Search

Snake venoms are natural sources of biologically active molecules that are able to act selectively and specifically on different cellular targets, modulating physiological functions. Thus, these mixtures, composed mainly of proteins and peptides, provide ample and challenging opportunities and a diversified molecular architecture to design and develop tools and agents of scientific and therapeutic interest. Among these components, peptides and small proteins play diverse roles in numerous physiological processes, exerting a wide range of pharmacological activities, such as antimicrobial, antihypertensive, analgesic, antitumor, analgesic, among others. The pharmaceutical and cosmetic industries have recognized the huge potential of these privileged frameworks and believe them to be a promising alternative to contemporary drugs. A number of natural or synthetic peptides from snake venoms have already found preclinical or clinical applications for the treatment of pain, hypertension, cardiovascular diseases and aging skin. A well-known example is captopril, whose natural peptide precursor was isolated from Bothrops jararaca snake venom, which is a peptide-based drug that inhibits the angiotensin-converting enzyme, producing an anti-hypertensive effect. The present review looks at the main peptides (natriuretic peptides, bradykinin-potentiating peptides and sarafotoxins) and low mass proteins (crotamine, disintegrins and three-Finger toxins) from snake venoms, as well as synthetic peptides inspired by them, describing their biochemical, structural and physiological features, as well as their applications as research tools and therapeutic agents.

Tolerance is a developmentally acquired property of the vertebrate immune system, in part ensured by regulatory CD4+ lymphocytes (Treg cells) expressing the Foxp3 transcription factor. Recent work has shown that thymic emigrants are the preferential source of peripherally generated Treg cells. A new report in this issue of the European Journal of Immunology [Eur. J. Immunol. 2013. 43: 2598–2604] describes a cell autonomous defect in Foxp3 induction in aged CD4+ cells in mice. Immune homeostasis becomes progressively less robust as ontogeny gives way to aging, and a key feature of senescence is thymic involution and the impaired T-cell turnover that follows. In this Commentary, we discuss the implications of these recent findings for our understanding of the induction of tolerance to peripheral antigens in aging.

A bradykinin-potentiating peptide (BPP) from Amazon Bothrops atrox venom with m/z 1384.7386 was identified and characterized by collision induced dissociation (CID) using an ESI-MS/MS spectra obtained in positive ion mode on a hybrid Qq-oaTOF mass spectrometer, Xevo G2 QTof MS (Waters, Manchester, UK). De novo peptide sequence analysis of the CID fragmentation spectra showed the amino acid sequence ZKWPRPGPEIPP, with a pyroglutamic acid and theoretical monoisotopic m/z 1384.7378, which is similar to experimental data, showing a mass accuracy of 0.6 ppm. The peptide is homologous to other BPP from Bothrops moojeni and was named as BPP-BAX12.

Objective: To evaluate the frequency of primary immunodeficiencies (PID) in juvenile systemic lupus erythematosus (JSLE) patients. Methods: Some 72 JSLE patients were analyzed for levels of immunoglobulin classes and IgG subclasses and early components of the classical complement pathway. Determination of C4 gene copy number (GCN) and detection of type I C2 deficiency (D) were also performed. Results: PID was identified in 16 patients (22%): C2D in three, C4D in three, C1qD in two, IgG2D ( 0.05). Remarkably, the median of Systemic Lupus International Collaborating Clinics/ACR-damage index (SLICC/ACR-DI) was significantly higher in JSLE patients with PID compared with patients without these abnormalities ( p = 0.0033), likewise the high frequency of SLICC/ACR-DI > 1 ( p = 0.023). Conclusions: A high frequency of PID was observed in JSLE patients, suggesting that these defects may contribute to lupus development. Our findings indicate that these two groups of PID should be investigated in severe pediatric lupus.

Snake Venom L-amino acid oxidases (LAAOs E.C. 1.4.3.2) are flavoenzymes broadly found in various snake venom compositions. LAAOs have become an attractive subject for molecular biology, biochemistry, physiology and medicine due to their actions on various cells and biological effects on platelets, apoptosis, hemorrhage and others. In this review we try to summarize some of these reports, with special emphasis on apoptosis, anti-protozoa, bactericidal and anti-viral activities.

Early interactions between blood-stage Plasmodium parasites and cells of the innate immune system are very important in shaping the adaptive immune response to malaria, and a number of studies have suggested that DC are responsible for this phenomenon. Therefore, we examined the capacity of murine BM-derived DC to internalize parasites, be activated and produce cytokines upon in vitro interaction with murine erythrocytes infected with two different strains of rodent malaria parasites (Plasmodium berghei and Plasmodium chabaudi chabaudi). We show that the increased expression of MHC class II and co-stimulatory molecules and increased production of cytokines by DC following Plasmodium infection involves internalization of infected RBC. Such DC activation not only requires direct cell-to-cell contact and internalization of infected RBC by DC but also involves TLR4, TLR9, MyD88 and signaling via NF-κB; however, TLR involvement in survival to Plasmodium infection was found to be negligible.

SUMMARYWhile BALB/c mice survive infection with blood stages ofPlasmodium chabaudi chabaudi(AS), 70% of DBA/2 mice die by day 9–11 of infection, both strains controlling parasitaemia. We describe here that infection of DBA/2 mice results in extensive, multifocal hepatocyte death. Antibody neutralization of TNF-α prevents both liver damage and death.

Background Haemoglobin S (HbS) is the gene known to confer the strongest advantage against malaria morbidity and mortality. Multiple HbS effects have been described resulting in protection against parasitaemia and reduction of severe malaria risk. This study aimed to explore HbS protection against severe malaria and Plasmodium falciparum parasitaemia in Angolan children exhibiting different severe malaria syndromes. Methods A case–control study was designed with 430 malaria cases (n = 288 severe malaria and n = 142 uncomplicated malaria) and 319 uninfected controls, attending a central paediatric hospital in Luanda. Severe malaria syndromes were cerebral malaria (n = 130), severe malaria anaemia (n = 30) and hyperparasitaemia (n = 128). Quantitative trait locus analysis was carried out to study HbS association to parasite densities. Results Previously reported HbS protection against severe malaria was confirmed in case–control analysis (P = 2 × 10−13) and corroborated by transmission disequilibrium test (P = 4 × 10−3). High parasite density protection conferred by HbS was detectable within severe malaria patients (P = 0.04). Stratifying severe malaria patients according parasite densities, it was found that HbS was highly associated to hyperparasitaemia protection (P = 1.9 × 10−9) but did not protect non-hyperparasitaemic children against severe malaria complications, namely cerebral malaria and severe malaria anaemia. Many studies have shown that HbS protects from severe malaria and controls parasite densities but the analysis further suggests that HbS protection against severe malaria syndromes was at a large extent correlated with control of parasitaemia levels. Conclusions This study supports the hypothesis that HbS confers resistance to hyperparasitaemia in patients exhibiting severe malaria syndromes and highlights that parasitaemia should be taken into account when evaluating HbS protection in severe malaria.

