Lambda Family Research Articles

Reduced IFNL1 and/or IFNL2, but not IFNL3 is associated with worse outcome in patients with COVID-19.

The recent pandemic was caused by the emergence of a new human pathogen, SARS-CoV-2. While the rapid development of many vaccines provided an end to the immediate crisis, there remains an urgent need to understand more about this new virus and what constitutes a beneficial immune response in terms of successful resolution of infection. Indeed, this is key for development of vaccines that provide long lasting protective immunity. The interferon lambda (IFNL) family of cytokines are produced early in response to infection and are generally considered anti-viral and beneficial. However, data regarding production of IFNL cytokines in COVID-19 patients is highly variable, and generally from underpowered studies. In this study, we measured all three IFNL1, IFNL2 and IFNL3 cytokines in plasma from a well characterised, large COVID-19 cohort (n=399) that included good representation from patients with a more indolent disease progression, and hence a beneficial immune response. While all three cytokines were produced, they differed in both the frequency of expression in patients, and the levels produced. IFNL3 was produced in almost all patients but neither protein level nor IFNL3/IFNL4 SNPs were associated with clinical outcome. In contrast, both IFNL1 and IFNL2 levels were significantly lower, or absent, in plasma of patients that had a more severe disease outcome. These data are consistent with the concept that early IFNL1 and IFNL2 cytokine production is protective against SARS-CoV-2 infection.

Cardiotoxicity in light chain amyloidosis: Insights from the first murine model with intramyocardial injection of patient-derived amyloidogenic light chains

Abstract Background Light chain amyloidosis (AL) is a plasma cell dyscrasia in which cardiac involvement determines the prognosis. Cardiotoxicity of light chains (LCs) complicates the management of heart failure in AL and has not been addressed in the clinical praxis. We have previously genetically identified and biotechnologically produced full-length LCs from patients with AL and cardiac involvement and isotype LCs (WT) from healthy volunteers. We characterized the LCs’ toxicity on primary adult ventricular murine cardiomyocytes (CMs) and our investigation led to the selection of three cardiotoxic and amyloidogenic patient-derived LCs (one from the kappa and two from the lambda family) for further characterization. Purpose We aimed to 1) generate a murine model that recapitulates LCs’ cardiotoxicity in vivo, 2) characterize the mechanism of cardiotoxicity and 3) identify cardioprotective therapies. Methods C57BL6 mice were randomized into 6 groups as follows: 1) PBS (vehicle), 2) WT_kappa, 3) AL_kappa, 4) WT_lambda, 5) AL_lambda1 and 6) AL_lambda2. The animals received the LCs (1 mg/mouse) or vehicle via intramyocardial injection (IMI). Echocardiography evaluation and blood sampling was performed weekly. The animals were sacrificed on the 4th week post IMI for histology, electron microscopy and investigation of the cardiotoxicity mechanism. Results On the 4th week, the AL-lambda LCs significantly reduced the fractional shortening and ejection fraction compared to the vehicle and isotype control groups. The AL_lambda2 also increased mortality of the mice. The AL-kappa did not alter the systolic function of the mice. The circulating lactate dehydrogenase was significantly increased on the 2nd and 4th week in the AL-LCs treated mice indicative of the LCs’ toxicity. The IMI of AL_kappa led to signs of apoptosis including the shrinkage of cardiomyocytes and increased condensation of nuclear chromatin to the nuclear membranes. The IMI of AL_lambda LCs led to increased cardiomyocyte size indicative of hypertrophy, interstitial fibrosis, and increased number of autophagosomes. None of the animals bared amyloid deposits in the heart. The AL-LCs induced different molecular responses in vivo and confirmed the histology findings. The AL-kappa increased apoptosis via the CHOP-Bax pathway, and the ΑL-lambda increased Beclin-1 mediated autophagy without inducing apoptosis. We identified that the common upstream mediator of AL-LCs cardiotoxicity in vivo is the overexpression of the endoplasmic reticulum stress (ERS) markers, IRE-1a and Bip. In vitro, we confirmed that the AL-LCs induce cell death in CMs through ERS. Treatment with the IRE-1a inhibitor STF-083010 and the molecular chaperone tauroursodeoxycholic acid restored the LCs’ induced cardiotoxicity. Conclusions The IMI of AL-LCs stands as a method for the investigation of the LCs cardiotoxicity in vivo in the absence of amyloid deposits via which we identified ERS as a target to alleviate AL-LCs toxicity.

