Clinical and economic burden of systemic lupus erythematosus in Colombia.

AIMS: Our cost-of-illness (COI) model adopted payer and societal perspectives over five years to measure the economic burden of Systemic Lupus Erythematosus (SLE) in Colombia. MATERIALS AND METHODS: A prevalence-based model was constructed to estimate costs and economic consequences for SLE patients in Colombia. The model included four health states: three phenotypes of SLE representing mild, moderate, and severe states and death. The clinical inputs were captured from the published literature and validated by the Delphi panel. Our model measured direct medical and indirect costs, including disease management, transient events, and indirect costs. One-way sensitivity analysis was also performed. RESULTS: The number of Colombian SLE patients was 37,498. The number of SLE patients with mild, moderate, and severe phenotypes was 5343, 28757 and 3,397, respectively. SLE-patients with moderate (Colombian pesos; COP 146 billion) and severe phenotypes (COP276 billion) incurred higher costs than those with mild phenotypes (COP2 billion), over 5 years. The total SLE cost in Colombia over five years from the payer and societal perspectives was estimated to be COP 915 billion and 8 trillion, respectively. The costs per patient per year from the payer and societal perspectives were COP 4,881,902 ($3,510) and COP 46,637,054 ($33,528), respectively. CONCLUSION: The burden of SLE in Colombia over five years is substantially high, mainly due to the consequences of economic loss because it affects women and men of working age, in addition to the costs of SLE management and its consequences, such as flares, infection, and organ damage. Our COI indicated that disease management costs among patients with moderate and severe SLE were substantially higher than those among patients with a mild phenotype. Therefore, more attention should be paid to limiting the progression of SLE and the occurrence of flares, with the need for further economic evaluation of novel treatment strategies that help in disease control.

Lower female survival from an opportunistic infection reveals progesterone-driven sex bias in trained immunity.

Immune responses differ between females and males, although such sex-based variance is incompletely understood. Observing that bacteremia of the opportunistic pathogen Burkholderia gladioli caused many more deaths of female than male mice bearing genetic deficiencies in adaptive immunity, we determined that this was associated with sex bias in the innate immune memory response called trained immunity. Female attenuation of trained immunity varies with estrous cycle stage and correlates with serum progesterone, a hormone that decreases glycolytic capacity and recall cytokine secretion induced by antigen non-specific stimuli. Progesterone receptor antagonism rescues female trained immune responses and survival from controlled B. gladioli infection to magnitudes similar to those of males. These data demonstrate progesterone-dependent sex bias in trained immunity where attenuation of female responses is associated with survival outcomes from opportunistic infection.

Computational investigation of IP3 diffusion.

Inositol 1,4,5-trisphosphate (IP3) plays a key role in calcium signaling. After stimulation, it diffuses from the plasma membrane where it is produced to the endoplasmic reticulum where its receptors are localized. Based on in vitro measurements, IP3 was long thought to be a global messenger characterized by a diffusion coefficient of ~ 280 µm2s-1. However, in vivo observations revealed that this value does not match with the timing of localized Ca2+ increases induced by the confined release of a non-metabolizable IP3 analog. A theoretical analysis of these data concluded that in intact cells diffusion of IP3 is strongly hindered, leading to a 30-fold reduction of the diffusion coefficient. Here, we performed a new computational analysis of the same observations using a stochastic model of Ca2+ puffs. Our simulations concluded that the value of the effective IP3 diffusion coefficient is close to 100 µm2s-1. Such moderate reduction with respect to in vitro estimations quantitatively agrees with a buffering effect by non-fully bound inactive IP3 receptors. The model also reveals that IP3 spreading is not much affected by the endoplasmic reticulum, which represents an obstacle to the free displacement of molecules, but can be significantly increased in cells displaying elongated, 1-dimensional like geometries.

STING controls T cell memory fitness during infection through T cell-intrinsic and IDO-dependent mechanisms.