Copyright © 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim., CD4+ CD25+ Foxp3+ regulatory T cells (Treg) are natural suppressors of autoimmunity but they can also dampen the effective clearance of infectious organisms. These cells have the potential to be exploited to prevent transplant rejection and to treat autoimmune disease. A paper in this issue of the European Journal of Immunology details a method to selectively expand antigen-specific Treg from a polyclonal Treg population, by using a specific dendritic cell (DC) subset. Furthermore, the authors show that such Treg can be used to prevent experimental type I diabetes; however, as Treg are positively selected by thymic epithelial cells (TEC) on the basis of self-reactivity, they would systematically suppress protective immune responses unless their repertoire is devoid of recognition towards peripheral antigen-presenting cells. This may be achieved by negative selection of developing Treg on thymic DC, thus creating a 'blind-spot' corresponding to DC-self-antigens in the mature Treg repertoire. Therefore, therapeutic use of DC subsets for the expansion of rare Treg populations should take into account this blind-spot, as peptides that are not accessible to thymic DC may be significantly more effective for the expansion of Treg.

Ever since the foundations of Immunology, "self-tolerance" has remained a central issue in this field, pertaining to basic and clinical questions alike. Burnet and Medawar shared the Nobel Prize in 1960 for proposing that tolerance is induced by tissue antigens, if present during the development of the immune system during the embryonic/neonatal period. Very elegant experiments by Le Douarin and colleagues in the 1980's demonstrated that this is not the case; rather, the establishment of tolerance to peripheral tissues requires thymic epithelium which selects CD4 T lymphocytes mediating "dominant tolerance". The recent wealth of work on "regulatory T cells", as well as observations on the selective regulation of "tissue-specific" gene expression in thymic epithelial cells, confirm the critical relevance of those seminal findings in modern immunology.

The fundamental concepts of autopoiesis, which emphasize the circular organization underlying both living organisms and cognition, have been criticized on the grounds that since they are conceived as a tight logical chain of definitions and implications, it is often not clear whether they are indeed a scientific theory or rather just a potential scientific vocabulary of doubtful utility to working scientists. This article presents the deployment of the concepts of autopoiesis in the field of immunology, a discipline where working biologists themselves spontaneously have long had recourse to “cognitive” metaphors: “recognition”; a “repertoire” of recognized molecular shapes; “learning” and “memory”; and, most striking of all, a “self versus non-self” distinction. It is shown that in immunology, the concepts of autopoiesis can be employed to generate clear novel hypotheses, models demonstrating these ideas, testable predictions, and novel therapeutic procedures. Epistemologically, it is shown that the self–non-self distinction, while quite real, is misleadingly named. When a real mechanism for generating this distinction is identified, it appears that the actual operational distinction is between (a) a sufficiently numerous set of initial antigens, present from the start of ontogeny, in conditions that allow for their participation in the construction of the system's organization and operation, and (b) single antigens that are first presented to the system after two successive phases of maturation. To call this a self–non-self distinction obscures the issue by presupposing what it ought to be the job of scientific investigation to explain.

Maternal antibodies provide offspring with passive immunity and exert various active immunostimulatory functions, such as (i) antimicrobial protection through non-antigen-reactive antibodies, namely anti-idiotypes, (ii) allergen-specific suppression of IgE responsiveness and (iii) under pathological conditions, transfer of autoimmune diseases. As products of mainly T cell-dependent immune responses with long-lived antigen-independent plasma cells, maternal IgG molecules have undergone immune maturation by somatic hypermutations and are therefore acquired immunological phenotypes representing the collective immunological experience of the mother. The inductive function of maternal IgG, although limited to a neonatal imprinting period, exerts a life-long determinative influence, which can dominate over seemingly genetic predispositions. Hence, the functional impact of maternal IgG in offspring appears phenotypically as a non-genetic inheritance, which thus reveals a Lamarckian dimension of the immune system.

Natural Abs (NAbs) are Igs present in the serum and body fluids of healthy vertebrate animals, without any previous intentional immunization. NAbs often exhibit autoreactivity but also play an essential role in immunity, being a first line of defense against infectious microorganisms. We have previously analyzed the natural serum IgM Ab repertoire of normal mice, characterizing their reactivity with hundreds/thousands of self Ags; a significant similarity among different individuals was observed, and it was found that many reactivities of NAbs stably kept during adulthood were established early in life, implicating that period as a critical time window in the physiology of NAb repertoire selection. In the work reported here, experiments were conducted to address the role of normal lymphocyte ontogeny to the formation and stability of adult NAb repertoire. The massive destruction of the lymphoid system was promoted in adult mice with gamma-irradiation, and regeneration of hemopoietic tissues was granted by bone marrow or fetal liver inoculum. NAb repertoire regeneration was followed for 60 days after gamma-irradiation in bone marrow or fetal liver chimeric animals. The analysis of serum IgM reactivity with hundreds/thousands of self Ags showed that the NAb repertoire regenerated most of its original format after massive destruction of lymphoid compartments, characterizing autoreactive repertoire selection as a robust biological process. The data also show that regeneration of the NAb repertoire occurred similarly in fetal liver and bone marrow chimeras, although the latter animals poorly reconstituted their CD5+ B1 cell compartment, suggesting that B1 cells are not essential for natural Ab regeneration.

Nitric oxide (NO) is a proposed component of malaria pathogenesis, and the inducible nitric oxide synthase gene ( NOS2 ) has been associated to malaria susceptibility. We analyzed the role of NOS2 polymorphisms on NO bioavailability and on susceptibility to infection, Plasmodium carrier status and clinical malaria. Two distinct West African sample collections were studied: a population-based collection of 1,168 apparently healthy individuals from the Príncipe Island and a hospital-based cohort of 269 Angolan children. We found that two NOS2 promoter single-nucleotide polymorphism (SNP) alleles associated to low NO plasma levels in noninfected individuals were also associated to reduced risk of pre-erythrocytic infection as measured anti-CSP antibody levels (6.25E–04 < P < 7.57E–04). In contrast, three SNP alleles within the NOS2 cistronic region conferring increased NO plasma levels in asymptomatic carriers were strongly associated to risk of parasite carriage (8.00E–05 < P < 7.90E–04). Notwithstanding, three SNP alleles in this region protected from cerebral malaria (7.90E–4 < P < 4.33E–02). Cohesively, the results revealed a dual regimen in the genetic control of NO bioavailability afforded by NOS2 depending on the infection status. NOS2 promoter variants operate in noninfected individuals to decrease both NO bioavailability and susceptibility to pre-erythrocytic infection. Conversely, NOS2 cistronic variants (namely, rs6505469) operate in infected individuals to increase NO bioavailability and confer increased susceptibility to unapparent infection but protect from cerebral malaria. These findings corroborate the hypothesis that NO anti-inflammatory properties impact on different steps of malaria pathogenesis, explicitly by favoring infection susceptibility and deterring severe malaria syndromes.

