Presence Of CTX Research Articles

Low Dose Cyclophosphamide in Combination with Elotuzumab - a Novel Immunotherapeutic Strategy for Multiple Myeloma

IntroductionCyclophosphamide (CTX) is a widely used anti-neoplastic, performing as an alkylating agent at high doses and immunomodulatory agent at low doses 1.. Combining CTX with monoclonal antibody (mAb) therapy has proven beneficial in potentiating relapsed and/or refractory multiple myeloma (RRMM) therapies, with daratumumab-directed MM cell death enhanced in the presence of CTX 2,3.. Elotuzumab (ELO), the second mAb approved for treating RRMM, promotes MM cell clearance by enhancing macrophage-mediated phagocytosis and CD16- and SLAMF7-directed NK cell cytotoxicity. ELO has been approved for use alongside dexamethasone and lenalidomide 4 or pomalidomide (POM) 5.. However, potential therapeutic benefits of ELO in combination with immunomodulatory drugs such as CTX and POM have yet to be examined. Our research investigates, the efficacy of combining low-dose CTX, alone or in combination with POM, and ELO in enhancing macrophage and NK cell infiltration and function in the MM tumour microenvironment.Materials and MethodsMultiple myeloma cells (MM1S and H929) were treated with low-dose CTX and/or POM for 24hrs, washed to remove residual drug and resuspended in fresh media for tumour cell secretome (TCS) generation. Direct effects of CTX and/or POM on surface expression of checkpoint proteins (PD-1 and CD47) on MM cells was assessed by mean fluorescent intensity (MFI) flow cytometry. CD32/CD64 receptor expression on THP-1 macrophages, NKG2D, CD2, DNAM-1, CD96 and KIR2DL1 receptors on KHYG1 and primary NK cells, were measured using flow cytometry as a measure of activation. Migration of serum-starved, CFSE-labelled macrophages and NK cells towards CTX and/or POM TCS was assessed after 4hrs, with total number of migrated cells quantified using the Accuri flow cytometer. Immune cell function following indirect conditioning of macrophages/NK cells with MM cell TCS was measured by quantifying antibody-directed cellular phagocytosis (ADCP) or antibody-directed cellular cytotoxicity (ADCC), respectively. Conditioned immune cells were co-cultured with MM cells in a 2:1 effector to target ratio for 4hrs in the absence/presence of mAbs (ELO, nivolumab and anti-CD47), after which MM cell clearance was quantified by flow cytometry and presented as relative uptake (ADCP) and cytotoxicity (ADCC). One-way ANOVA statistical analysis was performed, followed by Tukey post hoc tests, with significance recognized at p<0.05.ResultsDirect treatment of MM cells with CTX increased surface expression of immune evading checkpoint proteins PD-1 and CD47 (p<0.05,n=3). POM monotherapy did not alter PD-1/CD47 expression, however dual therapy of CTX and POM supported the CTX-driven effect (p<0.001,n=3). Expression of CD32/CD64 macrophage activation markers was significantly increased on THP-1 cells following CTX-TCS conditioning (p<0.001,n=3). POM altered CD32, but not CD64, however dual treatment with CTX and POM significantly increased expression of both CD32 and CD64 (p<0.001, n=3). Migration of macrophages towards CTX-TCS was enhanced in a dose-dependent manner (p<0.01,n=3). CTX and POM dual therapy supported this CTX driven effect (p<0.001,n=3). Migration trends of both primary and KHYG1 NK cells were also increased towards the secretome from CTX treated MM cells. ADCP and ADCC were increased by CTX alone or in combination with POM (p<0.05, n=3). Effects of CTX on ADCP were not significantly enhanced by ELO, however ELO did significantly augment ADCC by CTX-conditioned primary NK cells (p<0.05,n=3). Given the increased expression of PD-1 and CD47, we investigated if the inclusion of nivolumab and anti-CD47 mAbs potentiated ADCC. Although ADCC was increased in all combinations, there was no significant difference between ELO alone versus ELO in combination with either nivolumab or anti-CD47.ConclusionsLow-dose CTX and POM potentiated the immunomodulatory effects of ELO, with NK-directed cytotoxicity of MM cells enhanced in the presence of this mAb. Our data therefore indicates that the inclusion of low-dose CTX and or POM in combination with ELO could be a novel immunotherapeutic strategy for treating RRMM.