Special features of interferonlambda contents in blood serum of the patients with HIV infection

Studies in HIV infection remain an important issue for modern medicine, which, becomes controlled due to widespread usage of antiretroviral therapy. At the same time, however, it cannot be cured completely, and there is a number of “white spots” in understanding immunopathogenesis of disorders complicating this disease. The in-depth studies of interferon system, in particular from the lambda family, are desirable, because of their antiviral activity in HIV-infected patients. The aim of our study was to evaluate the content of interferons-lambda: IFNλ1 (IL-29) and IFNλ3 (IL-28B) in patients with HIV infection.Blood serum of 120 patients with HIV infection (average age 49.7±6.2 years) who were treated in the outpatient setting at the Center for AIDS Prevention and Control of Regional Clinical Hospital No. 2 in Vladivostok was subjected to laboratory testing. HIV infection 4A was the main clinical diagnosis in all the patients, i.e., the stage of secondary diseases, remission phase on the background of antiretroviral therapy (ARVT). In 52 patients, chronic viral hepatitis C was found as a concomitant disease. The content of IFNλ1 (IL-29) and IFNλ3 (IL-28B) in venous blood serum was determined by ELISA technique using a “Multiscan” analyzer. The ELISA reagents were from RD systems, catalog numbers DY5259; DY7246.In the groups of patients with HIV infection, both with and without viral hepatitis C, the levels of IFNλ1 (IL-29) and IFNλ3 (IL-28B) were significantly reduced in comparison with control group. When comparing the IFN values between the groups, a more pronounced decrease in IFNλ1 (IL-29) was revealed among the patients with viral hepatitis C. When analyzing the level of IFNλ3 (IL-28B), an opposite pattern was observed, i.e., its values in the patients with HIV infection and viral hepatitis C were higher than in the group without hepatitis, but still did not reach appropriate values of the control group. Based on the data on IFNλ3 (IL-28B) antiviral effect upon HIV transmission via macrophages One may assume that induction and maintenance of higher type 3 interferon levels can favorably affect the course of HIV infection. The registered changes in IFNλ1 (IL-29) and IFNλ3 (IL-28B) levels in the patients with HIV and HCV suggest some viral effect upon innate immunity characterized by multidirectional changes, depending on presence or absence of hepatitis C virus. The studies of changes in innate immunity and role of type 3 interferons may extend our knowledge on the interaction between the human body and HIV, and will promote preventive measures and rehabilitation in the patients with HIV infection.

A concise review of the red macroalgae Chondracanthus teedei (Mertens ex Roth) Kützing and Chondracanthus teedei var. lusitanicus (J.E. De Mesquita Rodrigues) Bárbara & Cremades

Chondracanthus teedei is a small red alga, whose fronds are cartilaginous membranous, dark crimson to red black, turning yellow-green due to decay. The main frond axes, due to their broad branches, reach 1 cm in the oldest portions in the case of the lusitanicus variety. Originally described in Portugal, C. teedei is found widespread in the Atlantic, Mediterranean, Black Sea and Indian Ocean. This cosmopolitan species is typical of lower intertidal and shallow subtidal habitats, of sciophilous habitats in semi-exposed or protected areas, and tolerates the presence of mud and sand. Its composition may vary according to the geographical location of origin, and the time of year when it is collected. It has relatively high levels of protein (14.66%), ash (28.68%), fibres (2.21%), lipids (1.82%) and moisture (86.73%), making this alga able to be considered for its implementation as food. In addition, C. teedei var. lusitanicus produces hybrid carrageenans belonging to the lambda family (xi-theta hybrid carrageenans), in the case of tetrasporophytes, and kappa-iota-mu-nu hybrid carrageenans, in the case of female gametophytes and non-fruited thalli. The carrageenans extracted from this species have antifungal and antiviral activities, and the dry ground biomass of C. teedei has potential to be used in cosmetic formulations.