Summary: Little progress has been achieved over the last 20 years on the clinical management of several conditions that relate to self-tolerance and to the regulation of immune responses: autoimmune diseases, transplantation tolerance, tumor immunity, allergy and vaccine development in chronic infections. These failures, it is argued, are due to the inability of the prevalent “recessive tolerance” concepts to accommodate physiological autoreactivity and the regulatory potential it embodies. In this review, the advantages of “dominant tolerance” models are underlined in the light of critical evidence and in the general context of the natural autoimmune activities. The role of regulatory T cells is discussed, notably in the regulation of inflammatory reactions and, more generally, in the “quality control” of immune responses. It is anticipated that progress will be brought about by dominant tolerance approaches, and through an increased knowledge of the differentiative pathways, repertoires, mechanisms of activation and effector functions of autoreactive, regulatory T cells.

The Xid mutation predominantly affects the development of B cells and consequently the levels and composition of natural antibodies in sera. In contrast to the congenic and susceptible BALB/c strain, immunodeficient BALB.Xid mice display a resistant phenotype both to acute Trypanosoma cruzi infection and to the development of severe cardiopathy. Because natural antibodies are known to be basically self-antigen driven, IgM and IgG natural antibody repertoires (NAR) were compared before and during infection in these two strains. The analysis revealed fundamental alterations of IgM and IgG NAR in pre- and post-infected Xid mice. In particular, relatively increased natural (pre-existing) autoreactive IgG, dominated by the unique recognition of a single band in autologous heart extracts, was typical for uninfected Xid mice. This natural autoreactive IgG directed to heart antigens disappeared early after infection not only in Xid, but also in individual BALB/c mice that survived the acute infection. Conversely, the subgroup of BALB/c mice that died early after infection presented the most pronounced instances of the rapid, relative increase of IgM reactivities to self and non-self proteins. These results suggest that self-reactive NAR may play a role in an immunoregulatory mechanism relevant for the determination of susceptibility/resistance to infections. This may act either by influencing specific responses, or by modulating the self-aggressive components responsible for pathology.

International audience; Lymphocyte polyclonal activation is a generalized mechanism of immune evasion among pathogens. In a mouse model of Trypanosoma cruzi infection (American trypanosomiasis), reduced levels of polyclonal lymphocyte responses correlate with resistance to infection and cardiopathy. We report here the characterization of a parasite protein with B-cell mitogenic properties in culture supernatants of infective forms, the cloning of the corresponding gene and the analysis of the biological properties of its product. We characterized the protein as a co-factor-independent proline racemase, and show that its expression as a cytoplasmic and/or membrane-associated protein is life-stage specific. Inhibition studies indicate that availability of the racemase active site is necessary for mitogenic activity. This is the first report to our knowledge of a eukaryotic amino acid racemase gene. Our findings have potential consequences for the development of new immune therapies and drug design against pathogens.

Locally produced type I interferon (IFN-I) enhances the sensitivity of bone marrow B cell to IgM receptor ligation. The establishment of B cell repertoires, on the other hand, seems to involve selective processes that are critically dependent on B cell receptor (BCR) ligation. In order to assess the importance of BCR triggering thresholds on the selection of polyclonal unmanipulated B cell populations, we compared VH gene expression and reactivity repertoires in various B cell compartments of wild-type and IFN-I receptor-deficient mice (IFN-I-R ‐/‐ ). These analyses demonstrate that increased B cell sensitivity to BCR ligation mediated by IFN-I in the bone marrow (BM) has consequences on the stringency of B cell repertoire selection. Thus, the normal counterselection of both VH7183 gene family expression and multireactivity was impaired among immature BM B cells from mutant mice. Furthermore, as a result of reduced efficiency of BCR ligationdependent inhibition of terminal differentiation, IFN-I-R ‐/‐ animals produce, in BM and thymus, higher numbers of plasma cells secreting antibodies that are more multireactive than wild-type animals. Finally, mutant serum IgM natural antibodies display a more reactive repertoire than controls, a likely reflection of the BM resident plasma cell repertoire. The present observations demonstrate, therefore, that local modulation of BCR triggering thresholds leads to important modifications in the generation and/or selection of normal B cell populations.

Global analysis of natural antibody repertoires has revealed a marked conservation of reactivity patterns within inbred mouse strains, and characteristic strain-specific differences. We have now analyzed the genetic control of reactivity repertoires, aiming at identifying the respective selection mechanisms. Multiparametric statistics of a large number of serum antibody reactivities scored by quantitative Western blot analyses using extracts from homologous tissues and bacteria readily distinguish the reactivity patterns of C57BL/6 and BALB/c, revealing homogeneity among genetically identical individuals. Antibody repertoires in the prototype strains can also be segregated from those expressed by the respective IgH congenics, BC.8 and CB.20, demonstrating that IgH-linked genes contribute to determining natural antibody repertoires. Conversely, strains sharing IgH haplotype also express distinct reactivity patterns, indicating that other genes participate in the selection of serum IgM repertoires. Two such non-IgH loci were now identified. Thus, analysis of four MHC-congenic strains demonstrated that MHC-linked control of natural antibody repertoires is likely to operate through differential selection of T cell repertoires, since (1) mice that are congenic at the TCR beta locus, and (2) BALB/c nude mice grafted at birth with pure thymic epithelium from either C57BL/6 or BALB/c also differ in their natural antibody repertoires.

Compartmentation of B lymphocyte populations is associated with differences in both development stage and sensitivity to Ig (sIg)-dependent triggering. In order to characterize the factors that contribute in setting the level of sensitivity of a B cell, we quantified sIgM-dependent regulation of Ig secretion in purified mature and immature B cells after ex vivo and in vivo modification of their environment. These analyses formally demonstrate that the bone marrow (BM) microenvironment locally induces high B cell sensitivity to sIgM ligation irrespective of differentiation stage. We further provide evidence that BM macrophages create a dominant environment that enhances B cell sensitivity to B cell receptor triggering. Finally, using ex vivo assays as well as type I IFN receptor-deficient mice we show that IFN-beta produced by resident BM macrophages is necessary and sufficient to define B cell sensitivity. Implications of these findings for the understanding of B cell selection processes are discussed.

