Tumor-Specific Delivery of 5-Fluorouracil-Incorporated Epidermal Growth Factor Receptor-Targeted Aptamers as an Efficient Treatment in Pancreatic Ductal Adenocarcinoma Models.

BACKGROUNDS & AIMS: Fluoropyrimidine c (5-fluorouracil [5FU]) increasingly represents the chemotherapeutic backbone for neoadjuvant, adjuvant, and palliative treatment of pancreatic ductal adenocarcinoma (PDAC). Even in combination with other agents, 5FU efficacy remains transient and limited. One explanation for the inadequate response is insufficient and nonspecific delivery of 5FU to the tumor. METHODS: We designed, generated, and characterized 5FU-incorporated systematic evolution of ligands by exponential enrichment (SELEX)-selected epidermal growth factor receptor (EGFR)-targeted aptamers for tumor-specific delivery of 5FU to PDAC cells and tested their therapeutic efficacy in vitro and in vivo. RESULTS: 5FU-EGFR aptamers reduced proliferation in a concentration-dependent manner in mouse and human pancreatic cancer cell lines. Time-lapsed live imaging showed EGFR-specific uptake of aptamers via clathrin-dependent endocytosis. The 5FU-aptamer treatment was equally effective in 5FU-sensitive and 5FU-refractory PDAC cell lines. Biweekly treatment with 5FU-EGFR aptamers reduced tumor burden in a syngeneic orthotopic transplantation model of PDAC, in an autochthonously growing genetically engineered PDAC model (LSL-KrasG12D/+;LSL-Trp53flox/+;Ptf1a-Cre [KPC]), in an orthotopic cell line-derived xenograft model using human PDAC cells in athymic mice (CDX; Crl:NU-Foxn1nu), and in patient-derived organoids. Tumor growth was significantly attenuated during 5FU-EGFR aptamer treatment in the course of follow-up. CONCLUSIONS: Tumor-specific targeted delivery of 5FU using EGFR aptamers as the carrier achieved high target specificity; overcame 5FU resistance; and proved to be effective in a syngeneic orthotopic transplantation model, in KPC mice, in a CDX model, and in patient-derived organoids and, therefore, represents a promising backbone for pancreatic cancer chemotherapy in patients. Furthermore, our approach has the potential to target virtually any cancer entity sensitive to 5FU treatment by incorporating 5FU into cancer cell-targeting aptamers as the delivery platform.

Homeopathic Treatment as an Add-On Therapy May Improve Quality of Life and Prolong Survival in Patients with Non-Small Cell Lung Cancer: A Prospective, Randomized, Placebo-Controlled, Double-Blind, Three-Arm, Multicenter Study.

LESSONS LEARNED: Conventional medicine and homeopathy work well together. Quality of life improves with additive homeopathy in patients with non-small cell lung cancer (NSCLC). Survival improves with additive homeopathy in patients with NSCLC. BACKGROUND: Patients with advanced non-small cell lung cancer (NSCLC) have limited treatment options. Alongside conventional anticancer treatment, additive homeopathy might help to alleviate side effects of conventional therapy. The aim of the present study was to investigate whether additive homeopathy might influence quality of life (QoL) and survival in patients with NSCLC. METHODS: In this prospective, randomized, placebo-controlled, double-blind, three-arm, multicenter, phase III study, we evaluated the possible effects of additive homeopathic treatment compared with placebo in patients with stage IV NSCLC, with respect to QoL in the two randomized groups and survival time in all three groups. Treated patients visited the outpatients' centers every 9 weeks: 150 patients with stage IV NSCLC were included in the study; 98 received either individualized homeopathic remedies (n = 51) or placebo (n = 47) in a double-blinded fashion; and 52 control patients without any homeopathic treatment were observed for survival only. The constituents of the different homeopathic remedies were mainly of plant, mineral, or animal origin. The remedies were manufactured by stepwise dilution and succussion, thereby preparing stable Good Manufacturing Practice grade formulations. RESULTS: QoL as well as functional and symptom scales showed significant improvement in the homeopathy group when compared with placebo after 9 and 18 weeks of homeopathic treatment (p < .001). Median survival time was significantly longer in the homeopathy group (435 days) versus placebo (257 days; p = .010) as well as versus control (228 days; p < .001). Survival rate in the homeopathy group differed significantly from placebo (p = .020) and from control (p < .001). CONCLUSION: QoL improved significantly in the homeopathy group compared with placebo. In addition, survival was significantly longer in the homeopathy group versus placebo and control. A higher QoL might have contributed to the prolonged survival. The study suggests that homeopathy positively influences not only QoL but also survival. Further studies including other tumor entities are warranted.