Stimulator of interferon genes (STING) signaling has been extensively studied in inflammatory diseases and cancer, while its role in T cell responses to infection is unclear. Using Listeria monocytogenes strains engineered to induce different levels of c-di-AMP, we found that high STING signals impaired T cell memory upon infection via increased Bim levels and apoptosis. Unexpectedly, reduction of TCR signal strength or T cell-STING expression decreased Bim expression, T cell apoptosis, and recovered T cell memory. We found that TCR signal intensity coupled STING signal strength to the unfolded protein response (UPR) and T cell survival. Under strong STING signaling, Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibition also reduced apoptosis and led to a recovery of T cell memory in STING sufficient CD8 T cells. Thus, STING signaling regulates CD8 T cell memory fitness through both cell-intrinsic and extrinsic mechanisms. These studies provide insight into how IDO and STING therapies could improve long-term T cell protective immunity.

Early expression of mature αß TCR in CD4-CD8- T cell progenitors enables MHC to drive development of T-ALL bearing NOTCH mutations.

During normal T cell development in mouse and human, a low-frequency population of immature CD4-CD8- double-negative (DN) thymocytes expresses early, mature αß T cell antigen receptor (TCR). We report that these early αß TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αß T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αß TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

Three-Dimensional Model of Sub-Plasmalemmal Ca2+ Microdomains Evoked by T Cell Receptor/CD3 Complex Stimulation.

Ca2+ signalling plays an essential role in T cell activation, which is a key step to start an adaptive immune response. During the transition from a quiescent to a fully activated state, Ca2+ microdomains of reduced spatial and temporal extents develop in the junctions between the plasma membrane and the endoplasmic reticulum (ER). These microdomains rely on Ca2+ entry from the extracellular medium, via the ORAI1/STIM1/STIM2 system that mediates store operated Ca2+ entry Store operated calcium entry. The mechanism leading to local store depletion and subsequent Ca2+ entry depends on the activation state of the cells. The initial, smaller microdomains are triggered by D-myo-inositol 1,4,5-trisphosphate (IP3) signalling in response to T cell adhesion. T cell receptor (TCR)/CD3 stimulation then initiates nicotinic acid adenine dinucleotide phosphate signalling, which activates ryanodine receptors (RYR). We have recently developed a mathematical model to elucidate the spatiotemporal Ca2+ dynamics of the microdomains triggered by IP3 signalling in response to T cell adhesion (Gil et al., 2021). This reaction-diffusion model describes the evolution of the cytosolic and endoplasmic reticulum Ca2+ concentrations in a three-dimensional ER-PM junction and was solved using COMSOL Multiphysics. Modelling predicted that adhesion-dependent microdomains result from the concerted activity of IP3 receptors and pre-formed ORAI1-STIM2 complexes. In the present study, we extend this model to include the role of RYRs rapidly after TCR/CD3 stimulation. The involvement of STIM1, which has a lower KD for Ca2+ than STIM2, is also considered. Detailed 3D spatio-temporal simulations show that these Ca2+ microdomains rely on the concerted opening of ∼7 RYRs that are simultaneously active in response to the increase in NAADP induced by T cell stimulation. Opening of these RYRs provoke a local depletion of ER Ca2+ that triggers Ca2+ flux through the ORAI1 channels. Simulations predict that RYRs are most probably located around the junction and that the increase in junctional Ca2+ concentration results from the combination between diffusion of Ca2+ released through the RYRs and Ca2+ entry through ORAI1 in the junction. The computational model moreover provides a tool allowing to investigate how Ca2+ microdomains occur, extend and interact in various states of T cell activation.

Current experimental methods to investigate the impact of specialized pro-resolving lipid mediators on Sjögren's syndrome.

Sjögren's syndrome is a chronic inflammatory autoimmune disease characterized by diminished secretory function of the exocrine glands. Although extensive investigation has been done to understand Sjögren's syndrome, the causes of the disease are as yet unknown and treatments remain largely ineffective, with established therapeutic interventions being limited to use of saliva substitutes with modest effectiveness. A primary feature of Sjögren's syndrome is uncontrolled inflammation of exocrine tissues and previous studies have demonstrated that lipid-based specialized pro-resolving mediators reduce inflammation and restores tissue integrity in salivary glands. However, these studies are limited to a single specialized pro-resolving lipid mediator's family member resolvin D1 or RvD1 and its aspirin-triggered epimer, AT-RvD1. Consequently, additional studies are needed to explore the potential benefits of other members of the specialized pro-resolving lipid mediator's family and related molecules (e.g., additional resolvin subtypes as well as lipoxins, maresins and protectins). In support of this goal, the current review aims to briefly describe the range of current experimental methods to investigate the impact of specialized pro-resolving lipid mediators on Sjögren's syndrome, including both strengths and weaknesses of each approach where this information is known. With this article, the possibilities presented by specialized pro-resolving lipid mediators will be introduced to a wider audience in immunology and practical advice is given to researchers who may wish to take up this work.

