Anti-CD25 Treatment Depletes Treg Cells and Decreases Disease Severity in Susceptible and Resistant Mice Infected with Paracoccidioides brasiliensis

Regulatory T (Treg) cells are fundamental in the control of immunity and excessive tissue pathology. In paracoccidioidomycosis, an endemic mycosis of Latin America, the immunoregulatory mechanisms that control the progressive and regressive forms of this infection are poorly known. Due to its modulatory activity on Treg cells, we investigated the effects of anti-CD25 treatment over the course of pulmonary infection in resistant (A/J) and susceptible (B10.A) mice infected with Paracoccidioides brasiliensis. We verified that the resistant A/J mice developed higher numbers and more potent Treg cells than susceptible B10.A mice. Compared to B10.A cells, the CD4(+)CD25(+)Foxp3(+) Treg cells of A/J mice expressed higher levels of CD25, CTLA4, GITR, Foxp3, LAP and intracellular IL-10 and TGF-beta. In both resistant and susceptible mice, anti-CD25 treatment decreased the CD4(+)CD25(+)Foxp3(+) Treg cell number, impaired indoleamine 2,3-dioxygenase expression and resulted in decreased fungal loads in the lungs, liver and spleen. In A/J mice, anti-CD25 treatment led to an early increase in T cell immunity, demonstrated by the augmented influx of activated CD4(+) and CD8(+) T cells, macrophages and dendritic cells to the lungs. At a later phase, the mild infection was associated with decreased inflammatory reactions and increased Th1/Th2/Th17 cytokine production. In B10.A mice, anti-CD25 treatment did not alter the inflammatory reactions but increased the fungicidal mechanisms and late secretion of Th1/Th2/Th17 cytokines. Importantly, in both mouse strains, the early depletion of CD25(+) cells resulted in less severe tissue pathology and abolished the enhanced mortality observed in susceptible mice. In conclusion, this study is the first to demonstrate that anti-CD25 treatment is beneficial to the progressive and regressive forms of paracoccidioidomycosis, potentially due to the anti-CD25-mediated reduction of Treg cells, as these cells have suppressive effects on the early T cell response in resistant mice and the clearance mechanisms of fungal cells in susceptible mice.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Pesquisas (CNPq)Conselho Nacional de Pesquisas (CNPq

The intrinsically disordered, epigenetic factor RYBP binds to the citrullinating enzyme PADI4 in cancer cells

14 p.-6 fig.-1 tab.RYBP (Ring1 and YY 1 binding protein) is a multifunctional, intrinsically disordered protein (IDP), best described as a transcriptional regulator. It exhibits a ubiquitin-binding functionality, binds to other transcription factors, and has a key role during embryonic development. RYBP, which folds upon binding to DNA, has a Zn-finger domain at its N-terminal region. By contrast, PADI4 is a well-folded protein and it is one the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. As both proteins intervene in signaling pathways related to cancer development and are found in the same localizations within the cell, we hypothesized they may interact. We observed their association in the nucleus and cytosol in several cancer cell lines, by using immunofluorescence (IF) and proximity ligation assays (PLAs). Binding also occurred in vitro, as measured by isothermal titration calorimetry (ITC) and fluorescence, with a low micromolar affinity (~1 μM). AlphaFold2-multimer (AF2) results indicate that PADI4's catalytic domain interacts with the Arg53 of RYBP docking into its active site. As RYBP sensitizes cells to PARP (Poly (ADP-ribose) polymerase) inhibitors, we applied them in combination with an enzymatic inhibitor of PADI4 observing a change in cell proliferation, and the hampering of the interaction of both proteins. This study unveils for the first time the possible citrullination of an IDP, and suggests that this new interaction, whether it involves or not citrullination of RYBP, might have implications in cancer development and progression.This research was funded by Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033/ and “ERDF A way of Making Europe” [PID2021-127296OB-I00 to AVC; and PDC2022-133952-I00 to EF]; by Instituto de Salud Carlos III co-funded by European Social Fund “Investing in your future” [CP19/00095 to CdJ] [PI22/00824 to MS and CdJ] [PI18/00394 to OA]; by Diputación General de Aragón [“Protein targets and Bioactive Compounds group” E45-20R to AVC, and “Digestive Pathology Group” B25-20R to OA], and by Consellería de Innovación, Universidades, Ciencia y Sociedad Digital (Generalitat Valenciana) [CAICO 2021/0135 to CdJ and JLN].Peer reviewe