We often cite Melvin Cohn as saying that ‘‘it is better to be clearthan right’’. We were not surprised, therefore, with Langman &Cohn’s (L&C) recent Editorial discussion [1], for they continueto be rare examples of serious thinkers in the field. Ironically, wewere not surprised either that they treat the ‘time and space’problems in ways that are radically different from those we took,in a paper published some 5 years ago [2].Let us first welcome L&C’s statement that ‘‘the conceptualhorizon in immunology has narrowed to details of geneexpression and chemistry’’. We could not agree more on how‘‘imperative’’ it is ‘‘to retain sight of an immune system that isthe co-ordinated collection of all these subsystems and provides acoherent, strong selective advantage’’ (underlinings are ours).This is precisely what we have been repeating, ever since welearned it from Jerne.Let us next welcome a major shift in the thinking of L&C,namely that their view of tolerance and immunity now requiresthat ‘‘anti-S(elf) cells must be present at some level’’. This isalso what we have been saying for some time: there is no wayto explain tolerance by ‘recessive’ mechanisms only, and ‘domi-nant’ tolerance must be based on productive self-recognition.Moreover, L&C come to the conclusion that ‘‘there can be noexclusion of self by the definition of nonself’’. This is alsosimilar to Avrameas’ ‘‘know thyself’’ [3] or our soundbite‘‘tolerance by self assertion’’ [4]. L&C seem to have evolvedaway from recent writings, where this was all considered to benonsense, from ‘‘an articulate group of immunologists of theFrench heretic school’’ [5]. Finally, we also agree that ‘‘thedefinition of self can only be based on the persistence ofantigen’’, but here we have set the time (problem) a little moreprecisely and underlined the fact that persistence in the adultdoes not lead to tolerance, but to maintenance of immunity. Fordefining self, it is necessary that persistence starts during devel-opment (see, for example, the work of Hannahan et al. ref. 3 in[2]), thus introducing a radical difference between ‘persistence’and ‘history’, and between L&C’s time problem—any time, iflong enough, and ours—developmental time only, maybe even ifshort. This is precisely the reason why ‘dominant tolerance’ isnecessary: newly formed lymphocytes must ‘‘discriminate’’ notbetween ‘‘antigens that are there or not’’ when they are produced,but between ‘‘antigens that were there and those that were not’’in development, much before these lymphocytes themselveswere produced. Incidentally, this is also the reason why tolerancemust be a ‘supraclonal’ property, since single lymphocytes cannottell of differences that existed before they were produced. In otherwords, the ‘system’, not the individual cells, must tell thedifference. The same system that L&C now also suggest is‘co-ordinated’ and provides a ‘‘coherent selective advantage’’.Let us now welcome L&C’s present argument against Leder-berg’s popular model. It is worth underlining that, essentially, allimmunology of the recent ‘tolerance-by-deletion’ period hasbeen implicitly or explicitly based on that model. Let us thenadd, however, that in Bretscher & Cohn’s model, now presentedas the first alternative to Lederberg’s, the difference betweenan ‘antigen-sensitive’ and an ‘effector’ lymphocyte (i-state ande-state cells, in L&C’s current designation) does not go muchbeyond Lederberg’s postulate of differential responses of‘immature’ and ‘mature’ lymphocytes. Not surprisingly, there-fore, L&C recognize that ‘‘having rejected the Lederberg model,no satisfactory answer was forthcoming’’ (in Bretscher & Cohn’smodel). As L&C also point out, Lederberg makes ‘‘tolerance andinduction each epitope-by-epitope level events’’, but ‘‘toleranceremained an epitope-by-epitope level event’’ in Bretscher &Cohn’s model. In other words, in our reading, the latter modelcontributed to the understanding of tolerance little more than ahandful of ‘soundbites’. Coming back to being ‘‘clear and/orright’’, this critique does not at all imply that we discard asirrelevant the efforts of Cohn and his associates in establishingrigorous thinking in the discipline. Their contribution has cer-tainly been immense. We just do not agree with the solutionsthey propose.Let us finally welcome L&C’s attempts to introduce somerigour into the ‘‘hybrid time and space model as proposed byMatzinger’’ and their respective critique. We also agree that itwould be very dangerous to have ‘danger’ alone controlling thewhole thing. Even the grim years of the ‘cold war’ went furtherthan that.First, the definition of the problems. The time problem arisesfrom the fact that natural tolerance is predominantly or exclu-sively acquired in development (before adult life) while newlymphocytes are produced throughout life. It makes a lot of sense(teleologically, that is) that tolerance is exclusively acquiredduring development, for this is the only period in life that theorganism is exclusively exposed to self antigens. Burnet, natu-rally not aware of the continuous lymphocyte production later inlife, had actually based on this restriction the whole tolerance/deletion hypothesis of the clonal selection theory [6]. After birth,the organism is exposed to external antigens and colonized bymicroorganisms, such that newly formed lymphocytes will meetboth self and non-self. Hence, self can only be ‘historicallyidentified’ by cellular and molecular mechanisms that are

Spleen and bone marrow cells from normal or Rag-1-deficient donors are equally competent in their ability to induce neonatal transplantation tolerance in semi-allogeneic hosts, and the latter are also capable of tolerizing fully allogeneic recipients. Both types of donor cells resulted in comparable levels of haemopoietic chimerism in tolerant animals. Lymphoid hyperactivity, however, was absent in animals tolerized with Rag-1-deficient cells. The authors conclude that neonatal tolerance induced with haemopoietic cells requires no donor lymphocytes, and is thus not the result of deficient antigen presentation. Furthermore, the state of tolerance can be dissociated from the lymphoid hyperactivity that requires donor lymphocytes and is regularly scored in conventionally tolerized animals.

To examine the physiological role of maternal natural IgG antibodies on the development of B lineage cells of the progeny, we have bred homozygous muMT/muMT or heterozygous muMT/+ females to muMT/muMT or muMT/+ males, respectively. We could thus compare normal or B cell-deficient mice born from Ig-deprived (Ig-) or phenotypically normal mothers (Ig+). B cell-deficient progeny of heterozygous mothers contain no detectable serum IgA or IgM, but IgG concentrations that peak at 2 mg/ml by 7-21 days of age, decay after weaning with a half-life of 7 days, and remain detectable for 2 months after birth. At 7 days after birth, muMT/+ progeny born of Ig+ mothers contain two- to threefold higher numbers of bone marrow (BM) pre-B and B cells, and of splenic B cells, compared to mice of the same age born from Ig mothers. In contrast, the former progeny exhibit two to four times lower numbers of Ig-secreting plasma cells in spleen and thymus, and contain sixfold lower serum IgM concentrations. A similar maternal IgG-dependent stimulation of BM B cell precursors is also observed in muMT/muMT progeny. No significant differences were detected between the groups on day 3 after birth, suggesting the requirement for a minimal IgG concentration in the serum.

We have generated a mutant mouse in which the most D-proximal V(H) gene (V(H)81X) has been disrupted by introducing a neomycin-resistance gene into the V(H)81X exon by means of gene targeting in embryonic stem cells. The mutant mice generated are unable to express the V(H)81X gene but appear to display a normal pattern of B cell differentiation as well as normal numbers of bone marrow and peripheral B cells from fetal life all through ontogeny. They mount normal immune responses to several different antigens tested. In contrast, the distribution of V(H) gene rearrangements in the V(H)7183 family is altered in homozygous mutant mice. Thus, the antibody repertoire of the targeted mice is modified, at least as far as the expression of V(H)7183 genes is concerned.