The Antiviral and Virucidal Activities of Voacangine and Structural Analogs Extracted from Tabernaemontana cymosa Depend on the Dengue Virus Strain.

Currently, no specific licensed antiviral exists for treating the illness caused by dengue virus (DENV). Therefore, the search for compounds of natural origin with antiviral activity is an important area of research. In the present study, three compounds were isolated and identified from seeds of Tabernaemontana cymosa plants. The in vitro antiviral effect of those compounds and voacangine against different DENV strains was assessed using different experimental approaches: compounds added before the infection (Pre), at the same time with the virus (Trans), after the infection (Post) or compounds present in all moments of the experiment (Pre-Trans-Post, Combined treatment). In silico studies (docking and molecular dynamics) were also performed to explain the possible antiviral mechanisms. The identified compounds were three structural analogs of voacangine (voacangine-7-hydroxyindolenine, rupicoline and 3-oxo-voacangine). In the Pre-treatment, only voacangine-7-hydroxyindolenine and rupicoline inhibited the infection caused by the DENV-2/NG strain (16.4% and 29.6% infection, respectively). In the Trans-treatment approach, voacangine, voacangine-7-hydroxyindolenine and rupicoline inhibited the infection in both DENV-2/NG (11.2%, 80.4% and 75.7% infection, respectively) and DENV-2/16681 infection models (73.7%, 74.0% and 75.3% infection, respectively). The latter strain was also inhibited by 3-oxo-voacangine (82.8% infection). Moreover, voacangine (most effective virucidal agent) was also effective against one strain of DENV-1 (DENV-1/WestPac/74) and against the third strain of DENV-2 (DENV-2/S16803) (48.5% and 32.4% infection, respectively). Conversely, no inhibition was observed in the post-treatment approach. The last approach (combined) showed that voacangine, voacangine-7-hydroxyindolenine and rupicoline inhibited over 90% of infections (3.5%, 6.9% and 3.5% infection, respectively) of both strains (DENV-2/NG and DENV-2/16681). The free energy of binding obtained with an in silico approach was favorable for the E protein and compounds, which ranged between -5.1 and -6.3 kcal/mol. Finally, the complex formed between DENV-2 E protein and the best virucidal compound was stable for 50 ns. Our results show that the antiviral effect of indole alkaloids derived from T. cymose depends on the serotype and the virus strain.

In Vitro and In Silico Anti-Arboviral Activities of Dihalogenated Phenolic Derivates of L-Tyrosine.

Despite the serious public health problem represented by the diseases caused by dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses, there are still no specific licensed antivirals available for their treatment. Here, we examined the potential anti-arbovirus activity of ten di-halogenated compounds derived from L-tyrosine with modifications in amine and carboxyl groups. The activity of compounds on VERO cell line infection and the possible mechanism of action of the most promising compounds were evaluated. Finally, molecular docking between the compounds and viral and cellular proteins was evaluated in silico with Autodock Vina®, and the molecular dynamic with Gromacs®. Only two compounds (TDC-2M-ME and TDB-2M-ME) inhibited both ZIKV and CHIKV. Within the possible mechanism, in CHIKV, the two compounds decreased the number of genome copies and in the pre-treatment strategy the infectious viral particles. In the ZIKV model, only TDB-2M-ME inhibited the viral protein and demonstrate a virucidal effect. Moreover, in the U937 cell line infected with CHIKV, both compounds inhibited the viral protein and TDB-2M-ME inhibited the viral genome too. Finally, the in silico results showed a favorable binding energy between the compounds and the helicases of both viral models, the NSP3 of CHIKV and cellular proteins DDC and ß2 adrenoreceptor.

Three-Dimensional Model of Sub-Plasmalemmal Ca2+ Microdomains Evoked by the Interplay Between ORAI1 and InsP3 Receptors.