Environmental Triggers of lrgA Expression in Streptococcus mutans.

The cidAB and lrgAB operons of Streptococcus mutans encode proteins that are structurally similar to the bacteriophage lambda family of holin-antiholin proteins, which are believed to facilitate cell death in other bacterial species. Although their precise function is not known, cidAB and lrgAB are linked to multiple virulence traits of S. mutans, including oxidative stress tolerance, biofilm formation, and autolysis. Here we investigate the regulation of lrgAB which in S. mutans shows a complex dependence on growth conditions that is not fully understood. By combining single-cell imaging of a fluorescent gene reporter with microfluidic control of the extracellular environment, we identify specific environmental cues that trigger lrgA expression and characterize cell-to-cell heterogeneity in lrgA activity. We find that the very abrupt activation of lrgA at stationary phase is tightly synchronized across the population. This activation is controlled by a small number of inputs that are sensitive to growth phase: extracellular pyruvate, glucose, and molecular oxygen. Activation of lrgA appears to be self-limiting, so that strong expression of lrgA is confined to a short interval of time. lrgA is programmed to switch on briefly at the end of exponential growth, as glucose and molecular oxygen are exhausted and extracellular pyruvate is available. Our findings are consistent with studies of other bacteria showing that homologs of lrgAB participate, with input from lytST, in the reimport of pyruvate for anaerobic fermentative growth.

Characterization of immunoglobulin light chain utilization and variable family diversity in rainbow trout

This study characterizes immunoglobulin light chain (IgL) expression and variable family usage in rainbow trout. IgL transcripts were generated by 5’ RACE from both immune and TNP-KLH immunized fish. Phylogenetic analysis revealed that the IgL variable regions clustered into seven different families: three kappa families (two newly described in this study), three sigma families, and a single lambda family. IgL1 and IgL3 transcripts expressing identical variable regions were identified and genomic analysis revealed that the two isotypes are co-localized on chromosomes 7, 15, 18, and 21 allowing for potential rearrangement between clusters. Fish were immunized with TNP-KLH (n = 5) and percent expression of IgL1, IgL2, IgL3, and IgL4 measured by qRT-PCR from immune tissues and magnetically sorted TNP-specific lymphocyte populations. In all samples IgL1 constituted 80–95% of the transcripts. The percentage of anti-TNP specific IgL1 transcripts was measured in naïve, unsorted, and TNP-specific cell populations of TNP-KLH fish (n = 3) and found to be significantly higher in the TNP positive cell population (21%) compared to the naïve population (1%; p = 0.02) suggesting that there is a selection of TNP specific IgL sequences.

Interferon Lambda Family along with HTLV-1 Proviral Load, Tax, and HBZ Implicated in the Pathogenesis of Myelopathy/Tropical Spastic Paraparesis

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic neuroinflammatory disease related to human T lymphotropic virus type 1 (HTLV-1) infection. Interferon type III (IFN-λ), which includes IL28, IL29, and IL28R, and affects the outcome of viral infections, might be complicated in the progression of HAM/TSP. Here, we investigated the host-virus interactions in the manifestation of HAM/TSP, using IL28B, IL29, IL28R, HTLV-1 Tax, HTLV-1 basic leucine zipper factor (HBZ), and proviral load (PVL). The study groups consisted of 20 patients with HAM/TSP, 20 asymptomatic HTLV-1 carriers (ACs), and 20 healthy controls (HCs). The means of PVL, Tax, and HBZ gene expressions in the HAM/TSP group (p = 0.004, 0.006, and < 0.0001, respectively) were significantly higher than in the AC group. The comparison of IL28B, IL29, and IL28R expression in the HAM/TSP, AC, and HC groups revealed no significant difference between the first 2, but lower concentrations in the HCs (IL28B: p = 0.03, 0.01; IL29: p = 0.07, 0.01; and IL28R: p < 0.0001, respectively). In the HAM/TSP group, correlations were seen between Tax and HBZ (R = 0.61, p = 0.004) and between Tax and IL29 (R = 0.45, p = 0.04). Negative correlations were observed between Tax and IL28B (R = –0.49, p = 0.02) and between HBZ and IL28R (R = –0.43, p = 0.06). In the ACs, an inverse correlation was found between Tax and IL28B (R = –0.42, p = 0.06). These findings suggest that IL29, IL28B, and IL28R interfere in the infection of HAM/TSP, mainly via Tax activation.