This publication hasn't any creative commons license associated. There is no public supplementary material available. Development of cerebral malaria (CM), a severe and fatal form of clinical Plasmodium falciparum infection, results from a damaging cascade of vascular, inflammatory, and immunological host responses that leads to brain injury. Progression to CM can be modified by host genetic factors. Our case-control study in Angolan children aimed at highlighting the role of IFN (α, β) receptor 1 (IFNAR1) in progression to CM. We report a robust association between IFNAR1 and CM protection, as well as detailed studies showing analogous protection from experimental CM in Ifnar1(-/-) mice infected with P. berghei ANKA. We developed a novel cell-transfer protocol that enables spleen cell priming in the absence of disease. This led to the discovery that IFNAR1 expression in CD8(+) T cells is crucial and can abrogate resistance to experimental CM in Ifnar1(-/-) mice. Splenic CD8(+) T cells from Ifnar1(-/-) mice are functionally activated upon infection, yet are unable to mediate experimental CM development within the brain tissue. Our findings prove that IFNAR1 signaling unleashes CD8(+) T cell effector capacity, which is vital for CM, and raises the hypothesis that the cohesive role of IFNAR1 in both human and mouse CM operates through CD8(+) T cell triggering. FCT fellowships: (SFRH/BD/33564/2008, SFRH/BPD/29354/2006).

This paper is based on a new model of the immune network which explicitly incorporates B-T cell co-operation. A major feature of this model is the simplifying assumption that inhibition by anti-TCR soluble Ig is the only possible down-regulatory influence on activated T-cells. This model is capable of coupling with antigens in both an “immune response” mode and a “tolerant” mode. In the present paper, we simulate the ontogenesis of the immune system by metadynamical recruitment of T- and B-cell clones from the thymus and the bone marrow, seeking to identify the conditions under which each of these modes of antigen coupling occurs. Achieving the tolerant mode depends principally on four parameters: a high value of S B , the rate of bone-marrow production of B-cells; a relatively high efficiency of T-help through mIg-TCR recognition compared with (MHC + peptide)-TCR interaction; and a relatively high value of the product P R .N A , where P R is the average probability that an Ig recognizes another molecule and N A is the number of antigens which are present throughout ontogeny. Analysis of the conditions under which these two modes can coexist, shows that this is possible when a sufficiently numerous set of founder antigens couple in a tolerant mode, whereas isolated antigens first presented once development is completed couple in an immune response mode. The present model thus provides a possible mechanism for the distinction (hitherto purely descriptive) between a Central Immune System organized as a network and responsible for tolerance, and a Peripheral Immune System responsible for immune responses.

Analyses of bone marrow (BM) lymphocytes in C57BL/6 mice homozygous for the lpr mutation (B6.lpr) disclosed low numbers of pre-B and B cells, as compared with age-matched control B6 mice. BM depletion in B6.lpr mice was selective for B-lineage cells, appeared in young adults, and developed markedly with age and disease progression, contrasting with the peripheral lymphocyte hypercellularity. Normalization of pre-B and B cellularity in BM of B6.lpr mice was observed after administration of polyclonal Ig, that also markedly improved the clinical condition. Isolated pre-B (B220+ IgM-) cells from B6 or B6.lpr mice, however, showed essentially the same rates of IL-7-dependent proliferation and differentiation to B (IgM+) cells in culture, indicating that the BM B-lineage deficit is not the result of an intrinsic defect in B cell generation.

Athymic mice grafted at birth with allogeneic thymic epithelium (TE) display life-long tolerance to tissue grafts of the TE donor strain, in spite of harboring peripheral T cells capable of rejecting those grafts. Tolerance is maintained in these chimeras by TE-specific regulatory CD4 T cells. We presently address the quantification and the mechanisms of this dominant tolerance process. C57BL/6 mice containing variable but defined numbers of peripheral, resident T cells received cell transfers of graded numbers of peripheral T cells from B6(BALB E10) chimeras (C57BL/6 nude mice grafted with TE from 10-day-old BALB/c embryos), resulting in a series of animals containing a wide range of donor (tolerant) versus host (non-tolerant) T cell chimerism. Increasing the relative representation of donor T cells results in a progressive delay in the rejection of BALB/c skin grafts, life-long tolerance being achieved at a ratio of tolerant and non-tolerant T cell populations of 1. In recipients displaying full tolerance, graft-reactive non-tolerant T cells were not deleted, anergized or committed to noninflammatory functions. Thus, sorted host T cells from tolerant recipients readily rejected BALB/c skin grafts upon transfer to immunodeficient animals. Finally, measurements of "helper" and inflammatory activities, as well as interleukin-4 and interferon-gamma production, failed to discriminate between T cell populations from tolerant and non-tolerant animals after specific in vitro stimulation. We conclude that: (a) TE-selected regulatory T cells can suppress, in a quantitative manner, in vivo T cell responses against major and minor histocompatibility antigens expressed by the TE and, (b) this suppressive activity neither inactivates mature non-tolerant T cells, nor does it seem to drive their differentiation along noninflammatory pathways.

Athymic mice grafted at birth with allogeneic thymic epithelium (TE) from day 10 embryos before hematopoietic cell colonization reconstitute normal numbers of T cells and exhibit full life-long tolerance to skin grafts of the TE haplotype. Intravenous transfers of splenic cells, from these animals to adult syngeneic athymic recipients, reconstitute T-cell compartments and the ability to reject third-party skin grafts. The transfer of specific tolerance to skin grafts of the TE donor strain, however, is not observed in all reconstituted recipients, and the fraction of nontolerant recipients increases with decreasing numbers of cells transferred. Furthermore, transfers of high numbers of total or CD4+ T cells from TE chimeras to T-cell receptor-anti-H-Y antigen transgenic immunocompetent syngeneic hosts specifically hinder the rejection of skin grafts of the TE haplotype that normally occurs in such recipients. These observations demonstrate (i) that mice tolerized by allogeneic TE and bearing healthy skin grafts harbor peripheral immunocompetent T cells capable of rejecting this very same graft; and (ii) that TE selects for regulatory T cells that can inhibit effector activities of graft-reactive cells.

