Imidazole Carbamates as a Promising Alternative for Treating Trichomoniasis: In Vitro Effects on the Growth and Gene Expression of Trichomonas vaginalis.

Metronidazole (MTZ) is the most common drug used against Trichomonas vaginalis (T. vaginalis) infections; however, treatment failures and high rates of recurrence of trichomoniasis have been reported, suggesting the presence of resistance in T. vaginalis to MTZ. Therefore, research into new therapeutic options against T. vaginalis infections has become increasingly urgent. This study investigated the trichomonacidal activity of a series of five imidazole carbamate compounds (AGR-1, AGR-2, AGR-3, AGR-4, and AGR-5) through in vitro susceptibility assays to determine the IC50 value of each compound. All five compounds demonstrated potent trichomonacidal activity, with IC50 values in the nanomolar range and AGR-2 being the most potent (IC50 400 nM). To gain insight into molecular events related to AGR-induced cell death in T. vaginalis, we analyzed the expression profiles of some metabolic genes in the trophozoites exposed to AGR compounds and MTZ. It was found that both AGR and MTZ compounds reduced the expression of the glycolytic genes (CK, PFK, TPI, and ENOL) and genes involved in metabolism (G6PD, TKT, TALDO, NADHOX, ACT, and TUB), suggesting that disturbing these key metabolic genes alters the survival of the T. vaginalis parasite and that they probably share a similar mechanism of action. Additionally, the compounds showed low cytotoxicity in the Caco-2 and HT29 cell lines, and the results of the ADMET analysis indicated that these compounds have pharmacokinetic properties similar to those of MTZ. The findings offer significant insights that can serve as a basis for future in vivo studies of the compounds as a potential new treatment against T. vaginalis.

The Role of Bacterial Extracellular Vesicles in the Immune Response to Pathogens, and Therapeutic Opportunities.

Pathogenic bacteria have several mechanisms to evade the host's immune response and achieve an efficient infection. Bacterial extracellular vesicles (EVs) are a relevant cellular communication mechanism, since they can interact with other bacterial cells and with host cells. In this review, we focus on the EVs produced by some World Health Organization (WHO) priority Gram-negative and Gram-positive pathogenic bacteria; by spore-producing bacteria; by Mycobacterium tuberculosis (a bacteria with a complex cell wall); and by Treponema pallidum (a bacteria without lipopolysaccharide). We describe the classification and the general properties of bacterial EVs, their role during bacterial infections and their effects on the host immune response. Bacterial EVs contain pathogen-associated molecular patterns that activate innate immune receptors, which leads to cytokine production and inflammation, but they also contain antigens that induce the activation of B and T cell responses. Understanding the many effects of bacterial EVs on the host's immune response can yield new insights on the pathogenesis of clinically important infections, but it can also lead to the development of EV-based diagnostic and therapeutic strategies. In addition, since EVs are efficient activators of both the innate and the adaptive immune responses, they constitute a promising platform for vaccine development.

Nitazoxanide Inhibits the Bifunctional Enzyme GlG6PD::6PGL of Giardia lamblia: Biochemical and In Silico Characterization of a New Druggable Target.

Giardiasis, which is caused by Giardia lamblia infection, is a relevant cause of morbidity and mortality worldwide. Because no vaccines are currently available to treat giardiasis, chemotherapeutic drugs are the main options for controlling infection. Evidence has shown that the nitro drug nitazoxanide (NTZ) is a commonly prescribed treatment for giardiasis; however, the mechanisms underlying NTZ's antigiardial activity are not well-understood. Herein, we identified the glucose-6-phosphate::6-phosphogluconate dehydrogenase (GlG6PD::6PGL) fused enzyme as a nitazoxanide target, as NTZ behaves as a GlG6PD::6PGL catalytic inhibitor. Furthermore, fluorescence assays suggest alterations in the stability of GlG6PD::6PGL protein, whereas the results indicate a loss of catalytic activity due to conformational and folding changes. Molecular docking and dynamic simulation studies suggest a model of NTZ binding on the active site of the G6PD domain and near the structural NADP+ binding site. The findings of this study provide a novel mechanistic basis and strategy for the antigiardial activity of the NTZ drug.

