Genomic V exons from whole genome shotgun data in reptiles.

Reptiles and mammals diverged over 300 million years ago, creating two parallel evolutionary lineages amongst terrestrial vertebrates. In reptiles, two main evolutionary lines emerged: one gave rise to Squamata, while the other gave rise to Testudines, Crocodylia, and Aves. In this study, we determined the genomic variable (V) exons from whole genome shotgun sequencing (WGS) data in reptiles corresponding to the three main immunoglobulin (IG) loci and the four main T cell receptor (TR) loci. We show that Squamata lack the TRG and TRD genes, and snakes lack the IGKV genes. In representative species of Testudines and Crocodylia, the seven major IG and TR loci are maintained. As in mammals, genes of the IG loci can be grouped into well-defined IMGT clans through a multi-species phylogenetic analysis. We show that the reptilian IGHV and IGLV genes are distributed amongst the established mammalian clans, while their IGKV genes are found within a single clan, nearly exclusive from the mammalian sequences. The reptilian and mammalian TRAV genes cluster into six common evolutionary clades (since IMGT clans have not been defined for TR). In contrast, the reptilian TRBV genes cluster into three clades, which have few mammalian members. In this locus, the V exon sequences from mammals appear to have undergone different evolutionary diversification processes that occurred outside these shared reptilian clans. These sequences can be obtained in a freely available public repository (http://vgenerepertoire.org).

Mode of action of trabectedin in myxoid liposarcomas.

To elucidate the mechanisms behind the high sensitivity of myxoid/round cell liposarcoma (MRCL) to trabectedin and the suggested selectivity for specific subtypes, we have developed and characterized three MRCL xenografts, namely ML017, ML015 and ML004 differing for the break point of the fusion gene FUS-CHOP, respectively of type I, II and III. FUS-CHOP binding to the promoters of some target genes such as Pentraxin 3 or Fibronectin 1, assessed by chromatin immunoprecipitation, was strongly reduced in the tumor 24 h after the first or the third weekly dose of trabectedin, indicating that the drug at therapeutic doses causes a detachment of the FUS-CHOP chimera from its target promoters as previously shown in vitro. Moreover, the higher sensitivity of MRCL types I and II appears to be related to a more prolonged block of the transactivating activity of the fusion protein. Doxorubicin did not affect the binding of FUS-CHOP to target promoters. Histologically, the response to trabectedin in ML017 and ML015 was associated with a marked depletion of non-lipogenic tumoral cells and vascular component, as well as lipidic maturation as confirmed by PPARγ2 expression in western Blot. By contrast, in ML004 no major changes either in the cellularity or in the amount of mature were found, and consistently PPARγ2 was null. In conclusion, the data support the view that the selective mechanism of action of trabectedin in MRCL is specific and related to its ability to cause a functional inactivation of the oncogenic chimera with consequent derepression of the adypocytic differentiation.

Tracking T and B cells from two-photon microscopy imaging using constrained SMC clusters.

This paper describes a novel software algorithm, called constrained Sequential Monte Carlo (SMC) clusters, for tracking a large collection of individual cells from intra-vital two-photon microscopy image sequences. We show how our method and software tool, implemented in python, is useful for quantifying the motility of T and B lymphocytes involved in an immune response vs lymphocytes under non immune conditions. We describe the theory behind our algorithm and briefly discuss the architecture of our software. Finally, we demonstrate both the functionality and utility of software by applying it to two practical examples from videos displaying lymphocyte motility in B cell zones (follicles) and T cell zones of lymph nodes.

L-ficolin binding and lectin pathway activation by acetylated low-density lipoprotein.