Ca2+ signaling plays an essential role in T cell activation, which is a key step to start an adaptive immune response. During the transition from a quiescent to a fully activated state, Ca2+ microdomains characterized by reduced spatial and temporal extents are observed in the junctions between the plasma membrane (PM) and the endoplasmic reticulum (ER). Such Ca2+ responses can also occur in response to T cell adhesion to other cells or extracellular matrix proteins in otherwise unstimulated T cells. These non-TCR/CD3-dependent Ca2+ microdomains rely on d-myo-inositol 1,4,5-trisphosphate (IP3) signaling and subsequent store operated Ca2+ entry (SOCE) via the ORAI/STIM system. The detailed molecular mechanism of adhesion-dependent Ca2+ microdomain formation remains to be fully elucidated. We used mathematical modeling to investigate the spatiotemporal characteristics of T cell Ca2+ microdomains and their molecular regulators. We developed a reaction-diffusion model using COMSOL Multiphysics to describe the evolution of cytosolic and ER Ca2+ concentrations in a three-dimensional ER-PM junction. Equations are based on a previously proposed realistic description of the junction, which is extended to take into account IP3 receptors (IP3R) that are located next to the junction. The first model only considered the ORAI channels and the SERCA pumps. Taking into account the existence of preformed clusters of ORAI1 and STIM2, ORAI1 slightly opens in conditions of a full ER. These simulated Ca2+ microdomains are too small as compared to those observed in unstimulated T cells. When considering the opening of the IP3Rs located near the junction, the local depletion of ER Ca2+ allows for larger Ca2+ fluxes through the ORAI1 channels and hence larger local Ca2+ concentrations. Computational results moreover show that Ca2+ diffusion in the ER has a major impact on the Ca2+ changes in the junction, by affecting the local Ca2+ gradients in the sub-PM ER. Besides pointing out the likely involvement of the spontaneous openings of IP3Rs in the activation of SOCE in conditions of T cell adhesion prior to full activation, the model provides a tool to investigate how Ca2+ microdomains extent and interact in response to T cell receptor activation.

Insights into the functions and RNA binding of Trypanosoma brucei ZC3H22, RBP9 and DRBD7.

Trypanosoma brucei is unusually reliant on mRNA-binding proteins to control mRNA fate, because its protein-coding genes lack individual promoters. We here focus on three trypanosome RNA-binding proteins. ZC3H22 is specific to Tsetse fly forms, RBP9 is preferentially expressed in bloodstream forms; and DRBD7 is constitutively expressed. Depletion of RBP9 or DRBD7 did not affect bloodstream-form trypanosome growth. ZC3H22 depletion from procyclic forms caused cell clumping, decreased expression of genes required for cell growth and proliferation, and increased expression of some epimastigote markers. Apart from decreases in mRNAs encoding enzymes of glucose metabolism, levels of most ZC3H22-bound transcripts were unaffected by ZC3H22 depletion. We compared ZC3H22, RBP9 and DRBD7 RNA binding with that of 16 other RNA-binding proteins. ZC3H22, PUF3 and ERBP1 show a preference for ribosomal protein mRNAs. RBP9 preferentially binds mRNAs that are more abundant in bloodstream forms than in procyclic forms. RBP9, ZC3H5, ZC3H30 and DRBD7 prefer mRNAs with long coding regions; UBP1-associated mRNAs have long 3'-untranslated regions; and RRM1 prefers mRNAs with long 3'or 5'-untranslated regions. We suggest that proteins that prefer long mRNAs may have relatively short or degenerate binding sites, and that preferences for A or U increase binding in untranslated regions.

Public and private human T-cell clones respond differentially to HCMV antigen when boosted by CD3 copotentiation.

Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(-) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

Levels of satisfaction with rheumatoid arthritis treatment and associated alignment between physicians and patients across Latin America.