Aspergillus Myosin-V Supports Polarized Growth in the Absence of Microtubule-Based Transport

In the filamentous fungus Aspergillus nidulans, both microtubules and actin filaments are important for polarized growth at the hyphal tip. Less clear is how different microtubule-based and actin-based motors work together to support this growth. Here we examined the role of myosin-V (MYOV) in hyphal growth. MYOV-depleted cells form elongated hyphae, but the rate of hyphal elongation is significantly reduced. In addition, although wild type cells without microtubules still undergo polarized growth, microtubule disassembly abolishes polarized growth in MYOV-depleted cells. Thus, MYOV is essential for polarized growth in the absence of microtubules. Moreover, while a triple kinesin null mutant lacking kinesin-1 (KINA) and two kinesin-3s (UNCA and UNCB) undergoes hyphal elongation and forms a colony, depleting MYOV in this triple mutant results in lethality due to a severe defect in polarized growth. These results argue that MYOV, through its ability to transport secretory cargo, can support a significant amount of polarized hyphal tip growth in the absence of any microtubule-based transport. Finally, our genetic analyses also indicate that KINA (kinesin-1) rather than UNCA (kinesin-3) is the major kinesin motor that supports polarized growth in the absence of MYOV

The Functions of Myosin II and Myosin V Homologs in Tip Growth and Septation in Aspergillus nidulans

Because of the industrial and medical importance of members of the fungal genus Aspergillus, there is considerable interest in the functions of cytoskeletal components in growth and secretion in these organisms. We have analyzed the genome of Aspergillus nidulans and found that there are two previously unstudied myosin genes, a myosin II homolog, myoB (product = MyoB) and a myosin V homolog, myoE (product = MyoE). Deletions of either cause significant growth defects. MyoB localizes in strings that coalesce into contractile rings at forming septa. It is critical for septation and normal deposition of chitin but not for hyphal extension. MyoE localizes to the Spitzenkörper and to moving puncta in the cytoplasm. Time-lapse imaging of SynA, a v-SNARE, reveals that in myoE deletion strains vesicles no longer localize to the Spitzenkörper. Tip morphology is slightly abnormal and branching occurs more frequently than in controls. Tip extension is slower than in controls, but because hyphal diameter is greater, growth (increase in volume/time) is only slightly reduced. Concentration of vesicles into the Spitzenkörper before incorporation into the plasma membrane is, thus, not required for hyphal growth but facilitates faster tip extension and a more normal hyphal shape

CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Integrative neurobiology of metabolic diseases, neuroinflammation, and neurodegeneration

Alzheimer’s disease (AD) is a complex, multifactorial disease with a number of leading mechanisms, including neuroinflammation, processing of amyloid precursor protein (APP) to amyloid β peptide, tau protein hyperphosphorylation, relocalization and deposition. These mechanisms are propagated by obesity, the metabolic syndrome and type-2 diabetes mellitus. Stress, sedentariness, dietary overconsumption of saturated fat and refined sugars, and circadian derangements/disturbed sleep contribute to obesity and related metabolic diseases, but also accelerate age-related damage and senescence that all feed the risk of developing AD too. The complex and interacting mechanisms are not yet completely understood and will require further analysis. Instead of investigating AD as a mono- or oligocausal disease we should address the disease by understanding the multiple underlying mechanisms and how these interact. Future research therefore might concentrate on integrating these by systems biology approaches, but also to regard them from an evolutionary medicine point of view. The current review addresses several of these interacting mechanisms in animal models and compares them with clinical data giving an overview about our current knowledge and puts them into an integrated framework

TNF-alpha and CD8(+) T Cells Mediate the Beneficial Effects of Nitric Oxide Synthase-2 Deficiency in Pulmonary Paracoccidioidomycosis