Extracellular vesicles from Mycobacterium tuberculosis-infected neutrophils induce maturation of monocyte-derived dendritic cells and activation of antigen-specific Th1 cells.

Tuberculosis remains one of the leading public health problems in the world. The mechanisms that lead to the activation of the immune response against Mycobacterium tuberculosis have been extensively studied, with a focus on the role of cytokines as the main signals for immune cell communication. However, less is known about the role of other signals, such as extracellular vesicles, in the communication between immune cells, particularly during the activation of the adaptive immune response. In this study, we determined that extracellular vesicles released by human neutrophils infected with M. tuberculosis contained several host proteins that are ectosome markers. In addition, we demonstrated that extracellular vesicles released by human neutrophils infected with M. tuberculosis released after only 30 min of infection carried mycobacterial antigens and pathogen-associated molecular patterns, and we identified 15 mycobacterial proteins that were consistently found in high concentrations in extracellular vesicles released by human neutrophils infected with M. tuberculosis; these proteins contain epitopes for CD4 T-cell activation. We found that extracellular vesicles released by human neutrophils infected with M. tuberculosis increased the expression of the costimulatory molecule CD80 and of the coinhibitory molecule PD-L1 on immature monocyte-derived dendritic cells. We also found that immature and mature dendritic cells treated with extracellular vesicles released by human neutrophils infected with M. tuberculosis were able to induce IFN-γ production by autologous M. tuberculosis antigen-specific CD4 T cells, indicating that these extracellular vesicles acted as antigen carriers and transferred mycobacterial proteins to the antigen-presenting cells. Our results provide evidence that extracellular vesicles released by human neutrophils infected with M. tuberculosis participate in the activation of the adaptive immune response against M. tuberculosis.

Pyridyl Methylsulfinyl Benzimidazole Derivatives as Promising Agents against Giardia lamblia and Trichomonas vaginalis.

Protozoan parasites, such as Giardia lamblia and Trichomonas vaginalis, cause the most prevalent infections in humans in developing countries and provoke significant morbidity and mortality in endemic countries. Despite its side-effects, metronidazole is still the drug of choice as a giardiacidal and trichomonacidal tissue-active agent. However, the emergence of metronidazole resistance and its evolved strategies of parasites to evade innate host defenses have hindered the identification and development of new therapeutic strategies against these parasites. Here, we tested five synthesized benzimidazole derivatives as possible drugs for treating giardiasis and trichomoniasis, probing the bifunctional enzyme glucose 6-phosphate dehydrogenase::6-phosphogluconolactone from G. lamblia (GlG6PD::6PGL) and T. vaginalis (TvG6PD::6PGL) as a drug target. The investigated benzimidazole derivatives were H-B2M1, H-B2M2, H2N-BZM6, O2N-BZM7, and O2N-BZM9. The recombinant enzymes were used in inhibition assays, and in silico computational predictions and spectroscopic studies were applied to follow the structural alteration of the enzymes and identify the possible mechanism of inhibition. We identified two potent benzimidazole compounds (O2N-BZM7 and O2N-BZM9), which are capable of inhibiting both protozoan G6PD::6PGL enzymes and in vitro assays with these parasites, showing that these compounds also affect their viability. These results demonstrate that other therapeutic targets of the compounds are the enzymes GlG6PD::6PGL and TvG6PD::6PGL, which contribute to their antiparasitic effect and their possible use in antigiardial and trichomonacidal therapies.

Liposomes Bearing Non-Bilayer Phospholipid Arrangements Induce Specific IgG Anti-Lipid Antibodies by Activating NK1.1+, CD4+ T Cells in Mice.

