RESUMEN
DNAM-1 (CD226) is an activating receptor expressed in CD8+ T cells, NK cells, and monocytes. It has been reported that two SNPs in the DNAM-1 gene, rs763361 C>T and rs727088 G>A, have been associated with different autoimmune diseases; however, the role of DNAM-1 in ankylosing spondylitis has been less studied. For this reason, we focused on the study of these two SNPs in association with ankylosing spondylitis. For this, 34 patients and 70 controls were analyzed using endpoint PCR with allele-specific primers. Our results suggest that rs763361 C>T is involved as a possible protective factor under the CT co-dominant model (OR = 0.34, 95% CI = 0.13-0.88, p = 0.022) and the CT + TT dominant model (OR = 0.39, 95% CI = 0.17-0.90, p = 0.025), while rs727088 G>A did not show an association with the disease in any of the inheritance models. When analyzing the relationships of the haplotypes, we found that the T + A haplotype (OR = 0.31, 95% CI = 0.13-0.73, p = 0.0083) is a protective factor for developing the disease. In conclusion, the CT and CT + TT variants of rs763361 C>T and the T + A haplotype were considered as protective factors for developing ankylosing spondylitis.
RESUMEN
The NKp30 receptor is one of the three natural cytotoxic receptors reported in NK cells. This receptor is codified by the NCR3 gene, which encodes three isoforms, a consequence of the alternative splicing of exon 4. A greater expression of the three isoforms (A, B, and C), along with low levels of the NKp30 ligand B7H6, has been reported as a positive prognostic factor in different cancer types. Here, in patients with cervical cancer and precursor lesions, we report an altered immune-phenotype, characterized by non-fitness markers, that correlated with increased disease stage, from CIN 1 to FIGO IV. While overall NK cell numbers increased, loss of NKp30+ NK cells, especially in the CD56dim subpopulation, was found. Perforin levels were decreased in these cells. Decreased expression of the NKp30 C isoform and overexpression of soluble B7H6 was found in cervical cancer patients when compared against healthy subjects. PBMCs from healthy subjects downregulated NKp30 isoforms after co-culture with B7H6-expressing tumour cells. Taken together, these findings describe a unique down-modulation or non-fitness status of the immune response in cervical cancer, the understanding of which will be important for the design of novel immunotherapies against this disease.
Asunto(s)
Neoplasias del Cuello Uterino , Humanos , Femenino , Perforina/genética , Células Asesinas Naturales , Isoformas de Proteínas/genética , Empalme Alternativo , Receptor 3 Gatillante de la Citotoxidad Natural/genéticaRESUMEN
INTRODUCTION: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer in the world, which is associated with a wide spectrum of factors that play an important role in epidemiology, risk stratification, and therapeutic intervention. Several studies have shown the role of microRNAs (miRNAs) in the development of the disease. Genetic variations such as single-nucleotide polymorphisms (SNPs) in miRNAs can alter their function and lead to alter the expression of their target genes. OBJECTIVE: The aim of this study was to evaluate the association of rs12402181 in MIR3117 and rs12803915 in MIR612 with the risk of childhood preB-ALL in Mexican population. MATERIAL AND METHODS: DNA from 148 children (<18 years old) diagnosed with preB-ALL and 172 samples from participants in control group were included in the present study. Genotyping of the rs12402181 and rs12803915 polymorphisms was carried out by Real-Time PCR. To estimate the risk factor, the multiple genetic models co-dominant, dominant, and recessive were determined in both polymorphisms. RESULTS: In dominant genetic model from rs12402181, a high risk of susceptibility to ALL was observed (OR = 2.03, 95% CI = 1.27-3.22, p = 0.003). In the analysis adjusted for gender, a significant increase in the risk of ALL was maintained (OR = 2.03, 95% CI = 1.28-3.24, p = 0.003). The rs12803915 polymorphism was no associated with the risk of susceptibility to preB-ALL in any of the genetic models using in this study. CONCLUSIONS: Our data indicated that the A allele of the rs12402181 polymorphism may be considered as a genetic biomarker of preB-ALL susceptibility. Likewise, it was identified that the A allele of the rs12402181 polymorphism is an independent risk factor for ALL.
