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IgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linked glycan located in the IgG Fc domain. Here, we compared the anti-Spike (S) IgG1 Fc glycosylation profiles in response to mRNA, adenoviral, and protein-based COVID-19 vaccines by mass spectrometry (MS). All vaccines induced a transient increase of antigen-specific IgG1 Fc galactosylation and sialylation. An initial, transient increase of afucosylated IgG was induced by membrane-encoding S protein formulations. A fucose-sensitive ELISA for antigen-specific IgG (FEASI) exploiting FcγRIIIa affinity for afucosylated IgG was used as an orthogonal method to confirm the LC-MS-based afucosylation readout. Our data suggest that vaccine-induced anti-S IgG glycosylation is dynamic, and although variation is seen between different vaccine platforms and individuals, the evolution of glycosylation patterns display marked overlaps.
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BACKGROUND: Afucosylated IgG1 responses have only been found against membrane-embedded epitopes, including anti-S in SARS-CoV-2 infections. These responses, intrinsically protective through enhanced FcγRIIIa binding, can also trigger exacerbated pro-inflammatory responses in severe COVID-19. We investigated if the BNT162b2 SARS-CoV-2 mRNA also induced afucosylated IgG responses. METHODS: Blood from vaccinees during the first vaccination wave was collected. Liquid chromatography-Mass spectrometry (LC-MS) was used to study anti-S IgG1 Fc glycoprofiles. Responsiveness of alveolar-like macrophages to produce proinflammatory cytokines in presence of sera and antigen was tested. Antigen-specific B cells were characterized and glycosyltransferase levels were investigated by Fluorescence-Activated Cell Sorting (FACS). FINDINGS: Initial transient afucosylated anti-S IgG1 responses were found in naive vaccinees, but not in antigen-experienced ones. All vaccinees had increased galactosylated and sialylated anti-S IgG1. Both naive and antigen-experienced vaccinees showed relatively low macrophage activation potential, as expected, due to the low antibody levels for naive individuals with afucosylated IgG1, and low afucosylation levels for antigen-experienced individuals with high levels of anti-S. Afucosylation levels correlated with FUT8 expression in antigen-specific plasma cells in naive individuals. Interestingly, low fucosylation of anti-S IgG1 upon seroconversion correlated with high anti-S IgG levels after the second dose. INTERPRETATION: Here, we show that BNT162b2 mRNA vaccination induces transient afucosylated anti-S IgG1 responses in naive individuals. This observation warrants further studies to elucidate the clinical context in which potent afucosylated responses would be preferred. FUNDING: LSBR1721, 1908; ZonMW10430012010021, 09150161910033, 10430012010008; DFG398859914, 400912066, 390884018; PMI; DOI4-Nr. 3; H2020-MSCA-ITN 721815.
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Vacunas contra la COVID-19 , COVID-19 , Humanos , Vacuna BNT162 , Inmunoglobulina G , COVID-19/prevención & control , SARS-CoV-2 , Anticuerpos Antivirales , VacunaciónRESUMEN
BACKGROUND: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (FcγR). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell FcγRs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. METHODS: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives FcγRIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (FcγR-IgG ELISA). FINDINGS: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R2=0.93, p < 0.0001). The FEASI method was then used to quantify IgG levels and fucosylation in COVID-19 convalescent plasma which was independently validated by LC-MS. INTERPRETATION: FEASI can be reliably implemented to measure relative and absolute IgG Fc fucosylation and quantify binding of antigen-specific IgG to FcγR in a high-throughput manner accessible to all diagnostic and research laboratories. FUNDING: This work was funded by the Stichting Sanquin Bloedvoorziening (PPOC 19-08 and SQI00041) and ZonMW 10430 01 201 0021.
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Fucosa , Inmunoglobulina G , Receptores de IgG , COVID-19/diagnóstico , COVID-19/terapia , Ensayo de Inmunoadsorción Enzimática/métodos , Fucosa/química , Fucosa/metabolismo , Humanos , Inmunización Pasiva , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Receptores de IgG/química , Sueroterapia para COVID-19RESUMEN
Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.