Background: Nitric oxide (NO), a key antimicrobial molecule, was previously shown to exert a dual role in paracoccidioidomycosis, an endemic fungal infection in Latin America. in the intravenous and peritoneal models of infection, NO production was associated with efficient fungal clearance but also with non-organized granulomatous lesions. Because paracoccidioidomycosis is a pulmonary infection, we aimed to characterize the role of NO in a pulmonary model of infection.Methodology/Principal Findings: C57Bl/6 wild type (WT) and iNOS(-/-) mice were i.t. infected with 1x10(6) Paracoccidioides brasiliensis yeasts and studied at several post-infection periods. Unexpectedly, at week 2 of infection, iNOS(-/-) mice showed decreased pulmonary fungal burdens associated with an M2-like macrophage profile, which expressed high levels of TGF-beta impaired ability of ingesting fungal cells. This early decreased fungal loads were concomitant with increased DTH reactions, enhanced TNF-alpha synthesis and intense migration of activated macrophages, CD4(+) and CD8(+) T cells into the lungs. By week 10, iNOS(-/-) mice showed increased fungal burdens circumscribed, however, by compact granulomas containing elevated numbers of activated CD4(+) T cells. Importantly, the enhanced immunological reactivity of iNOS(-/-) mice resulted in decreased mortality rates. in both mouse strains, depletion of TNF-alpha led to non-organized lesions and excessive influx of inflammatory cells into the lungs, but only the iNOS(-/-) mice showed increased mortality rates. in addition, depletion of CD8(+) cells abolished the increased migration of inflammatory cells and decreased the number of TNF-alpha and IFN-gamma CD4(+) and CD8(+) T cells into the lungs of iNOS(-/-) mice.Conclusions/Significance: Our study demonstrated that NO plays a deleterious role in pulmonary paracoccidioidomycosis due to its suppressive action on TNF-alpha production, T cell immunity and organization of lesions resulting in precocious mortality of mice. It was also revealed that uncontrolled fungal growth can be overcome by an efficient immune response.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Inst Ciencias Biomed, Dept Imunol, BR-05508 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilHosp Sirio Libanes São Paulo, Dept Patol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 04/14518-2FAPESP: 2011/51258-2Web of Scienc

Treg cells from resistant mice have a higher suppressive potency than those of susceptible mice.

<p>CFSE-labeled responder CD4⁺CD25⁻ T cells from naïve mice were stimulated by irradiated naïve APCs plus anti-CD3 antibodies and cultured in the presence or absence of several ratios of CD25⁺ T cells obtained from lungs of resistant and susceptible mice at weeks 2 (A) and 10 (B) after infection with 1×10⁶<i>P.brasiliensis</i> yeasts. Cells were cultured for 5 days and the proliferative response of CFSE-labeled cells was measured by flow cytometry. The proliferation index (PI) was calculated as describe in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051071#s2" target="_blank">materials and methods</a> and the percentage of inhibition considered as 100% the PI of APC-stimulated CD4⁺CD25⁻ responder cells in the absence of CD4⁺CD25⁺ cells. The data represent the mean ± SEM of the results from 6 mice per group and are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with A/J mice.</p

Anti-CD25 treatment increases the influx of inflammatory cells to the lungs of resistant but not susceptible mice to <i>P. brasiliensis</i> infection.

<p>Anti-CD25-treated and untreated A/J and B10.A mice were inoculated i.t. with 1×10⁶<i>P. brasiliensis</i> yeast cells. At weeks 2 and 10 of infection lungs of both mouse strains (n = 6) were excised, minced, and digested enzymatically. Lung cells suspensions were obtained, counted, stained for CD3 (T cells), CD19 (B cells) and GR1 (myeloid cells, including neutrophils and monocytes) by flow cytometry. Anti-CD25 treatment significantly alters the number (A) but not the frequency of inflammatory cells in the lungs (B) of infected mice. At week 2, anti-CD25-treated A/J mice showed increased influx of T cells, B cells and myeloid cells, whereas at week 10 these populations appeared in decreased numbers (C, D). In B10.A mice only GR1⁺ cells appeared in decreased numbers at week 10 of infection (C, D). The data represent the mean ± SEM of the results from 6–8 mice per group and are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG-treated mice.</p