Target For Therapeutic Action Research Articles

Bronchial asthma resistant to pharmacotherapy: why there is no response to the treatment?

Despite the obvious and simple diagnosis, in practice we meet patients who do not respond to standard therapy. Why does drug resistance occur? The answer to this question is not unambiguous and requires careful investigation in each clinical case. Here we present a clinical case of a patient with bronchial asthma, who had no bad habits and occupational hazards and received adequate drug therapy. However, her condition worsened from attack to attack. Whole exome sequencing by NGS allowed us to determine the spectrum of pathogenic mutations which contribute to the pathological process and drug resistance. Atopy due to dysfunction of filaggrin gene (FLG) triggered the disease and supported the pathological process that led to bronchial asthma. Furthermore, the patient’s body is not able to neutralize the bacterial flaggelin. The inflammatory response is reduced due to а Toll-like receptor 5 (TLR5) deficiency. This is one of the mechanisms underlying development of allergic bronchial asthma. In addition, the patient has reduced cross-presentation of antigens by dendritic cells, that is, a reduced immune response in the absence of infection, due to the complete loss of UNC93B1 gene function. Conclusion. Thus, an atopic reaction based on reduced adaptive immunity led to severe IgE allergy and torpid course of bronchial asthma. This conclusion supports atopic sensitization as the target for therapeutic action and the main core of pathological processes. For this purpose, we used a monoclonal antibody omalizumab that is capable of binding and reducing the amount of IgE. Targeted treatment of bronchial asthma made it possible to interrupt the symptoms and achieve complete remission.

Characterization and functional analysis of the proteins Prohibitin 1 and 2 in Trypanosoma cruzi.

BackgroundChagas disease is the third most important neglected tropical disease. There is no vaccine available, and only two drugs are generally prescribed for the treatment, both of which with a wide range of side effects. Our study of T. cruzi PHBs revealed a pleiotropic function in different stages of the parasite, participating actively in the transformation of the non-infective replicative epimastigote form into metacyclic trypomastigotes and also in the multiplication of intracellular amastigotes.Methodology/principal findingsTo obtain and confirm our results, we applied several tools and techniques such as electron microscopy, immuno-electron microscopy, bioinformatics analysis and molecular biology. We transfected T. cruzi clones with the PHB genes, in order to overexpress the proteins and performed a CRISPR/Cas9 disruption to obtain partially silenced PHB1 parasites or completely silenced PHB2 parasites. The function of these proteins was also studied in the biology of the parasite, specifically in the transformation rate from non-infective forms to the metacyclic infective forms, and in their capacity of intracellular multiplication.Conclusion/significanceThis research expands our understanding of the functions of PHBs in the life cycle of the parasite. It also highlights the protective role of prohibitins against ROS and reveals that the absence of PHB2 has a lethal effect on the parasite, a fact that could support the consideration of this protein as a possible target for therapeutic action.

Mice Selected for Acute Inflammation Present Altered Immune Response during Pristane-Induced Arthritis Progression.

Mouse lines selected for maximal (AIRmax) or minimal acute inflammatory reaction (AIRmin) were used to characterize the immune response and the influence of genetic background during pristane-induced arthritis (PIA). Susceptible AIRmax mice demonstrated exacerbated cellular profiles during PIA, with intense infiltration of lymphocytes, as well as monocytes/macrophages and neutrophils, producing higher levels of IL-1β, IFN-γ, TNF-α, IL-10, total IgG3, and chemokines. Resistant AIRmin mice controlled cell activation more efficiently than the AIRmax during arthritis progression. The weight alterations of the spleen and thymus in the course of PIA were observed. Our data suggest that selected AIRmax cellular and genetic immune mechanisms contribute to cartilage damage and arthritis severity, evidencing many targets for therapeutic actions.

Pharmacological Profile of 2-Bromoterguride at Human Dopamine D2, Porcine Serotonin 5-Hydroxytryptamine 2A, and α2C-Adrenergic Receptors, and Its Antipsychotic-Like Effects in Rats

Dopaminergic, serotonergic, and adrenergic receptors are targets for therapeutic actions in schizophrenia. Dopamine D2 receptor partial agonists such as aripiprazole represent a treatment option for patients with this severe disorder. The ineffectiveness of terguride, another D2 receptor partial agonist, in treating schizophrenia was recently attributed to its considerably high intrinsic activity at D2 receptors. In this study, we used functional assays for recombinant D2 receptors and native 5-hydroxytryptamine 2A (5-HT2A), α2C-adrenergic, and histamine H1 receptors to compare the pharmacological properties of terguride and three of its halogenated derivatives (2-chloro-, 2-bromo-, 2-iodoterguride) with those of aripiprazole. Subsequently, we studied the antidopaminergic effects of 2-bromoterguride using amphetamine-induced locomotion (AIL). Its influence on spontaneous behavior was tested in the open field. Extrapyramidal side effect (EPS) liability was evaluated by catalepsy test. In a guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding assay, 2-chloro-, 2-bromo-, and 2-iodoterguride produced intrinsic activities at human D2short (hD2S) receptors that were half as high as the intrinsic activity for terguride; aripiprazole lacked agonist activity. 2-Bromoterguride and aripiprazole activated D2S receptor-mediated inhibition of cAMP accumulation to the same extent; intrinsic activity was half as high as that of terguride. All compounds tested behaved as antagonists at human D2long/Gαo (hD2L/Gαo) receptors. Compared with aripiprazole, terguride and its derivatives displayed higher affinity at porcine 5-HT2A receptors and α2C-adrenoceptors and lower affinity at H1 receptors. 2-Bromoterguride inhibited AIL and did not induce catalepsy in rats. Because of its in vitro and in vivo properties, 2-bromoterguride may be a strong candidate for the treatment of schizophrenia with a lower risk to induce EPS.