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Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Inmunoglobulina G/inmunología , SARS-CoV-2/inmunología , Adulto , Anciano , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/química , COVID-19/fisiopatología , Células Cultivadas , Enfermedad Crítica , Citomegalovirus/inmunología , Femenino , Fucosa/análisis , Glicosilación , VIH/inmunología , Vacunas contra Hepatitis B/inmunología , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/química , Inflamación , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Parvovirus B19 Humano/inmunología , Índice de Severidad de la Enfermedad , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas de Subunidad/inmunología , Adulto JovenRESUMEN
Intrinsic and acquired resistances are major hurdles preventing the effective use of MEK inhibitors for treatment of colorectal cancer (CRC). Some 35-45% of colorectal cancers are KRAS-mutant and their treatment remains challenging as these cancers are refractory to MEK inhibitor treatment, because of feedback activation of receptor tyrosine kinases (RTKs). We reported previously that loss of ERN1 sensitizes a subset of KRAS-mutant colon cancer cells to MEK inhibition. Here we show that the loss of RUNX2 or its cofactor CBFB can confer MEK inhibitor resistance in CRC cells. Mechanistically, we find that cells with genetically ablated RUNX2 or CBFB activate multiple RTKs, which coincides with high SHP2 phosphatase activity, a phosphatase that relays signals from the cell membrane to downstream pathways governing growth and proliferation. Moreover, we show that high activity of SHP2 is causal to loss of RUNX2-induced MEK inhibitor resistance, as a small molecule SHP2 inhibitor reinstates sensitivity to MEK inhibitor in RUNX2 knockout cells. Our results reveal an unexpected role for loss of RUNX2/CBFB in regulating RTK activity in colon cancer, resulting in reduced sensitivity to MEK inhibitors.
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Androgen receptor (AR) inhibitors represent the mainstay of prostate cancer treatment. In a genome-wide CRISPR-Cas9 screen using LNCaP prostate cancer cells, loss of co-repressor TLE3 conferred resistance to AR antagonists apalutamide and enzalutamide. Genes differentially expressed upon TLE3 loss share AR as the top transcriptional regulator, and TLE3 loss rescued the expression of a subset of androgen-responsive genes upon enzalutamide treatment. GR expression was strongly upregulated upon AR inhibition in a TLE3-negative background. This was consistent with binding of TLE3 and AR at the GR locus. Furthermore, GR binding was observed proximal to TLE3/AR-shared genes. GR inhibition resensitized TLE3KO cells to enzalutamide. Analyses of patient samples revealed an association between TLE3 and GR levels that reflected our findings in LNCaP cells, of which the clinical relevance is yet to be determined. Together, our findings reveal a mechanistic link between TLE3 and GR-mediated resistance to AR inhibitors in human prostate cancer.
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Proteínas Co-Represoras/genética , Factor Nuclear 3-alfa del Hepatocito/genética , Neoplasias de la Próstata/genética , Receptores Androgénicos/genética , Antagonistas de Receptores Androgénicos/farmacología , Benzamidas , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Masculino , Nitrilos , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Receptores de Glucocorticoides/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
BACKGROUND: Mutations in KRAS are frequent in human cancer, yet effective targeted therapeutics for these cancers are still lacking. Attempts to drug the MEK kinases downstream of KRAS have had limited success in clinical trials. Understanding the specific genomic vulnerabilities of KRAS-driven cancers may uncover novel patient-tailored treatment options. METHODS: We first searched for synthetic lethal (SL) genetic interactions with mutant RAS in yeast with the ultimate aim to identify novel cancer-specific targets for therapy. Our method used selective ploidy ablation, which enables replication of cancer-specific gene expression changes in the yeast gene disruption library. Second, we used a genome-wide CRISPR/Cas9-based genetic screen in KRAS mutant human colon cancer cells to understand the mechanistic connection between the synthetic lethal interaction discovered in yeast and downstream RAS signaling in human cells. RESULTS: We identify loss of the endoplasmic reticulum (ER) stress sensor IRE1 as synthetic lethal with activated RAS mutants in yeast. In KRAS mutant colorectal cancer cell lines, genetic ablation of the human ortholog of IRE1, ERN1, does not affect growth but sensitizes to MEK inhibition. However, an ERN1 kinase inhibitor failed to show synergy with MEK inhibition, suggesting that a non-kinase function of ERN1 confers MEK inhibitor resistance. To investigate how ERN1 modulates MEK inhibitor responses, we performed genetic screens in ERN1 knockout KRAS mutant colon cancer cells to identify genes whose inactivation confers resistance to MEK inhibition. This genetic screen identified multiple negative regulators of JUN N-terminal kinase (JNK) /JUN signaling. Consistently, compounds targeting JNK/MAPK8 or TAK1/MAP3K7, which relay signals from ERN1 to JUN, display synergy with MEK inhibition. CONCLUSIONS: We identify the ERN1-JNK-JUN pathway as a novel regulator of MEK inhibitor response in KRAS mutant colon cancer. The notion that multiple signaling pathways can activate JUN may explain why KRAS mutant tumor cells are traditionally seen as highly refractory to MEK inhibitor therapy. Our findings emphasize the need for the development of new therapeutics targeting JUN activating kinases, TAK1 and JNK, to sensitize KRAS mutant cancer cells to MEK inhibitors.