L-ficolin, like mannan-binding lectin (MBL), is a lectin pathway activator present in normal human plasma. Upon binding ligand, l-ficolin similarly initiates C4 cleavage via the serine protease MBL-associated serine protease-2 (MASP-2). We sought further insight into l-ficolin binding reactions and MASP-2 activation by passing plasma through GlcNAc-derivatized Sepharose. l-Ficolin bound in 1.0 M NaCl-ethylenediamine tetraacetic acid (EDTA), and remained bound in NaCl-free EDTA, while MASP-2 eluted in proenzyme form ( approximately 20% yield, > 40 000-fold purification). L-Ficolin was eluted with GlcNAc in 1.0 M NaCl ( approximately 10% yield, > 3000-fold purification), with trace amounts of C3, alpha(2)-macroglobulin and both native and activated MASP-2. These preparations were utilized to investigate l-ficolin reactivities with acetylated low-density lipoprotein (A-LDL) as a model ligand in albumin-free systems. L-Ficolin bound strongly to A-LDL in the absence as well as presence of calcium, including saline-EDTA, and was optimal in 1.0 M NaCl-EDTA, but binding failed to occur in EDTA in the absence of NaCl. The addition of l-ficolin to immobilized A-LDL resulted in activation of MASP-2 in unmodified but not ficolin-depleted plasma unless l-ficolin was restored. We conclude that A-LDL is a useful ligand for investigation of l-ficolin function; both binding and activation are optimally examined in systems free of albumin; and ligand binding in 1.0 M NaCl in EDTA can be useful in the isolation of l-ficolin and native MASP-2.

Defense factors of vaginal lactobacilli.

OBJECTIVE: To determine the antagonistic relationship between vaginal lactobacilli and endogenous vaginal microflora. STUDY DESIGN: Twenty-two Lactobacillus strains were studied for the production of lactic acid, hydrogen peroxide, and bacteriocin. RESULTS: Under standardized growth conditions, most strains increased their biomass by more than 4 times. Lactobacillus species grew best at a pH > or = 4.5, and growth was retarded at a pH < 4.5. Lactic acid levels were 0.68 to 2.518 mg/mL and were not related to the number of cells or the pH of media. The pH of the media was caused by the secretion of lactic and other organic acids. Approximately 80% of the strains produced H(2)O(2) and were graded as 2+ in one third of the strains and 1+ in others. No statistical correlation was found between H(2)O(2) lactic acid and bacteriocin production. Bacteriocin activity was tested on 4 strains of Gardnerella vaginalis. Approximately 80% of the lactobacilli tested produced bacteriocin that inhibited growth of G vaginalis. Six of the strains did not produce bacteriocin. Thirteen strains produced all 3 defense factors, whereas the others lacked 1 or 2 properties. CONCLUSIONS: Lactobacillus species grow best at a pH > 4.5. The pH of the media is dependent on the cell mass and on all organic acids produced by Lactobacillus species. Although all species produce organic acids, not all produce H(2)O(2) and bacteriocin. Not all strains of G vaginalis can be inhibited by lactobacilli-producing bacteriocin.

Effect of ecteinascidin-743 on the interaction between DNA binding proteins and DNA.

Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, a tunicate growing in mangrove roots in Caribbean. It has been shown to bind in the minor groove of DNA forming covalent adducts by reaction of the N2 of guanine with the carbinolamine moiety. We investigated ET-743 ability to inhibit the binding of different transcription factors to their consensus sequences by using gel shift assays. We have selected three types of factors: (i) oncogene products such as MYC, c-MYB and Maf; (ii) transcriptional activators regulated during the cell cycle as E2F and SRF; and (iii) general transcription factors such as TATA binding protein (TBP), Sp1 and NF-Y. We observed no inhibition of the binding of Sp1, Maf, MYB and MYC. Inhibition of DNA binding was observed for TBP, E2F, SRF at ET-743 concentrations ranging from 50 to 300 microM. The inhibition of binding of NF-Y occurs at even lower concentrations (i.e. 10-30 microM) when the recombinant subunits of NF-Y are preincubated with the drug, indicating that the inhibition of NF-Y binding does not require previous ET-743 DNA binding. Since NF-Y is a trimer containing two subunits with high resemblance to histones H2B and H2A, we have investigated the effect of ET-743 on nucleosome reconstitution. ET-743 caused a decrease of the nucleosomal band at 100 nM, with the complete disappearance of the band at 3-10 microM. These data suggest that the mode of action of this novel anticancer drug is related to its ability to modify the interaction between some DNA binding proteins and DNA.

Further studies on the problem of immune network modelling.