Liposomes are artificial models of cellular membranes that are used as delivery systems for genes, drugs and protein antigens. We have previously used them to study the antigenic properties of their phospholipids. Here, we used them to induce the production of IgG anti-non-bilayer phospholipid arrangements (NPAs) antibodies in mice; these antibodies cause cell lysis and trigger a lupus-like disease in mice. We studied the mechanisms that lead to the production of these antibodies, and provide evidence that NK1.1+, CD4+ T cells respond to NPA-bearing liposomes and deliver the help required for specific B cell activation and antibody class-switching to IgG. We found increased numbers of IL-4-producing NK1.1+, CD4+ T cells in the secondary lymphoid organs of mice administered with NPAs, and these cells also expressed CD40L, which is required for B cell activation. Additionally, we isolated and purified NK1.1+, CD4+ T cells from spleens and determined that they over-expressed 40 genes, which are key players in inflammatory processes and B cell stimulation and have TRAF6 and UNC39B1 as key nodes in their network. These results show that liposomes are membrane models that can be used to analyze the immunogenicity of lipids.

Kinetic and Molecular Docking Studies to Determine the Effect of Inhibitors on the Activity and Structure of Fused G6PD::6PGL Protein from Trichomonas vaginalis.

Trichomoniasis is a sexually transmitted disease with a high incidence worldwide, affecting 270 million people. Despite the existence of a catalog of available drugs to combat this infection, their extensive use promotes the appearance of resistant Trichomonas vaginalis (T. vaginalis), and some side effects in treated people, which are reasons why it is necessary to find new alternatives to combat this infection. In this study, we investigated the impact of an in-house library comprising 55 compounds on the activity of the fused T. vaginalis G6PD::6PGL (TvG6PD::6PGL) protein, a protein mediating the first reaction step of the pentose phosphate pathway (PPP), a crucial pathway involved in the parasite's energy production. We found four compounds: JMM-3, CNZ-3, CNZ-17, and MCC-7, which inhibited the TvG6PD::6PGL protein by more than 50%. Furthermore, we determined the IC50, the inactivation constants, and the type of inhibition. Our results showed that these inhibitors induced catalytic function loss of the TvG6PD::6PGL enzyme by altering its secondary and tertiary structures. Finally, molecular docking was performed for the best inhibitors, JMM-3 and MCC-7. All our findings demonstrate the potential role of these selected hit compounds as TvG6PD::6PGL enzyme selective inhibitors.

Effect of B-NIPOx in Experimental Trypanosoma cruzi Infection in Mice.

Chagas disease is caused by Trypanosoma cruzi and represents a major public health problem, which is endemic in Latin America and emerging in the rest of the world. The two drugs that are currently available for its treatment, Benznidazole and Nifurtimox, are partially effective in the chronic phase of the disease. In this study, we designed and synthesized the benzyl ester of N-isopropyl oxamic acid (B-NIPOx), which is a non-polar molecule that crosses cell membranes. B-NIPOx is cleaved inside the parasite by carboxylesterases, releasing benzyl alcohol (a molecule with antimicrobial activity), and NIPOx, which is an inhibitor of α-hydroxy acid dehydrogenase isozyme II (HADH-II), a key enzyme in T. cruzi metabolism. We evaluated B-NIPOx cytotoxicity, its toxicity in mice, and its inhibitory activity on purified HADH-II and on T. cruzi homogenates. We then evaluated the trypanocidal activity of B-NIPOx in vitro and in vivo and its effect in the intestine of T. cruzi-infected mice. We found that B-NIPOx had higher trypanocidal activity on epimastigotes and trypomastigotes than Benznidazole and Nifurtimox, that it was more effective to reduce blood parasitemia and amastigote nests in infected mice, and that, in contrast to the reference drugs, it prevented the development of Chagasic enteropathy.

Pro-inflammatory monocytes are increased in Duchenne muscular dystrophy and suppressed with omega-3 fatty acids: A double-blind, randomized, placebo-controlled pilot study.

