Bcl-2 Knockdown Accelerates T Cell Receptor-Triggered Activation-Induced Cell Death in Jurkat T Cells

Cell death and survival are tightly controlled through the highly coordinated activation/inhibition of diverse signal transduction pathways to insure normal development and physiology. Imbalance between cell death and survival often leads to autoimmune diseases and cancer. Death receptors sense extracellular signals to induce caspase-mediated apoptosis. Acting upstream of CED-3 family proteases, such as caspase-3, Bcl-2 prevents apoptosis. Using short hairpin RNAs (shRNAs), we suppressed Bcl-2 expression in Jurkat T cells, and this increased TCR-triggered AICD and enhanced TNFR gene expression. Also, knockdown of Bcl-2 in Jurkat T cells suppressed the gene expression of FLIP, TNF receptor-associated factors 3 (TRAF3) and TRAF4. Furthermore, suppressed Bcl-2 expression increased caspase-3 and diminished nuclear factor kappa B (NF-kappaB) translocation.

Concentración de TNF Alfa, óxido nítrico e interleukinas 1 y 6 en pacientes con sepsis, spsis severa y shock séptico / TNF alpha concentration, nitric oxide and interleukins 1 and 6 in patients with sepsis, septic shock and severe spsis

Sepsis, sepsis severa y shock séptico son cuadros clínicos cuya mortalidad se mantiene elevada a pesar de los avances tecnológicos, y primera causa de muerte en terapia intensiva. Sus manifestaciones clínicas ymorbimortalidad se atribuyen a un incremento desmesurado de citoquinas proinflamatorias, como son el TNF alfa y las IL-1 y 6. El Óxido Nítrico (ON) es quien genera el shock séptico por vasodilatación. El objetivo de este trabajo es evaluar la capacidad de predecir la mortalidad por estas citoquinas, la Proteína C Reactiva (PCR), la metaloproteasa 3 (MMP-3) y el Óxido Nítrico. Se han incluido pacientes varones y mujeres mayores de 18 años que ingresan al Hospital Nacional de Clínicas con criterios de sepsis, sepsis severa y shock séptico. Todos los pacientes han firmado el consentimiento informado y esteprotocolo ha sido aprobado por el Comité de Ética del Hospital. La cuantificación de citoquinas al ingreso de los pacientes al hospital se llevo a cabo por ELISA y el ON por la Reacción de Griess. Hemos hallado que la IL-1 en pacientes que fallecieron fue de 274 pg/ml, estadísticamente superior a los pacientes que sobrevivieron, con 202 pg/ml, con una p=0,028. Los valores de PCR para los sobrevivientes fue de 42,29 mg/dl mientras para los pacientes fallecidos de 104,05 mg/dl, diferencia estadísticamente significativa (p=0,02).

SUMMARY: Sepsis, severe sepsis and septic shock are clinical conditions where mortality remains high despite advances in technology, and leading cause of death in intensive care. Clinical manifestations and morbidity attributable to a disproportionate increase in proinflammatory cytokines. Nitric Oxide (NO) is one who generates vasodilation septic shock. The aim of this study was to evaluate the ability to predict mortality from these cytokines, C-reactive protein (CRP), metalloproteinase 3 (MMP-3) and Nitric Oxide. We included male and female patients over 18 admitted to the Hospital Nacional de Clínicas with sepsis, severe sepsis and septic shock. All patients signed informed consent and this protocol has been approved by the Hospital Ethics Committee. Quantification of cytokines on admission of patients to the hospital was carried out by ELISA and NO by the reaction of Griess. We found that IL-1 in patients who died was 274 pg / ml, statistically higher than patients who survived, with 202 pg / ml, p =0.028. CRP for survivors was 42.29 mg / dl while for patients who died of 104.05 mg / dl, statistically significant difference (p = 0.02).

MCP-1-induced protein-1, an immune regulator

MCP-1-induced protein-1 (MCPIP1) is a newly identified protein that is crucial to immune regulation. Mice lacking MCPIP1 gene suffer from severe immune disorders, and most of them cannot survive longer than 12 weeks. Considerable progress has been made in revealing the mechanism underlying the immune regulatory function of MCPIP1. MCPIP1 can act as an RNase to promote the mRNA degradation of some inflammatory cytokines, such as IL-6 and IL-1. Pre-microRNAs are also confirmed to be the substrate of MCPIP1 RNase. The structure of MCPIP1 N-terminal conserved domain shows a PilT N-terminus-like RNase structure, further supporting the notion that MCPIP1 has RNase activity. MCPIP1 can also deubiquitinate TNF receptor-associated factor family proteins, which are known to mediate immune and inflammatory responses. In this review, we summarize recent progress on the immune regulatory role of MCPIP1 and discuss the mechanisms underlying its function.

Síndrome de hiper-IgM en miembros de 2 familias Chilenas no relacionadas: análisis genético- molecular / Hyper-IgM syndrome in members of two unrelated Chilean families: molecular and mutation analysis

BACKGROUND: Hyper-IgM syndronie (HIGM) is a rare primary immunodeficiency used to describe a heterogeneous group of disorders characterized by recurrey bacterial infrctions, normal or elevated serum IgM levels and low or absent serum IgG, IgA and IgE. AIM: To make definitive diagnosis, detect mutations in carriers and perform genetic counseling in patients with HIGM. PATIENTS AND METHODS: We studied the expression of CD40L, CD40 and made a mutation analysis of the CD40L gene in 3 males of 2 unrelated Chilean families diagnosed as a possible syndrome of hyper-IgM and 3 relatives. RESULTS: We identified a deletion frameshift in the exon 2 (delA225) of the extracellular domain of GD40L gene in one patient and verified the carrier stains of his mother and sister. The other patients showed a low expression of GD40L in activated T cells (65.3% ammd 65.5%) and a normal expressiomi of CD40. No alterations were found in the single strand conformation polymorphism analysis of the CD40L. CONCLUSIONS: These result allowed us to make a definite diagnosis of HIGM1 of a patient, detect female carriers and suggest a HIGM of recessive inheritance with normal CD40 expression in the patients of the second family.