Antibody-Based Protective Immunity against Helminth Infections: Antibody Phage Display Derived Antibodies against BmR1 Antigen.

Helminth parasite infections are significantly impacting global health, with more than two billion infections worldwide with a high morbidity rate. The complex life cycle of the nematodes has made host immune response studies against these parasites extremely difficult. In this study, we utilized two phage antibody libraries; the immune and naïve library were used to identify single chain fragment variable (scFv) clones against a specific filarial antigen (BmR1). The V-gene analysis of isolated scFv clones will help shed light on preferential VDJ gene segment usage against the filarial BmR1 antigen in healthy and infected states. The immune library showed the usage of both lambda and kappa light chains. However, the naïve library showed preferential use of the lambda family with different amino acid distributions. The binding characteristics of the scFv clones identified from this work were analyzed by immunoassay and immunoaffinity pull down of BmR1. The work highlights the antibody gene usage pattern of a naïve and immune antibody library against the same antigen as well as the robust nature of the enriched antibodies for downstream applications.

In Silico Signature Prediction Modeling in Cytolethal Distending Toxin-Producing Escherichia coli Strains.

In this study, cytolethal distending toxin (CDT) producer isolates genome were compared with genome of pathogenic and commensal Escherichia coli strains. Conserved genomic signatures among different types of CDT producer E. coli strains were assessed. It was shown that they could be used as biomarkers for research purposes and clinical diagnosis by polymerase chain reaction, or in vaccine development. cdt genes and several other genetic biomarkers were identified as signature sequences in CDT producer strains. The identified signatures include several individual phage proteins (holins, nucleases, and terminases, and transferases) and multiple members of different protein families (the lambda family, phage-integrase family, phage-tail tape protein family, putative membrane proteins, regulatory proteins, restriction-modification system proteins, tail fiber-assembly proteins, base plate-assembly proteins, and other prophage tail-related proteins). In this study, a sporadic phylogenic pattern was demonstrated in the CDT-producing strains. In conclusion, conserved signature proteins in a wide range of pathogenic bacterial strains can potentially be used in modern vaccine-design strategies.

Association of Serum Interleukin-28B with Clinical Features, Laboratory Values and Radiographic Score in Rheumatoid Arthritis Patients.

Interleukin-28B (IL-28B) is a member of the interferon lambda family (also known as type III interferons), it has already been studied in many diseases, but it has almost never been studied in rheumatoid arthritis (RA). With this background, we determined the serum levels of IL-28B in RA patients and investigated its clinical significances. IL-28B levels were measured by enzyme-linked immunosorbent assay (ELISA) in 80 RA patients and 80 healthy controls. Radiographs were scored for total Sharp score (TSS). Serum IL-28B levels were significantly lower in the RA than in the control (p = 0.02). Anticyclic citrullinated peptide (anti-CCP) antibodies and rheumatoid factor (RF) were negatively correlated with serum IL-28B levels. There was no association between serum IL-28B levels and TSS at baseline, but there was a significant difference in change of IL-28B during 6 months follow up between progressors and non-progressors. The disease activity of RA patients decreased, but no difference was observed in serum IL-28B levels between before and after disease-modifying anti-rheumatic drugs (DMARDs) therapy. These findings indicated a role of IL-28B in RA and it may contribute to avoiding osteoclasia in RA patients.