BACKGROUND: Omega-3 long chain polyunsaturated fatty acids (LCPUFA) reduce circulating cytokines produced by monocytes. Nevertheless, whether the omega-3 LCPUFA regulate the monocytes and their cytokines in Duchenne muscular dystrophy (DMD) is unknown. The aim of this study was to evaluate whether circulating pro-inflammatory monocytes are increased and whether omega-3 LCPUFA selectively suppress these monocytes and their cytokines in patients with DMD. METHODS: This was a double-blind, randomized, placebo-controlled pilot study carried out in patients with DMD supplemented with omega-3 LCPUFA (n = 6) or sunflower oils (placebo, n = 6) for 6 months. Monocytes and their cytokines were measured at baseline and after 1, 2, 3, and 6 months of supplementation. RESULTS: The anti-inflammatory monocytes (median, [95% CI]) are increased at month 3 (-0.46 [-13.5-9.5] vs. 8.4 [5.5-12.5], p = 0.05) in the omega-3 LCPUFA group compared with the placebo group. The pro-inflammatory monocytes (-5.7 [-63.8-114.1] vs. -51.9 [-91.2 to -25.4], p = 0.026 and -16.4 [-50.8-50.6] vs. -57.9 [-86.9 to -18.5], p = 0.045 at months 3 and 6, respectively) and their cytokine interleukin 6 (-11.9 [-93.5-148.9] vs. -64.7 [-77.8 to -42.6], p = 0.019 at month 6) decreased in the omega-3 LCPUFA group compared with the placebo group. Pro-inflammatory monocytes decreased and anti-inflammatory monocytes were augmented (p < 0.05) during the 6 months of supplementation with omega-3 LCPUFA. CONCLUSIONS: This pilot study suggests that supplementation with omega-3 LCPUFA could have a selective reductive effect on pro-inflammatory monocytes and their cytokines in patients with DMD. These findings also support the performance of studies in a significant population to explore the role of omega-3 LCPUFA on monocyte populations and their cytokines in patients with DMD. This research was registered at clinicaltrials.gov (NCT018264229).

Validation and Selection of New Reference Genes for RT-qPCR Analysis in Pediatric Glioma of Different Grades.

Gliomas are heterogeneous, solid, and intracranial tumors that originate from glial cells. Malignant cells from the tumor undergo metabolic alterations to obtain the energy required for proliferation and the invasion of the cerebral parenchyma. The alterations in the expression of the genes related to the metabolic pathways can be detected in biopsies of gliomas of different CNS WHO grades. In this study, we evaluated the expression of 16 candidate reference genes in the HMC3 microglia cell line. Then, statistical algorithms such as BestKeeper, the comparative ΔCT method, geNorm, NormFinder, and RefFinder were applied to obtain the genes most suitable to be considered as references for measuring the levels of expression in glioma samples. The results show that PKM and TPI1 are two novel genes suitable for genic expression studies on gliomas. Finally, we analyzed the expression of genes involved in metabolic pathways in clinical samples of brain gliomas of different CNS WHO grades. RT-qPCR analysis showed that in CNS WHO grade 3 and 4 gliomas, the expression levels of HK1, PFKM, GAPDH, G6PD, PGD1, IDH1, FASN, ACACA, and ELOVL2 were higher than those of CNS WHO grade 1 and 2 glioma biopsies. Hence, our results suggest that reference genes from metabolic pathways have different expression profiles depending on the stratification of gliomas and constitute a potential model for studying the development of this type of tumor and the search for molecular targets to treat gliomas.

Identification and In Silico Characterization of Novel Helicobacter pylori Glucose-6-Phosphate Dehydrogenase Inhibitors.