Predominant Api m 10 sensitization as risk factor for treatment failure in honey bee venom immunotherapy.

BACKGROUND: Component resolution recently identified distinct sensitization profiles in honey bee venom (HBV) allergy, some of which were dominated by specific IgE to Api m 3 and/or Api m 10, which have been reported to be underrepresented in therapeutic HBV preparations. OBJECTIVE: We performed a retrospective analysis of component-resolved sensitization profiles in HBV-allergic patients and association with treatment outcome. METHODS: HBV-allergic patients who had undergone controlled honey bee sting challenge after at least 6 months of HBV immunotherapy (n = 115) were included and classified as responder (n = 79) or treatment failure (n = 36) on the basis of absence or presence of systemic allergic reactions upon sting challenge. IgE reactivity to a panel of HBV allergens was analyzed in sera obtained before immunotherapy and before sting challenge. RESULTS: No differences were observed between responders and nonresponders regarding levels of IgE sensitization to Api m 1, Api m 2, Api m 3, and Api m 5. In contrast, Api m 10 specific IgE was moderately but significantly increased in nonresponders. Predominant Api m 10 sensitization (>50% of specific IgE to HBV) was the best discriminator (specificity, 95%; sensitivity, 25%) with an odds ratio of 8.444 (2.127-33.53; P = .0013) for treatment failure. Some but not all therapeutic HBV preparations displayed a lack of Api m 10, whereas Api m 1 and Api m 3 immunoreactivity was comparable to that of crude HBV. In line with this, significant Api m 10 sIgG4 induction was observed only in those patients who were treated with HBV in which Api m 10 was detectable. CONCLUSIONS: Component-resolved sensitization profiles in HBV allergy suggest predominant IgE sensitization to Api m 10 as a risk factor for treatment failure in HBV immunotherapy.

Nitrosative stress triggers microtubule reorganization in Arabidopsis thaliana.

Microtubules (MTs) are essential components of the cytoskeleton and fulfil multiple cellular functions in developmental processes, readily responding to intrinsic and external cues. Nitric oxide signalling is well established in plants, and the MT cytoskeleton is one of its potential targets. To mimic low level nitrosative stress, growth medium was supplemented with 3-nitro-L-tyrosine (NO2-Tyr), a nitrated form of the amino acid tyrosine, and concentration-dependent changes in root growth rate and a reduction in cell division frequencies in Arabidopsis thaliana were observed. In addition, it is reported that exposure to low NO2-Tyr concentrations was not detrimental to plant health and caused subtle and reversible defects. In contrast, growth defects caused by high NO2-Tyr concentrations could not be reversed. Live cell imaging of an MT reporter line revealed that treatment with a low concentration of NO2-Tyr correlated with disorganized cortical MT arrays and associated non-polar cell expansion in the elongation zone. NO2-Tyr treatment antagonized the effects of taxol and oryzalin, further supporting the association of NO2-Tyr with MTs. Furthermore, oblique division plane orientations were observed which were probably induced prior to cytokinesis.

The microtubule-associated kinase-like protein RUNKEL functions in somatic and syncytial cytokinesis.

The microtubule (MT)-associated putative kinase RUNKEL (RUK) is an important component of the phragmoplast machinery involved in cell plate formation in Arabidopsis somatic cytokinesis. Since loss-of-function ruk mutants display seedling lethality, it was previously not known whether RUK functions in mature sporophytes or during gametophyte development. In this study we utilized RUK proteins that lack the N-terminal kinase domain to further examine biological processes related to RUK function. Truncated RUK proteins when expressed in wild-type Arabidopsis plants cause cellularization defects not only in seedlings and adult tissues but also during male meiocyte development, resulting in abnormal pollen and reduced fertility. Ultrastructural analysis of male tetrads revealed irregular and incomplete or absent intersporal cell walls, caused by disorganized radial MT arrays. Moreover, in ruk mutants endosperm cellularization defects were also caused by disorganized radial MT arrays. Intriguingly, in seedlings expressing truncated RUK proteins, the kinesin HINKEL, which is required for the activation of a mitogen-activated protein kinase signaling pathway regulating phragmoplast expansion, was mislocalized. Together, these observations support a common role for RUK in both phragmoplast-based cytokinesis in somatic cells and syncytial cytokinesis in reproductive cells.

