Overcoming trastuzumab resistance in HER2-positive breast cancer using combination therapy

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) comprises around 20–30% of all BC subtypes and is correlated with poor prognosis. For many years, trastuzumab, a monoclonal antibody, has been used to inhibit the HER2 activity. Though, the main resistance to trastuzumab has challenged the use of this drug in the management of HER2-positive BC. Therefore, the determination of resistance mechanisms and the incorporation of new agents may lead to the development of a better blockade of the HER family receptor signaling. During the last few years, some therapeutic drugs have been developed for treating patients with trastuzumab-resistant HER2-positive BC that have more effective influences in the management of this condition. In this regard, the present study aimed at reviewing the mechanisms of trastuzumab resistance and the innovative therapies that have been investigated in trastuzumab-resistant HER2-positive BC subjects.     

Developmental capacity of ferret embryos by nuclear transfer using G0/G1-phase fetal fibroblasts

With the ultimate goal of establishing experimental protocols necessary for cloning ferrets, the present study has established parameters for the reconstruction of ferret embryos by nuclear transfer (NT) using G0/G1-phase donor fetal fibroblasts. Cumulus-oocyte complexes were harvested from superovulated ferrets and cultured in maturation medium for 24 h. Matured oocytes were then enucleated and injected with the fibroblast nuclei derived from 14-16-h serum-starved cells. Reconstructed embryos were then activated by a combination of electric pulses and chemical stimulations. Subsequently, the reconstructed and activated embryos were either cultured in vitro or transferred to pseudopregnant ferrets to evaluate their developmental capacity in vitro and in vivo. Our results demonstrated that 56.3% of reconstructed embryos (n = 187) cleaved, while 26.0% and 17.6% developed to morula and blastocyst phases in vitro, respectively. The blastocysts derived from NT embryos demonstrated normal morphology by differentially staining as compared to normal blastocysts developed in vivo following fertilization. In vivo developmental studies at 21 days posttransplantation demonstrated 8.8% of reconstructed embryos (n = 91) implanted into the uterine lining of recipients, while 3.3% formed fetuses. However, reconstructed embryos (n = 387) failed to develop to term (42 days). These results demonstrate donor nuclei of G0/G1-phase fetal fibroblast cells can be reprogrammed to support the development of reconstructed ferret embryos in vitro and in vivo; however, a significant third-trimester block occurs preventing full-term development.     

Cu(II) induces small-size aggregates with amyloid characteristics in two alleles of recombinant ovine prion proteins

One of symptoms of transmissible spongiform encephalopathies is associated with the transformation of normal cellular prion protein, PrP, in its amyloid isoform resistant to proteolytic cleavage. The present study shows that interaction with copper ions converts both monomeric recombinant scrapie-susceptible PrP-VRQ and scrapie-resistant PrP-ARR variants into protease-resistant soluble oligomers with amyloid characteristics -- dominant beta-sheet secondary structure and interaction with thioflavine S. In contrast, binding of zinc ions resulting in the same resistance to proteolysis does not provoke transformation of alpha-helical monomeric structure of both PrP polymorphic variants. Cleavage of PrP N-terminus destabilises soluble form of such aggregates, and N-truncated PrPrec complexed with metal cations precipitate. N-truncated PrPrec complexed with Zn precipitated much faster than N-truncated PrPrec complexed with Cu. According to the hypothesis about the key role of small PrP oligomers in PrP(C)-PrP(Sc) transformation, formation of soluble oligomers of PrP complexed with Cu can constitute an additional element in TSE propagation. Identical metal-chelating behaviour of two studied polymorphic PrPrec variants conferring different susceptibilities of sheep to scrapie could indicate their different capabilities to form fibrils. This could imply also that other factors than physico-chemical differences between PrP-VRQ and PrP-ARR and the differences in PrP transformation are responsible for the onset of TSE.     

Mechanisms of metabisulfite-induced bronchoconstriction: evidence for bradykinin B2-receptor stimulation.

Sodium metabisulfite (MBS) is a food preservative that can trigger bronchoconstriction in asthmatic subjects. Previous studies designed to identify the mechanisms involved in this response have yielded conflicting results. We noted certain similarities between the pharmacology of MBS-induced airway responses and those elicited by bradykinin (BK), another provocating agent in asthmatic subjects. Therefore we used allergic sheep to determine whether MBS-induced bronchoconstriction 1) had a pharmacology similar to that previously seen with BK in this model, including protection by a BK B2-receptor antagonist, NPC-567, and 2) was associated with increased concentrations of immunoreactive kinins in bronchoalveolar lavage. We measured specific lung resistance before and immediately after inhaled buffer and increasing concentrations of MBS (30 breaths of 25, 50, and 100 mg/ml) and calculated the concentration producing 100% increase in specific lung resistance over baseline (PC100). In seven sheep, geometric mean control PC100 was 33.1 mg/ml. Pretreatment with either the anticholinergic agent ipratropium bromide (180 micrograms; PC100 87.1 mg/ml) or the antiasthma drug nedocromil sodium (1 mg/kg aerosol; PC100 97.7 mg/ml) blocked the MBS-induced bronchoconstriction (P less than 0.05), whereas the histamine H1-receptor antagonist chlorpheniramine (2 mg/kg iv) was ineffective. Furthermore the MBS-induced bronchoconstriction was not affected by the neutral endopeptidase inhibitor thiorphan (40 breaths of a 1 mg/ml solution) or the angiotensin-converting enzyme inhibitor enalaprilat (2.5 mg aerosol). In six sheep the MBS-induced bronchoconstriction was completely blocked by NPC-567 (20 breaths, 5 mg/ml aerosol): after treatment with NPC-567 mean PC100 was 100 mg/ml compared with 57.5 mg/ml in the control trial (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3–4 weeks by using Gateway® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.     