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MicroARNs , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Niño , Humanos , Biomarcadores , Estudios de Casos y Controles , Predisposición Genética a la Enfermedad , Genotipo , MicroARNs/genética , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMEN
AIM: Nonalcoholic fatty liver disease (NAFLD) is a complex metabolic condition in which both lifestyle and genetic factors have a pathogenic role. The LEP gene encodes leptin, which regulates appetite, body weight, and several metabolic functions. Proopiomelanocortin (POMC), regulates food intake and energy balance. The aim of the study was to determine partial or complete deletions of genes associated with obesity in patients diagnosed with NAFLD. MATERIAL AND METHODS: Blood samples and DNA from 43 individuals diagnosed with NAFLD by ultrasonographic technique (Fibroscan) were obtained. The partial or complete deletions of genes were determined by MLPA (Multiplex Ligation-dependent Probe Amplification) using the SALSA probemix P220-B2 Obesity only on 43 individuals. Fifty blood samples from healthy individuals were included. RESULTS: Eleven out of 43 individuals analyzed by MLPA presented some deletion of the genes analyzed: six were female and five were male. The partial or complete deletion of the LEPR and POMC genes was observed in eight patients (18.6%), SIM1 in six patients (13.9%), GRIK2 and SH2B1 in two patients (4.7%), SEZGL2 in four patients (9.3%), and MCR4 in one patient (2.3%). CONCLUSION: Partial deletion was observed in LEPR, POMC, SIM1, GRIK2, SH2B1, SEZGL2, and MCR4 genes in 26% of the cases, and we suggest that these alterations probably has a potential relationship for the development of NAFLD.
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Enfermedad del Hígado Graso no Alcohólico , Proteínas Adaptadoras Transductoras de Señales , Femenino , Humanos , Masculino , México , Enfermedad del Hígado Graso no Alcohólico/genética , Obesidad/complicaciones , Obesidad/genética , Proopiomelanocortina/genéticaRESUMEN
ABSTRACT: Rheumatoid arthritis (RA) is an autoimmune disease characterized by an inflammatory process that affects mainly synovial tissue in joints, and by the production of cyclic citrullinated peptides (anti-CCP) antibodies. In the inflammatory process the regulation of the nuclear factor kappa B (NFkB) transcription factor activation is a key point in the production of inflammatory cytokines. On the other hand, polymorphisms in several genes could contribute to the promotion of the inflammatory process observed in RA, and the association of the rs28362491 polymorphism in the NFkB gene with RA has been studied in different population. Therefore, it could be one of the interest targets to analyze their association with RA in a Mexican population.This is a case-control study to determine the influence of rs28362491 in the NFkB gene on RA and on clinical features of this disease, such as anti-CCP antibody levels, Disease Activity Score, and Health Assessment Questionnaire-Disability Index.The genotype of rs28362491 in the NFkB gene was determined in 140 RA patients and 135 healthy controls using the polymerase chain reaction-restriction fragment length polymorphism method with the enzyme PflMI. The following clinical variables were also determined: anti-CCP levels, Disease Activity Score, and Spanish version of the Health Assessment Questionnaire Disability-Index.Although no association of the polymorphism as a risk/protection factor with RA was found, the RA patients who carried the Ins/Ins genotype showed higher anti-CCP levels, while those with the Del/Del genotype showed higher Spanish version of the Health Assessment Questionnaire-Disability Index levels, compared to the other genotypes.The NFkB -94 Ins/Del ATTG (rs28362491) polymorphism is, therefore, associated with higher levels of anti-CCP antibodies, though no significant association as a risk or protection factor in RA cases was identified.