Evaluation of a porcine whole-limb heterotopic autotransplantation model.

BACKGROUND: Recently performed vascularized composite tissue allotransplantations (CTAs) stimulate the ongoing research in the area of whole-limb transplantation. A reliable in vivo animal model is required for investigations in vascularized whole-limb CTA. The model should allow in vivo assessment in whole-limb preservation, allograft and xenograft response, and host immunomodulation. The goal of this study is to describe and evaluate the in vivo feasibility and reproducibility of a whole-limb porcine model as a basis for future research in this field. MATERIALS AND METHODS: In seven large white pigs, one forelimb was amputated under anesthesia and autotransplanted heterotopically with an arc of rotation of 180° and partially placed in a subcutaneous pocket. Clinical parameters were monitored and muscle biopsies were analyzed using ultrastructural morphological assessment of mitochondria quality after an observation period of 7 days. RESULTS: All animals could fully mobilize postoperatively without restrictions. At sacrifice, the anastomosed pedicle vessels of the limb were patent in six animals. In one pig, venous thrombosis could be observed. Muscle response was triggered following direct electrostimulation in six replanted limbs. The replanted extremities gained 12.97% weight within 7 days postreplantation compared with the amputation baseline values (P = 0.464 while maintaining normal compartment pressures at sacrifice (8.25 ± 5.31 cmH(2)O, P = 0.60). The ultrastructural evaluation of mitochondria morphology revealed intact mitochondria without signs of ischemia/reperfusion damage. CONCLUSION: This porcine model proved feasible, reliable, and reproducible for whole-limb autotransplantation. It presents significant potential in future preclinical research of whole-limb CTA transplantation.

Effectiveness of additional self-care acupressure for women with menstrual pain compared to usual care alone: using stakeholder engagement to design a pragmatic randomized trial and study protocol.

BACKGROUND: Self-care acupressure might be successful in treating menstrual pain, which is common among young women. There is a need for comparative effectiveness research with stakeholder engagement in all phases seeking to address the needs of decision-makers. Our aim was to design a study on the effectiveness of additional self-care acupressure for menstrual pain comparing usual care alone using different methods of stakeholder engagement. METHODS: The study was designed using multiple mixed methods for stakeholder engagement. Based on the results of a survey and focus group discussion, a stakeholder advisory group developed the study design. RESULTS: Stakeholder engagement resulted in a two-arm pragmatic randomized trial. Two hundred and twenty women aged 18 to 25 years with menstrual pain will be included in the study. Outcome measurement will be done using electronic questionnaires provided by a study specific mobile application (App). Primary outcome will be the mean pain intensity at the days of pain during the third menstruation after therapy start. CONCLUSION: Stakeholder engagement helped to develop a study design that better serves the needs of decision makers, including an App as a modern tool for both intervention and data collection in a young target group. TRIAL REGISTRATION: Clinicaltrials.gov identifier http://NCT01582724.

Modulation of human motor cortex excitability by valproate.

RATIONALE: Valproate is widely used in the treatment of epilepsy, bipolar disorder, and chronic pain disorders, but its exact mechanisms of action is still incompletely understood. OBJECTIVES: Here we used transcranial magnetic stimulation to explore effects of a single dose of 800 mg valproate on motor cortex excitability in healthy volunteers. METHODS: Motor threshold, peripheral maximum M-wave, cortical silent period short intracortical inhibition, intracortical facilitation, and motor evoked potential recruitment were assessed before and 1.5 h after the administration of valproate in 15 (eight male, seven female) healthy volunteers. RESULTS: None of the measures of cortical excitability were found to be altered significantly after valproate. CONCLUSION: These results are in line with previous findings of unaffected intracortical excitability after a single dose of valproate, suggesting that valproate's immediate in vivo actions do not resemble the effects of classic GABAergic compounds.