Differential Signaling via Tumor Necrosis Factor-Associated Factors (TRAFs) by CD27 and CD40 in Mouse B Cells

BACKGROUND: CD27 is recently known as a memory B cell marker and is mainly expressed in activated T cells, some B cell population and NK cells. CD27 is a member of tumor necrosis factor receptor family. Like CD40 molecule, CD27 has (P/S/T/A) X(Q/E)E motif for interacting with TNF receptor-associated factors (TRAFs), and TRAF2 and TRAF5 bindings to CD27 in 293T cells were reported. METHODS: To investigate the CD27 signaling effect in B cells, human CD40 extracellular domain containing mouse CD27 cytoplamic domain construct (hCD40-mCD27) was transfected into mouse B cell line CH12.LX and M12.4.1. RESULTS: Through the stimulation of hCD40-mCD27 molecule via anti-human CD40 antibody or CD154 ligation, expression of CD11a, CD23, CD54, CD70 and CD80 were increased and secretion of IgM was induced, which were comparable to the effect of CD40 stimulation. TRAF2 and TRAF3 were recruited into lipid-enriched membrane raft and were bound to CD27 in M12.4.1 cells. CD27 stimulation, however, did not increase TRAF2 or TRAF3 degradation. CONCLUSION: In contrast to CD40 signaling pathway, TRAF2 and TRAF3 degradation was not observed after CD27 stimulation and it might contribute to prolonged B cell activation through CD27 signaling.

Functional proteomics of receptor-Ck in the developmental stages of human atherosclerotic arterial wall.

BACKGROUND: The study was undertaken to understand the relationship between the functional proteomics of receptor-Ck and developmental stages of human atherosclerotic aortic wall. METHODS AND RESULTS: Gene expression study of 25 aortas was undertaken and the results revealed a gradual increase in receptor-Ck gene expression paralleled by the regulatory response of its effector genes coding for sterol response element-binding protein, p27, cyclin D, interleukin-6 and CD40 from a normal to atherosclerotic arterial wall (viz. fatty streak and fibrofatty/fibrous plaque). CONCLUSIONS: Based upon this and our earlier studies, we propose that cholesterol-specific receptor-Ck-dependent gene regulation may be of crucial importance in atherogenesis.

Screening of new binding partners of CIKS with yeast two-hybrid system / 生物工程学报

The NF-kappaB transcription factor plays important regulatory roles in inflammation, apoptosis, immune and stress responses. IkappaB kinase (IKK) composed of two catalytic subunits and a regulator subunit, acts as a key component of NF-kappaB activation pathway through phosphorylation of IkappaB, the inhibitor of NF-kappaB. CIKS (connection to IKK and SAPK), a newly identified cellular protein, is involved in NF-kappaB and JNK activation. Although it has been known that CIKS interacts with IKK complex, and activates both IKK and SAPK when overexpressed; the underling mechanisms are poorly understood. To better understand the physiological roles of CIKS, we have screened human HeLa MATCHMAKER cDNA library for new binding partners of CIKS by using the yeast two-hybrid system with truncated CIKS (151-574) as the bait. The yeast strain AH109 was sequentially transformed with the bait plasmid and the library. The transformants were screened on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal)selective plates. Positive clones were restreaked on SD(-Leu/-Trp / + X-alpha-gal)plates three times to allow loss of some of the AD/library plasmids while maintaining selective pressure on both the DNA-BD and AD vectors. After 3 screenings on SD(-Leu/-Trp / + X-alpha-gal), the positive clones were further verified on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal) plates. The inserts in AD/library plasmids were amplified by PCR and PCR products were characterized by Hae III digestion to eliminate the duplicates. After screening in selective plates, the positive AD/library plasmids were rescued via transformation of E. coli HB101 and the interactions of CIKS (151-574) with positive AD/library plasmids were further assessed by yeast two-hybrid analysis. Finally, the DNA sequences of the positive AD/library plasmids were determined and BLAST analysis against the databases was performed. The BLAST results indicate that the inserts in the positive plasmids encode RIKEN cDNA 473340F03, PLAC8, CD27BP (Siva-1), CDC5L, SnRNP smB, and DVL2. CDC5L is a key component of the multi-protein complex essential for the formation of pre-mRNA splicing product and is not required for spliceosome assembly. A role for CDC5L in the cell division cycle has been precious suggested as its overexpression of this protein in mammalian cells leads to a shortening G2 phase of the cell cycle, and a negative-dominant mutant of CDC5L lacking the N-terminal activation domain delays the G2 phase cell's entry into the mitosis. It has been reported that SnRNP smB participates in pre-mRNA splicing and CD27BP (Siva-1) binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis and regulates T cell homeostasis. The functions of RIKEN cDNA 473340F03, PLAC8 and DVL2 are unknown. It has been suggested that CIKS functions as a critical component for cross-talk between NF-kappaB and JNK signaling pathways. IKK subunits, which have been demonstrated to interact with CIKS, were not shown up in this experiment. We speculate that the truncated CIKS (151-574) bait may not contain the binding domain that mediates the interaction of IKK subunits with CIKS. Taken together, the above results suggest that CIKS may play a role in cell regulation through interacting with various cellular proteins. Further investigations are required to characterize these interactions.