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Anticuerpos Antiproteína Citrulinada/sangre , Artritis Reumatoide/genética , FN-kappa B/genética , Adulto , Anciano , Artritis Reumatoide/sangre , Artritis Reumatoide/epidemiología , Autoanticuerpos , Estudios de Casos y Controles , Femenino , Predisposición Genética a la Enfermedad , Humanos , Mutación INDEL , Masculino , México/epidemiología , Persona de Mediana Edad , Péptidos Cíclicos , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Regiones Promotoras GenéticasRESUMEN
BACKGROUND: IKZF1 is a relevant gene associated with the pathogenesis of acute lymphoblastic leukemia, and the rs4132601 (T>G) and rs11978267 (A>G) polymorphisms have been associated with the development of this disease in several populations. The aim of this study was to determine the allelic and genotypic frequencies of the rs4132601 and rs11978267 polymorphisms in two indigenous Mexican groups (Cora and Huichol) and Mestizo populations from Nayarit, Mexico, and compare them with the frequencies of both polymorphisms in other populations of the world. METHODS: One hundred, 116, and 100 subjects from the Mestizo, Huichol, and Cora populations, respectively, all of them residents of the state of Nayarit, Mexico, were analyzed. The frequencies of rs4132601 and rs11978267 were determined by allelic discrimination using TaqMan assays. RESULTS: The allelic frequencies of rs4132601 were as follows: Mestizo group T = 0.74, G = 0.26; Cora T = 0.745, G = 0.255; and Huichol T = 0.47, G = 0.53. In the case of the rs11978267 polymorphism, the allelic frequencies were Mestizo A = 0.745, G = 0.255; Cora A = 0.735, G = 0.265; and Huichol A = 0.457, G = 0.543. For each population, both polymorphisms were in Hardy-Weinberg equilibrium. CONCLUSION: The Huichol population from Nayarit presented the highest frequencies of the risk allele reported to date in the whole world for both rs4132601 and rs11978267 polymorphisms.
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Frecuencia de los Genes , Factor de Transcripción Ikaros/genética , Pueblos Indígenas/genética , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adulto , Femenino , Humanos , Masculino , MéxicoRESUMEN
AIM: Ankylosing spondylitis (AS) pathogenesis has focused on the adaptive immune response; however, innate immune responses may also play a role in the inflammatory response of AS. Dysregulated neutrophil activation can induce tissue damage and contribute to the pathogenesis of immune-related diseases. Hence, the aim of this study was to assess the effect of immune complexes formed with the p30 of Salmonella typhimurium and anti-p30 antibodies present in the sera of AS patients and controls in inducing the release of neutrophil extracellular traps (NETs) and the secretion of pro-inflammatory cytokines. METHODS: We collected polymorphonuclear leukocytes (PMNs) from healthy donors. The PMNs isolated were stimulated with p30 alone or in immunocomplexes formed with antibodies presents in sera of AS patients or control subjects. Then, the NETs were analyzed by fluorescence microscopy. Concentrations of interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1ß, IL-8 and IL-10, were determined using the Cytometric Bead Array kit. RESULTS: Significant difference was observed in the release of NETs between the neutrophils stimulated with p30 + AS (70.52 ± 16.24) those unstimulated neutrophils (9.94 ± 12.12; P = .0095), stimulated with phorbol 12-myristate 13-acetate (39.78 ± 14.50; P = .0190), stimulated with control serum (CS) (10.85 ± 5.33; P = .0082) and serum of AS patient (10.28 ± 6.15; P = .0087). The stimulation of neutrophils with p30 alone induced a relatively low production of IL-6 (64.5 pg/mL), IL-8 (2658.3 pg/mL), IL-1ß (31.11 pg/mL), and TNF-α (3.8 pg/mL), compared to p30 + AS and p30 + CS groups. CONCLUSION: Our results show that neutrophils release NETs and pro-inflammatory cytokines in response to p30 in immunocomplexes. These findings could improve our understanding of the role of innate immunity in the initiation and/or maintenance of inflammatory responses, and in the progression of AS.
