showed a comparable short-term action but a different long-term effect on the HS response in yeast; this response was not related to any regulation by prednisolone or 17beta-estradiol (while 17beta-estradiol was unable to modify the response, the action of prednisolone in both the stress response and the cell cycle was equivocal).

Review article: pharmacological aspects of anti-TNF biosimilars in inflammatory bowel diseases.

BACKGROUND: Anti-tumour necrosis factor (anti-TNF) monoclonal antibodies have shown efficacy in inflammatory bowel diseases (IBD). As these therapies lose patent protection, biosimilar versions of the originator products are being developed, such as the infliximab biosimilar CT-P13; however, some uncertainty exists regarding their pharmacology in IBD. AIM: To review the literature on anti-TNF biosimilars focusing on pharmacokinetics, pharmacodynamic properties and comparative effectiveness, related to their use in IBD. METHODS: A PubMed literature search was performed using the following terms individually or in combination: 'biosimilars,' 'CT-P13,' 'Crohn's disease,' 'inflammatory bowel disease,' 'ulcerative colitis,' 'anti-TNFα therapy,' 'infliximab,' 'adalimumab,' 'pharmacokinetics,' 'immunogenicity.' RESULTS: Bioequivalence of CT-P13 and infliximab was shown in ankylosing spondylitis (AS) and therapeutic equivalence in rheumatoid arthritis (RA). Preliminary results of CT-P13 in IBD come from small post-marketing registries and case series with a relatively short-term follow-up period and suggest comparable efficacy and safety to infliximab. Inter- and intra-individual differences in exposure and response are well known for the original molecules but dosing regimens and concomitant medications are different for RA compared to IBD, limiting the ability to translate some of the pharmacology data in RA to IBD. Uncertainty exists about cross-reactivity of anti-drug antibodies and whether similar exposure-response relationships will be observed for biosimilars and efficacy thresholds for therapeutic drug monitoring can be used interchangeably. CONCLUSIONS: It is likely that biosimilars will be widely used for the treatment of IBD due to their cost savings and comparable efficacy. Nevertheless, robust post-marketing studies and pharmacovigilance are warranted in the coming years.

Anticancer drugs as inducers of thermotolerance in yeast.

Yeast cell viability was evaluated microscopically following exposure to heat shock for 30 min at 53 degrees C. The cells were previously grown in the presence of potential stressors (anticancer drugs; e.g., 5-fluorouracil, methotrexate, cisplatin, bleomycin, mitomycin-C and camptothecin-11). The induction of thermotolerance was documented by significantly increased viability after heat shock. This effect, which was reversed by cycloheximide, was comparable to that observed following exposure to a mild heat stress. These data demonstrate that pretreatment with sub-toxic concentrations of some of the clinically used antineoplastic agents conferres thermotolerance to yeast, possibly through the synthesis of protein components.

Similar response to adalimumab in patients with active Crohn's disease either naive to biologic agents or with prior loss of response or intolerance to infliximab.

UNLABELLED: The aim of this study was to investigate the efficacy of adalimumab, in patients with moderately active Crohn's disease (CD), either naive to biologic agents or with prior loss of response or intolerance to infliximab. MATERIAL AND METHOD: A total number of 30 patients with moderately active CD (14 men, 16 women, aged 38.5 +/- 14.4 yr) either naive to biologic agent treatment (19 pts (65%)) or with loss of response or intolerance to infliximab (11 pts (35%)), were enrolled to 4-wk trial with treatment with subcutaneous adalimumab 160 mg injection at week 0, 80 mg at week 2 and then 40 mg every other week. Outcome measures included the ability to tolerate adalimumab and clinical remission (defined as a CDAI score < or =150 points) and clinical response (defined as a decrease in the CDAI) > or =70 points). Eleven patients (37%) were smokers, 5(16%) ex-smokers and 14 (47%) non-smokers. Five patients (16%) had a positive family history for IBD. Duration of disease was 10.7 +/- 8.1 yr. Coexistence of extraintestinal manifestations was noticed in 12 (40%) patients. Vienna Classification of CD was A1=24 (80%), A2=6 (20%), L1=8 (26.7%), L2=6 (20%), L3=15 (50%), L4=1 (3.3%), B1=15 (50%), B2=5 (16.7%), B3=10 (33.3%). RESULTS: Remission was observed in 19 (63.3%) and clinical response in 9 (30%) patients. Two patients (6.7%) showed no response. No significant differences between patients with loss of response or intolerance to infliximab and the group of naive patients were noticed. Comparison between smokers and non smokers revealed significant difference in the response rate in favour of non-smokers (P < 0.002). A trend (P = 0.064) towards a significant difference in the response rate of the group of smokers according to the number of cigarettes smoked per day was observed. Patients with short duration of disease (<10 yr) had significantly better response compared to the group of patients with long (>10 yr) duration of disease. Similarly, patients with extraintestinal manifestations showed significantly better response (P = 0.044). None of the patients in both groups experienced acute or delayed hypersensitivity reactions during treatment with adalimumab. CONCLUSION: Adalimumab is well tolerated and appears to be a beneficial option for patients with CD who have not previously treated with biologic agents or have lost their response to, or cannot tolerate infliximab, with non-smokers, patients with short duration of CD, and patients with extraintestinal manifestations having a better clinical response.

Molybdate induces thermotolerance in yeast.

Application of a mild heat pretreatment, performed by shifting cells from 27 degrees C to 37 degrees C led to the protection of yeast cells from death due to a subsequent extreme heat shock at 53 degrees C. The presence of cycloheximide inhibited this induction of thermotolerance, indicating the involvement of de novo protein. The phosphatase inhibitor sodium molybdate induced thermotolerance to the non-pretreated yeast cells. This induction of thermotolerance did not seem to depend upon de novo protein synthesis. Thus, acquisition of thermotolerance in yeast may involve a number of cellular mechanisms depending on the conditions the organism encounters at any particular time.