In a previous work we have analysed a family of antibody and B-cell network models (basic AB models) of the immune system. This analysis focused principally on the physiological interpretation of their parameters. Our approach consisted in building a detailed and general mathematical model (referred to as the GIB model) and then simplifying it formally to a version (named the RIB model) that belongs to the family of AB models, but which is more general than the basic AB models. From that study it was clear that some of the assumptions necessary to simplify the GIB model into the RIB one, as well as to recover the basic AB models from the RIB one, are quite unrealistic from a physiological point of view. All this raised the issue of the reliability, or even the heuristic value, of theoretical studies based on current network models for experimental immunologists. One approach to clarify this issue is to ask whether the unrealism of the assumptions implicit in the RIB and AB models entails qualitatively different behaviours between them compared to the GIB one. We initiate here such a work by performing a comparative study of a two-clone system of the AB and RIB models, and a variant of the GIB model in which the different molecular compartments were merged into a single one (labelled IGB model). Because all those models rely critically on certain B-cell activation functions, which constitute the core of an implicit model of individual B-cell reactivity or "local rules", we focused the present numerical study, to a great extent, on two parameters determining those activation functions (Hill coefficient and thresholds). Our results indicate that: (1) the RIB and IGB models display in general a much larger diversity of steady states than the AB models; (2) only under a very restricted parameter regime did all studied models behave similarly; (3) the parameter regime under which the AB and IGB models, but not the RIB one, behave similarly is still rather restricted through not as much as in (2); and (4) even relatively small quantitative changes (within reasonable values) in the postulated "local rules" can induce very large quantitative changes in the behaviour of the AB and RIB models but not the IGB model. In the light of the present results, we discuss the need of postulating a set of "local rules" solidly based on experimental evidence as a necessary condition for the reliability of current network models.

A model of the immune network with B-T cell co-operation. I--Prototypical structures and dynamics.

Hitherto, "second generation" network models of the immune system have all been restricted to B-lymphocytes and the Ig molecules they produce. These models have not so far been able to provide a convincing mechanism for the distinction between a "Central Immune System" (CIS) composed of a connected network of lymphocyte clones which couple with "self" antigens in a tolerant mode, and a "Peripheral Immune System" (PIS) composed of clones with little or no supra-clonal organization and which produce classical immune responses when interacting with "non-self" antigens. Here, we present a new network model which explicitly incorporates B-T cell co-operation. In this model, B-cell activation is dependent on T-cell help, and activated T-cells are down-regulated by engagement of their TCRs by soluble Ig. We discuss the underlying biology on which we base the system of ordinary differential equations which defines the present network model. We then illustrate some basic features of the model by examining several prototypical situations with a small number of clones. Depending on the idiotypic connectivity structure, the model exhibits two distinct modes of coupling with antigens: an "immune response" mode in which T- and B-cell clones grow exponentially; and a "tolerant" mode in which T-cell clones are controlled by inclusion of all TCRs in the repertoire of an idiotypic B-cell network. Finally, we discuss the simplifying assumptions of the present model and argue that its range of validity is indeed the region of the state-space of the system where the discrimination between the CIS and the PIS take place.

Evidence for recovery of body mass and haptoglobin values of river otters following the Exxon Valdez oil spill.

Levels of blood haptoglobin (Hp) and interleukin-6 immunoreactive protein (IL-6 ir) were significantly elevated in river otters (Lutra canadensis) inhabiting oiled areas of Prince William Sound, Alaska (USA) following the Exxon Valdez oil spill in 1989. By May and June 1992, however, such differences were not apparent. Mean body mass of otters, adjusted for sex, age-class, and total length with analysis of covariance, differed between oiled and non-oiled areas from 1990 to 1992, but were nearly identical by May and June 1992. We propose that river otters may be recovering from chronic effects that we observed in 1990 and 1991 following the 1989 Exxon Valdez oil spill, but further research is necessary to test this hypothesis.

Numerical analysis of a model of ligand-induced B-cell antigen-receptor clustering. Implications for simple models of B-cell activation in an immune network.