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Antineoplásicos/farmacología , Neoplasias del Colon/genética , Endorribonucleasas/genética , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Bencimidazoles/farmacología , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Estrés del Retículo Endoplásmico , Células HEK293 , Humanos , Quinasas Quinasa Quinasa PAM/genética , Proteínas Proto-Oncogénicas c-jun/genética , Piridonas/farmacología , Pirimidinonas/farmacología , Respuesta de Proteína Desplegada , Levaduras/genéticaRESUMEN
Activation of the mitogen-activated protein kinase (MAPK) pathway is frequent in cancer. Drug development efforts have been focused on kinases in this pathway, most notably on RAF and MEK. We show here that MEK inhibition activates JNK-JUN signaling through suppression of DUSP4, leading to activation of HER Receptor Tyrosine Kinases. This stimulates the MAPK pathway in the presence of drug, thereby blunting the effect of MEK inhibition. Cancers that have lost MAP3K1 or MAP2K4 fail to activate JNK-JUN. Consequently, loss-of-function mutations in either MAP3K1 or MAP2K4 confer sensitivity to MEK inhibition by disabling JNK-JUN-mediated feedback loop upon MEK inhibition. In a panel of 168 Patient Derived Xenograft (PDX) tumors, MAP3K1 and MAP2K4 mutation status is a strong predictor of response to MEK inhibition. Our findings suggest that cancers having mutations in MAP3K1 or MAP2K4, which are frequent in tumors of breast, prostate and colon, may respond to MEK inhibitors. Our findings also suggest that MAP3K1 and MAP2K4 are potential drug targets in combination with MEK inhibitors, in spite of the fact that they are encoded by tumor suppressor genes.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , MAP Quinasa Quinasa 4/genética , Quinasa 1 de Quinasa de Quinasa MAP/genética , Neoplasias de la Próstata/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Bencimidazoles/farmacología , Bencimidazoles/uso terapéutico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Femenino , Xenoinjertos , Humanos , Mutación con Pérdida de Función , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Quinasa 1 de Quinasa de Quinasa MAP/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias de la Próstata/genética , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.
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Proteína de Unión a CREB/deficiencia , Proteína de Unión a CREB/metabolismo , Transformación Celular Neoplásica/metabolismo , Células Progenitoras Linfoides/metabolismo , Linfoma/metabolismo , Células Madre Neoplásicas/metabolismo , Acetilación , Animales , Proteína de Unión a CREB/genética , Proliferación Celular , Autorrenovación de las Células , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células Cultivadas , Daño del ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Histonas/metabolismo , Linfangiogénesis , Células Progenitoras Linfoides/patología , Linfoma/genética , Linfoma/patología , Linfopoyesis , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Células Madre Neoplásicas/patología , Fenotipo , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Activating mutations and translocations of the FGFR3 gene are commonly seen in urothelial cell carcinoma (UCC) of the bladder and urinary tract. Several fibroblast growth factor receptor (FGFR) inhibitors are currently in clinical development and response rates appear promising for advanced UCC. A common problem with targeted therapeutics is intrinsic or acquired resistance of the cancer cells. To find potential drug targets that can act synergistically with FGFR inhibition, we performed a synthetic lethality screen for the FGFR inhibitor AZD4547 using a short hairpin RNA library targeting the human kinome in the UCC cell line RT112 (FGFR3-TACC3 translocation). We identified multiple members of the phosphoinositide 3-kinase (PI3K) pathway and found that inhibition of PIK3CA acts synergistically with FGFR inhibitors. The PI3K inhibitor BKM120 acted synergistically with inhibition of FGFR in multiple UCC and lung cancer cell lines having FGFR mutations. Consistently, we observed an elevated PI3K-protein kinase B pathway activity resulting from epidermal growth factor receptor or Erb-B2 receptor tyrosine kinase 3 reactivation caused by FGFR inhibition as the underlying molecular mechanism of the synergy. Our data show that feedback pathways activated by FGFR inhibition converge on the PI3K pathway. These findings provide a strong rationale to test FGFR inhibitors in combination with PI3K inhibitors in cancers harboring genetic activation of FGFR genes.