A Winsorized Adaptive Rank Test for Location when Sampling from Asymmetric Distributions

&lt;p&gt;Se propone una prueba de rangos adaptativa winsorizada para la alternativa de localización en distribuciones asimétricas que provienen de la Familia Lambda Generalizada. Se da un método para calcular la distribución exacta y expresiones analíticas para la distribución asintótica, y los dos primeros momentos de la estadística de prueba. Por medio de un estudio de Monte Carlo, se muestran que para varias selecciones de parámetros de winsorización la prueba propuesta es más potente que la prueba del signo y que la prueba original para selecciones apropiadas del parámetro de winsorización, de la cual la prueba propuesta fue adaptada. © 2016. Acad. Colomb. Cienc. Ex. Fis. Nat.&lt;/p&gt;

Modeling censored data using modified lambda family

ABSTRACTIn the present article we propose the modified lambda family (MLF) which is the Freimer, Mudholkar, Kollia, and Lin (FMKL) parametrization of generalized lambda distribution (GLD) as a model for censored data. The expressions for probability weighted moments of MLF are derived and used to estimate the parameters of the distribution. We modified the estimation technique using probability weighted moments. It is shown that the distribution provides reasonable fit to a real censored data.

Sequence and structural models of a bovine monoclonal antibody Fv region specific for bovine herpesvirus 1 glycoprotein D (VET2P.1037)

Abstract The cDNA of heterohybridoma mRNA encoding the variable domains of the light (VL) and heavy (VH) chains of an IgG1 bovine monoclonal antibody (MAb) neutralizing bovine herpesvirus 1 was commercially synthesized, cloned and sequenced. The variable (V) light chain sequence had highest identity with members of the V lambda family, 1a subfamily, and the joining (J) gene JL3, both commonly expressed in cattle. The V heavy chain sequence had highest identity with members of the VH1 gene family and JH1, also both commonly expressed in cattle. The VH nucleotide sequence had highest identity with the bovine diversity (D) gene DH3. The derived amino acid sequences were used to examine the complementarity-determining regions (CDRs) according to multiple systems. CDRL1-3 and CDRH1-2 were identified as belonging to or being similar to known canonical classes, and results from “H3 rules” were determined for CDRH3. Models of the Fv structure were derived using three on-line software packages. Overlapping sets of rodent and human MAb were used by the packages as templates for each framework and CDR structure individually. Predictions of the CDRL3 and CDRH3 configurations were more variable than that of other CDRs, and resulted in different paratope models. The results suggest sequences and crystal structures of MAb from a variety of species may be useful in describing additional canonical structures of immunoglobulins.

IFN-λ4: the paradoxical new member of the interferon lambda family.

Interferons (IFNs) are generally considered antiviral cytokines, yet the newly discovered IFN-λ4 is linked with the failure to clear hepatitis C virus (HCV) infection either spontaneously or in response to treatment. IFN-λ4 can be generated only by individuals who carry the IFNL4-ΔG allele (rs368234815), which is the strongest known host factor for predicting clearance of HCV. The ancestral IFNL4-ΔG allele is the major variant in Africans while the minor variant in Asians, suggesting very strong negative genetic selection for this allele-most likely driven by an infectious agent other than HCV. IFN-λ4 most closely resembles IFN-λ3, but these proteins share only 29% amino-acid identity, and, in contrast to IFN-λ3, IFN-λ4 is only weakly secreted. Nevertheless, IFN-λ4 signals through the IFN-λ receptor complex and induces expression of IFN-stimulated genes via the Janus kinase-signal transducer and activator of transcription signaling pathway. Although the IFNL4-ΔG variant is strongly associated with the failure to clear HCV infection, HCV-infected patients who carry this allele have lower baseline HCV RNA levels in the absence of treatment. Resolving the paradoxical functions of IFN-λ4, which appears to induce antiviral activity yet impair effective clearance of HCV, may yield critical new insights into the immunologic response to HCV infection and IFN biology.

