Fecal microbiota transplantation from female donors restores gut permeability and reduces liver injury and inflammation in middle-aged male mice exposed to alcohol.

Background: Alcohol misuse, binge drinking pattern, and gender-specific effects in the middle-aged population has been clearly underestimated. In the present study, we focused on understanding gender-specific effects of alcohol exposure on the gut-liver axis and the role of gut microbiota in modulating gender-specific responses to alcohol consumption. Methods: Fifty-two-week-old female and male C57BL/6 mice were fasted for 12 h, and then administered a single oral dose of ethanol (EtOH) (6 g/kg). Controls were given a single dose of PBS. Animals were sacrificed 8 h later. Alternatively, fecal microbiota transplantation (FMT) was performed in 52-week-old male mice from female donors of the same age. Permeability of the large intestine (colon), gut microbiota, liver injury, and inflammation was thoroughly evaluated in all groups. Results: Middle-aged male mice exposed to EtOH showed a significant increase in gut permeability in the large intestine, evaluated by FITC-dextran assay and ZO-1, OCCLUDIN and MUCIN-2 immuno-staining, compared to PBS-treated animals, whilst female mice of the same age also increased their gut permeability, but displayed a partially maintained intestinal barrier integrity. Moreover, there was a significant up-regulation of TLRs and markers of hepatocellular injury, cell death (AST, TUNEL-positive cells) and lipid accumulation (ORO) in male mice after EtOH exposure. Interestingly, FMT from female donors to male mice reduced gut leakiness, modified gut microbiota composition, ameliorated liver injury and inflammation, TLR activation and the senescence phenotype of middle-aged mice. Conclusion: Our findings highlighted the relevance of gender in middle-aged individuals who are exposed to alcohol in the gut-liver axis. Moreover, our study revealed that gender-specific microbiota transplantation might be a plausible therapy in the management of alcohol-related disorders during aging.

State of the Art in CAR-T Cell Therapy for Solid Tumors: Is There a Sweeter Future?

Chimeric antigen receptor (CAR)-T cell therapy has proven to be a powerful treatment for hematological malignancies. The situation is very different in the case of solid tumors, for which no CAR-T-based therapy has yet been approved. There are many factors contributing to the absence of response in solid tumors to CAR-T cells, such as the immunosuppressive tumor microenvironment (TME), T cell exhaustion, or the lack of suitable antigen targets, which should have a stable and specific expression on tumor cells. Strategies being developed to improve CAR-T-based therapy for solid tumors include the use of new-generation CARs such as TRUCKs or bi-specific CARs, the combination of CAR therapy with chemo- or radiotherapy, the use of checkpoint inhibitors, and the use of oncolytic viruses. Furthermore, despite the scarcity of targets, a growing number of phase I/II clinical trials are exploring new solid-tumor-associated antigens. Most of these antigens are of a protein nature; however, there is a clear potential in identifying carbohydrate-type antigens associated with tumors, or carbohydrate and proteoglycan antigens that emerge because of aberrant glycosylations occurring in the context of tumor transformation.

Neuroblastoma RAS viral oncogene homolog (N-RAS) deficiency aggravates liver injury and fibrosis.

Progressive hepatic damage and fibrosis are major features of chronic liver diseases of different etiology, yet the underlying molecular mechanisms remain to be fully defined. N-RAS, a member of the RAS family of small guanine nucleotide-binding proteins also encompassing the highly homologous H-RAS and K-RAS isoforms, was previously reported to modulate cell death and renal fibrosis; however, its role in liver damage and fibrogenesis remains unknown. Here, we approached this question by using N-RAS deficient (N-RAS-/-) mice and two experimental models of liver injury and fibrosis, namely carbon tetrachloride (CCl4) intoxication and bile duct ligation (BDL). In wild-type (N-RAS+/+) mice both hepatotoxic procedures augmented N-RAS expression in the liver. Compared to N-RAS+/+ counterparts, N-RAS-/- mice subjected to either CCl4 or BDL showed exacerbated liver injury and fibrosis, which was associated with enhanced hepatic stellate cell (HSC) activation and leukocyte infiltration in the damaged liver. At the molecular level, after CCl4 or BDL, N-RAS-/- livers exhibited augmented expression of necroptotic death markers along with JNK1/2 hyperactivation. In line with this, N-RAS ablation in a human hepatocytic cell line resulted in enhanced activation of JNK and necroptosis mediators in response to cell death stimuli. Of note, loss of hepatic N-RAS expression was characteristic of chronic liver disease patients with fibrosis. Collectively, our study unveils a novel role for N-RAS as a negative controller of the progression of liver injury and fibrogenesis, by critically downregulating signaling pathways leading to hepatocyte necroptosis. Furthermore, it suggests that N-RAS may be of potential clinical value as prognostic biomarker of progressive fibrotic liver damage, or as a novel therapeutic target for the treatment of chronic liver disease.

Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress.

Introduction: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. Methods: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. Results: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. Conclusion: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.

Manufacture of Mesoporous Silicon Microparticles (MSMPs) as Adjuvants for Vaccine Delivery.

The advent of computational approaches has accelerated the identification of vaccine candidates like epitope peptides. However, epitope peptides are usually very poorly immunogenic and adequate platforms are required with adjuvant capacity to verity immunogenicity and antigenicity of vaccine subunits in vivo. Silicon microparticles are being developed as potential new adjuvants for vaccine delivery due to their physicochemical properties. This chapter explains the methodology to fabricate and functionalize mesoporous silicon microparticles (MSMPs) which can be loaded with antigens of different nature, such as viral peptides, proteins, or carbohydrates, and this strategy is particularly suitable for delivery of epitopes identified by computer.

Role of mTOR inhibitor in the cellular and humoral immune response to a booster dose of SARS-CoV-2 mRNA-1273 vaccine in kidney transplant recipients.

Background: Immunocompromised patients have an increased risk of developing severe COVID disease, as well as a tendency to suboptimal responses to vaccines. The objective of this study was to evaluate the specific cellular and humoral adaptive immune responses of a cohort of kidney transplant recipients (KTR) after 3 doses of mRNA-1273 vaccine and to determinate the main factors involved. Methods: Prospective observational study in 221 KTR (149 non infected), 55 healthy volunteers (HV) and 23 dialysis patients (DP). We evaluated anti-spike (by quantitative chemiluminescence immunoassay) and anti-nucleocapsid IgG (ELISA), percentage of TCD4+ and TCD8+ lymphocytes producing IFNγ against S-protein by intracellular flow cytometry after Spike-specific 15-mer peptide stimulation and serum neutralizing activity (competitive ELISA) at baseline and after vaccination. Results: Among COVID-19 naïve KTR, 54.2% developed cellular and humoral response after the third dose (vs 100% in DP and 91.7% in HV), 18% only showed cell-mediated response, 22.2% exclusively antibody response and 5.6% none. A correlation of neutralizing activity with both the IgG titer (r=0.485, p<0.001) and the percentage of S-protein-specific IFNγ-producing CD8-T cells (r=0.198, p=0.049) was observed. Factors related to the humoral response in naïve KTR were: lymphocytes count pre-vaccination >1000/mm3 [4.68 (1.72-12.73, p=0.003], eGFR>30 mL/min [7.34(2.72-19.84), p<0.001], mTOR inhibitors [6.40 (1.37-29.86), p=0.018]. Infected KTR developed a stronger serologic response than naïve patients (96.8 vs 75.2%, p<0.001). Conclusions: KTR presented poor cellular and humoral immune responses following vaccination with mRNA-1273. The immunosuppression degree and kidney function of these patients play an important role, but the only modifiable factor with a high impact on humoral immunogenicity after a booster dose was an immunosuppressive therapy including a mTOR inhibitor. Clinical trials are required to confirm these results.

Novel therapeutic avenues for the study of chronic liver disease and regeneration: The foundation of the Iberoamerican Consortium for the study of liver Cirrhosis.