Summary Self-tolerance is acquired in the embryonic/perinatal period, but new lymphocytes (that will have to distinguish between self and nonself) continue to be produced throughout life, after both self and nonself are present. This makes it impossible for natural tolerance to rely on recessive mechanisms. Recent observations on “dominant tolerance” have led to the hypothesis that natural tolerance is established as a consequence of simple developmental programs for gene expression and cellular composition of primary lymphoid organs. In development, the cellular composition of the thymus is predominantly epithelial, allowing for the positive selection and activation of “high avidity” self-reactive T cells that are not deleted because antigen presentation by haemopoietic cells is limiting. Such T cells, activated in that environment, display effector functions of a regulatory type that are maintained in the periphery upon restimulation by tissue peptides shared with the thymic epithelium. Recent thymic emigrants with specificity for tissue-specific antigens that are absent from the thymus will first encounter their ligands in the context of the “regulatory cell” recognition of ubiquitous peptides and are thus recruited into similar regulatory activities. In contrast, thymic emigrants with specificity for nonself antigens (absent during the perinatal period) are not activated intrathymically, reach the periphery as resting cells, and move out of the time window of susceptibility to functional recruitment. These will react “de novo” upon encounter of the respective antigens and will acquire the class of effector functions determined by the peripheral microenvironment in which they are activated. This strategy, which explains the thymic dependence of peripheral tissue-specific tolerance, may be re-inforced by developmental restrictions in cytokine gene expression, and it will ensure the establishment and maintenance of T-cell tolerance through the dynamic storage of a distributed memory of the embryonic self. B lymphocytes that are produced in the embryonic/perinatal period characteristically rearrange and express a few V-genes very predominantly. These V-genes encode antibodies with unique properties of “connectivity” to other V-regions, making it possible to establish a network that limits clonal expansions and/or terminal differentiation to antibody production. Self-reactive B cells are thus recruited into such a network which, by contributing to the molecular environment of the body and to the selection of emergent repertoires, leads to deletion of connected cells and to the “normalization” of the adult antibody repertoires. Natural autoantibody repertoires in the adult are thus recursively maintained, stable and continuously adjusted to the thresholds of single cell deletion and to the alterations in the body composition. The activity of self-specific regulatory T cells contributes to limiting clonal expansion and inhibiting somatic mutation of self-reactive B cells. This model explains a number of observations that were not included in the “clonal” and “recessive” tolerance views, and offers suggestions on mechanisms in physiological autoimmunity and pathology.

The differential class-regulation of CD4+ T lymphocyte populations is believed to play a major role in determining the qualitative behaviour of the immune system, and in the fate of immune responses in particular. In this article we propose a model for the dynamics of the Th1 and Th2 subpopulations. We put forward the concept of an 'antigenic niche' which allows us to postulate that the key feature underlying the regulation of Th differentiation pathways is the population dynamics of the lymphocytes themselves. Using this model we are able to account for a number of well established experimental observations which were hitherto apparently unrelated and poorly understood. This suggests that our simplified model might be capturing some essential features of the immune system.

OBJECTIVE: The aim of this study was to assess the IgE serum levels in juvenile systemic lupus erythematosus patients and to evaluate possible associations with clinical and laboratory features, disease activity and tissue damage. METHODS: The IgE serum concentrations in 69 consecutive juvenile systemic lupus erythematosus patients were determined by nephelometry. IgG, IgM and IgA concentrations were measured by immunoturbidimetry. All patients were negative for intestinal parasites. Statistical analysis methods included the Mann-Whitney, chi-square and Fisher's exact tests, as well as the Spearman rank correlation coefficient. RESULTS: Increased IgE concentrations above 100 IU/mL were observed in 31/69 (45%) juvenile systemic lupus erythematosus patients. The mean IgE concentration was 442.0 ± 163.4 IU/ml (range 3.5-9936.0 IU/ml). Fifteen of the 69 patients had atopic disease, nine patients had severe sepsis and 56 patients presented with nephritis. The mean IgE level in 54 juvenile systemic lupus erythematosus patients without atopic manifestations was 271.6 ± 699.5 IU/ml, and only nine of the 31 (29%) patients with high IgE levels had atopic disease. The IgE levels did not statistically differ with respect to the presence of atopic disease, severe sepsis, nephritis, disease activity, or tissue damage. Interestingly, IgE concentrations were inversely correlated with C4 levels (r = -0.25, p = 0.03) and with the SLICC/ACR-DI score (r = -0.34, p = 0.005). The IgE concentration was also found to be directly correlated with IgA levels (r = 0.52, p = 0.03). CONCLUSIONS: The present study demonstrated for the first time that juvenile systemic lupus erythematosus patients have increased IgE serum levels. This increase in IgE levels was not related to allergic or parasitic diseases. Our results are in line with the hypothesis that high IgE levels can be considered a marker of immune dysregulation.

We have previously shown that in IgH congenic mice VH-gene family usage in neonatal spleen B cells and adult Ig-secreting cells is entirely determined by the IgH locus, while in adult resting B cells it is regulated by genetic element(s) located outside the IgH locus. Two observations reported here demonstrate that the differential expression of VH genes is an intrinsic property of the respective cell populations, determined by both the IgH locus and by a cis element(s) operating independently in the same animal. First, the study of F1 hybrids between the IgH congenic B6a and CB.20 strains demonstrates that cis elements control VH-gene family expression. Second, studies in irradiation chimeras showed that the environment in which cell differentiation proceeds is unable to overcome those controls. In chimeras of IgH congenic donors, VH-gene expression in fetal liver-derived splenic B cells and Ig-secreting cells is dictated by the IgH haplotype, while in bone marrow-derived B cells is entirely determined by the cis element(s). These results show a developmental and cell lineage-related restriction in VH-gene expression, and suggest that most adult splenic Ig-secreting cells may originate from precursors originally present in fetal liver, but which are rare among adult bone marrow precursors and CD5+ B cells.

The number of peripheral T cells in mice increases up to 100-fold in the first few weeks of life. We have followed the fate of Thy-1 congenic T cells transferred into newborn recipients, to evaluate the relative contribution of thymic output versus peripheral expansion in the constitution of peripheral T cell pools during post-natal development. The results show that in normal animals there is essentially no peripheral expansion of T cells, which show slow turnover rates (1 to 2 months) along that time period. The rates of cell accumulation in the periphery require, therefore, an average of 1 x 10(6)-2 x 10(6) mature thymic emigrants/day for the first 3 weeks of life.

The cellular composition and VH-gene family repertoire were compared in different B-cell compartments from young adult (8-12 weeks) and old (18-24 months) C57BL/6 and BALB/c mice. Ageing mice were found to have a higher frequency of peripheral mature B cells utilizing genes from a single VH-gene family. While in each individual old C57BL/6 mice cells expressing the VH J558 gene family consistently were over-represented, a marked individual variation was observed in old BALB/c mice with increased frequency of either the VH J558, Q52 or J606 families. Aged mice were found also to have a reduced number of bone-marrow pre-B cells and an augmented number of splenic Ig-secreting cells. These results suggest that old mice express less diversified antibody repertoires possibly as a consequence of reduced input from precursors and increased peripheral selection, which may be responsible for the progressive establishment of immunodeficiency.

This paper describes a procedure for analysing multiple antibody reactivities that explores a commercially available immunoblot system, and is based on a double staining of nitrocellulose membranes, revealing both antibody reactivities and the migration position of the blotted proteins in the membrane. Quantification of both stainings by densitometry allowed the accurate superposition of the immunoreactivity and total protein profiles of each lane. Moreover, the protein stainings of the different lanes could be adjusted with a simple-scale transformation algorithm, correcting for possible distortions during electrophoretic migration, and allowing for the precise comparison of the immunoreactivity profiles in different lanes. The procedure is discriminatory enough to identify unique reactivity patterns in random pools of 10(4) activated B cells, and to define strain-specific natural antibody repertoires. The utility of this immunoblot method as an assay for simultaneously scoring multiple reactivities to hundreds of antigens in complex mixtures of antibodies, and thus defining antibody repertoires in a global manner, is discussed.