A modified runs test for symmetry

We propose a modification of a Modarres–Gastwirth test for the hypothesis of symmetry about a known center. By means of a Monte Carlo Study we show that the modified test overtakes the original Modarres–Gastwirth test for a wide spectrum of asymmetrical alternatives coming from the lambda family and for all assayed sample sizes. We also show that our test is the best runs test among the runs tests we have compared.

The Regulation, Targets and Clinical Relevance of Microrna Mir-650 in Chronic Lymphocytic Leukemia (CLL),

Abstract 3874It is known that the biology of CLL and other B-cell malignancies is driven by processes dependent on immunoglobulin structure and BCR-signaling. Recently, our group and others have described the importance of miRNAs in CLL and their involvement in BCR-signaling, IgG production and V(D)J recombination. Considering the important functions of miRNAs it is remarkable that the human locus for immunoglobulin lambda light chain (IgL) contains a miR gene. The miR-650 gene is localized in exon 1 of IgL variable subgene (1st exon of V2 family members). The aim of this study was to reveal the regulation and expression of miR-650 in CLL and its relation to disease prognosis.CLL samples were separated by RosetteSep Human B Cell Enrichment Cocktail (obtained purity ≥95% of CD5+19+ cells). The expression of light chain surface immunoglobulin chain (lambda vs kappa) was determined by flow cytometry. The utilized IgL variable (V) segment was determined using BIOMED-2 protocol and sequencing. To study the relation of miR-650 expression and IgL rearrangement the surface expression of Ig light chain and the utilized V segment was determined in a CLL cohort containing 47 patients (λ n=27, κ n=19, λ+κ n=1). The gene expression was analyzed by TaqMan Assays (ABI) for mature miR-650 and protein-coding genes: CDK1, EBF3 and ING4. The western-blot for miR-650 targets was performed after transfection (48hrs/96hrs) of B-cell lines (NALM-6, MEC-1) with a short RNA mimicking miR-650 (Dharmacon).The analyses of miR-650 expression revealed that cells utilizing V2-8, V2-5, V2-14, V2-23 subgenes for IgL (n=14) had ∼10 fold higher expression of miR-650 (p<0.005) when compared to samples utilizing different V lambda family (n=13) or expressing kappa Ig (n=20). This suggests a unique mechanism for coordinate expression of miR-650 and immunoglobulin light chain (for IgL utilizing the V2 family members). This observation is partially surprising because miR-650 was originally identified in colorectal and breast cells and it was believed to be regulated independently of immunoglobulin genes. Our data demonstrate that miR-650 expression is likely regulated at least partially by immunoglobulin light chain promoter.We next studied the possible targets for miR-650 in B-cells. Recently, two targets were identified in solid tumors - ING4 (Inhibitor of Growth 4) and CDK1 (cyclin dependent kinase 1) (Zhang, 2010; Chien, 2010). It has been demonstrated that miR-650 is involved in the p16INK4-mediated pathway and directly regulates the CDK1. This publication suggested that up-regulation of miR-650 leads to inhibition of cell cycle progression. Moreover, the putative targets predicted by software tools (TargetScan, miRanda) include genes important for B-cell biology like EBF3 (early B-cell factor3), CLLU1, Bcl2 and cyclin D1. We therefore studied correlation between miR-650 expression and the expression of mRNAs for CDK1, ING4 and EBF3 (a predicted target with the highest score). The expression of miR-650 was not significantly associated with the expression of any of these genes on mRNA level. The lack of available material did not allow us to study the expression of CDK1, ING4, EBF3 protein levels in the original cohort. However, the transfection of B-cells with short RNA mimicking miR-650 led to down-regulation of protein levels of CDK1 and EBF3. This confirms the relevance of CDK1 as a target in B-cells and identifies a new target - EBF3, which is known to be important for B-cells development. Moreover, the expression of miR-650 was associated with overall survival (OS) and treatment free survival (TFS) in CLL (n=82). In this analysis patients were divided in two groups (based on the median of miR-650 expression). The higher expression of miR-650 was associated with statistically significant (p<0.05) longer OS (not-reached vs. 161 months) and TFS (60 vs. 34 months). This is in line with the observation that miR-650 inhibits CDK1 and cell cycle progression.In conclusion, we have described a mechanism regulating miR-650 expression, identified its relevant targets in B-cells and demonstrated the association of miR-650 expression with CLL prognosis.Supported by IGA MZCR NT11218-6/2010, MSMTMSM0021622430, NS10439-3/2009, FR-TI2/254 Disclosures:Mayer:Roche: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Astellas: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Novartis: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Fresenius Medical Care: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Pfizer: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Genzyme: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; GSK: Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Amgen:.