Alternative splicing: regulation of HIV‐1 multiplication as a target for therapeutic action

The retroviral life cycle requires that significant amounts of RNA remain unspliced and perform several functions in the cytoplasm. Thus, the full-length RNA serves both the viral genetic material that will be encapsulated in viral particles and the mRNA encoding structural and enzymatic proteins required for viral replication. Simple retroviruses produce one single-spliced env RNA from the full-length precursor RNA, whereas complex retroviruses, such as HIV, are characterized by the production of multiple-spliced RNA species. In this review we will summarize the current acknowledge about the HIV-1 alternative splicing mechanism and will describe how this malleable process can help further understanding of infection, spread and dissemination through splicing regulation. Such studies coupled with the testing of splicing inhibitors should help the development of new therapeutic antiviral agents.

Production and characterization of active hepatitis C virus RNA-dependent RNA polymerase

The non-structural protein 5B (NS5B) is an essential component for the genome replication of hepatitis C virus (HCV). Thus, its activity holds the potential of being a target for therapeutic actions against HCV. The availability of large amount of functionally active NS5B enzyme may facilitate the identification of NS5B inhibitors via high-throughput screening (HTS). Here, we expressed the C-terminal 20-amino acids truncated NS5B in a bacterial system using the N-terminal domain of Escherichia coli lysyl-tRNA synthetase (LysN) as a solubility enhancer. The fusion protein (LysN-NS5B) was purified in a yield of 6.2 mg/L. The activity of LysN-NS5B was confirmed by in vitro RNA-dependent RNA polymerase (RdRp) activity assay, and the biochemical properties of LysN-NS5B were further characterized by kinetic analysis. The optimal RdRp activity was shown at 30 °C with 5 mM of Mg 2+ or 10 mM of Mn 2+, while the K m value for UTP was determined as 5 μM. The RdRp activity of LysN-NS5B was strongly inhibited by phenyldiketoacid, a specific inhibitor of HCV NS5B activity. Our results suggest that the LysN fusion system is a suitable approach for producing an active form of NS5B that can be used for HTS of NS5B inhibitors.

Adrenergic targets for the treatment of cognitive deficits in schizophrenia.

The cognitive functions of the prefrontal cortex (PFC) are profoundly impaired in schizophrenic patients. Although dopamine has been the major focus of schizophrenia research, norepinephrine (NE) also has marked influences on PFC cognitive functioning. This review aims to identify the adrenergic receptors which may be appropriate targets for therapeutic actions in schizophrenia. Studies of adrenergic mechanisms influencing PFC function in animals and humans were reviewed. Modest levels of NE engage postsynaptic alpha(2A)-adrenergic receptors and strengthen working memory. These beneficial effects have been observed at both the behavioral and cellular levels in animals, and have translated to the clinic in patients with PFC impairments. Thus, the alpha(2A)-adrenergic receptor is a proven molecular target. In contrast, high levels of NE released during stress impair PFC cognitive function via activation of protein kinase C intracellular signaling, a pathway increasingly associated with the etiology of schizophrenia. Blockade of alpha(1) adrenoceptors or inhibition of protein kinase C helps to protect PFC cognitive function in animals, and may have similar therapeutic actions in humans. Blockade of the alpha(2C) receptor may also be helpful in enhancing catecholamine release while blocking detrimental DA actions in striatum. Highly selective adrenergic agents may be useful for enhancing PFC function in schizophrenic patients

Visualization of beta-amyloid peptide (Abeta) phagocytosis by human mononuclear phagocytes: dependency on Abeta aggregate size.

Previous studies from this laboratory have shown that human monocytes exposed to beta-amyloid peptide (Abeta) exert a graded neurotoxic response in an organotypic brain culture paradigm. Moreover, this toxicity can be reduced by compounds that inhibit cell motility and phagocytosis, suggesting that internalization of Abeta may be a requirement for neurotoxic action. To confirm that Abeta is indeed phagocytosed by monocytes and to further lay the groundwork for resolving the possible linkage between this process and neurotoxicity, we examined Abeta:monocyte interactions using immunocytochemistry and fluorescence histochemistry followed by confocal microscopy and three-dimensional image reconstruction. Internalization of Abeta was detected by 24 hr following exposure of monocytes to the purified peptide, and the relative efficacy of this process appeared to be influenced by the size of the Abeta aggregates. Specifically, smaller aggregates were observed to be more efficiently internalized, while larger Abeta masses tended to reside only on the monocyte surface, apparently bound to several monocytes at once. Both colchicine and cytochalasin D, cytoskeletal perturbants that block phagocytosis, caused Abeta to accumulate in deep pits within monocytes and inhibited complete envelopment by monocyte cytoplasm. These results suggest that monocytes can indeed phagocytose aggregates of Abeta and that this process may be critical in activating these cells to a neurocytopathic state. Accordingly, interference of Abeta phagocytosis by monocytes or monocyte-derived cells may be a novel target for therapeutic action.