Helicobacter pylori (H. pylori) is a pathogen that can remain in the stomach of an infected person for their entire life. As a result, this leads to the development of severe gastric diseases such as gastric cancer. In addition, current therapies have several problems including antibiotics resistance. Therefore, new practical options to eliminate this bacterium, and its induced affections, are required to avoid morbidity and mortality worldwide. One strategy in the search for new drugs is to detect compounds that inhibit a limiting step in a central metabolic pathway of the pathogen of interest. In this work, we tested 55 compounds to gain insights into their possible use as new inhibitory drugs of H. pylori glucose-6-phosphate dehydrogenase (HpG6PD) activity. The compounds YGC-1; MGD-1, MGD-2; TDA-1; and JMM-3 with their respective scaffold 1,3-thiazolidine-2,4-dione; 1H-benzimidazole; 1,3-benzoxazole, morpholine, and biphenylcarbonitrile showed the best inhibitory activity (IC50 = 310, 465, 340, 204 and 304 µM, respectively). We then modeled the HpG6PD protein by homology modeling to conduct an in silico study of the chemical compounds and discovers its possible interactions with the HpG6PD enzyme. We found that compounds can be internalized at the NADP+ catalytic binding site. Hence, they probably exert a competitive inhibitory effect with NADP+ and a non-competitive or uncompetitive effect with G6P, that of the compounds binding far from the enzyme's active site. Based on these findings, the tested compounds inhibiting HpG6PD represent promising novel drug candidates against H. pylori.

Interleukin 4 deficiency limits the development of a lupus-like disease in mice triggered by phospholipids in a non-bilayer arrangement.

Non-bilayer phospholipids arrangements (NPAs) are transient molecular associations different from lipid bilayers. When they become stable, they can trigger a disease in mice resembling human lupus, which is mainly characterized by the production of anti-NPA IgG antibodies. NPAs are stabilized on liposomes or cell bilayers by the drugs procainamide or chlorpromazine, which produce drug-induced lupus in humans. Here, we evaluated the participation of the TH 2 response, through its hallmark cytokine IL-4, on the development of the lupus-like disease in mice. Wild-type or IL-4 knockout BALB/c mice received liposomes bearing drug-induced NPAs, the drugs alone, or an anti-NPA monoclonal antibody (H308) to induce the lupus-like disease (the last two procedures stabilize NPAs on mice cells). IL-4 KO mice showed minor disease manifestations, compared to wild-type mice, with decreased production of anti-NPA IgG antibodies, no anti-cardiolipin, anti-histones and anticoagulant antibodies, and no kidney or skin lesions. In these mice, H308 was the only inducer of anti-NPA IgG antibodies. These findings indicate that IL-4 has a central role in the development of the murine lupus-like disease induced by NPA stabilization.

Muscle function and age are associated with loss of bone mineral density in Duchenne muscular dystrophy.

INTRODUCTION: Patients with Duchenne muscular dystrophy (DMD) demonstrate decreased bone mineral density (BD). It is not clear which factors exert the greatest impact on BD loss in these patients. METHODS: In 63 patients with DMD, serum cytokines (interleukin [IL]-1, IL-6, and tumor necrosis factor-beta [TNF-ß]), C-reactive protein (CRP), creatine kinase (CK), muscle function (by Vignos scale), body composition, and total BD (the latter 2 measured by dual-energy X-ray absorptiometry, or DEXA) were determined. RESULTS: The main factors associated with BD loss were muscle function (34.0%; ß = -0.139; P < 0.023) and age (36.7%; ß = -0.151; P = 0.004). Cytokines, CRP, body fat mass, and CK did not contribute to BD loss. DISCUSSION: Muscle function and age contribute to BD loss in DMD. We propose that a cut-off of at least 6 points for the Vignos scale and at least 10.5 years of age predict a Z-score of less than or equal to -2.0. Muscle Nerve 59:417-421, 2019.

Lupresan, a new drug that prevents or reverts the formation of nonbilayer phospholipid arrangements that trigger a murine lupus resembling human lupus.

