Manipulating Immune Responses Review with Immunosuppressive Agents that Target NFAT iting a remarkable gain of function relative to the drugs themselves (Schreiber and Crabtree, 1992). In the immune system, calcineurin and NFAT, the ma- jor targets of CsA

, 1999). this normally protective system overreacts and directs The main drawback of these immunosuppressive regiits forces at the organism itself. The clinical syndrome mens, however, is that they produce major toxic side of septic shock results from overactive host responses effects and therefore are rarely employed except in serito gram-negative bacterial infections and can be fatal ous clinical situations. because of an overwhelming production of proinflamOur thesis in this review is that since NFAT is a valimatory cytokines. Transplantation of solid organs is dated target for two clinically important immunosupconfounded by the immunologic problem of graft rejecpressive drugs, interference with NFAT regulation might tion, while graft-versus-host-disease is one of the major be expected to yield additional avenues for immunosupcomplications of transplantation with allogeneic bone pression. Moreover, since NFAT is only one of many marrow or peripheral blood stem cells, severely limiting calcineurin substrates, compounds that interfere selecthe application of these potentially life-saving therapeutively with NFAT regulation might be expected to have tic options. Other examples of inappropriate immune fewer side effects than CsA and FK506. Unfortunately, responses include allergic asthma and chronic autoimthis simple premise is complicated by the existence of mune diseases such as rheumatoid arthritis. A major multiple NFAT proteins, our incomplete knowledge of challenge for clinicians is how to suppress deleterious their regulation and function in immune and nonimmune immune responses in these diverse clinical settings. cells, and our lack of information about the mechanisms The fungal metabolites cyclosporin A (CsA) and tacrolunderlying the toxic effects of CsA and FK506. imus (FK506) are among the most potent immunosupHere we review our current understanding of these pressive drugs available today. CsA revolutionized the points. We consider what features of NFAT regulation field of organ transplantation following its discovery in might be exploited to develop selective inhibitors of 1976 (Borel et al., 1976); it was approved for clinical use NFAT, whether such drugs would be as immunosupin 1983. Tacrolimus was described soon after (Kino et pressive as CsA or FK506, whether they would lack the al., 1987) and introduced into clinical trials in 1989. Detoxic effects of CsA and FK506, and whether they might spite their lack of structural similarity, the modes of have unexpected side effects in immune and nonimmune action of these two drugs are very similar: at the low organs. An informed understanding of these issues might concentrations relevant to their immunosuppressive efspur the development of better, less toxic immunosupfects, they inhibit the calcium-dependent serine/threopressive agents that target the NFAT pathway. nine phosphatase calcineurin and its substrate, the transcription factor NFAT (Schreiber and Crabtree, 1992). The drugs do not interact directly with calcineurin but What Features of NFAT Regulation Might Be Exploited rather form complexes with intracellular receptors known to Develop Selective Inhibitors of NFAT? as immunophilins. The immunophilin receptors for cyclo- The NFAT family, consisting of the "classical" members sporin A are the cyclophilins, while those for FK506 are NFAT1 (p, c2), NFAT2 (c, c1), NFAT3 (c4), and NFAT4 the FK506-binding proteins (FKBPs). Both classes of (x, c3), has recently been extended by a fifth member, immunophilin receptors are widely expressed, abundant NFAT5/TonEBP ( , 1997a, 1997b). For NFAT4, the results were less consistent: in one study, mutation of two residues be envisioned to explain this result; neither has yet been tested experimentally. Calcineurin might be needed as in the serine-rich region of NFAT4 resulted in nuclear localization (Chow et al., 1997), while in another report, an integral part of the NFAT transcriptional complex to maintain NFAT in an active and dephosphorylated form, deletion of the entire serine-rich region rendered the protein hypersensitive to calcium ionophore, a result particularly if DNA-bound NFAT present in active transcriptional complexes is still capable of being inactiinterpreted by the authors as the effect of deleting the docking site for CK1␣, a proposed NFAT kinase (Zhu vated by rephosphorylation. Alternatively, the DNAbound, transcriptionally active fraction of NFAT might et al., 1998; see below). The latter study located the phosphoserine residues responsible for nuclear import not be accessible to calcineurin or nuclear kinases; if so, the ongoing requirement for calcineurin indicates in the first SP motif of NFAT4 (Zhu et al., 1998). While, overall, the results implicate both the serine-rich region that this fraction of NFAT would be in continuous equilibrium with free nuclear NFAT. These possibilities are not and the SP motifs in regulating nuclear import, the specific discrepancies are puzzling given the high degree mutually exclusive: the detailed structure of NFAT-containing transcriptional complexes is likely to vary deof sequence conservation in the NFAT regulatory domain and the fact that all four NFATs are regulated by pending on the regulatory region involved, and hence the accessibility of NFAT to modifying enzymes could calcineurin. These discrepancies will need to be resolved before pharmacological strategies targeting debe different in each case. Paradoxically, it has been suggested that calcineurin phosphorylation-regulated nuclear import can be devised. activity is not necessary for transcription mediated by NFAT2 and NFAT4 that have been artificially localized to DNA Binding and Transcriptional Activity Have Undesired Side Effects? Functions of NFAT in Nonimmune Cells This is an area in dire need of more specific information. There is much evidence for NFAT expression and funcAs discussed in previous sections, the contribution of tion in cells outside the immune system. NFAT2-defi-NFAT to CsA and FK506 toxicity is not known. However, cient mice die in utero because of defects in the morpho- family member may be useful include allergic asthma they lack the toxicity of CsA and FK506, and whether (NFAT2) and cardiac hypertrophy (NFAT3). The developthey have unexpected side expects of their own. Indeed, ment of such specific inhibitors will require a greater a transgenic mouse has been generated that expresses appreciation of shared versus unique regulatory mechaa "dominant-negative" NFAT, whose effectiveness is at nisms, and shared versus unique functions, of individual least partly due to inhibition of NFAT-calcineurin binding NFAT proteins within and outside the immune system