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Citocinas/metabolismo , Trampas Extracelulares/inmunología , Inmunidad Celular , Inflamación/inmunología , Neutrófilos/inmunología , Espondilitis Anquilosante/inmunología , Biomarcadores/metabolismo , Progresión de la Enfermedad , Trampas Extracelulares/metabolismo , Humanos , Inflamación/metabolismo , Neutrófilos/patología , Espondilitis Anquilosante/patologíaRESUMEN
BACKGROUND: Although great progress has been made in treatment regimens, cervical cancer remains as one of the most common cancer in women worldwide. Studies focusing on molecules that regulate carcinogenesis may provide potential therapeutic strategies for cervical cancer. B7-H6, an activating immunoligand expressed by several tumor cells, is known to activate NK cell-mediated cytotoxicity once engaged with its natural receptor NKp30. However, the opposite, that is, the effects in the tumor cell triggered by B7-H6 after interacting with NKp30 has not yet been well explored. METHODS: In this study, we evaluated the surface expression of B7-H6 by flow cytometry. Later, we stimulated B7-H6 positive cervical cancer derived-cell lines (HeLa and SiHa) with recombinant soluble NKp30 (sNKp30) protein and evaluated biological effects using the impedance RTCA system for cell proliferation, the scratch method for cell migration, and flow cytometry for apoptosis. Cellular localization of B7-H6 was determined using confocal microscopy. RESULTS: Notably, we observed that the addition of sNKp30 to the cervical cancer cell lines decreased tumor cell proliferation and migration rate, but had no effect on apoptosis. We also found that B7-H6 is selectively maintained in tumor cell lines, and that efforts to sort and purify B7-H6 negative or positive cells were futile, as negative cells, when cultured, regained the expression of B7-H6 and B7-H6 positive cells, when sorted and cultivated, lost a percentage of B7-H6 expression. CONCLUSIONS: Our results suggest that B7-H6 has an important, as of yet undescribed, role in the biology of the cervical tumor cells themselves, suggesting that this protein might be a promising target for anti-tumor therapy in the future.
Asunto(s)
Apoptosis , Antígenos B7/metabolismo , Proliferación Celular , Receptor 3 Gatillante de la Citotoxidad Natural/metabolismo , Neoplasias del Cuello Uterino/patología , Movimiento Celular , Femenino , Humanos , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/metabolismoRESUMEN
BACKGROUND: B7-H6 has been revealed as an endogenous immunoligand expressed in a variety of tumors, but not expressed in healthy tissues. Heretofore, no studies have been reported describing B7-H6 in women with cervical cancer. To investigate this question, our present study was conducted. RESULTS: This retrospective study comprised a total of 62 paraffinized cervical biopsies, which were distributed in five groups: low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), squamous cervical carcinoma (SCC), uterine cervical adenocarcinoma (UCAC), and a group of cervicitis (as a control for non-abnormal/non-transformed cells). Cervical sections were stained by immunohistochemistry to explore the expression of B7-H6, which was reported according to the immunoreactive score (IRS) system. We observed a complete lack of B7-H6 in LSIL abnormal epithelial cells. Interestingly, B7-H6 began to be seen in HSIL abnormal epithelial cells; more than half of this group had B7-H6 positive cells, with staining characterized by a cytoplasmic and membranous pattern. B7-H6 in the SCC group was also seen in the majority of the sections, showing the same cytoplasmic and membranous pattern. Strong evidence of B7-H6 was notably found in UCAC tumor columnar cells (in 100% of the specimens, also with cytoplasmic and membranous pattern). Moreover, consistent B7-H6 staining was observed in infiltrating plasma cells in all groups. CONCLUSIONS: B7-H6 IRS positively correlated with disease stage in the development of cervical cancer; additionally, B7-H6 scores were found to be even higher in the more aggressive uterine cervical adenocarcinoma, suggesting a possible future therapeutic target for this cancer type.
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Antígenos B7/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Células Epiteliales/metabolismo , Queratinocitos/metabolismo , Células Plasmáticas/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Adulto , Carcinogénesis , Carcinoma de Células Escamosas/patología , Progresión de la Enfermedad , Células Epiteliales/patología , Femenino , Humanos , Inmunohistoquímica , Queratinocitos/patología , Persona de Mediana Edad , Células Plasmáticas/patología , Estudios Retrospectivos , Neoplasias del Cuello Uterino/patologíaRESUMEN
Rheumatoid arthritis (RA) is a chronic inflammatory disease whose association with SNPs has led to the identification of biomarkers in different populations. To determine the association of the -857C/T SNP of the TNFA gene with RA and clinical parameters, 233 RA patients and 237 healthy controls were included in this study. The -857C/T polymorphism was determined using the TaqMan® system and clinical features were also determined. We found that the -857C/T SNP was in Hardy-Weinberg equilibrium. Our results showed no association of the -857C/T SNP with RA; however, RA patients carrying the TT genotype showed lower anti-CCP levels than other groups. Therefore, the TT genotype could be a risk factor for developing anti-CCP-negative RA. Our results suggest that the T allele of the TNFA -857C/T SNP exerts an influence on anti-CCP levels and could be a candidate marker for anti-CCP-negative RA.