Antibiotic resistance pattern and frequency of some beta lactamase genes in Klebsiella pneumoniae isolated from raw milk samples in Iran

Klebsiella pneumoniae have become an important cause of mastitis in dairy cows. Resistance to beta lactam antibiotics resulted from beta lactamases enzyme production. The aim of this study was to investigate the antibiotic resistance pattern and frequency of some beta lactamase genes in Klebsiella pneumoniae isolated from raw milk samples in Iran. 200 raw cow milk samples were collected from different villages of north west of Iran. The samples were cultured and biochemical tests were performed for phenotypic diagnosis. Then, antibiotic resistance pattern was determined by antibiogram test. Finally, the presence of CTX, SHV and TEM genes in Klebsiella pneumoniae isolates was found by PCR method. Of total 200 raw cow milk samples, 80 samples (40%) contained Klebsiella pneumoniae. The frequency of CTX, SHV and TEM genes in Klebsiella pneumoniae isolates was 50 (62.5%), 34 (42.5%) and 70 (87.5%), respectively. 14 Klebsiella pneumoniae isolates (17.5%) possessed all three intended genes simultaneously. All strains of Klebsiella pneumoniae (100%) were resistant to ampicillin. The most strains were resistant to ceftriaxone (75%), gentamicin (70%) and nitrofurantoin (70%). 4 Klebsiella pneumoniae strains (5%) were resistant to all of tested antibiotics. The results showed high frequency of ESBLs and antibiotic resistance in Klebsiella pneumonia samples isolated from raw milk. It may occur exchange of resistance genes within and across species and with commensal bacteria of the human and animals.

Abstract CT282: KEYNOTE-975: A randomized, double-blind, placebo-controlled phase 3 trial of pembrolizumab vs placebo in participants with esophageal carcinoma receiving concurrent definitive chemoradiotherapy

Abstract Background: For patients with unresectable esophageal cancer (EC), definitive chemoradiotherapy (dCRT) is a standard treatment option. Platinum plus fluoropyrimidine-based regimens are comparable in dCRT and are considered standard options; however, overall survival (OS) is still poor, indicating a need for more effective therapies. The programmed cell death 1 inhibitor pembrolizumab showed promising response as third- and second-line monotherapy in patients with advanced, unresectable EC in the KEYNOTE-180 and KEYNOTE-181 studies, respectively. In the phase 3 KEYNOTE-181 trial, pembrolizumab extended OS vs chemotherapy in patients with a PD-L1 combined positive score (CPS) ≥10. KEYNOTE-975 is a randomized, multicenter, double-blind, phase 3 study investigating pembrolizumab in combination with dCRT. Methods: Key patient eligibility criteria are age ≥18 years; presence of cTX N+M0 or cT2-T4a NXM0, locally advanced esophageal squamous cell carcinoma (SCC) or adenocarcinoma or Siewert type 1 adenocarcinoma of the esophagogastric junction; ineligible for curative surgery; no previous chemotherapy or radiation for EC; and suitable for dCRT. Patients will be randomly assigned 1:1 to pembrolizumab or placebo added to dCRT, administered as pembrolizumab 200 mg or placebo Q3W for 8 cycles followed by pembrolizumab 400 mg or placebo Q6W for 5 cycles (13 cycles total). The dCRT regimen will be the site's choice of continuous infusion 5-FU + cisplatin (FP) with radiotherapy (RT) 50 Gy, FP with RT 60 Gy, or FOLFOX with RT 50 Gy. Randomization will be stratified by PD-L1 positivity (CPS ≥10 vs CPS &amp;lt;10), RT dose (50 Gy vs 60 Gy), and region/histology (SCC East Asia vs SCC rest of world and adenocarcinoma regardless of region). The primary objectives of KEYNOTE-975 are to compare pembrolizumab and placebo added to dCRT in OS and event-free survival within the prespecified analysis cohorts: patients with CPS ≥10, patients with SCC, and all patients (intention-to-treat population). The secondary objective is to assess the safety and tolerability profile of pembrolizumab vs placebo added to dCRT (adverse events, overall and leading to study drug discontinuation). Exploratory objectives include comparing time to deterioration and change from baseline in quality of life measures, characterizing health utility scores, and identifying molecular biomarkers that may be determinants of response. Citation Format: Manish A. Shah, Jaafar Bennouna, Toshihiko Doi, Lin Shen, Ken Kato, Antoine Adenis, Harvey Mamon, Markus Moehler, Xiaolong Fu, Byoung Chul Cho, Pooja Bhagia, Chie-Schin Shih, Anjali Desai, Peter Enzinger. KEYNOTE-975: A randomized, double-blind, placebo-controlled phase 3 trial of pembrolizumab vs placebo in participants with esophageal carcinoma receiving concurrent definitive chemoradiotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT282.