From experimental observations on induction of transplantation tolerance, we discuss a model that accounts for tissue-specific tolerance to antigens not expressed inside the thymus. It is postulated that antigens presented to differentiating T cells by thymic epithelium (or at large within the thymic environment) positively select and activate self-reactive T cells. A developmental program and/or prevalent conditions in the thymic environment restrict the proliferative potential and the class of effector functions that can be exerted by differentiating T cells activated in the thymus. These do not mediate inflammatory or cytolytic activities, but instead will produce the appropriate mediators to inhibit aggressive effector activities by other T cells activated in their proximity. Such "regulatory" functions will be locally expressed at the periphery upon recognition of tissue antigens shared with the thymus, towards newly formed thymic emigrants directed at tissue-specific antigens expressed by the same "target" cells. This mechanism imposes "dominant tolerance", based on specific self-recognition and predominantly established in the embryonic and neonatal period. Throughout life, the process of thymic positive selection results in all newly-formed T cells being susceptible to such suppressive mechanisms, but becoming increasingly refractory with time in the resting, post-differentiative stage. Absence of antigen (nonself) in the embryonic and neonatal life therefore allows for the accumulation of such "suppression-resistant" antigen-reactive T cells that will mount aggressive responses upon antigenic exposure. Tolerance or immunity thus represent two classes of specific immune responses, the relative predominance of which is determined by the frequency of each type of effector T cell, representing the antigenic overlap between thymic and peripheral tissues, as well as the frequency of tissue-specific T-cell generation, and the kinetics of peripheral antigenic exposure. Tolerance induced by hemopoietic cells to all other tissues is also "dominant" and based on thymic colonization and persistence of antigenic cells, with the consequent positive selection of regulatory T cells and peripheral conditions for the establishment of suppression. Upon this simple model, that ensures "interclonal class regulation" by "bridging" regulatory and effector T cells through the recognition of different antigens on the same target cell, other mechanisms which are based on V-region interactions among T cells (Ben-Nun et al. 1981, Pereira et al. 1989, Webb & Sprent 1990, Gaur et al. 1993) might well operate to ensure "dominant tolerance" by self-reactivity and class regulation.(ABSTRACT TRUNCATED AT 400 WORDS)

The brain-derived neurotrophic factor (BDNF), a neurotrophin fundamental for brain development and function, has previously been implicated in autism. In this study, the levels of BDNF in platelet-rich plasma were compared between autistic and control children, and the role of two genetic factors that might regulate this neurotrophin and contribute to autism etiology, BDNF and NTRK2, was examined. We found that BDNF levels in autistic children (n = 146) were significantly higher (t = 6.82; P < 0.0001) than in control children (n = 50) and were positively correlated with platelet serotonin distribution (r = 0.22; P = 0.004). Heritability of BDNF was estimated at 30% and therefore candidate genes BDNF and NTRK2 were tested for association with BDNF level distribution in this sample, and with autism in 469 trio families. Genetic association analysis provided no evidence for BDNF or NTRK2 as major determinants of the abnormally increased BDNF levels in autistic children. A significant association with autism was uncovered for six single nucleotide polymorphisms (SNPs) [0.004 (Z((1df)) = 2.85) < P < 0.039 (Z((1df)) = 2.06)] and multiple haplotypes [5 × 10(-4) (χ((3df)) = 17.77) < P < 0.042 (χ((9df)) = 17.450)] in the NTRK2 gene. These results do not withstand correction for multiple comparisons, however, reflect a trend toward association that supports a role of NTRK2 as a susceptibility factor for the disorder. Genetic variation in the BDNF gene had no impact on autism risk. By substantiating the previously observed increase in BDNF levels in autistic children in a larger patient set, and suggesting a genetic association between NTRK2 and autism, this study integrates evidence from multiple levels supporting the hypothesis that alterations in BDNF/tyrosine kinase B (TrkB) signaling contribute to an increased vulnerability to autism.

BACKGROUND: Cerebral malaria (CM) represents a severe outcome of the Plasmodium falciparum infection. Recent genetic studies have correlated human genes with severe malaria susceptibility, but there is little data on genetic variants that increase the risk of developing specific malaria clinical complications. Nevertheless, susceptibility to experimental CM in the mouse has been linked to host genes including Transforming Growth Factor Beta 2 (TGFB2) and Heme oxygenase-1 (HMOX1). Here, we tested whether those genes were governing the risk of progressing to CM in patients with severe malaria syndromes. METHODOLOGY/PRINCIPAL FINDINGS: We report that the clinical outcome of P. falciparum infection in a cohort of Angolan children (n = 430) correlated with nine TGFB2 SNPs that modify the risk of progression to CM as compared to other severe forms of malaria. This genetic effect was explained by two haplotypes harboring the CM-associated SNPs (Pcorrec. = 0.035 and 0.036). In addition, one HMOX1 haplotype composed of five CM-associated SNPs increased the risk of developing the CM syndrome (Pcorrec. = 0.002) and was under-transmitted to children with uncomplicated malaria (P = 0.036). Notably, the HMOX1-associated haplotype conferred increased HMOX1 mRNA expression in peripheral blood cells of CM patients (P = 0.012). CONCLUSIONS/SIGNIFICANCE: These results represent the first report on CM genetic risk factors in Angolan children and suggest the novel hypothesis that genetic variants of the TGFB2 and HMOX1 genes may contribute to confer a specific risk of developing the CM syndrome in patients with severe P. falciparum malaria. This work may provide motivation for future studies aiming to replicate our findings in larger populations and to confirm a role for these genes in determining the clinical course of malaria.

This paper discusses current evidence for the relationship between polyclonal lymphocyte activation, specific immunosuppression with decreased resistance, and autoimmune pathology, that are all often found associated with infections by a variety of virus, bacteria and parasites. The central question of class determination of immune effector activities is considered in the context of the cellular targets for nonspecific mitogenic activities associated with infection. A model is presented to integrate these findings: mitogens produced by the microorganism or the infected cells are preferentially active on CD5 B cells; the resulting over-production of IL-10 will tend to bias all immune activities into a Th2-mode of effector functions, with high titers of polyclonal antibodies and little or no production of gamma IFN and other "inflammatory" lymphokines that often mediate resistance. In turn, these conditions allow for parasite persistence and the corresponding long-term disregulation of self-directed immune reactivities, resulting in autoimmunity in the chronic phase. This model would predict that selective immunization with the mitogenic principles involved in deregulation, could stand better chances than strategies of vaccination based on immunopotentiation against other, functionally neutral antigenic epitopes. It is argued, however, that the complexity of immune responses and their regulation, together with our ignorance on the genetic controls of class-determination, offer poor prospects for a scientifically-based, rational development of vaccines in the near future. It is suggested that empirically-based and technologically developed vaccines might succeed, while basic scientific approaches are reinforced and given the time to provide a better understanding of those processes.