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Aminopiridinas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzamidas/farmacología , Carcinoma/tratamiento farmacológico , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Morfolinas/farmacología , Mutación , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Urotelio/efectos de los fármacos , Animales , Carcinoma/enzimología , Carcinoma/genética , Carcinoma/patología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Ratones Desnudos , Terapia Molecular Dirigida , Interferencia de ARN , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Carga Tumoral/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/enzimología , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Urotelio/enzimología , Urotelio/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This study aimed to determine protein expression levels of fibroblast growth factor receptors (FGFR) 1, 2 and 3 in early stage non-small cell lung cancer (NSCLC). Additionally, a screen to define the frequency of FGFR3-TACC3 translocation and FGFR3 amplification was performed. Archived tissues from 653 NSCLC samples (adenocarcinoma (AC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC)) were analysed with immunohistochemistry (IHC) for expression of FGFR1, 2 and 3. Expression levels of FGFR1, 2 and 3 were correlated with clinicopathological features. The presence of FGFR3-TACC3 translocation was detected by RT-PCR and FGFR3 amplification was detected by fluorescence in situ hybridization. FGFR1, 2 and 3 proteins were highly expressed in 64 (10.6%), 76 (12.9%) and 20 (3.3%) NSCLC tumour samples, respectively. Protein expression of FGFR1 was significantly related to worse overall survival in NSCLC. Furthermore, FGFR1 protein expression was associated with light smoking and histological subtype (AC), FGFR2 protein expression with female gender, younger age, histological subtype (AC) and lower tumour stage, and FGFR3 protein was significantly overexpressed in tumours of older patients and SCC histology. The FGFR3-TACC3 fusion was detected in 3.0% (6/200) of NSCLC samples and the FGFR3 gene was amplified in 4.7% of IHC positive NSCLC samples (2/43). FGFR1, 2 and 3 proteins are expressed in a high number of early stage NSCLC and FGFR1 protein expression may serve as a prognostic biomarker. Recurrent translocations and amplifications in FGFR3 can be found in NSCLC. This study shows that FGFR family members are frequently aberrant in NSCLC and could be interesting therapeutic targets for the treatment of NSCLC.
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BACKGROUND: Fatty acid ethyl esters (FAEE), nonoxidative products of ethanol metabolism, are formed by the esterification of fatty acids and ethanol. Alcoholic subjects have high levels of FAEE in the circulation as well as in organs and tissues, especially those most often damaged by ethanol abuse. Our previous studies showed a significant synthesis of FAEE by human mononuclear cells within seconds of exposure to physiologic doses of ethanol. In addition, FAEE inhibited phytohemagglutinin (PHA)-stimulated interleukin-2 production and calcium (Ca(2+)) influx into human mononuclear cells. FAEE also caused a rapid increase in the intracellular cAMP. The mechanism by which alcohol suppresses the immune system remains undetermined. OBJECTIVES: To evaluate the morphological and physiological effects of FAEE on human mononuclear cells and to study the impact of FAEE on cell viability. METHODS: Mononuclear cell fractions of human white blood cells (WBC) were incubated with physiological doses (25 and 50 microM) of ethyl oleate, a representative FAEE, for 15, 30, 60, 120 or 180 minutes. Morphological changes were evaluated by light and transmission electron microscopy (TEM). Lactate dehydrogenase (LDH) release was measured as a physiological indicator of necrosis. Physiological changes were also evaluated by western blots performed on whole-cell lysates of treated and untreated cells and by DNA electrophoresis. RESULTS: Significant morphological changes were detected in cells exposed to FAEE by both light and TEM. Concentration and time-dependent increases in the rates of apoptosis and necrosis were found by light microscopy and by LDH release, respectively, following 60 minutes exposure to 25 or 50 microM FAEE. One-hour 50 microM FAEE exposure caused activation of the caspase cascade, as demonstrated by Poly (ADP-ribose) Polymerase (PARP) cleavage, and significant DNA damage as a result of necrosis in human mononuclear cells. CONCLUSIONS: These studies provide evidence to support the toxic effects of FAEE on intact human mononuclear cells. The results from our studies also show that both apoptosis and necrosis are modes of cell death in FAEE-treated human mononuclear cells. This may be an important mechanism in alcohol-induced immunosuppression.