B-cell activation driven by ligand-induced crosslinking of membrane immunoglobulin (mIg) is one of the most important processes in experimental and theoretical immunology. Although the activation of B cells through mIgs involves a complex series of intracellular processes, in immune network models it is usually assumed that there is a correlation between the degree of mIg crosslinking and the probability of B-cell activation. We explore the implications of this hypothesis by studying a model of ligand-induced B-cell receptor clustering proposed by Bell and further elaborated by Delisi and Perelson (BDP model). In this model, a critical time (tc) is defined at which the probability of infinite size complex formation (i.e., percolation) becomes non-zero. We use this variable, tc, as a means to characterize the degree of mIg crosslinking. To study the dependence of tc with respect to ligand valence, kinetic constants and ligand-receptor affinity x ligand concentration (K x C), we perform a systematic numerical study of the BDP model for parameter ranges including current empirical estimates for the kinetic constants. Concerning tc, we find that, for ranges of immunological interest (namely, those including current estimates of dissociation and receptor crosslinking rate constants), the curves obtained by plotting 1/tc vs. log(K x C) shift sensibly towards higher values of log(K x C), broadening and increasing its maximum amplitude, as the dissociation rate constant increases. As this finding suggests important consequences for immune network models, we further study the BDP model in an extended version for the case of two different ligands interacting simultaneously with a given B cell.3+

Crosslinking of membrane immunoglobulins and B-cell activation: a simple model based on percolation theory.

In immune network models it is assumed that membrane immunoglobulin (mIg) crosslinking leads to B-cell activation. To analyse further the implications of this idea, a model of B-cell activation by ligand-induced mIg crosslinking in the absence of cell-to-cell interactions is proposed. The present model, based on a simple crosslinking mechanism previously proposed by other authors, assumes that activation of B-cells is possible once crosslinks of mIgs percolate and that percolation of crosslinks can only happen within a relatively short time tau. Given a lattice (regular or not), a molecular cluster is said to percolate or to become a percolating cluster if it spans the whole lattice (this is the case, for instance, of a polymer in a gel phase). From this model of B-cell activation we define the activation function fa (LK) as the fraction of B-cells activated after tau minutes of interaction with a ligand at concentration L and with affinity K. Numerical calculations show that, for current estimates of kinetic constants involved in the interaction of a given ligand with a B-cell clone, the activation function fa shifts when k-, the dissociation rate constant, is varied below 10(-3) s-1, this shift being linearly proportional to the variation of k-. This result contradicts and, therefore, challenges the assumption in immune network models that the activation function is identical for all ligands. This is important because the behaviour of at least some of those immune network models is quite sensitive to the relative values of the activation function thresholds.

Studies on a recent class of network models of the immune system.

It is argued that the realism of computer simulations of network models of the immune system depends basically on the coherence of these models with the essentials of the known physiology of the cells and molecules selected to be modelled and on the incorporation in them of the different compartments of activated B cells. Focusing on these two aspects, here we analyse the simplifications and assumptions that go implicit in the formulation of a recently developed new class of network models that distinguish between immunoglobulins and B cells. This is approached by first building a general model which incorporates explicitly the kinetics of different B-cell compartments as well as a splenic compartment and a peripheric one for immunoglobulins, and then formally studying the simplifications on this model that are necessary to recover the initial simpler models. Following this procedure, it is shown that the effective coefficients of the different rate terms in the simpler models are particular combinations of the elementary rates obtained empirically. These relations reflect the particular assumptions associated with each simplification step. Also, it is shown that the usual biological interpretation of some of the coefficients in the ordinary differential equations of the simpler models is inconsistent with the more exact general model, unless one makes certain unreasonable assumptions about B-cell physiology. The relevance of this approach in providing variables with a biologically identifiable reality and for realistic, testable, computer simulations is discussed.

Differences in blood haptoglobin and length-mass relationships in river otters (Lutra canadensis) from oiled and nonoiled areas of Prince William Sound, Alaska.

Significant differences in levels of blood haptoglobin occurred between river otters (Lutra canadensis) inhabiting oiled (mean = 361 mg/100 ml, SD = 38, n = 6) and nonoiled (mean = 306 mg/100 ml, SD = 87, n = 8) areas of Prince William Sound, Alaska (USA) following the Exxon Valdez oil spill in 1989. Additionally, male river otters from oiled areas had significantly lower body mass (1.13 kg) than male otters from nonoiled areas. We propose oil-related causes for these differences.