Preliminary Characterization of Carrageenan from the Red Seaweed Halymenia floresii

Increasing worldwide demand and development of new applications of carrageenan have added urgency to the search for new or additional raw material sources. Native and alkali-treated carrageenan from Halymenia floresii from Yucatán was extracted and preliminarily characterized. Results showed that this species contains a highly sulfated carrageenan, with minor amounts of 3,6-anhydro-galactose. Native sulfated galactan seems to be rheologically and chemically similar to the lambda-family carrageenan with appropriate thickening ability for food applications. Further studies are needed to precisely define the structure of these complex polymers and its potential use as a source of industrial lambda family.

Le Juvénal des Carolingiens à la lumière du Ms Cambridge King’s College 52

Since the nineteenth century, Heiric ‘of Auxerre’, as he is usually called, has been credited with a commentary on Juvenal’s Satires attributed in some late manuscripts to a Cornutus. No earlier manuscript evidence has ever been found for Heiric’s authorship. The manuscript that is now Cambridge King’s College 52, privately owned until 1954 and ignored by all editors of Juvenal, furnishes the oldest testimony for the Nicaean subscription and contains one of the richest sets of glosses in the ninth-century manuscripts. On paleographical grounds it can be considered a product of the scriptorium of Hincmar of Rheims, and its text belongs to Knoche’s lambda family, held to go back to Antiquity. Its marginal commentary, copied along with the text, contains, in condensed form, most of the information found in the vulgate ‘Cornutus commentary’. Some of the glosses, added later, can be attributed to the hand of Heiric. Most of this material also appears in the vulgate commentary. The King’s manuscript makes it possible to recognize the Carolingian contribution to what is mostly a late-antique commentary. Thus, it advances our understanding of the Carolingian approach to Roman satire, which in fact seems quite limited and largely concerned with prosody.

Detail Characterization of 22q11 Deletion and Mir-650 Role in CLL Patients.