Anti-myeloma activity of the novel 2-aminothienopyrimidine Hsp90 inhibitor NVP-BEP800.

The 90 kD heat shock protein (Hsp90) molecular chaperone sustains multiple components of oncogenic pathways and has recently emerged as a therapeutic target that is now being clinically tested in a number of malignancies. In order to address formulation issues and to deal with possible resistance mechanisms against small molecule Hsp90 inhibitors, a range of compounds based on different molecular scaffolds are now being developed. The present study preclinically tested the effects of the novel 2-aminothienopyrimidine class Hsp90 inhibitor NVP-BEP800, which is suitable for oral formulations, on multiple myeloma cells from established cell lines and on a larger cohort (n = 40) of primary myeloma samples. The drug effectively and specifically killed the majority of primary myeloma cells in coculture with bone marrow stromal cells and reliably entailed molecular consequences of Hsp90 blockade - such as survival pathway breakdown and client protein depletion - in multiple myeloma cells from cell lines as well as from patients. Collectively, the properties of this novel drug support clinical testing in multiple myeloma.

Magnetic fluid hyperthermia therapy for malignant brain tumors--an ethical discussion.

This paper describes the worldwide first nanoparticle-based therapy against brain tumors termed magnetic fluid hyperthermia (MFH) or Nano-Cancer therapy, which now is under investigation in a phase II study. The preliminary results show evidence of a local effectiveness and with only minor to moderate therapy-associated side effects. The evaluation, according to the principles of medical ethics (autonomy, beneficence, nonmaleficence, justice), supports the further development of the MFH therapy. FROM THE CLINICAL EDITOR: This paper describes the worlwide first nanoparticle-based therapy of brain tumors through local hyperthermia now under investigation in a phase II study. The preliminary results show effectiveness and only minor to moderate side effects. Further development of this novel therapy is justified.

A role for receptor tyrosine phosphatase zeta in glioma cell migration.

Glioblastomas (GBM) are the most frequent and malignant human brain tumor type. Typically striking in adulthood, tumor progression is rapid, relentless, and ultimately leads to the patient's death within a year of diagnosis. The identification of transcriptionally regulated genes can lead to the discovery of targets for antibody or small-molecule-mediated therapy, as well as diagnostic markers. We prepared cDNA arrays that are specifically enriched for genes expressed in human brain tumors and profiled gene expression patterns in 14 individual tumor samples. Out of 25,000 clones arrayed, greater than 200 genes were found transcriptionally induced in glioblastomas compared to normal human brain tissue including the receptor tyrosine phosphatasezeta (RPTPzeta) and one of its ligands, pleiotrophin (Ptn). We confirmed by Northern blot analysis and immunohistochemistry that RPTPzeta is enriched in tumor samples. Knockdown of RPTPzeta by RNA interference studies established a functional role of RPTPzeta in cell migration. Our results suggest a novel function for RPTPzeta in regulating glioblastoma cell motility and point to the therapeutic utility of RPTPzeta as a target for antibody-mediated therapy of brain tumors.

Evaluation of a homemade SYBR green I reaction mixture for real-time PCR quantification of gene expression.

Real-time PCR is an accurate method that can be used for the quantification of specific DNA molecules. Here we provide a protocol for SYBR Green I in real-time PCR applications using plastic reaction tubes. We report that SYBR Green I is alkali labile and once degraded inhibits the PCR. In our optimized protocol, diluted aliquots of SYBR Green I remain stable for at least two weeks. We also evaluated different cDNA synthesis protocols for the quantification of multiple genes from the same cDNA preparation. The best result was obtained with cDNAs synthesized by OmniScript reverse transcriptase from 2.5 microg total RNA using oligo d(T)18 primers. The cDNA reactions could be diluted 1:25, allowing the quantification of up to 125 different medium expressed genes of Arabidopsis. Extension times ranged between 20 and 40 bp/s for accurate quantification of PCR products up to approximately 400 bp in the Rotor-Gene 2000 system. Using our optimized real-time PCR protocol, the reproducibility and amplification efficiency was high and comparable to a commercially available SYBR Green I kit. Furthermore, the sensitivity allowed us to quantify 10-20 copies of mRNA and dsDNA. Thus, the protocol eliminates the need for expensive pre-made kits.