Unfortunately, there is a gap of understanding in the pathophysiology of chronic liver disease due to the lack of experimental models that exactly mimic the human disease. Additionally, the diagnosis of patients is very poor due to the lack of biomarkers than can detect the disease in early stages. Thus, it is of utmost interest the generation of a multidisciplinary consortium from different countries with a direct translation. The present reports the meeting of the 2021 Iberoamerican Consortium for the study of liver Cirrhosis, held online, in October 2021. The meeting, was focused on the recent advancements in the field of chronic liver disease and cirrhosis with a specific focus on cell pathobiology and liver regeneration, molecular and cellular targets involved in non-alcoholic hepatic steatohepatitis, alcoholic liver disease (ALD), both ALD and western diet, and end-stage liver cirrhosis and hepatocellular carcinoma. In addition, the meeting highlighted recent advances in targeted novel technology (-omics) and opening therapeutic avenues in this field of research.

Genetic and pharmacological inhibition of XBP1 protects against APAP hepatotoxicity through the activation of autophagy.

Acetaminophen (APAP) hepatotoxicity induces endoplasmic reticulum (ER) stress which triggers the unfolded protein response (UPR) in hepatocytes. However, the mechanisms underlying ER stress remain poorly understood, thus reducing the options for exploring new pharmacological therapies for patients with hyperacute liver injury. Eight-to-twelve-week-old C57BL/6J Xbp1-floxed (Xbp1f/f) and hepatocyte-specific knockout Xbp1 mice (Xbp1∆hepa) were challenged with either high dose APAP [500 mg/kg] and sacrificed at early (1-2 h) and late (24 h) stages of hepatotoxicity. Histopathological examination of livers, immunofluorescence and immunohistochemistry, Western blot, real time (RT)-qPCR studies and transmission electron microscopy (TEM) were performed. Pharmacological inhibition of XBP1 using pre-treatment with STF-083010 [STF, 75 mg/kg] and autophagy induction with Rapamycin [RAPA, 8 mg/kg] or blockade with Chloroquine [CQ, 60 mg/kg] was also undertaken in vivo. Cytoplasmic expression of XBP1 coincided with severity of human and murine hyperacute liver injury. Transcriptional and translational activation of the UPR and sustained activation of JNK1/2 were major events in APAP hepatotoxicity, both in a human hepatocytic cell line and in a preclinical model. Xbp1∆hepa livers showed decreased UPR and JNK1/2 activation but enhanced autophagy in response to high dose APAP. Additionally, blockade of XBP1 splicing by STF, mitigated APAP-induced liver injury and without non-specific off-target effects (e.g., CYP2E1 activity). Furthermore, enhanced autophagy might be responsible for modulating CYP2E1 activity in Xbp1∆hepa animals. Genetic and pharmacological inhibition of Xbp1 specifically in hepatocytes ameliorated APAP-induced liver injury by enhancing autophagy and decreasing CYP2E1 expression. These findings provide the basis for the therapeutic restoration of ER stress and/or induction of autophagy in patients with hyperacute liver injury.

Fat: Quality, or Quantity? What Matters Most for the Progression of Metabolic Associated Fatty Liver Disease (MAFLD).

OBJECTIVES: Lately, many countries have restricted or even banned transfat, and palm oil has become a preferred replacement for food manufacturers. Whether palm oil is potentially an unhealthy food mainly due to its high content of saturated Palmitic Acid (PA) is a matter of debate. The aim of this study was to test whether qualitative aspects of diet such as levels of PA and the fat source are risk factors for Metabolic Syndrome (MS) and Metabolic Associated Fatty Liver Disease (MAFLD). METHODS: C57BL/6 male mice were fed for 14 weeks with three types of Western diet (WD): 1. LP-WD-low concentration of PA (main fat source-corn and soybean oils); 2. HP-WD-high concentration of PA (main fat source-palm oil); 3. HP-Trans-WD-high concentration of PA (mainly transfat). RESULTS: All types of WD caused weight gain, adipocyte enlargement, hepatomegaly, lipid metabolism alterations, and steatohepatitis. Feeding with HP diets led to more prominent obesity, hypercholesterolemia, stronger hepatic injury, and fibrosis. Only the feeding with HP-Trans-WD resulted in glucose intolerance and elevation of serum transaminases. Brief withdrawal of WDs reversed MS and signs of MAFLD. However, mild hepatic inflammation was still detectable in HP groups. CONCLUSIONS: HP and HP-Trans-WD play a crucial role in the genesis of MS and MAFLD.