INTRODUCTION: Discordance (misalignment) regarding treatment satisfaction may exist in real-life clinical practice between patients and their physicians. We aimed to assess physician and patient treatment satisfaction levels and associated degree of misalignment in rheumatoid arthritis (RA). METHOD: A point-in-time, multinational survey of patients and physicians was conducted in Latin America from December 2014 to October 2015. Physician- and patient-reported satisfaction levels with current RA treatment, alignment levels in satisfaction perception, and factors associated with satisfaction misalignment were assessed through bivariate and logistic regression analyses. RESULTS: Participating physicians (N = 114) completed 555 patient record forms (PRFs); 372 patients completed self-complete questionnaires (PSC). A total of 346 physician-patient pairs were analyzed. Physicians reported satisfaction with current disease control in 270/346 (78.0%) PRFs; patients reported such satisfaction in 286/346 (82.7%) PSCs. Physician-patient alignment was observed in 78.6% of pairs. Compared with aligned patients, misaligned patients were younger, more likely to have moderate or severe disease (physician subjectively defined), deteriorating or unstable disease (physician subjectively defined), been exposed to a greater number of advanced therapy lines (biologic or Janus kinase inhibitor), greater current pain, a current acute episode, poorer health, and greater disability and impairment. Misaligned patients were less likely to be in remission. Logistic regression analysis revealed that misaligned patients were more likely to experience greater activity impairment. CONCLUSIONS: High treatment satisfaction and alignment were observed among RA patients and their physicians in Latin America. Misaligned patients were more likely to report more severe disease and were less likely to be in remission. Addressing misalignment may lead to improved RA disease control.Key Points• High treatment satisfaction was observed among RA patients and their treating physicians in Latin America.• One-fifth of physician-patient pairs were misaligned in treatment satisfaction.• Patients misaligned with their physicians reported higher disease activity, lower quality of life, and greater disability than those who were aligned with their physicians.• Understanding and addressing misalignment in treatment satisfaction may improve outcomes in this patient population.

Physician-patient alignment in satisfaction with psoriatic arthritis treatment in Latin America.

INTRODUCTION: Physician-patient misalignment may exist in real-life clinical practice. We aimed to assess physician and patient treatment satisfaction levels and associated degree of misalignment in psoriatic arthritis (PsA). METHOD: Data from a cross-sectional survey of patients and their physicians conducted in Latin America were analyzed. Physician-reported and patient-reported satisfaction levels with current PsA treatment, alignment in satisfaction levels, and factors associated with satisfaction misalignment were assessed through bivariable and multivariable regression analyses. RESULTS: A total of 179 physician-patient pairs were analyzed. Physicians reported satisfaction with current disease control in 87.7% (n = 157) of cases; patients reported satisfaction in 91.1% (n = 163 of cases). A total of 82.1% of physician-patient pairs were aligned. Compared with aligned patients, misaligned patients were older and more likely to have moderate or severe disease, deteriorating or unstable disease, a past hospital procedure, current or past psoriasis symptoms, greater current pain, a current acute episode, poorer health and quality of life, greater impairment, poorer medication compliance, to consider PsA a major daily burden, and to believe that PsA treatments were ineffective. Misaligned patients were less likely to be in remission. Logistic regression analysis revealed that misaligned patients were older, and more likely to consider PsA a major daily burden and PsA treatments as ineffective. CONCLUSIONS: High levels of treatment satisfaction and alignment were observed among PsA patients and their physicians in Latin America. Patients in this study nevertheless experienced a considerable clinical and quality-of-life burden, especially the misaligned patients. Addressing misalignment may lead to improved PsA disease control.Key points• High treatment satisfaction was observed among PsA patients and their treating physicians in Latin America.• Patients experienced a considerable clinical and quality-of-life burden, especially the misaligned patients.• One-fifth of physician-patient pairs were misaligned regarding satisfaction.• Understanding and addressing misalignment may improve outcomes in this patient population.

In vitro and in silico anti-dengue activity of compounds obtained from Psidium guajava through bioprospecting.