Identification of Natural Killer (NK) Cells in Lesions of Human Cutaneous Graft-Versus-Host Disease: Expression of a Novel NK-Associated Surface Antigen (Kp43) in Mononuclear Infiltrates

We performed an immunohistochemical analysis of skin biopsies from 13 allogeneic bone marrow transplant (BMT) recipients, undergoing either acute graft-versus-host-disease (aGVHD, n = 8) or chronic GVHD (cGVHD, n = 5). A panel of different monoclonal antibodies (MoAb) was employed including anti-CD2, -CD3, -CD4, -CD8, -CD11b, -CD16, -CD56, and -CD57, as well as a recently described reagent (HP-3B1) specific for a novel natural killer (NK)-associated cell-surface antigen (Kp43). Our data indicate that in a GVHD lesions the proportions of CD2+ cells often exceeded those detected with anti-CD3 MoAb. Double labeling confirmed the presence of CD2+ CD3- lymphocytes and suggested the coexpression in some cells of CD2 and CD11b. When MoAb specific for non-lineage-restricted NK-associated markers were employed, anti-CD56 and -CD57 occasionally stained variable numbers of lymphocytes (x¯ = 14.6% of mononuclear cells in 0.05mm2, range <1-48% and x¯ = 10.3%, range 2–25%, respectively), whereas no CD16+lymphocytes were observed. In contrast, most samples consistently displayed substantial proportions of Kp43+cells (&xsline = 32.8%, range 12–63%), which appeared CD3-and were mainly located at the dermoepidermal junction. On the other hand, sections from most (four of five) cGVHD lichenoid lesions analyzed displayed lower proportions of Kp43 + and CD56 + cells. Our data point out the interest of the anti-Kp43 MoAb to identify NK cells in aGVHD lesions, suggesting their pathogenetic participation

Supplementary data for the article: García-Fernández, P.; Aramburu, J. A.; Moreno, M.; Zlatar, M.; Gruden-Pavlović, M. A Practical Computational Approach to Study Molecular Instability Using the Pseudo-Jahn-Teller Effect. Journal of Chemical Theory and Computation 2014, 10 (4), 1824–1833. https://doi.org/10.1021/ct4011097

Supplementary material for: [https://doi.org/10.1021/ct4011097]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1765

The Transcription Factor NFAT5 Is Required for Cyclin Expression and Cell Cycle Progression in Cells Exposed to Hypertonic Stress

Background: Hypertonicity can perturb cellular functions, induce DNA damage-like responses and inhibit proliferation. The transcription factor NFAT5 induces osmoprotective gene products that allow cells to adapt to sustained hypertonic conditions. Although it is known that NFAT5-deficient lymphocytes and renal medullary cells have reduced proliferative capacity and viability under hypertonic stress, less is understood about the contribution of this factor to DNA damage responses and cell cycle regulation. Methodology/Principal Findings: We have generated conditional knockout mice to obtain NFAT5−/− T lymphocytes, which we used as a model of proliferating cells to study NFAT5-dependent responses. We show that hypertonicity triggered an early, NFAT5-independent, genotoxic stress-like response with induction of p53, p21 and GADD45, downregulation of cyclins, and cell cycle arrest. This was followed by an NFAT5-dependent adaptive phase in wild-type cells, which induced an osmoprotective gene expression program, downregulated stress markers, resumed cyclin expression and proliferation, and displayed enhanced NFAT5 transcriptional activity in S and G2/M. In contrast, NFAT5−/− cells failed to induce osmoprotective genes and exhibited poorer viability. Although surviving NFAT5−/− cells downregulated genotoxic stress markers, they underwent cell cycle arrest in G1/S and G2/M, which was associated with reduced expression of cyclins E1, A2 and B1. We also show that pathologic hypertonicity levels, as occurring in plasma of patients and animal models of osmoregulatory disorders, inhibited the induction of cyclins and aurora B kinase in response to T cell receptor stimulation in fresh NFAT5−/− lymphocytes. Conclusions/Significance: We conclude that NFAT5 facilitates cell proliferation under hypertonic conditions by inducing an osmoadaptive response that enables cells to express fundamental regulators needed for cell cycle progression.Molecular and Cellular Biolog