Abnormal Liver Function Test in Patients Infected with Coronavirus (SARS-CoV-2): A Retrospective Single-Center Study from Spain.

The outbreak of the novel coronavirus SARS-CoV-2 epidemic has rapidly spread and still poses a serious threat to healthcare systems worldwide. In the present study, electronic medical records containing clinical indicators related to liver injury in 799 COVID-19-confirmed patients admitted to a hospital in Madrid (Spain) were extracted and analyzed. Correlation between liver injury and disease outcome was also evaluated. Serum levels of Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyltransferase (GGT), Alkaline phosphatase (ALP), Lactate dehydrogenase (LDH) and AST/ALT ratio were elevated above the Upper Limit of Normal (ULN) in 25.73%, 49.17%, 34.62%, 24.21%, 55.84% and 75% of patients, respectively. Interestingly, significant positive correlation between LDH levels and the AST/ALT ratio with disease outcome was found. Our data showed that SARS-CoV-2 virus infection leads to mild, but significant changes in serum markers of liver injury. The upregulated LDH levels as well as AST/ALT ratios upon admission may be used as additional diagnostic characteristic for COVID-19 patients.

Activation of the Unfolded Protein Response (UPR) Is Associated with Cholangiocellular Injury, Fibrosis and Carcinogenesis in an Experimental Model of Fibropolycystic Liver Disease.

Fibropolycystic liver disease is characterized by hyperproliferation of the biliary epithelium and the formation of multiple dilated cysts, a process associated with unfolded protein response (UPR). In the present study, we aimed to understand the mechanisms of cyst formation and UPR activation in hepatocytic c-Jun N-terminal kinase 1/2 (Jnk1/2) knockout mice. Floxed JNK1/2 (Jnkf/f) and Jnk∆hepa animals were sacrificed at different time points during progression of liver disease. Histological examination of specimens evidenced the presence of collagen fiber deposition, increased α-smooth muscle actin (αSMA), infiltration of CD45, CD11b and F4/80 cells and proinflammatory cytokines (Tnf, Tgfß1) and liver injury (e.g., ALT, apoptosis and Ki67-positive cells) in Jnk∆hepa compared with Jnkf/f livers from 32 weeks of age. This was associated with activation of effectors of the UPR, including BiP/GRP78, CHOP and spliced XBP1. Tunicamycin (TM) challenge strongly induced ER stress and fibrosis in Jnk∆hepa animals compared with Jnkf/f littermates. Finally, thioacetamide (TAA) administration to Jnk∆hepa mice induced UPR activation, peribiliary fibrosis, liver injury and markers of biliary proliferation and cholangiocarcinoma (CCA). Orthoallografts of DEN/CCl4-treated Jnk∆hepa liver tissue triggered malignant CCA. Altogether, these results suggest that activation of the UPR in conjunction with fibrogenesis might trigger hepatic cystogenesis and early stages of CCA.

Intestinal Epithelial Cell-Derived Extracellular Vesicles Modulate Hepatic Injury via the Gut-Liver Axis During Acute Alcohol Injury.

Binge drinking, i.e., heavy episodic drinking in a short time, has recently become an alarming societal problem with negative health impact. However, the harmful effects of acute alcohol injury in the gut-liver axis remain elusive. Hence, we focused on the physiological and pathological changes and the underlying mechanisms of experimental binge drinking in the context of the gut-liver axis. Eight-week-old mice with a C57BL/6 background received a single dose (p.o.) of ethanol (EtOH) [6 g/kg b.w.] as a preclinical model of acute alcohol injury. Controls received a single dose of PBS. Mice were sacrificed 8 h later. In parallel, HepaRGs and Caco-2 cells, human cell lines of differentiated hepatocytes and intestinal epithelial cells intestinal epithelial cells (IECs), respectively, were challenged in the presence or absence of EtOH [0-100 mM]. Extracellular vesicles (EVs) isolated by ultracentrifugation from culture media of IECs were added to hepatocyte cell cultures. Increased intestinal permeability, loss of zonula occludens-1 (ZO-1) and MUCIN-2 expression, and alterations in microbiota-increased Lactobacillus and decreased Lachnospiraceae species-were found in the large intestine of mice exposed to EtOH. Increased TUNEL-positive cells, infiltration of CD11b-positive immune cells, pro-inflammatory cytokines (e.g., tlr4, tnf, il1ß), and markers of lipid accumulation (Oil Red O, srbep1) were evident in livers of mice exposed to EtOH, particularly in females. In vitro experiments indicated that EVs released by IECs in response to ethanol exerted a deleterious effect on hepatocyte viability and lipid accumulation. Overall, our data identified a novel mechanism responsible for driving hepatic injury in the gut-liver axis, opening novel avenues for therapy.