Cross-suppression of EGFR ligands amphiregulin and epiregulin and de-repression of FGFR3 signalling contribute to cetuximab resistance in wild-type KRAS tumour cells

BACKGROUND:In addition to the mutational status of KRAS, the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG) might function as bona fide biomarkers of cetuximab (Ctx) sensitivity for most EGFR-driven carcinomas.METHODS:Lentivirus-delivered small hairpin RNAs were employed to specifically reduce AREG or EREG gene expression in wild-type KRAS A431 squamous cell carcinoma cells. Colony-forming assays were used to monitor the impact of AREG and EREG knockdown on Ctx efficacy. Amphiregulin and EREG protein expression levels were assessed by quantitative ELISA in parental A431 cells and in pooled populations of A431 cells adapted to grow in the presence of Ctx. A phosphoproteomic platform was used to measure the relative level of phosphorylation of 42 distinct receptor tyrosine kinases before and after the acquisition of resistance to Ctx.RESULTS:Stable gene silencing of either ligand was found to notably reduce the expression of the other ligand. Parental A431 cells with normal expression levels of AREG/EREG exhibited significantly increased growth inhibition in response to Ctx, compared with derivatives that are engineered to produce minimal AREG/EREG. The parental A431 cells acutely treated with Ctx exhibited reduced basal expression levels of AREG/EREG. Pooled populations of Ctx-resistant A431 cells expressed significantly lower levels of AREG/EREG and were insensitive to the downregulatory effects of Ctx. Phosphoproteomic screen identified a remarkable hyperactivation of FGFR3 in Ctx-resistant A431 cells, which gained sensitivity to the cytotoxic and apoptotic effects of the FGFR3 TK inhibitor PD173074. The A431 parental cells acutely treated with Ctx rapidly activated FGFR3 and their concomitant exposure to Ctx and PD173074 resulted in synergistic apoptosis.CONCLUSION:Cross-suppression of AREG/EREG expression may explain the tight co-expression of AREG and EREG, as well as their tendency to be more highly expressed than other EGFR ligands to determine Ctx efficacy. The positive selection for Ctx-resistant tumour cells exhibiting AREG/EREG cross-suppression may have an important role in the emergence of Ctx resistance. As de-repression of FGFR3 activity rapidly replaces the loss of EGFR-ligand signalling in terms of cell proliferation and survival, combinations of Ctx and FGFR3-targeted drugs may be a valuable strategy to enhance the efficacy of single Ctx while preventing or delaying acquired resistance to Ctx.

English

Objective: Production of Extended Spectrum β- Lactamase is an important mechanism of antimicrobial resistance exhibited by Enterobacteriaceae. There are many types of ESBLs among which CTX M is a latest emerged one which carries the bla CTX M gene. CTX M lactamase producing Enterobacteriaceae are present in the intestinal flora without harming the host causing infection in extra intestinal sites. Transferring the gene through plasmids is responsible for the dissemination of antimicrobial resistant strains in the community. This study is designed to detect bla CTX-M gene in Escherichia coli isolates. Method: A total of 532 Escherichia coli isolates were screened for susceptibility testing. Isolates with decreased resistance to third generation Cephalosporins (3GC) were subjected to double disk synergy test for phenotypic confirmation of ESBL in which 178 were positive. Multiplex PCR was done for these 178 isolates to detect the four groups I, II, III & IV of CTX M. Results: Multiplex PCR yielded the products with predicted size for group 1 CTX M in 152 (28.57%) isolates. None of the isolates were positive for other groups of CTX M (Group II, III & IV) Conclusion: The presence of CTX M ESBL producing E. coli cause enormous problems in therapeutic interventions of infections which can be prevented only by continuous surveillance and prudent use of antibiotics.

A charybdotoxin-insensitive conductance in human T lymphocytes: T cell membrane potential is set by distinct K+ channels.