Application of the SMED method to increase the efficiency of the sealing process in a Peruvian company that produces flexible plastic

Currently, in the Peruvian industrial sector, most companies are affected by the low efficiency provided by the staff when carrying out their respective work. This arises because most companies are looking for the cheapest labor they can get and not all these people have the economic capacity to receive a good level of education. In manufacturing companies, it is almost always possible to observe "the law of minimum effort in the operators" which causes operations without added value to arise in the processes, unnecessary delays and all this translates into processes with low efficiency. What we want to demonstrate in the following investigation is how, with adequate training for the personnel, all these mentioned defects can be reduced and with a not very high investment for the company. There are many tools that we can use that the Lean Six Sigma methodology gives us depending on the objective to improve. This article mentions the implementation of the SMED tool in a flexible plastic production company where we were able to achieve an 8% increase in the efficiency of the sealing area with the reduction of product change over time

Implicit Subspace Iteration to Improve the Stability Analysis in Grinding Processes

An alternative method is devised for calculating dynamic stability maps in cylindrical and centerless infeed grinding processes. The method is based on the application of the Floquet theorem by repeated time integrations. Without the need of building the transition matrix, this is the most efficient calculation in terms of computation effort compared to previously presented time-domain stability analysis methods (semi-discretization or time-domain simulations). In the analyzed cases, subspace iteration has been up to 130 times faster. One of the advantages of these time-domain methods to the detriment of frequency domain ones is that they can analyze the stability of regenerative chatter with the application of variable workpiece speed, a well-known technique to avoid chatter vibrations in grinding processes so the optimal combination of amplitude and frequency can be selected. Subspace iteration methods also deal with this analysis, providing an efficient solution between 27 and 47 times faster than the abovementioned methods. Validation of this method has been carried out by comparing its accuracy with previous published methods such as semi-discretization, frequency and time-domain simulations, obtaining good correlation in the results of the dynamic stability maps and the instability reduction ratio maps due to the application of variable speed

Characterization of Tomato spotted wilt virus isolates that overcome the Sw-5 resistance gene in tomato and fitness assays

Resistance-breaking (RB) isolates of Tomato spotted wilt virus (TSWV) that overcome the resistance conferred by the Sw-5 gene in tomato have had only a limited spread since they were first detected in north-eastern Spain in 2002. Symptom expression, homogeneity, stability and the transmission capacity of RB and non-resistance breaking (NRB) isolates were biologically compared. The fitness of both types of isolates infecting tomato plants was determined in competition assays. All TSWV isolates induced similar systemic symptoms in a wide range of plant species, except RB isolates in tomato carrying the Sw-5 resistance gene and pepper carrying the Tsw resistance gene. The mechanical transmission of RB isolates to tomato plants with the Sw-5 gene failed in some trials, although NRB isolates did not differ noticeably in transmission efficiency when tested with the thrips Frankliniella occidentalis. Biological clones from individual local lesions obtained by mechanically inoculating Nicotiana glutinosa in some TSWV field samples showed that they were biologically homogeneous. Mixed infections of RB and wilt-type isolates were not found. The RB isolates were relatively stable because no reversion to NRB isolates was seen after serial passages in susceptible tomato plants. In competition assays between RB and NRB isolates, after serial passages in susceptible tomato plants, the prevalence of a particular isolate was not related to its capacity to overcome Sw-5 gene resistance. The low spread of the RB isolates in Spain does not seem to be related to a loss of fitness in tomato plants or to differences in transmission capacity by thrips, but it could be related to the reduction of the selection pressure of RB isolates as consequence of the gradual replacement of susceptible tomato plants by resistant tomato plants by growers

Complete Genome Sequence of a Tomato Isolate of Parietaria Mottle Virus from Italy

We report here the complete genome sequence of isolate T32 of parietaria mottle virus (PMoV) infecting tomato plants in Turin, Italy, obtained by Sanger sequencing. T32 shares 90.48 to 96.69% nucleotide identity with other two PoMV isolates, CR8 and Pe1, respectively, whose complete genome sequences are available

Dabrafenib for cutaneous melanoma infiltrating the vitreous: regression of metastasis and occurrence of uveitis as a secondary effect

Intraocular metastasis of cutaneous melanoma is extremely infrequent. This typically occurs in advanced metastatic disease and has a poor survival prognosis. The most frequent reported treatment is radiotherapy. BRAF inhibitors are new, orally administered and very effective drugs used for metastatic cutaneous melanoma. Herein, we report a case of a 58-year-old patient with a recent diagnosis of cutaneous melanoma who consulted for floaters and presented vitreous opacities in both eyes. A diagnostic vitrectomy of his left eye was performed and pathologic analysis disclosed infiltrating melanoma cells in the vitreous. Treatment with dabrafenib (a type of BRAF inhibitor) achieved the regression of the intraocular metastasis in the right eye. Moreover, the patient presented a severe anterior uveitis due to dabrafenib, a well-known secondary effect of this drug