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Alelos , Artritis Reumatoide/epidemiología , Artritis Reumatoide/etiología , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Factor de Necrosis Tumoral alfa/genética , Adulto , Anciano , Anticuerpos Antiproteína Citrulinada/sangre , Anticuerpos Antiproteína Citrulinada/inmunología , Artritis Reumatoide/sangre , Artritis Reumatoide/terapia , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Masculino , México/epidemiología , Persona de Mediana Edad , Oportunidad Relativa , Vigilancia de la PoblaciónRESUMEN
The IKZF1 gene is formed by 8 exons and encodes IKAROS, a transcription factor that regulates the expression of genes that control cell cycle progression and cell survival. In general, 15-20% of the patients with preB acute lymphoblastic leukemia (preB ALL) harbor IKZF1 deletions, and the frequency of these deletions increases in BCR-ABL1 or Ph-like subgroups. These deletions have been associated with poor treatment response and the risk of relapse. The aim of this descriptive study was to determine the frequency of IKZF1 deletions and the success of an induction therapy response in Mexican pediatric patients diagnosed with preB ALL in 2 hospitals from 2017 to August 2018. Thirty-six bone marrow samples from patients at the Instituto Nacional de Pediatría in Mexico City and the Centro Estatal de Cancerología in Tepic were analyzed. The IKZF1 deletion was identified by MLPA using the SALSA MLPA P335 ALL-IKZF1 probemix. Deletions of at least 1 IKZF1 exon were observed in 7/34 samples (20.6%): 3 with 1 exon deleted; 1 with 2 exons, 1 with 5 exons, 1 with 6 exons, and 1 patient with a complete IKZF1 deletion. This study was descriptive in nature; we calculated the frequency of the IKZF1 gene deletion in a Mexican pediatric population with preB ALL as 20.6%.
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Factor de Transcripción Ikaros/genética , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Médula Ósea/química , Médula Ósea/patología , Niño , Preescolar , Exones/genética , Femenino , Frecuencia de los Genes , Genes Relacionados con las Neoplasias , Predisposición Genética a la Enfermedad , Humanos , Lactante , Recién Nacido , Masculino , México , Tipificación de Secuencias Multilocus , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/etnología , Inducción de Remisión , Eliminación de Secuencia , Resultado del TratamientoRESUMEN
Acute hepatopancreatic necrosis disease (AHPND) was first reported in China in 2009 and afterwards in Mexico in 2013. AHPND is caused by Vibrio parahaemolyticus and affects Penaeus monodon and Litopenaeus vannamei shrimp cultures. The bacterium contains the pirA- and pirB-like genes in 69- to 70-Kb plasmids, which encode the toxins that produce the disease. The aim of this study was to determine whether pirA- and pirB-like genes existed in bacterial genera distinct from Vibrio before the first cases of AHPND were documented in Mexico. Two bacterial isolates were selected from shrimp farms in Nayarit in 2006 and analysed by nested-PCR to determine the presence of pirA- and pirB-like genes. The two isolates chosen did indeed show the presence of these genes, and those findings were confirmed by sequencing. Both strains matched to the bacterial species Micrococcus luteus. Results revealed two important situations: (a) the pirA- and pirB-like genes were present in a bacterial species that has not been reported previously (Micrococcus luteus); and (b) pirA- and pirB-like bacterial genes were present in Mexico before the first AHPND outbreak was reported in China.