1. Changes in the membrane potential (Vm) of human T lymphocytes upon K+ channel block were inferred from alterations in K+ current reversal potential in cell-attached patches. It was found that a high concentration of charybdotoxin (100 nM, CTX), which blocks both voltage-gated (K(V)) and Ca(2+)-activated (K(Ca)) potassium channels in these cells, depolarizes Vm of lymphocytes only partially. Subsequent whole-cell measurements of the same cells showed that 39 +/- 25% of the voltage-gated current remains in the presence of CTX. 2. The CTX-resistant current reverses at potentials between -80 and -90 mV, indicating that it is K+ selective. The current is activated at more depolarized potentials compared with the unblocked IK(V) current with a threshold between -40 and -20 mV and a half-maximal activation at +50 mV. Inactivation during prolonged depolarization is slow. Steady-state inactivation is half-maximal at -45 mV and complete at potentials > -20 mV. The CTX-resistant IK(V) is completely blocked by nifedipine and is not sensitive to dendrotoxin. 3. The effect of nifedipine on the Vm of lymphocytes varies between cells depending on the contribution of the nifedipine-sensitive current to whole-cell IK(V). Combined application of CTX and nifedipine completely depolarizes Vm. 4. The extent to which T cell receptor-evoked Ca2+ signals of resting cells are inhibited by K+ channel blockers correlates with the magnitude of the depolarization induced by the drugs. Complete suppression of the response is achieved only by combined block of the CTX-sensitive and -insensitive IK(V). The enhanced Ca2+ response of activated cells, which express increased numbers of K(Ca) channels, is in addition subject to modulation by blockers which prevent the hyperpolarization during the Ca2+ rise mediated by these channels.

Action of Taiwan cobra cardiotoxin on membranes: binding modes of a beta-sheet polypeptide with phosphatidylcholine bilayers.

The interaction of Taiwan cobra cardiotoxin (CTX A3), a basic polypeptide consisting of three-fingered loops and five-strand beta-sheet structure, with zwitterionic dipalmitoylphosphatidylcholine (DPPC) has been studied by 31P and 2H NMR to understand the binding modes of CTX in membrane bilayers. The results, in conjunction with DPH fluorescence anisotropy and differential scanning calorimetry studies, show that CTX may penetrate and lyse the bilayers into small aggregates at a lipid/protein molar ratio of about 20 in the ripple Pbeta' phase. Elevating the temperature to that of the liquid crystalline Lalpha phase leads to the fusion of the small aggregates into larger ones as evidenced by the change of the isotropic signal into a magnetically aligned 31P signal with a marked reduction in the chemical shift anisotropy. 2H NMR study on deuterium-labeled DPPC in the head group and fatty acyl region as a function of temperature and CTX concentration reveals a molecular model that CTX undergoes a redistribution between penetrating and peripheral binding states depending on the temperature studied. In addition, both the conformational and dynamic states of the phosphocholine head group of DPPC bilayers are significantly perturbed in the presence of CTX. Structural consideration of the CTX molecule indicates that the penetration binding mode of CTX with the DPPC bilayer may involve a novel membrane-binding motif identified recently in the three-fingered loops of P-type CTX. CTX can only bind to DPPC membrane peripherally in the Lalpha phase due to the mismatch of their hydrophobic lengths.

Production of IgE antibody and allergic sensitization of intestinal and peripheral tissues after oral immunization with protein Ag and cholera toxin.

Cholera toxin (CTX) is a potent oral adjuvant for the induction of mucosal IgA Ab responses protein Ags. We examined the Ab responses and allergic sensitization of several strains of mice to protein Ags, administered orally with CTX. The mice made strong IgA and IgG1 serum Ab responses, but little IgG2a Ab to Ags such as hen egg lysozyme (HEL) and OVA. However, when given a subsequent i.p. challenge with Ag alone, the same mice had immediate hypersensitivity reactions that included respiratory distress and death. Within 10 min of i.p. challenge, immunized mice had high levels of plasma histamine and extensive degranulation of mast cells in target tissues. These mice had detectable serum IgE Ab. Ag administered orally with the B subunit (CTB) of CTX did not sensitize mice. Intestinal tissues taken from these mice had Ag-specific ion-secretory responses in vitro, typical of intestinal anaphylaxis. Ag given s.c. without adjuvant could also sensitize for systemic and intestinal anaphylaxis. Sensitization with HEL given s.c. was dose dependent and correlated with a critical amount of HEL in the circulation. HEL was detected in the circulation after oral immunization, but CTX did not increase the uptake of HEL. Thus, oral immunization with a protein Ag in the presence of CTX can sensitize an animal for systemic and intestinal anaphylaxis. These results suggest a cautious approach to the use of CTX as an adjuvant in oral vaccines, and provide a new model to study immediate hypersensitivity reactions to intestinal Ag.