Infection by Plasmodium , the causative agent of malaria, is associated with hemolysis and therefore with release of hemoglobin from RBC. Under inflammatory conditions, cell-free hemoglobin can be oxidized, releasing its heme prosthetic groups and producing deleterious free heme. Here we demonstrate that survival of a Plasmodium -infected host relies strictly on its ability to prevent the cytotoxic effects of free heme via the expression of the heme-catabolyzing enzyme heme oxygenase-1 (HO-1; encoded by the Hmox1 gene). When infected with Plasmodium chabaudi chabaudi ( Pcc ), wild-type ( Hmox1 +/+ ) BALB/c mice resolved infection and restored homeostasis thereafter (0% lethality). In contrast, HO-1 deficient ( Hmox1 −/− ) BALB/c mice developed a lethal form of hepatic failure (100% lethality), similar to the one occurring in Pcc -infected DBA/2 mice (75% lethality). Expression of HO-1 suppresses the pro-oxidant effects of free heme, preventing it from sensitizing hepatocytes to undergo TNF-mediated programmed cell death by apoptosis. This cytoprotective effect, which inhibits the development of hepatic failure in Pcc -infected mice without interfering with pathogen burden, is mimicked by pharmacological antioxidants such as N-acetylcysteine (NAC). When administered therapeutically, i.e., after Pcc infection, NAC suppressed the development of hepatic failure in Pcc -infected DBA/2 mice (0% lethality), without interfering with pathogen burden. In conclusion, we describe a mechanism of host defense against Plasmodium infection, based on tissue cytoprotection against free heme and limiting disease severity irrespectively of parasite burden.

Differences in metabolism of drugs can lead to severe toxicity or therapeutic failure. In addition to cytochrome P450 2D6, which plays a critical role in drug metabolism, ABCB1 encoded P-glycoprotein (PGP) is also an important determinant in drug bioavailability. The genes encoding these molecules are highly variable among populations and, given their clinical importance in drug therapy, determining CYP2D6 and ABCB1 allele frequencies in specific populations is very important for useful application in clinical settings. In this study the frequency of the pharmacologically relevant CYP2D6*3, *4, *5, *6 allelic variants and gene duplication, and ABCB1 C1236T and C3435T gene polymorphisms and their haplotypes was determined in a population sample of 100 Portuguese healthy subjects. CYP2D6 allele frequencies were 1.4% (*3), 13.3% (*4), 2.8% (*5), 1.8% (*6) and 6.1% (gene duplication), with 5% of the individuals classified as PM and 8.4% as UM. The frequencies obtained for the non-functional alleles and for the CYP2D6 gene duplication are in agreement with other South European populations, and reinforce the previously suggested south/north gradient of CYP2D6 duplications. Allelic frequencies for the ABCB1 polymorphisms were 52% (3435C) and 54% (1236C) and the most common haplotype (1236C-3435C) occurred with a frequency of 45.5%. Although allele and haplotype frequency data for ABCB1 in Southern Europe is limited, some discrepancies were found with other European populations, with possible therapeutic implications for PGP substrate drugs. Copyright © 2009 John Wiley & Sons, Ltd.

Corticosteroids are commonly used in the therapy of autoimmune disease (AID), although they are rarely, if ever, curative. This failure may result from their deleterious effects on regulatory T cells (Treg). In this work, we directly tested the effects of hydrocortisone (HC) administration on Treg number and function in established mouse models of multiple sclerosis and colitis. Treatment with pertussis toxin (Ptx) or Cyclophosphamide (Cyp), two compounds known to affect Treg function served as controls. We first show that contrarily to Ptx, HC administration to mice transgenic for a TCR specific to myelin basic protein induces a mild lymphopenia, without selective depletion of Treg, nor induction of experimental autoimmune encephalomyelitis (EAE). We next report that HC administration to normal mice has no effect on Treg suppressive function tested in vitro. Moreover, we document that Treg isolated from HC-treated animals maintain their capacity to prevent T cell-induced colitis. In contrast, the combined administration of HC and Cyp, as is frequently used in the therapy of severe AID, dramatically enhanced the deleterious effect of Cyp on Treg number and function. Our analysis indicates that while a short course of corticosteroids alone is not deleterious to immune regulation, combined therapies, notably with Cyp, should be avoided.

The mechanisms regulating growth and differentiation of B-cell precursors in adult bone marrow (BM) and/or selecting immunocompetent cells for peripheral export are poorly understood. We report here that small numbers of activated peritoneal B cells selectively suppress the numbers of small pre-B (B220+IgM-) and B (B220+IgM+) cells in BM, if transferred into syngeneic adult mice. No significant alterations are detected in other BM cell lineages or in peripheral lymphocytes of recipient mice. Both CD5+ and CD5- peritoneal B cells display this activity, but the same or higher numbers of similarly activated splenic B cells have no effect. Suppression of B-lineage cells is independent of T lymphocytes but requires that both donor and recipient are matched for immunoglobulin allotypes. These findings provide evidence for regulation of BM B-cell production by peripheral B cells, especially when located in the peritoneal cavity, and ascribe regulatory roles to the peritoneal B-cell compartment. They also could contribute to understanding the control of total B-lymphocyte numbers in the organism.

In B-cell development, expression of immunoglobulin heavy-chain variable-region (VH) gene repertoires is determined by genetic mechanisms that favor rearrangement of the most D-proximal genes, resulting in overutilization of the VH7183 gene family early in ontogeny and in differentiating B cells of the adult bone marrow. Maturation of the immune system is accompanied by a decreased expression of VH7183 genes in the peripheral immunocompetent B-cell pool of adult animals. By comparing VH gene family expression in the bone marrow (emergent) and peripheral (available and actual) B-cell repertoires of germ-free and conventionally raised BALB/c mice, we found that peripheral selection of VH gene family utilization does not occur in germ-free animals. Reconstitution of germ-free mice with normal serum immunoglobulins purified from syngeneic donors reestablishes selection of VH7183-expressing B cells. Our results indicate that preimmune B-cell repertoires are selected in normal animals by environmental antigens and serum immunoglobulins.

The kinetics of postnatal intestinal colonization by T cells carrying gamma delta and alpha beta T-cell antigen receptors were studied in nude and normal mice by flow cytometry and immunohistology. Furthermore, gamma delta and alpha beta T-cell development was analyzed in lethally irradiated mice that were reconstituted by fetal liver precursors with or without a thymus. Our results establish that a major subpopulation of gamma delta intestinal intraepithelial lymphocytes is produced from uncommitted precursors at extrathymic sites. This work further shows that a small pool of T cells carrying alpha beta T-cell receptors can also differentiate extrathymically from CD3- fetal liver precursors but with rates of production and peripheral expansion much reduced as compared with those observed in thymus-bearing animals.