BACKGROUND: For decades, bioprospecting has proven to be useful for the identification of compounds with pharmacological potential. Considering the great diversity of Colombian plants and the serious worldwide public health problem of dengue-a disease caused by the dengue virus (DENV)-in the present study, we evaluated the anti-DENV effects of 12 ethanolic extracts derived from plants collected in the Colombian Caribbean coast, and 5 fractions and 5 compounds derived from Psidium guajava. METHODS: The cytotoxicity and antiviral effect of 12 ethanolic extracts derived from plants collected in the Colombian Caribbean coast was evaluated in epithelial VERO cells. Five fractions were obtained by open column chromatography from the ethanolic extract with the highest selectivity index (SI) (derived from P. guajava, SI: 128.2). From the fraction with the highest selectivity (Pg-YP-I-22C, SI: 35.5), five compounds were identified by one- and two-dimensional nuclear magnetic resonance spectroscopy. The antiviral effect in vitro of the fractions and compounds was evaluated by different experimental strategies (Pre- and post-treatment) using non-toxic concentrations calculated by MTT method. The DENV inhibition was evaluated by plate focus assay. The results were analyzed by means of statistical analysis using Student's t-test. Finally the antiviral effect in Silico was evaluated by molecular docking. RESULTS: In vitro evaluation of these compounds showed that three of them (gallic acid, quercetin, and catechin) were promising antivirals as they inhibit the production of infectious viral particles via different experimental strategies, with the best antiviral being catechin (100% inhibition with a pre-treatment strategy and 91.8% with a post-treatment strategy). When testing the interactions of these compounds with the viral envelope protein in silico by docking, only naringin and hesperidin had better scores than the theoretical threshold of - 7.0 kcal/mol (- 8.0 kcal/mol and - 8.2 kcal/mol, respectively). All ligands tested except gallic acid showed higher affinity to the NS5 protein than the theoretical threshold. CONCLUSION: Even though bioprospecting has recently been replaced by more targeted tools for identifying compounds with pharmacological potential, our results show it is still useful for this purpose. Additionally, combining in vitro and in silico evaluations allowed us to identify promising antivirals as well as their possible mechanisms of action.

Breaking tolerance with engineered class I antigen-presenting molecules.

Successful efforts to activate T cells capable of recognizing weak cancer-associated self-antigens have employed altered peptide antigens to activate T cell responses capable of cross-reacting on native tumor-associated self. A limitation of this approach is the requirement for detailed knowledge about the altered self-peptide ligands used in these vaccines. In the current study we considered allorecognition as an approach for activating CTL capable of recognizing weak or self-antigens in the context of self-MHC. Nonself antigen-presenting molecules typically contain polymorphisms that influence interactions with the bound peptide and TCR interface. Recognition of these nonself structures results in peptide-dependent alloimmunity. Alloreactive T cells target their inducing alloantigens as well as third-party alloantigens but generally fail to target self-antigens. Certain residues located on the alpha-1/2 domains of class I antigen-presenting molecules primarily interface with TCR. These residues are more conserved within and across species than are residues that determine peptide antigen binding properties. Class I variants designed with amino acid substitutions at key positions within the conserved helical structures are shown to provide strong activating signals to alloreactive CD8 T cells while avoiding changes in naturally bound peptide ligands. Importantly, CTL activated in this manner can break self-tolerance by reacting to self-peptides presented by native MHC. The ability to activate self-tolerant T cells capable of cross-reacting on self-peptide-MHC in vivo represents an approach for inducing autoimmunity, with possible application in cancer vaccines.

The early proximal αß TCR signalosome specifies thymic selection outcome through a quantitative protein interaction network.

During αß T cell development, T cell antigen receptor (TCR) engagement transduces biochemical signals through a protein-protein interaction (PPI) network that dictates dichotomous cell fate decisions. It remains unclear how signal specificity is communicated, instructing either positive selection to advance cell differentiation or death by negative selection. Early signal discrimination might occur by PPI signatures differing qualitatively (customized, unique PPI combinations for each signal), quantitatively (graded amounts of a single PPI series), or kinetically (speed of PPI pathway progression). Using a novel PPI network analysis, we found that early TCR-proximal signals distinguishing positive from negative selection appeared to be primarily quantitative in nature. Furthermore, the signal intensity of this PPI network was used to find an antigen dose that caused a classic negative selection ligand to induce positive selection of conventional αß T cells, suggesting that the quantity of TCR triggering was sufficient to program selection outcome. Because previous work had suggested that positive selection might involve a qualitatively unique signal through CD3δ, we reexamined the block in positive selection observed in CD3δ0 mice. We found that CD3δ0 thymocytes were inhibited but capable of signaling positive selection, generating low numbers of MHC-dependent αß T cells that expressed diverse TCR repertoires and participated in immune responses against infection. We conclude that the major role for CD3δ in positive selection is to quantitatively boost the signal for maximal generation of αß T cells. Together, these data indicate that a quantitative network signaling mechanism through the early proximal TCR signalosome determines thymic selection outcome.