Non-bilayer phospholipid arrangements (NPA) are lipid associations different from the bilayer, formed by the interactions of conic anionic lipids and divalent cations that produce an inverted micelle which is inserted between the lipid layers, so the polar heads of the outer lipids spread and expose new antigens. Since these structures are transient, they are not immunogenic, but if they are stabilized by drugs, such as chlorpromazine, they become immunogenic and induce anti-NPA antibodies that trigger a lupus-like disease in mice. Chloroquine is a drug used for the treatment of lupus; chloroquine has a quinoline ring and two positive charges that interact with conic anionic lipids and prevent or revert the formation of NPA. However, the polyamine spermidine is more effective, since it has three positive charges and interacts with more lipids, but polyamines cannot be used as drugs, because they are highly toxic. Here we report the design and synthesis of Lupresan, an analogous of chloroquine with its quinoline ring but with three positive charges. Lupresan is more effective in preventing or reverting the formation of NPA than chloroquine or spermidine, and as a consequence, it decreased auto-antibody titers and healed the malar rash in mice with lupus to a greater extent than chloroquine. A drug as Lupresan could be used for the treatment of human lupus.

Cytotaxonomic study of the Chilean endemic complex Alstroemeria magnifica Herb. (Alstroemeriaceae).

Alstroemeria L. (Alstroemeriaceae) represents one of the most diverse genera of vascular plants in Chile. It contains approximately 54 taxa, 40 of which are endemic. The "complex" Alstroemeria magnifica is endemic to Chile, and it comprises four varieties: A. magnifica var. magenta, A. magnifica var. magnifica, A. magnifica var. sierrae, and A. magnifica var. tofoensis. It is distributed from Coquimbo to the Valparaíso Region. We analyzed karyotypes of 10 populations along its natural distribution. All the populations presented an asymmetric karyotype, with 2n = 16 chromosomes but with three different karyotypic formulae. Alstroemeria magnifica var. magnifica and A. magnifica var. sierrae presented the same karyotypic fomula, and A. magnifica var. magenta, and A. magnifica var. tofoensis each had a different formula. The scatter plot among CVCL vs. MCA shows different groupings between populations of the four varieties. Based on the results, it is possible to consider raising Alstroemeria magnifica var. magenta to species level (A. magenta) and A. magnifica var. tofoensis to subspecies level (A. magnifica subsp. tofoensis); A. magnifica var. magnifica and A. magnifica var. sierrae should each remain as varieties. Nevertheless, these taxonomic changes should be considered tentative, as additional sources of evidence become available.

Strong indication of an extinction-based saturation of the flora on the Pacific Robinson Crusoe Islands.

Oceanic islands are vulnerable ecosystems and their flora has been under pressure since the arrival of the first humans. Human activities and both deliberately and inadvertently introduced biota have had and continue to have a severe impact on island endemic plants. The number of alien plants has increased nearly linearly on many islands, perhaps resulting in extinction-based saturation of island floras. Here, we provide evidence for such a scenario in Alejandro Selkirk, Robinson Crusoe Islands (Archipelago Juan Fernández, Chile). We compared species richness and species composition of historical vegetation samples from 1917 with recent ones from 2011. Changes in species' relative occurrence frequency were related to their taxonomic affiliation, dispersal mode, distribution status, and humidity and temperature preferences. While total species richness of vascular plants remained relatively similar, species composition changed significantly. Plants endemic to the Robinson Crusoe Islands declined, exotic species increased substantially within the period of ca. 100 years. Further, the relative occurrence frequency of plants with preferences for very warm and humid climate decreased, while the opposite was found for plants preferring drier and colder environments. Potential drivers responsible for this dramatic shift in the vegetation within only one century might have been the large goat population affecting especially small populations of endemic plants and climatic changes. Taking into account a substantial extinction debt, we expect further shifts in the vegetation of this small oceanic island toward alien plants. This would have significant negative consequences on global biodiversity, considering that island floras contribute substantially to global plant species richness due to their high proportion of endemics.