Theoretical investigations on the hydrolysis pathway of tin verdoheme complexes: elucidation of tin’s ring opening inhibition role

In order to obtain a better molecular understanding of inhibitory role of tin metal in the verdoheme ring opening process, hydrolysis of three possibly six, five, and four coordinate verdoheme complexes of tin(IV) and (II) have been studied using DFT method. The results of calculations indicate that, in excellent accord with experimental reports, hydrolysis of different possibly coordinated tin(IV) and (II) verdohemes does not lead to the opening of the macrocycle. Contrary to iron and zinc verdohemes, in five and four coordinate verdoheme complexes of tin(IV) and (II), formation of open ring helical complexes of tin are unfavorable both thermodynamically and kinetically. In these pathways, coordination of hydroxide nucleophile to tin metal due to the highly charged, exclusive oxophilicity nature of the Sn center, and high affinity of Sn to increase coordination state are proposed responsible as inhibiting roles of tin via the ring opening. While, in saturated six coordinate tin(IV) and (II) verdoheme complexes the ring opening of tin verdohemes is possible thermodynamically, but it is not predicted to occur from a kinetics point of view. In the six coordinate pathway, tin plays no coordination role and direct addition of hydroxide nucleophile to the positive oxo-carbon centers and formation of closed ring hydroxy compounds is proposed for preventing the verdoheme ring opening. These key points and findings have been corroborated by the results obtained from atomic charge analysis, geometrical parameters, and molecular orbital calculations. In addition, the results of inhibiting ring opening reaction of tin verdoheme complexes could support the great interest of tin porphyrin analogues as pharmacologic means of chemoprevention of neonatal jaundice by the competitive inhibitory action of tin porphyrins on heme oxygenase.     

Attract and kill for codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) control in Syria

The possibility of controlling the codling moth Cydia pomonella (Linnaeus) using an attract and kill approach as an alternative to chemical sprays with contact insecticides was investigated in widely separated orchards. The results of a 4‐year study have shown that, using an attract and kill approach, three applications/season kept infestation rates in treated orchards below the economic injury level except in one with a too high codling moth population density. The mean number of male codling moths/trap/week in attract and kill‐treated orchards was much lower in comparison with control orchards which were treated with the usual cover sprays of insecticides. The results also showed that the efficacy of attract and kill under orchard conditions decreased with time and the relationship between time effect and codling moth death rate was very strong. These data indicate that the attract and kill technique applied at a rate of three application per season resulted in good control of codling moth in well managed orchards in Syria.     

Activation of triggering receptor expressed on myeloid cells-1 on human neutrophils by marburg and ebola viruses

Marburg virus (MARV) and Ebola virus (EBOV), members of the viral family Filoviridae, cause fatal hemorrhagic fevers in humans and nonhuman primates. High viral burden is coincident with inadequate adaptive immune responses and robust inflammatory responses, and virus-mediated dysregulation of early host defenses has been proposed. Recently, a novel class of innate receptors called the triggering receptors expressed in myeloid cells (TREM) has been discovered and shown to play an important role in innate inflammatory responses and sepsis. Here, we report that MARV and EBOV activate TREM-1 on human neutrophils, resulting in DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory cytokines, and phenotypic changes. A peptide specific to TREM-1 diminished the release of tumor necrosis factor alpha by filovirus-activated human neutrophils in vitro, and a soluble recombinant TREM-1 competitively inhibited the loss of cell surface TREM-1 that otherwise occurred on neutrophils exposed to filoviruses. These data imply direct activation of TREM-1 by filoviruses and also indicate that neutrophils may play a prominent role in the immune and inflammatory responses to filovirus infections.     

Cryptococcus neoformans glycoantigens are captured by multiple lectin receptors and presented by dendritic cells

Cell-mediated immune responses to glycoantigens have been largely uncharacterized. Protective T cell responses to the pathogenic yeast Cryptococcus neoformans are dependent on heavily mannosylated Ags termed mannoproteins. In the work presented, the innate immune response to mannoprotein was determined. Purified murine splenic dendritic cells (DC), B cells, and macrophages were used to stimulate mannoprotein-specific T cells. Only DC were capable of any measurable stimulation. Depletion of DC resulted in the abrogation of the T cell response. Human and murine DC rapidly captured fluorescent-labeled mannoprotein by a mannose receptor-mediated process. Using transfected cell lines, the type II C-type lectin receptor DC-specific ICAM-3-grabbing nonintegrin (CD209) was determined to have affinity for mannoprotein. Taken together with prior work demonstrating that mannoprotein was captured by the macrophage mannose receptor (CD206), these data suggest that multiple mannose receptors on DC recognize mannoprotein. Pulsing experiments demonstrated that DC captured sufficient mannoprotein over 2 h to account for 50% of total stimulation. Capture appeared dependent on mannose receptors, as competitive mannosylated inhibitors and calcium chelators each interfered with T cell stimulation. By confocal microscopy, intracellular mannoprotein trafficked to an endo-lysosomal compartment in DC, and at later time points extended into tubules in a similar fashion to the degradation marker DQ-OVA. Mannoprotein colocalized intracellularly with CD206 and CD209. These data suggest that DC provide the crucial link between innate and adaptive immune responses to C. neoformans via a process that is dependent upon the efficient uptake of mannoprotein by mannose receptors.