The phorbol ester TPA potentiates cholera toxin- and isoproterenol-stimulated cyclic AMP-synthesis in primary astrocyte cultures

Cellular responses to changes in the extracellular environment are mediated by intracellular signaling systems. One of the most extensively studied systems is adenylate cyclase which generates the second messenger molecule cAMP. Another one is the phosphatidylinositol (PI) second messenger system giving rise to IP3 and diacylglycerol, the latter stimulating protein kinase C. Recently, a third potential signaling system has attracted increased scientific attention: the phospholipase A2 system which generates arachidonic acid. This substance may be used for eicosanoid synthesis or serve as a second messenger molecule. The present report gives more evidence about mechanisms how these signaling pathways interact in cultured astrocytes. Substances commonly used for stimulation of arachidonic acid release and prostaglandin synthesis in these cultures (A23187, TPA) had no influence on intracellular cAMP levels. Pertussis toxin that had previously been shown to inhibit prostaglandin synthesis, had no influence on cAMP levels either. Cholera toxin, however, raised intracellular cAMP significantly, although much less than the beta-adrenoceptor agonist isoproterenol. Cholera toxin also caused a marked change in astroglial morphology even at reduced concentrations (1-10 ng/ml). A23187 used in combination with Ctx had a moderate stimulatory effect on cAMP synthesis. In contrast, in the presence of Ctx, the PKC-activating phorbol ester TPA synergistically stimulated cAMP production, raising cAMP levels as high as isoproterenol-stimulated levels. The TPA effect was concentration-dependent. It was also dependent on an intact PKC since preincubation of cells with the phorbol ester completely abolished the synergistic effect. The synergistic effect of the phorbol ester was also observed at subthreshold concentrations of isoproterenol. The data reveal that the sole activation of most Gs molecules is a necessary but not sufficient prerequisite to achieve maximal adenylate cyclase activity. The fine-tuning of this activity apparently occurs at the catalytic subunit which is under the (partial) control of phosphorylation by PKC.

Omega conotoxin and prejunctional modulation of the biphasic response of the rat isolated urinary bladder to single pulse electrical field stimulation

1. Single pulse electrical field stimulation (EFS) produces a biphasic response of muscle strips of the rat isolated urinary bladder consisting of an early and a late contraction which were atropine-resistant and atropine-sensitive, respectively. Repeated application of desensitizing doses of the P2 purinoceptor agonist, alpha, beta-methylene ATP (mATP) inhibited the early response while leaving unaffected the late component. 2. Omega conotoxin (CTX, 0.1 microM) inhibited both the early and the late response either in control conditions or after enhancement by physostigmine (0.1 microM). The effect of CTX was, in both cases, more pronounced on the late than the early response to EFS. CTX (0.1 microM) failed to affect contraction produced by ATP or acetylcholine at concentrations (0.3 mM and 0.5 microM) which produced a response similar to that to EFS. 3. The effect of physostigmine was more intense for the late than the early response and was abolished by atropine. In the presence of CTX, physostigmine enhanced both the early and the late components of the mechanical response to EFS. 4. Nifedipine (0.1-1 microM) reduced to a similar extent both the early and late responses. Bay K 8644 (1 microM) produced a marked enhancement of the response to EFS, which, however, did not have a distinct late peak. In the presence of Bay K 8644, either atropine (3 microM) or tetrodotoxin (1 microM) had minor inhibitory effects indicating the myogenic origin of the response. 5. Neurokinin A (0.1-1 nM) enhanced both the early and late responses to EFS without affecting the contraction produced by exogenous acetylcholine or ATP. A consistent potentiation was evident also in the presence of CTX and for the early response, in the presence of atropine. Clonidine (3 microM) inhibited the response to EFS either in the absence or the presence of physostigmine. The inhibitory effect of clonidine, shown previously to depend upon activation of prejunctional alpha 2-adrenoceptors, was still observed in presence of CTX or atropine. 6. It is concluded that CTX-sensitive voltage dependent calcium channels play a more important role in determining the cholinergic rather than the non-cholinergic, putatively purinergic, component of the biphasic response of the rat bladder to single pulse EFS. The action of CTX is likely to be exerted on N-type rather than L-type (dihydropyridine-sensitive) calcium channels. Prejunctional modulation (enhancement by neurokinin A, inhibition by clonidine) occurs even in the presence of CTX-sensitive channels blockade.