Abstract 1231Poster Board I-253 BackgroundChromosomal abnormalities are present in the majority of CLL cases (>80%) and recently, a novel deletion of 22q11 was described in CLL patients. This locus encodes lambda immunoglobulin light chain (IgL) subgenes and several protein-coding genes, which distinguishes IgL from kappa Ig or heavy Ig locus. Gunn et al. (2008) previously characterized the minimally deleted region of 22q11 in CLL as 0,34Mb (containing PRAME, ZNF280A, ZNF280B, GGTLC2) and described the PRAME as a candidate gene with prognostic impact in CLL. Interestingly, the IgL variable segment (V2-8) located at 22q11 includes an annotated miR gene miR-650. According to the performed BLAST search also other subgenes of V2 family include homologues for miR-650, which overlay the leader exon of the V2 subgenes. This could be of particular importance because the expression of miRNAs was previously shown to be associated with known prognostic markers in CLL: 13q deletion, IgVH status (Calin, 2002, 2005) and p53 deletion/mutation (Mraz, 2009). Moreover, miRNA pathways are involved in the regulation of immune system functions including IgG production and V(D)J recombination (Vigorito, 2007; Koralov, 2008). AimWe analyzed genomes of 40 CLL patients using array-CGH (CGH Microarray 4×44 K, Agilent) and FISH (del13q14, del17p13, del11q23, +12), and in particular focused on detailed characterization of 22q11 deletion. An expression analyses (RT-qPCR, TaqMan miRNA Assay, ABI) was performed for protein-coding genes and miR-650 located at 22q11. ResultsUsing array-CGH additional aberrations to FISH were found in the majority of samples and among them, the deletion of 22q11 locus was observed most often (ranging in size up to ∼ 0,77 Mb). Altogether, 40% of patients (16/40) had detectable deletion in this locus and according to our finding, all observed deletions were a consequence of a VJ rearrangement event, which lead to an elimination of specific genes located between VJ elements. These rearrangements were characterized in detail by PCR detection (BIOMED-2 protocol) and sequencing. In our cohort, deletions of 22q11 containing PRAME gene constituted only ∼44% (n=7) and 56% patients (n=9) harbored smaller deletions not involving PRAME. To analyze the gene expression, we performed a Real-Time PCR analyses for previously mentioned protein-coding genes and miR-650. The Real-Time PCR assay for miR-650 reported higher expression of miR-650 in CLL cells utilizing V2-8, V2-5, V2-14, V2-23 subgenes for IgL (n=5) compared to samples utilizing different V lambda family or expressing kappa Ig (n=36). It remains to be elucidated how and whether all the V2 subgenes give rise to a mature and functional miRNA. The overlap between miR-650 and the leader exon of IgL gene could provide a unique mechanism for coordinated IgL and miRNA expression. To assess the function of miR-650 we performed a microarray (Human OneArray, Phalanx) expression profiling 24/48 hours post transfection of CLL cells with a short RNA mimicing miR-650 (miRIDIAN Mimic, Dharmacon). The transfection led to a significant down-regulation of dozens of mRNAs that could be considered as potential targets for miR-650. Interestingly, the predicted targets for miR-650 include early B cell factor (EBF3; target with highest total score – TargetScan) and VPREB1 (pre B lymphocyte gene 1) whose expression is important for immature phenotype of pre-B lymphocytes prior to light chain recombination. The coupled activation of miR-650 and lambda Ig expression following productive rearrangement of IgL locus could potentially affect the expression of VPREB1/EBF3. ConclusionDeletion of 22q11 is frequently detected in CLL patients and we suggest that it is caused by physiological process of lambda immunoglobulin variable locus rearrangement. This leads to the elimination of protein-coding genes located between VJ elements. Interestingly, the V2 family of variable segments for lambda light chain at 22q11 includes an annotated miR gene miR-650 and its homologues. The productive lambda locus rearrangement utilizing V2 family could be also coupled with activation of miR-650 expression with consequent relevance for B cell biology. This work is in progress and in further analyses we expect complex characterization of miR-650 functions in B cells and CLL. Supported by IGA MZ CR NR9293-3/2007 and IGA MZ CR NS10439-3/2009. DisclosuresMayer:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy; Fresenius: Consultancy; Roche: Research Funding; Pfizer: Research Funding.

Comparative ability of IL-12 and IL-28B to regulate Treg populations and enhance adaptive cellular immunity

AbstractImproving the potency of immune responses is paramount among issues concerning vaccines against deadly pathogens. IL-28B belongs to the newly described interferon lambda (IFNλ) family of cytokines, and has not yet been assessed for its potential ability to influence adaptive immune responses or act as a vaccine adjuvant. We compared the ability of plasmid-encoded IL-28B to boost immune responses to a multiclade consensus HIV Gag plasmid during DNA vaccination with that of IL-12. We show here that IL-28B, like IL-12, is capable of robustly enhancing adaptive immunity. Moreover, we describe for the first time how IL-28B reduces regulatory T-cell populations during DNA vaccination, whereas IL-12 increases this cellular subset. We also show that IL-28B, unlike IL-12, is able to increase the percentage of splenic CD8+ T cells in vaccinated animals, and that these cells are more granular and have higher antigen-specific cytolytic degranulation compared with cells taken from animals that received IL-12 as an adjuvant. Lastly, we report that IL-28B can induce 100% protection from mortality after a lethal influenza challenge. These data suggest that IL-28B is a strong candidate for further studies of vaccine or immunotherapy protocols.