A case of incidental infection of Hepatitis E virus (HEV) genotype 1 in a domestic pig.

Hepatitis E virus (HEV) infection involving zoonotic genotypes is a public health problem in high-income and non-endemic developing countries. Herein we report the detection of a human genotype 1 (HEV-1) strain infecting a domestic pig, which is not considered a natural reservoir of this genotype. Viral load was quantified in stool by Real-Time qPCR and sequence analyses were performed. Infectivity of the HEV-1 strain was assesed by in vitro isolation in A549 cell line. Results suggest that certain epidemiological settings might favour accidental spillover infection and thus influence the host range restriction of HEV.

Differences in the replicative capacities of clinical isolates of dengue virus in C6/36 cells and in urban populations of Aedes aegypti from Colombia, South America.

Dengue, the most prevalent arboviral disease worldwide, is caused by any of the four dengue virus (DENV) serotypes that co-circulate constantly in hyperendemic areas such as Medellin (Colombia), and these serotypes are transmitted by mosquitoes of the genus Aedes. In this study, we evaluated the replicative capacity of strains isolated in Medellin between 2003 and 2007 in C6/36 cells and in colonies of Aedes aegypti collected during 2010-2011 from high or low-incidence areas within the same city. The phylogenetic analysis grouped isolates according to the predominant genotypes found in the Americas, and the in vitro characterization showed differences in the morphological changes induced by the isolates of each of the isolated serotypes compared to the reference serotypes. In vitro replicative capacity studies demonstrated that genomic copy number increased at four days post-infection and that cell viability decreased significantly compared to the control for all serotypes. The largest number of genomic copies in C6/36 was produced by DENV-2, followed by DENV-1 and DENV-4; DENV-3 produced the smallest number of genomic copies and had the smallest negative effect on cell viability. Finally, differences in the in vivo replication of intercolonial serotypes between the Rockefeller colony and the field colonies and among the intracolonial serotypes were found. The replication of DENV-2 at 7 and 14 days in both high- and low-incidence colonies was higher than that of the other serotypes, and replication of DENV-3 in the mosquito colonies was the most stable on the days evaluated. Our results support the notion that replication and, possibly, DENV transmission and severity depend on many factors, including serotype and vector characteristics.

Differences in the replicative capacities of clinical isolates of dengue virus in C6/36 cells and in urban populations of Aedes aegypti from Colombia, South America

ABSTRACT Dengue, the most prevalent arboviral disease worldwide, is caused by any of the four dengue virus (DENV) serotypes that co-circulate constantly in hyperendemic areas such as Medellin (Colombia), and these serotypes are transmitted by mosquitoes of the genus Aedes. In this study, we evaluated the replicative capacity of strains isolated in Medellin between 2003 and 2007 in C6/36 cells and in colonies of Aedes aegypti collected during 2010-2011 from high or low-incidence areas within the same city. The phylogenetic analysis grouped isolates according to the predominant genotypes found in the Americas, and the in vitro characterization showed differences in the morphological changes induced by the isolates of each of the isolated serotypes compared to the reference serotypes. In vitro replicative capacity studies demonstrated that genomic copy number increased at four days post-infection and that cell viability decreased significantly compared to the control for all serotypes. The largest number of genomic copies in C6/36 was produced by DENV-2, followed by DENV-1 and DENV-4; DENV-3 produced the smallest number of genomic copies and had the smallest negative effect on cell viability. Finally, differences in the in vivo replication of intercolonial serotypes between the Rockefeller colony and the field colonies and among the intracolonial serotypes were found. The replication of DENV-2 at 7 and 14 days in both high- and low-incidence colonies was higher than that of the other serotypes, and replication of DENV-3 in the mosquito colonies was the most stable on the days evaluated. Our results support the notion that replication and, possibly, DENV transmission and severity depend on many factors, including serotype and vector characteristics.