Alcoholic liver disease: Utility of animal models.

Alcoholic liver disease (ALD) is a major cause of acute and chronic liver injury. Extensive evidence has been accumulated on the pathological process of ALD during the past decades. However, effective treatment options for ALD are very limited due to the lack of suitable in vivo models that recapitulate the full spectrum of ALD. Experimental animal models of ALD, particularly rodents, have been used extensively to mimic human ALD. An ideal animal model should recapitulate all aspects of the ALD process, including significant steatosis, hepatic neutrophil infiltration, and liver injury. A better strategy against ALD depends on clear diagnostic biomarkers, accurate predictor(s) of its progression and new therapeutic approaches to modulate stop or even reverse the disease. Numerous models employing rodent animals have been established in the last decades to investigate the effects of acute and chronic alcohol exposure on the initiation and progression of ALD. Although significant progress has been made in gaining better knowledge on the mechanisms and pathology of ALD, many features of ALD are unknown, and require further investigation, ideally with improved animal models that more effectively mimic human ALD. Although differences in the degree and stages of alcoholic liver injury inevitably exist between animal models and human ALD, the acquisition and translational relevance will be greatly enhanced with the development of new and improved animal models of ALD.

Monocyte-Derived Dendritic Cells Differentiated in the Presence of Lenalidomide Display a Semi-Mature Phenotype, Enhanced Phagocytic Capacity, and Th1 Polarization Capability.

Lenalidomide is an analog of thalidomide, with potent anticancer activity demonstrated in several hematological malignancies. It has immunomodulatory properties, being able to enhance the activation of different types of immune cells, which results in antitumor activities. Dendritic cells (DCs) are pivotal in the immune response, and different immunotherapeutic approaches targeting these cells are being developed. Since little is known about the effect of lenalidomide on DCs, the goal of the present work was to investigate the phenotype and function of human monocyte-derived DCs differentiated in the presence of lenalidomide (L-DCs). Our results showed that L-DCs display a unique phenotype, with increased cell surface expression of some maturation markers such as CD1d, CD83, CD86, and HLA-DR. This phenotype correlates with a lower expression of the E3 ubiquitin-ligase MARCH-I in L-DCs, upregulating the cell surface expression of CD86 and HLA-DR. In addition, immature L-DCs express higher amounts of DC-SIGN on the cell surface than control immature DCs. After LPS stimulation, production of IL-6 and TNF-α was severely decreased, whereas IL-12 and IL-10 secretion was dramatically upregulated in L-DCs, compared to that in the controls. Functionally, L-DCs are more effectively recognized by NKT cells in cytotoxicity experiments. Furthermore, L-DCs display higher opsonin-independent antigen uptake capability than control DCs. Mixed lymphocyte reaction experiments showed that L-DCs could stimulate naïve CD4 T-cells, polarizing them toward a predominant Th1 phenotype. In summary, DCs derived from monocytes in the presence of lenalidomide present a semi-mature phenotype, increased phagocytic capacity, reduced production of proinflammatory cytokines, and the ability to polarize T-cells toward predominant Th1-type responses; these are qualities that might be useful in the development of new immunotherapeutic treatments.

Dissecting the molecular pathophysiology of drug-induced liver injury.

Drug-induced liver injury (DILI) has become a major topic in the field of Hepatology and Gastroenterology. DILI can be clinically divided into three phenotypes: hepatocytic, cholestatic and mixed. Although the clinical manifestations of DILI are variable and the pathogenesis complicated, recent insights using improved preclinical models, have allowed a better understanding of the mechanisms that trigger liver damage. In this review, we will discuss the pathophysiological mechanisms underlying DILI. The toxicity of the drug eventually induces hepatocellular damage through multiple molecular pathways, including direct hepatic toxicity and innate and adaptive immune responses. Drugs or their metabolites, such as the common analgesic, acetaminophen, can cause direct hepatic toxicity through accumulation of reactive oxygen species and mitochondrial dysfunction. The innate and adaptive immune responses play also a very important role in the occurrence of idiosyncratic DILI. Furthermore, we examine common forms of hepatocyte death and their association with the activation of specific signaling pathways.

Intestinal epithelial cell endoplasmic reticulum stress promotes MULT1 up-regulation and NKG2D-mediated inflammation.

Endoplasmic reticulum (ER) stress is commonly observed in intestinal epithelial cells (IECs) and can, if excessive, cause spontaneous intestinal inflammation as shown by mice with IEC-specific deletion of X-box-binding protein 1 (Xbp1), an unfolded protein response-related transcription factor. In this study, Xbp1 deletion in the epithelium (Xbp1ΔIEC ) is shown to cause increased expression of natural killer group 2 member D (NKG2D) ligand (NKG2DL) mouse UL16-binding protein (ULBP)-like transcript 1 and its human orthologue cytomegalovirus ULBP via ER stress-related transcription factor C/EBP homology protein. Increased NKG2DL expression on mouse IECs is associated with increased numbers of intraepithelial NKG2D-expressing group 1 innate lymphoid cells (ILCs; NK cells or ILC1). Blockade of NKG2D suppresses cytolysis against ER-stressed epithelial cells in vitro and spontaneous enteritis in vivo. Pharmacological depletion of NK1.1+ cells also significantly improved enteritis, whereas enteritis was not ameliorated in Recombinase activating gene 1-/-;Xbp1ΔIEC mice. These experiments reveal innate immune sensing of ER stress in IECs as an important mechanism of intestinal inflammation.

The Role of Lipids in Development of Allergic Responses.

Most allergic diseases are caused by activation of Th2 type immune responses resulting in the production of specific IgE against proteins found in normally harmless substances such as pollen, mites, epithelia or food. Allergenic substances are composed, in addition to proteins, of other compounds such as carbohydrates and lipids. Those lipids are able to promote the development of Th2-type responses associated with allergy. There are lipids found in pollen, milk or insect venom that are specifically recognized by CD1 restricted unconventional T lymphocytes, which can promote allergic reactions. Furthermore, a large number of allergens are proteins containing hydrophobic parts that specifically bind lipids that are capable to favor allergenic immune responses. Also, lipids associated to substances like pollen, dander, epithelia or the bacteria can act on cells of the innate system, including dendritic cells, which in turn lead to the differentiation of Th2-type clones. Finally, lipids may also influence the ability of allergens to be exposed to the immune system within the oral, respiratory or intestinal mucosa where allergic response occurs with great frequency.

Human Invariant Natural Killer T Cells Respond to Antigen-Presenting Cells Exposed to Lipids from Olea europaea Pollen.

BACKGROUND: Allergic sensitization might be influenced by the lipids present in allergens, which can be recognized by natural killer T (NKT) cells on antigen-presenting cells (APCs). The aim of this study was to analyze the effect of olive pollen lipids in human APCs, including monocytes as well as monocyte-derived macrophages (Mϕ) and dendritic cells (DCs). METHODS: Lipids were extracted from olive (Olea europaea) pollen grains. Invariant (i)NKT cells, monocytes, Mϕ, and DCs were obtained from buffy coats of healthy blood donors, and their cell phenotype was determined by flow cytometry. iNKT cytotoxicity was measured using a lactate dehydrogenase assay. Gene expression of CD1A and CD1D was performed by RT-PCR, and the production of IL-6, IL-10, IL-12, and TNF-α cytokines by monocytes, Mϕ, and DCs was measured by ELISA. RESULTS: Our results showed that monocytes and monocyte-derived Mϕ treated with olive pollen lipids strongly activate iNKT cells. We observed several phenotypic modifications in the APCs upon exposure to pollen-derived lipids. Both Mϕ and monocytes treated with olive pollen lipids showed an increase in CD1D gene expression, whereas upregulation of cell surface CD1d protein occurred only in Mϕ. Furthermore, DCs differentiated in the presence of human serum enhance their surface CD1d expression when exposed to olive pollen lipids. Finally, olive pollen lipids were able to stimulate the production of IL-6 but downregulated the production of lipopolysaccharide- induced IL-10 by Mϕ. CONCLUSIONS: Olive pollen lipids alter the phenotype of monocytes, Mϕ, and DCs, resulting in the activation of NKT cells, which have the potential to influence allergic immune responses.

Orthologous Allergens and Diagnostic Utility of Major Allergen Alt a 1.

PURPOSE: Hypersensitivity to fungi is associated with rhinoconjunctivitis and asthma. For some fungi, such as Alternaria alternata (A. alternata), the symptoms of asthma are persistent, increasing disease severity and the risk of fatal outcomes. There are a large number of species of fungi but knowledge of them remains limited. This, together with the difficulties in obtaining adequate standardized extracts, means that there remain significant challenges in the diagnosis and immunotherapy of allergy associated with fungi. The type of indoor fungi related to asthma/allergy varies according to geographic, climatic, and seasonal factors, making their study difficult. The aim of this study was to determine hypersensitivity to indoor fungi in a population from Cuenca, Spain. METHODS: Thirty-five patients with symptoms compatible with rhinitis or asthma who showed clear worsening of their symptoms in their homes or workplace were included. In vivo and in vitro tests were made with a battery of fungal allergens, including the species isolated in the home or workplace. RESULTS: Ulocladium botrytis (U. botrytis) and A. alternata were the most representative species as a source of home sensitization. These species showed very high concordance in skin tests, specific IgE, and histamine release. The allergen Alt a 1, which was recognized in all patients, was detected in A. alternata, U. botrytis, and Stemphylium botryosum (S. botryosum). CONCLUSIONS: U. botrytis and A. alternata were the most representative species as a source of home sensitization. Alt a 1 was recognized in all patients and may be considered a non-species-specific allergen that could be used as a diagnostic source of sensitization to some species of the Pleosporaceae family.

Novel vaccines targeting dendritic cells by coupling allergoids to nonoxidized mannan enhance allergen uptake and induce functional regulatory T cells through programmed death ligand 1.

BACKGROUND: Allergen immunotherapy (AIT) is the only curative treatment for allergy. AIT faces pitfalls related to efficacy, security, duration, and patient compliance. Novel vaccines overcoming such inconveniences are in demand. OBJECTIVES: We sought to study the immunologic mechanisms of action for novel vaccines targeting dendritic cells (DCs) generated by coupling glutaraldehyde-polymerized grass pollen allergoids to nonoxidized mannan (PM) compared with glutaraldehyde-polymerized allergoids (P) or native grass pollen extracts (N). METHODS: Skin prick tests and basophil activation tests with N, P, or PM were performed in patients with grass pollen allergy. IgE-blocking experiments, flow cytometry, confocal microscopy, cocultures, suppression assays, real-time quantitative PCR, ELISAs, and ELISpot assays were performed to assess allergen capture by human DCs and T-cell responses. BALB/c mice were immunized with PM, N, or P. Antibody levels, cytokine production by splenocytes, and splenic forkhead box P3 (FOXP3)(+) regulatory T (Treg) cells were quantified. Experiments with oxidized PM were also performed. RESULTS: PM displays in vivo hypoallergenicity, induces potent blocking antibodies, and is captured by human DCs much more efficiently than N or P by mechanisms depending on mannose receptor- and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated internalization. PM endorses human DCs to generate functional FOXP3(+) Treg cells through programmed death ligand 1. Immunization of mice with PM induces a shift to nonallergic responses and increases the frequency of splenic FOXP3(+) Treg cells. Mild oxidation impairs these effects in human subjects and mice, demonstrating the essential role of preserving the carbohydrate structure of mannan. CONCLUSIONS: Allergoids conjugated to nonoxidized mannan represent suitable vaccines for AIT. Our findings might also be of the utmost relevance to development of therapeutic interventions in other immune tolerance-related diseases.