Inside the thymus, Mls antigen is exclusively presented by B lymphocytes.

The ability to stimulate an Mls-1 mixed lymphocyte reaction (MLR) is predominantly expressed by low density B lymphocytes in the spleen and peritoneal cavity of normal adult mice, and is absent in splenic B cells 1 month after lethal irradiation and reconstitution from autologous bone marrow. Coreconstitution of these mice with normal syngeneic peritoneal cells restores the stimulatory potential of splenic B cells, but sorted CD5+ or CD5- IgM+ lymphocytes from peritoneum are equally good stimulators, suggesting that functional Mls-1 expression may require long life spans and selection. Bone-marrow-reconstituted DBA/2 mice that fail to express Mls-1 antigens in the periphery nevertheless maintain T-cell receptor V beta 6 and 8.1 deletions among the newly formed T cells. These findings led us to directly investigate the Mls stimulatory ability of purified antigen-presenting cell populations inside the thymus. We report here that thymic B lymphocytes seem to represent the only intrathymic cell population able to stimulate Mls-1 MLR.

Ontogenic characterization of thymic B lymphocytes. Analysis in different mouse strains.

We have characterized a population of murine B lymphocytes present in the thymus (TBL). They are a minor subset (0.2%-1% of total thymocytes), present from perinatal periods onwards and constituted by activated cells with a high proportion of Ig-secreting cells. They represent the first B lymphocytes detected that secrete IgG after birth. Functional analysis reveals that the frequency of lipopolysaccharide-responding cells in TBL is 5- to 10-fold lower than in the spleen. TBL from adult mice did not show any significant difference in their VH repertoire expression when compared to peripheral B lymphocytes. Furthermore, we have been able to isolate a subpopulation of B220+IgM-CD3- thymocytes whose putative B cell precursor potential needs to be directly analyzed. These and other findings support the intrathymic resident characteristics of TBL and suggest new ways of elucidating its physiological role in the complex selective processes occurring inside the thymus.

[Anti-phosphorylcholine response of autoimmune NZB/W mice: variations with age of serum titers and homogeneity]. / Respuesta antifosforilcolina de ratones autoinmunes NZB/W: variaciones con la edad del título y homogenidad de los sueros.

Antibody titers and avidity of sera of autoimmune NZB/W mice in responses induced by different antigens were determined. Results show an age-dependent decrease of the antibody titer in sera from female mice immunized with phosphorylcholine coupled to keyhole limpet haemocyanin. This decrease was not detected when using as immunogen an antigenic preparation of Neisseria meningitidis that naturally induces anti-phosphorylcholine antibodies, but was detected with a modification of this antigen (heat inactivation and further coupling with the hapten). Determinations of inhibition profiles of antisera suggest that this loss of immune competence is paralleled by a decrease in avidity and homogeneity of antisera. This finding may be related to the loss of idiotypic clonal dominance recently reported to occur in these mice.

Immunoresponses to Neisseria meningitidis epitopes: in vivo analysis of immunocompetent cells involved in suppression of secondary response to phosphorylcholine.

The immune response to phosphorylcholine (PC) antigens has been extensively studied in recent years. Neisseria meningitidis serogroup B M986 (NMB) was recently reported to induce a PC-specific plaque-forming cell (PFC) immuno-response in mice, a characteristic useful for the study of immunomodulating properties of N. meningitidis. With this technique, priming mice with low doses of NMB has been shown greatly to impair their ability, one month after priming, to mount an anti-PC response induced by NMB; this suppression is permanent, does not involve switching from IgM to another immunoglobulin class, transiently affects the T15 idiotype expression and is carrier specific. We report, based on an analysis of spleen cells from NMB-primed mice in an adoptive transfer model, that this suppression does not appear to be mediated by B lymphocytes nor does it seem to be under the direct control of T lymphocytes; rather, it involves radio-resistant cells. Additionally, our results show that NMB modulates the idiotype composition of the anti-phosphorylcholine response, probably by enhancing the expression of so called hapten-augmentable PFC. These results demonstrate that NMB can interfere effectively with the immune response in a variety of ways.