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Proteínas Bacterianas/genética , Genes Bacterianos/genética , Micrococcus luteus/genética , Animales , México , Penaeidae/microbiología , Reacción en Cadena de la Polimerasa/veterinariaRESUMEN
Background and rationale for the study. Bacterial translocation is an important triggering factor of infection and mortality in cirrhosis. In a rat model using bile duct ligation (BDL), bacterial translocation appears within 24 h after ligation. The dynamic between TH1/TH2/TH17 cytokines and the integrity of the colonic mucosa in the context of cirrhosis is little known. This study aims to determine the link between bacterial translocation and intestinal inflammation in a cholestasis model. Additionally, alterations of the colonic mucus layer and the bacterial load were also addressed. RESULTS: Bacterial translocation detected by microbiological cultures and MALDI-TOF showed that Escherichia coli predominates in mesenteric lymph nodes of BDL rats. Intestinal bacterial load analyzed by qPCR indicates a dramatic Escherichia/Shigella overgrowth at 8 and 30 days post-BDL. IFN-γ, IL-4, and IL-17 evaluated by Western blotting were increased at 8 and 30 days in the small intestine. In the colon, in contrast, only IFN-γ was significantly increased. The colonic mucus layer and mucin-2 expression determined by Alcian blue staining and immunohistochemistry surprisingly showed an increase in the mucus layer thickness related to increased mucin-2 expression during the entire process of liver damage. Hepatic enzymes, as well as collagen I, collagen III, TNF-α, and IL-6 liver gene expression were increased. In conclusion, bacterial overgrowth associated with bacterial translocation is linked to the over-expression of IFN-γ, IL-4, IL-17 and mucin-2. These molecules might facilitate the intestinal permeability through exacerbating the inflammatory process and disturbing tight junctions, leading to the perpetuation of the liver damage.
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Traslocación Bacteriana , Colestasis/metabolismo , Colestasis/microbiología , Microbioma Gastrointestinal , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Intestinos/microbiología , Mucina 2/metabolismo , Animales , Colestasis/patología , Modelos Animales de Enfermedad , Hepatitis/metabolismo , Hepatitis/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Intestinos/patología , Hígado/metabolismo , Hígado/microbiología , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/microbiología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Masculino , Permeabilidad , Ratas Wistar , Factores de Tiempo , Regulación hacia ArribaRESUMEN
B7H6, an endogenous ligand expressed on tumor cell surfaces, triggers NKp30-mediated activation of human NK cells. In contrast, the release of soluble B7H6 has been proposed as a novel mechanism by which tumors might evade NK cell-mediated recognition. Since NK cells are critical for the maintenance of early pregnancy, it is not illogical that soluble B7H6 might also be an important factor in directing NK cell activity during normal pregnancy. Thus, this study was focused on the characterization of soluble B7H6 during the development of normal pregnancy. Serum samples were obtained from healthy pregnant women who were experiencing their second pregnancies (n=36). Additionally, 17 of these pregnant participants were longitudinally studied for the presence of B7H6 during their second and third trimesters. Age-matched healthy non-pregnant women served as controls (n=30). The presence of soluble B7H6 was revealed by Western blotting. A further characterization was performed using an immunoproteomic approach based on 2DE-Western blotting combined with MALDI-MS. The results show that sera from all pregnant women were characterized by the presence of two novel isoforms of B7H6, both with lower MW than the reported of 51kDa. These isoforms were either a heavy (â¼37kDa) or a light isoform (â¼30kDa) and were mutually exclusive. N-glycosylation did not completely explain the different molecular weights exhibited by the two isoforms, as was demonstrated by enzymatic deglycosylation with PNGase F. The confirmation of the identity and molecular mass of each isoform indicates that B7H6, while maintaining the C- and N-termini, is most likely released during pregnancy by a mechanism distinct from proteolytic cleavage. We found that both isoforms, but mainly the heavier B7H6, were released via exosomes; and that the lighter isoform was also released in an exosome-free manner that was not observed in the heavy isoform samples. In conclusion, we find that soluble B7H6 is constitutively expressed during pregnancy and that, moreover, the soluble B7H6 is present in two new isoforms, which are released by exosomal and exosome-free mechanisms.