Extracellular Vesicles Released from Mycobacterium tuberculosis-Infected Neutrophils Promote Macrophage Autophagy and Decrease Intracellular Mycobacterial Survival.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb). In the lungs, macrophages and neutrophils are the first immune cells that have contact with the infecting mycobacteria. Neutrophils are phagocytic cells that kill microorganisms through several mechanisms, which include the lytic enzymes and antimicrobial peptides that are found in their lysosomes, and the production of reactive oxygen species. Neutrophils also release extracellular vesicles (EVs) (100-1,000 nm in diameter) to the extracellular milieu; these EVs consist of a lipid bilayer surrounding a hydrophilic core and participate in intercellular communication. We previously demonstrated that human neutrophils infected in vitro with Mtb H37Rv release EVs (EV-TB), but the effect of these EVs on other cells relevant for the control of Mtb infection, such as macrophages, has not been completely analyzed. In this study, we characterized the EVs produced by non-stimulated human neutrophils (EV-NS), and the EVs produced by neutrophils stimulated with an activator (PMA), a peptide derived from bacterial proteins (fMLF) or Mtb, and observed that the four EVs differed in their size. Ligands for toll-like receptor (TLR) 2/6 were detected in EV-TB, and these EVs favored a modest increase in the expression of the co-stimulatory molecules CD80, a higher expression of CD86, and the production of higher amounts of TNF-α and IL-6, and of lower amounts of TGF-ß, in autologous human macrophages, compared with the other EVs. EV-TB reduced the amount of intracellular Mtb in macrophages, and increased superoxide anion production in these cells. TLR2/6 ligation and superoxide anion production are known inducers of autophagy; accordingly, we found that EV-TB induced higher expression of the autophagy-related marker LC3-II in macrophages, and the co-localization of LC3-II with Mtb inside infected macrophages. The intracellular mycobacterial load increased when autophagy was inhibited with wortmannin in these cells. In conclusion, our results demonstrate that neutrophils produce different EVs in response to diverse activators, and that EV-TB activate macrophages and promote the clearance of intracellular Mtb through early superoxide anion production and autophagy induction, which is a novel role for neutrophil-derived EVs in the immune response to Mtb.

Corrigendum: Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus.

[This corrects the article on p. 396 in vol. 7, PMID: 27746783.].

Dysregulation of miR-155-5p and miR-200-3p and the Anti-Non-Bilayer Phospholipid Arrangement Antibodies Favor the Development of Lupus in Three Novel Murine Lupus Models.

Systemic lupus erythematosus (SLE) is characterized by deregulated activation of T and B cells, autoantibody production, and consequent formation of immune complexes. Liposomes with nonbilayer phospholipid arrangements (NPA), induced by chlorpromazine, procainamide, or manganese, provoke a disease resembling human lupus when administered to mice. These mice produce anti-NPA IgM and IgG antibodies and exhibit an increased number of TLR-expressing spleen cells and a modified gene expression associated with TICAM1-dependent TLR-4 signaling (including IFNA1 and IFNA2) and complement activation. Additionally, they showed a diminished gene expression related to apoptosis and NK cell activation. We hypothesized that such gene expression may be affected by miRNAs and so miRNA expression was studied. Twelve deregulated miRNAs were found. Six of them were common to the three lupus-like models. Their validation by qRT-PCR and TaqMan probes, including miR-342-3p, revealed that miR-155-5p and miR-200a-3p expression was statistically significant. Currently described functions for these miRNAs in autoimmune diseases such as SLE reveal their participation in inflammation, interferon production, germinal center responses, and antibody maturation. Taking into account these findings, we propose miR-155-5p and miR-200a-3p, together with the anti-NPA antibodies, as key players in the murine lupus-like models and possible biomarkers of the human SLE.

Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus.

Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1-, CD19-, CD138+) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD-, CD19+, PNA+) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220+, Blimp1+) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers.