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Immune cells are especially dependent on the proper functioning of the actin cytoskeleton, and both innate and adaptive responses rely on it. Leukocytes need to adhere not only to substrates but also to cells in order to form synapses that pass on instructions or kill infected cells. Neutrophils literally squeeze their cell body during blood extravasation and efficiently migrate to the inflammatory focus. Moreover, the development of immune cells requires the remodeling of their cytoskeleton as it depends on, among other processes, adhesive contacts and migration. In recent years, the number of reports describing cytoskeletal defects that compromise the immune system has increased immensely. Furthermore, a new emerging paradigm points toward a role for the cellular actin content as an essential component of the so-called homeostasis-altering molecular processes that induce the activation of innate immune signaling pathways. Here, we review the role of critical actin-cytoskeleton-remodeling proteins, including the Arp2/3 complex, cofilin, coronin and WD40-repeat containing protein 1 (WDR1), in immune pathophysiology, with a special focus on autoimmune and autoinflammatory traits.

Autoinflammatory diseases are hyperinflammatory, immune dysregulatory diseases caused by innate immune cells dysregulation that present typically in the perinatal period with systemic and organ-targeted inflammation, but with improved genetic testing and the development of diagnostic criteria, milder and later-onset forms are being detected in adulthood. While the discovery of gain-of-function mutations in innate sensors linked to the production of proinflammatory cytokines provided the bases for anti-cytokine therapies that changed disease and patient outcomes, the field is expanding with the increasing discovery of disease-causing loss-of-function mutations in genes with cellular house-keeping functions that affect cell homeostasis and when dysregulated trigger innate inflammatory pathways. This review focuses on updates on molecular pathways and diseases that cause predominantly IL-1β and Type-I IFN-mediated autoinflammatory diseases.

Diarrheic diseases account for the annual death of approximately 1.9 million children under the age of 5 years, and it is a major cause of work absenteeism in developed countries. As diarrheagenic bacteria, enteropathogenic Escherichia coli (EPEC) attach to cells in the small intestine, causing local disappearance of microvilli and inducing the formation of actin-rich pedestals that disrupt the intestinal barrier and help EPEC adhere to and infect intestinal cells. Antibiotics and other bioactive compounds can often be found by analyzing traditional medicines. Here a crude aqueous extract of Hibiscus sabdariffa, which typically grows in subtropical and tropical areas and is a popular medicinal tisane in many countries, was analyzed for antibacterial activity against EPEC. In standard microdilution assays, the extract showed a minimum inhibitory concentration of 6.5 mg/ml against EPEC growth. Time-kill kinetics assays demonstrated significant 24 h bactericidal activity at 25 mg/ml. The extract is able to impede pedestal induction. Not only did the extract inhibit preformed pedestals but it prevented pedestal induction as well. Remarkably, it also promoted the formation of EPEC filaments, as observed with other antibiotics. Our results in vitro support the potential of Hibiscus sabdariffa as an antimicrobial agent against EPEC.

HLA genes (class I and II) have been studied in a Kurd population from Iran (North West towns of Saqqez and Baneh, close to Irak border). Kurds speak an Iranian language. HLA Kurd profile has been compared with those of Central Asians, Siberians, Mediterraneans and other worldwide populations; a total of 7746 chromosomes were used for computer comparisons. Both Neighbor-joining and correspondence genetic analyses place Kurds in the Mediterranean population cluster, close to Iranians, Europeans and Caucasus populations (Svan and Georgian). New extended HLA haplotypes are described, being A*02:01-B*35:01-DRB1*01:01-DQB1*05:01 and A*24:02-B*18:09-DRB1*11:01- DQB1*03:01 the most frequent ones; other Kurd extended haplotypes are also found in Azeris and Palestinians. This research work may be useful for: 1) future Iranian Kurds transplantation regional programs, 2) HLA pharmacogenomics in order to practise a preventive Medicine and drug side effects, and 3) Epidemiology of HLA-associated diseases in Kurds.

Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca2+ signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells.

Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area.

Mother-to-child transmission during pregnancy provides a unique system for studying the correlation between HLA phenotype and susceptibility to HIV infection. We studied this relationship in a Spanish cohort. We determined frequencies of HLA class I and II alleles in 120 infants born to HIV-infected mothers and 67 HIV-infected mothers. Although there was no statistical difference in the frequency of HLA-B35 between transmitting and non-transmitting mothers, the allele was more frequent in infected children than in uninfected children. HLA-B35 has been consistently reported as a risk factor in the progression to AIDS. In addition, it has been proposed that whether a given allele can confer susceptibility to, or protection against, progression depends on maternal versus paternal inheritance patterns, since the child inherits a virus that reflects the history of CTL encounters of the mother. Our results on vertical HIV transmission combine for the first time the 'HLA-B35 disadvantage' and the 'pattern of inheritance' theories.

Nineteen different new MHC-DMB complete cDNA sequences have been obtained in thirteen different individuals belonging to the following primate species/families: Hylobates lar, Papio hamadryas, Macaca mulatta, Macaca fascicularis, Cercopithecus aethiops and Saguinus oedipus . Exonic allelism has been recorded all along the DM molecule domains and analyses of the critical residues in the conformation of the MHC-DR peptide-binding site were done; it was found an evolutionary pressure over the putative peptide-binding region of the DMB molecule that favours synonymous changes. These results are in contrast with the ones found in the MHC class I and class II genes, where non-synonymous DNA base substitutions are favoured The immunoreceptor inhibition motif Tyr230-X231-X232-Leu233 (ITIM) is invariantly present in all extant studied primates since 40 million years ago. It confirms the important function for this molecule, directing DR molecules towards the endosomal/ lysosomal class II compartment and sending inhibitory signals to cells in order to stop synthesis of unnecessary MHC-DR molecules. Some Macaca individuals DMB molecules (appear on Earth more than ten million years ago) dobear both short (without ITIM) and long cytoplasmic tails (with ITIM), similarly to what has been found in human individuals. These differences may have important functional implications. Other molecules, like NK-cell receptors and Fc receptors, bear this type of tyrosine-based inhibitory motifs in order to switch off specific cell functions. MHC-DMB variations occurring within each species suggest that their polymorphism may have an intraspecific evolution, since alleles of the same species cluster together, as it occurs in other MHC related genes (Bf, C4d). Other MHC class I and class II molecules, phylogenetic trees show a trans-species pattern of evolution. Finally, a cluster grouping human and gorilla DMB cDNA sequences is obtained using a dendrogram (for the MHC genes, i.e.: C4d trees); this is in contrast to others' results that obtain a human/chimpanzee cluster using different DNA sequences. Keywords: MHC (Major Histocompatibility Complex), MHC-DMB, HLA-DMB, primates, evolution, ITIM, MHC Class II metabolism, gibbon, macaque, New World Monkeys.

HLA-G polymorphism has been found to be relatively low in all world populations. In the present paper two new HLA-G molecules are described in ancient American natives. A new HLA-G molecule from a Ecuador Amerindian individual (male) showed four codon changes with respect to HLA-G*010101. Silent changes at α1 domain (residue 57, Pro, CCG → CCA) and α2 domain (residue 93, His, CAC → CAT and residue 100, Gly, GGC → GGT) and one productive change in α3 domain (residue 219 changed from Arg to Trp). This α3 change may dramatically alter HLA-G interactions with beta-2 microglobulin, CD8, ILT-2 and ILT-4 ligands present in subsets of T, B, NK, monocytes, macrophages and dentritic cells. Another HLA-G new molecule was found in a woman from Hispaniola Island, Dominican Republic (Sto Domingo) it presented a silent change at α2 domain residue 107, Gly, GGA → GGT and non-silent change at residue 178, Met → Thr (with respect to HLA-G*010101) which is close to class I molecule/clonotypic T cell receptor interaction sites. Functional implications of these findings are discussed. © 2016 American Society for Histocompatibility and Immunogenetics

Background HLA-DMB proteins are important for intracellular microbial metabolism in order other major histocompatibility complex (MHC) molecules present peptides to lymphocytes. In addition, HLA-DMB alleles have been found linked to diseases in some ethnic groups and HLA-DMB molecules may be important to explain HLA disease association. Objective To detect HLA-DMB alleles profile in Amerindians for the first time and compare them to other populations. This will establish the bases to study HLA-DMB linkage to disease in Amerindians. Method A group of 168 voluntary Amerindians have been typed for HLA-DMB alleles. They have been characterized both, by genetic and genealogical bases. Cloning and automated HLA-DMB DNA (exons 2, 3 and 4) sequencing have been performed for allele assignation. Results HLA-DMB*01:01:01 and HLA-DMB*01:03:01 show the highest frequencies. These have been compared to other World wide populations. HLA-DMB*01:03:01 is tightly associated to certain specific HLA-DRB1 alleles in Amerindians. Conclusion The specific Amerindian HLA-DMB allele frequencies and their linkage disequilibrium with other MHC alleles may be crucial to determine HLA-DMB World wide variation, evolution and specific linkage to disease in Amerindians and other populations.

The Wiskott–Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement.

In a sense, the field of intracellular signaling began with the discovery of SH2 domains in 1989 by Tony Pawson’s group (Mayer and Baltimore 1993). Dr. Pawson wrote in a farsighted review in 1991 that “the SH3 domain, together with the SH2, may modulate interactions with the cytoskeleton and membrane” (Koch et al. 1991). This prediction expresses the motivation for the present chapter, which aims to review how SH domain-containing proteins regulate the cytoskeleton, while focusing on how such proteins interact directly with factors that control actin remodeling.

The Crk adaptor family of proteins comprises the alternatively spliced CrkI and CrkII isoforms, as well as the paralog Crk-like (CrkL) protein, which is encoded by a different gene. Initially thought to be involved in signaling during apoptosis and cell adhesion, this ubiquitously expressed family of proteins is now known to play essential roles in integrating signals from a wide range of stimuli. In this review, we describe the structure and function of the different Crk proteins. We then focus on the emerging roles of Crk adaptors during Enterobacteriaceae pathogenesis, with special emphasis on the important human pathogensSalmonella,Shigella,Yersinia, and enteropathogenicEscherichia coli. Throughout, we remark on opportunities for future research into this intriguing family of proteins.

Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). Here we show that WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant (Cdc42(V12)) and by N-WASP. Our results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella.

WIP, the Wiskott-Aldrich syndrome protein-interacting protein, is a human protein involved in actin polymerization and redistribution in lymphoid cells. The mechanism by which WIP reorganizes actin cytoskeleton is unknown. WIP is similar to yeast verprolin, an actin- and myosin-interacting protein required for polarized morphogenesis. To determine whether WIP and verprolin are functional homologues, we analyzed the function of WIP in yeast. WIP suppresses the growth defects of VRP1 missense and null mutations as well as the defects in cytoskeletal organization and endocytosis observed in vrp1-1 cells. The ability of WIP to replace verprolin is dependent on its WH2 actin binding domain and a putative profilin binding domain. Immunofluorescence localization of WIP in yeast cells reveals a pattern consistent with its function at the cortical sites of growth. Thus, like verprolin, WIP functions in yeast to link the polarity development pathway and the actin cytoskeleton to generate cytoskeletal asymmetry. A role for WIP in cell polarity provides a framework for unifying, under a common paradigm, distinct molecular defects associated with immunodeficiencies like Wiskott-Aldrich syndrome.

DRB genes have been studied for the first time in green monkeys (Cercopithecus aethiops). Eleven new DRB alleles (exon 2, exon 3) have been obtained and sequenced from cDNA. A limited number of lineages have been identified: DRB1*03 (4 alleles), DRB1*07(3 alleles), DRB5 (1 allele), DRB*w6 (1 allele), and DRB*w7(2 alleles). The existence of Ceae-DRB1 duplications is supported by the finding of 3 DRB1 alleles in 3 different individuals. Ceae-DRB1 *0701 may be non-functional because it bears serine at position 82, which hinders molecule surface expression in mice; the allele is only found in Ceae-DRB duplicated haplotypes. Base changes in cDNA Ceae-DRB alleles are consistent with the generation of polymorphism by point mutations or short segment exchanges between alleles. The eleven green monkey DRB alleles meet the requirements for functionality as antigen-presenting molecules (perhaps, excluding DRBl*0701), since: 1) they have been isolated from cDNA and do not present deletions, insertions or stop codons: 2) structural motifs necessary for a correct folding of the molecule, for the formation of DR/DR dimers and for CD4 interactions are conserved, and 3) the number of non-synonymous substitutions is higher than the number of synonymous substitutions in the peptide binding region (PBR), while the contrary holds true for the non-PBR region.

The cortical actin network is dynamically rearranged during secretory processes. Nevertheless, it is unclear how de novo actin polymerization and the disruption of the preexisting actin network control transmitter release. Here we show that in bovine adrenal chromaffin cells, both formation of new actin filaments and disruption of the preexisting cortical actin network are induced by Ca2+ concentrations that trigger exocytosis. These two processes appear to regulate different stages of exocytosis; whereas the inhibition of actin polymerization with the N-WASP inhibitor wiskostatin restricts fusion pore expansion, thus limiting the release of transmitters, the disruption of the cortical actin network with cytochalasin D increases the amount of transmitter released per event. Further, the Src kinase inhibitor PP2, and cSrc SH2 and SH3 domains also suppress Ca2+-dependent actin polymerization, and slow down fusion pore expansion without disturbing the cortical F-actin organization. Finally, the isolated SH3 domain of c-Src prevents both the disruption of the actin network and the increase in the quantal release induced by cytochalasin D. These findings support a model where a rise in the cytosolic Ca2+ triggers actin polymerization through a mechanism that involves Src kinases. The newly formed actin filaments would speed up the expansion of the initial fusion pore, whereas the preexisting actin network might control a different step of the exocytosis process.

Sequencing studies of HLA class II molecules have focused almost exclusively on its exon 2. The complete cDNA sequence of the DRB*1302 allele is reported here for the first time. A conservative polymorphism was detected outside the exon 2 at residue 206; these changes can only be recorded if complete cDNA sequences are studied, and may be of value in drawing evolutive inferences.

A diploid expression of class I and class II human leukocyte antigens (HLA) has been found in purified spermatozoa by using double fluorescence labeling cytofluorometry and relevant monoclonal antibodies; this expression has been confirmed for the first time by the analysis of specific HLA mRNA and metabolic 35 S labeling followed by immunoprecipitation, which demonstrates an active ongoing translation of HLA proteins in germinal cells. Long-living mRNA coming from diploid germinal cells may be translated to HLA molecules in spermatozoa. This translation is controlled (or at least inversely correlated) by a testicular hormone (inhibin) in a cyclic fashion. Remarkably, serum levels of inhibin, synthesized by Leydig and Sertoli cells, follow a 12- to 13-day cycle, with a peak level at Day 6; this is probably controlled by FSH (not cyclic in males) and other testicular and/or unknown hormones. Peak levels of inhibin concur with the lower density and percentage of spermatozoa expressing both HLA class I and II molecules (close to 3% by cytofluorometry); lowest levels of inhibin coincide with the highest numbers (35-40%) of spermatozoa positive for both HLA molecules and a higher surface density. These observations could put to an end a disconcerting and long-lasting controversy on the expression/non-expression of HLA antigens on spermatozoa. The possibility that HLA-bearing spermatozoa are more capacitated for fertilization than those that do not bear HLA, and the implications of our results on male fertility control are also discussed.

BACKGROUND: Cortactin is a key regulator of the actin cytoskeleton and is involved in pathogen-host cell interactions. Numerous pathogens exploit the phagocytic process and actin cytoskeleton to infect host cells. Coxiella burnetii, the etiologic agent of Q fever, is internalized by host cells through a molecular mechanism that is poorly understood. METHODOLOGY/PRINCIPAL FINDING: Here we analyzed the role of different cortactin motifs in the internalization of C. burnetii by non-phagocytic cells. C. burnetii internalization into HeLa cells was significantly reduced when the cells expressed GFP-cortactin W525K, which carries a mutation in the SH3 domain that renders the protein unable to bind targets such as N-WASP. However, internalization was unaffected when the cells expressed the W22A mutant, which has a mutation in the N-terminal acidic region that destroys the protein's ability to bind and activate Arp2/3. We also determined whether the phosphorylation status of cortactin is important for internalization. Expression of GFP-cortactin 3F, which lacks phosphorylatable tyrosines, significantly increased internalization of C. burnetii, while expression of GFP-cortactin 3D, a phosphotyrosine mimic, did not affect it. In contrast, expression of GFP-cortactin 2A, which lacks phosphorylatable serines, inhibited C. burnetii internalization, while expression of GFP-cortactin SD, a phosphoserine mimic, did not affect it. Interestingly, inhibitors of Src kinase and the MEK-ERK kinase pathway blocked internalization. In fact, both kinases reached maximal activity at 15 min of C. burnetii infection, after which activity decreased to basal levels. Despite the decrease in kinase activity, cortactin phosphorylation at Tyr421 reached a peak at 1 h of infection. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the SH3 domain of cortactin is implicated in C. burnetii entry into HeLa cells. Furthermore, cortactin phosphorylation at serine and dephosphorylation at tyrosine favor C. burnetii internalization. We present evidence that ERK and Src kinases play a role early in infection by this pathogen.

The HIV epidemic continues to be a major world-wide health and human problem as reflected in the AIDS epidemic updates from the World Health Organization (WHO). Highly active antiretroviral therapy (HAART) has improved health management, especially in developed countries, since it was first introduced in the mid-1990s. However, access to antiretroviral therapy in developing countries has been limited. Developing an effective vaccine is an on-going mission, the success of which depends on understanding key aspects of the immune response to HIV. Hence, studying the genetic components of the immune response to the HIV virus is essential. The existence of genetic factors that modulate immune response to infectious diseases was described more than 10 years ago (Hill, 1998). We recommend various recent and extensive reviews that have focused on the immunobiology of HIV infection (Tripathi and Agrawal, 2007), the immune response to HIV (Chakrabarti and Simon, 2010; Miyazawa, et al. 2008) and specifically the innate response (Borrow and Bhardwaj 2008). Innate and adaptive immune responses play a decisive role during the initial stages of HIV infection and will also affect the progression of the disease. Definitive evidence that host genetics plays a role in the immune response to HIV is the fact that HIV-1 infection does not always progress to AIDS. A small percentage (less than 0.2%) of HIV-1 sero-positive patients is able to control the infection, meaning that they can maintain a viral load of fewer than 50 copies of HIV-1 RNA per ml over 10 years. These patients have been given different names: HIV controllers, HIV elite controllers, long-term non-progressors (LTNPs) or natural virus suppressors (Chakrabarti and Simon, 2010). This chapter focuses on the influence exerted on HIV infection by the human major histocompatibility complex (MHC) system, also known as the human leukocyte antigen (HLA) system. The effect of the MHC system on HIV infection is crucial for development of an effective vaccine. We recommend several reviews on this subject that complement the present monograph (Carrington and O'Brien, 2003; Stephens, 2005; Miyazawa et al. 2008; Kaur and Mehra, 2009; Singh and Spector, 2009). In this chapter, we will first describe the most important and relevant characteristics of the MHC, then we will review the recent

SummaryCell migration and invasion require the coordinated regulation of cytoskeletal architectural changes by signaling factors, including the actin-binding protein cortactin. Bacterial and viral pathogens subvert these signaling factors to promote their uptake, spread and dissemination. We show that the gastric pathogen Helicobacter pylori (Hp) targets cortactin by two independent processes leading to its tyrosine dephosphorylation and serine phosphorylation to regulate cell scattering and elongation. The phosphorylation status of cortactin dictates its subcellular localization and signaling partners. Upon infection, cortactin was found to interact with and stimulate the kinase activity of focal adhesion kinase (FAK). This interaction required the SH3 domain and phosphorylation of cortactin at serine 405 and a proline-rich sequence in FAK. Using Hp as a model, this study unravels a previously unrecognized FAK activation pathway. We propose that Hp targets cortactin to protect the gastric epithelium from excessive cell lifting and ensure sustained infection in the stomach.

Mother-to-child transmission during pregnancy provides a unique system for studying the correlation between HLA phenotype and susceptibility to HIV infection. We studied this relationship in a Spanish cohort. We determined frequencies of HLA class I and II alleles in 120 infants born to HIV-infected mothers and 67 HIV-infected mothers. Although there was no statistical difference in the frequency of HLA-B35 between transmitting and non-transmitting mothers, the allele was more frequent in infected children than in uninfected children. HLA-B35 has been consistently reported as a risk factor in the progression to AIDS. In addition, it has been proposed that whether a given allele can confer susceptibility to, or protection against, progression depends on maternal versus paternal inheritance patterns, since the child inherits a virus that reflects the history of CTL encounters of the mother. Our results on vertical HIV transmission combine for the first time the 'HLA-B35 disadvantage' and the 'pattern of inheritance' theories.

Summary Many Gram-negative pathogenic bacteria possess type-III or type-IV secretion systems to inject ‘effector’ proteins directly into host cells to modulate cellular processes in their favour. A common target is the actin-cytoskeleton linked to the delivery of a single (CagA) effector by Helicobacter pylori and multiple effectors by enteropathogenic Escherichia coli (EPEC) respectively. Here we report co-infection as a powerful strategy for defining effector protein function and mechanisms by which they modulate cellular responses. This is exemplified by our finding that EPEC inhibits H. pylori-induced AGS cell elongation, a disease-related event linked to Rac1 activation. While this inhibitory process is dependent on the translocated Intimin receptor, Tir, and the outer-membrane protein, Intimin, it unexpectedly revealed evidence for Tir signalling independent of Intimin interaction and tyrosine phosphorylation of Tir. Furthermore, the work defined a long awaited role for protein kinase A (PKA)-mediated phosphorylation of Tir at serine-434 and serine-463. Our data are consistent with a model whereby EPEC activates PKA for Tir phosphorylation. Activated PKA then phosphorylates Rac1 at serine-71 associated with reduced GTP-load and inhibited cell elongation. Thus, the co-infection approach is a powerful strategy for defining novel effector function with important implications for characterizing mechanisms and regulatory signalling pathways in bacterial pathogenesis.

Background Cortactin activates the actin-related 2/3 (Arp2/3) complex promoting actin polymerization to remodel cell architecture in multiple processes (e.g. cell migration, membrane trafficking, invadopodia formation etc.). Moreover, it was called the Achilles' heel of the actin cytoskeleton because many pathogens hijack signals that converge on this oncogenic scaffolding protein. Cortactin is able to modulate N-WASP activation in vitro in a phosphorylation-dependent fashion. Thus Erk-phosphorylated cortactin is efficient in activating N-WASP through its SH3 domain, while Src-phosphorylated cortactin is not. This could represent a switch on/off mechanism controlling the coordinated action of both nucleator promoting factors (NPFs). Pedestal formation by enteropathogenic Escherichia coli (EPEC) requires N-WASP activation. N-WASP is recruited by the cell adapter Nck which binds a major tyrosine-phosphorylated site of a bacterial injected effector, Tir (translocated intimin receptor). Tir-Nck-N-WASP axis defines the current major pathway to actin polymerization on pedestals. In addition, it was recently reported that EPEC induces tyrosine phosphorylation of cortactin. Results Here we demonstrate that cortactin phosphorylation is absent on N-WASP deficient cells, but is recovered by re-expression of N-WASP. We used purified recombinant cortactin and Tir proteins to demonstrate a direct interaction of both that promoted Arp2/3 complex-mediated actin polymerization in vitro, independently of cortactin phosphorylation. Conclusion We propose that cortactin binds Tir through its N-terminal part in a tyrosine and serine phosphorylation independent manner while SH3 domain binding and activation of N-WASP is regulated by tyrosine and serine mediated phosphorylation of cortactin. Therefore cortactin could act on Tir-Nck-N-WASP pathway and control a possible cycling activity of N-WASP underlying pedestal formation.

First CanaryIslands(Spain) Inhabitants (“Guanches”) origin has been much debated. Lately, it has come popular the simplistic theory that they came from North Africa. In the present paper, we conclude that not only North Africans but also Iberian/Atlantic Europeans (and possibly others) must have been first Canarians. Debate whether North Africans or Iberians were the first “Guanches” is artificial since Iberian Peninsula-North African genes flow in ancient times was abundant and Iberians share a great part of genetic profile with North Africans. New genetic (HLA) and linguistic data shown in the present paper, is supported by diverse early anthropological and “Guanches” mummies characters which confirm existence of at least two “Guanches” types and a correct interpretation of R1b Y chromosome high frequency in Atlantic Europe (Ireland, British Isles, North Spain,Basque Coast and Portugal), and also, is present in Canary Islands (13.3%). Present paper HLA genes partial data and presence of abundant old Iberian language scripts (which show an easy translation proposal by using Basque) in Fuerteventura and also in Lanzarote and El Hierro Islands suggest that a present day dogma of a hypothetically North African single origin should be changed. Both Atlantic/Europeans and North Africans define origin of Canary Islands first inhabitants. Keywords: Basque, Canary Islands, El Hierro, Fuerteventura, genes, genetic markers, Guanche, HLA, Iberian, Language, Lanzarote, Latin Inscriptions, R1b, R1b1b, Rock scripts, Usko-Mediterranean, Y chromosome.

Sequencing studies of HLA class II molecules have focused almost exclusively on exon 2. In this study the complete cDNA sequence of the DRB1*09012 allele is reported for the first time. This sequence was previously only partially published. In the DR9 antigen, two synonymous allelic variants (DRB1*09011 and 09012) were officially recognized, though it was later found that the first one contained an error and both sequences were, thus, identical. Major histocompatibility complex (MHC) class II molecules are highly polymorphic cell surface glycoproteins that play a central role in the immune response by binding and presenting peptides to helper T lymphocytes (Brown et al., 1993). The use of DNA-based typing methods for the analysis of class II antigens has resulted in the continuous detection of new variants. DNA sequencing of these alleles has, however, focused mainly on exon 2, which codes for the most polymorphic β1 domain of the molecule, and complete cDNA sequences are scarce. A complete cDNA sequence (including all six exons) of the DRB1*09012 allele is reported in this paper for the first time; only its exon 2 sequence has been previously published elsewhere (Bell et al., 1987). HLA-DRB DNA typing by Amplicor low-resolution test (Roche, NJ, USA) of an infant born to an HIV-infected mother yielded an HLA-DR3, DR9 phenotype. To further assess the results, cDNA sequencing of all HLA-DRB alleles was carried out. RNA was obtained from PBLs using the NP-40 protocol. Cells were pelleted and resuspended in 250 μL of lysis buffer (10 mM Tris pH 8.6, 140 mM NaCl, 1.5 mM MgCl2 0.5% NP-40) with RNAsin 1000 U mL–1 (final concentration) (Promega, Madison, WI). After 1 minutes’ incubation at 4 °C, lysates were centrifuged and supernatants were transferred to 250 μL of proteinase K buffer 2X (200 mM Tris pH 7.5, 25 mM EDTA, 300 mM NaCl, 2% SDS) with 200 μg of proteinase K. These samples were vortexed and incubated for 30 min at room temperature. A standard phenol-chloroform extraction was done to obtain whole RNA. cDNA synthesis was performed using a reverse transcription system (Promega, Madison, WI) according to the manufacturer’s protocols, using an oligo (dT)15 primer. The cDNA was subjected to polymerase chain reaction (PCR) amplification using the DRB5'UT and DRB3'UT primers (Corell et al., 1991), in order to obtain the full cDNA sequence. The PCR products from two different amplifications were purified using Qiaquick gel extraction Kit (Qiagen, Germany) and inserted into the pMOSBlue T-vector (Amersham Life Science, UK). Ten clones for each of the two PCR amplifications were sequenced in an Applied Biosystem 373 A DNA sequencer (Foster City, CA), as previously described (Gomez-Casado et al., 1995). To assess base identification, direct (5'-3') and reverse (3'-5') sequencing was done. The complete cDNA sequence (exons 1–6) of the DRB1*09012 allele is described for the first time in the present work (Fig. 1). In the DR9 antigen two synonymous allelic variants, DRB1*09011 and DRB1*09012, were officially recognized. It was later found that the original DRB1*09011 allele, whose sequence was obtained from the DR9 homozygous cell line ISK, contained an error and was finally confirmed as being identical to the DRB1*09012 allele (Naruse et al., 1997). The DRB1*09012 allele presents a GTC codon, which codes for valine, at position 57 of this DRB1 molecule. Aspartate is commonly found at position 57 in most DRB1 molecules and forms a salt bridge with a conserved arginine residue at position 76 of the DRα chain (Brown et al., 1993). Furthermore, this same residue at the HLA-DQβ chain has been found to correlate with protection against insulin-dependent diabetes mellitus (IDDM) in different populations, including the Spanish population (Vicario et al., 1992). The residue 57 lies in the terminal helical β region forming part of the binding site of HLA-DR1 molecules (Brown et al., 1993). Furthermore, residue 57 belongs to pocket 9, and is directly in contact with the foreign peptide during antigen presentation (Stern et al., 1995). Thus, a change in it could influence the repertoire of the peptides bound to the DRB1 molecule. As a comparison, an alignment of the HLA-DRB1*09012 sequence with that of a close HLA-DRB allele (HLA-DRB1*0701) was carried out (see Fig. 1). It was observed that, besides the already well-known changes affecting exon 2, only one further change is detected outside this exon. In codon 217 (exon 4), CTG is found in HLA-DRB1*09012, whereas TTG is present in HLA-DRB1*0701. Both codons, however, code for leucine.

The Arp2/3 complex can be independently activated to initiate actin polymerization by the VCA domain of WASP family members and by the acidic N-terminal and F-actin-binding repeat region of cortactin, which possesses a C-terminal SH3 domain. Cortactin is a target for phosphorylation by Src tyrosine kinases and by serine/threonine kinases that include Erk. Here we demonstrate that cortactin binds N-WASP and WASP via its SH3 domain, induces in vitro N-WASP-mediated actin polymerization, and colocalizes with N-WASP and WASP at sites of active actin polymerization. Erk phosphorylation and a mimicking S405,418D double mutation enhanced cortactin binding and activation of N-WASP. In contrast, Src phosphorylation inhibited the ability of cortactin previously phosphorylated by Erk, and that of S405,418D double mutant cortactin, to bind and activate N-WASP. Furthermore, Y--D mutation of three tyrosine residues targeted by Src (Y421, Y466, and Y482) inhibited the ability of S405,418D cortactin to activate N-WASP. We propose that Erk phosphorylation liberates the SH3 domain of cortactin from intramolecular interactions with proline-rich regions, causing it to synergize with WASP and N-WASP in activating the Arp2/3 complex, and that Src phosphorylation terminates cortactin activation of N-WASP and WASP.

Wiskott-Aldrich syndrome (WAS) is an inherited immune deficiency that is marked by eczema, bleeding and recurrent infections. The lymphocytes and platelets of WAS patients display cytoskeletal abnormalities, and their T lymphocytes show a diminished proliferative response to stimulation through the T-cell receptor-CD3 complex (TCR-CD3). The product of the WAS gene, WAS protein (WASP), binds to the small GTPase Cdc42. Small GTPases of the Rho family are crucial for the regulation of the actin-based cytoskeleton. WASP and its relative NWASP might play an important role in regulating the actin cytoskeleton. Since both WASP and NWASP have the potential to bind to multiple proteins, they might serve as a hub to coordinate the redistribution of many cellular signals to the actin cytoskeleton. In this review, the authors discuss the possible role of WASP/NWASP and of the newly described protein WIP, which interacts with WASP and NWASP, in coupling signals from the T-cell receptor to the actin-based cytoskeleton.

Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization., Author Summary Infections by enteropathogenic Escherichia coli are an important cause of diarrhea linked to high infant mortality. Such bacteria attach to cells and form actin-rich structures called pedestals, which contain many proteins that play unknown functions during pedestal formation. Here we studied two nearly identical forms (isoforms) of Crk adaptor proteins, CrkII and CrkL, during pedestal formation. Eliminating both isoforms from the cell enhanced pedestal formation, while eliminating only one did not, implying that the isoforms are redundant inhibitors of pedestal formation. We also found that Crk proteins bind the bacterial protein Tir, which binds another adaptor, Nck, to promote actin polymerization in pedestals. We propose that Crk adaptor proteins inhibit actin polymerization by competing with Nck binding to Tir. This work opens the door to investigating how Crk adaptor proteins may participate in numerous actin polymerization pathways.

The high polymorphism of the HLA system has been used as a powerful genetic tool to single out individuals and populations. By studying characteristic allele frequencies and extended HLA haplotypes in different populations, it is possible to identify ethnic groups and establish the genetic relationships among them. In the present study, HLA-A, -B, -C, -DR and -DQ typing at the serological/antigenic and the DNA level has been used for the first time to assign specific HLA frequencies and haplotypes to Spaniards and Basques and compare them with frequencies in other populations, particularly with North Africans. Allelic frequencies do not significantly differ between Spaniards and Basques. HLA genetic distances and their respective dendrogram together with the results on complete HLA haplotypes place Basques and Spaniards closer to paleo-North African populations than to other Europeans. This goes in favour of the Basques being a relative genetic isolate coming from the primitive Iberian/paleo-North African people. In addition, a tentative assignment of the most common Spanish HLA haplotypes to the different people who populated Iberia according to historical records has been done.

TAP2 genes are placed within the HLA complex, have limited genetic variability and encode two main groups of peptide transporter proteins, the so-called TAP2*01 alleles, with a short ATP-binding domain, and the TAP2*0201 allele with a long domain. These transporters carry antigenic peptides from cytoplasm across the endoplasmic reticulum membrane to release them into nascent HLA class I molecules, which will then travel towards the plasma membrane. The shorter TAP2*01 alleles are present in 99% of diabetics and 90% of controls; these alleles may add slight, although significant and independent, susceptibility to diabetes, particularly in subjects carrying non-Asp 57 at beta DQ. Moreover, this increased susceptibility is not due to linkage disequilibrium with other HLA markers (i.e.: DR4), which does not exist in our Spanish population.

OBJECTIVE—To study the influence MHC class II and TAP2 alleles exert on systemic lupus erythematosus (SLE) susceptibility and on the clinical and serological manifestations of the disease, in a cohort of Spanish patients. METHODS—HLA-DR serological typing and HLA-DQA, DQB, and TAP2 DNA sequence specific oligotyping, were carried out in 85 unrelated Spanish SLE patients and 186 healthy controls. Autoantibodies detection was carried out by indirect immunofluorescence and counter immunoelectrophoresis. RESULTS—Total SLE group: the frequency of HLA-DR3 and HLA-DQA1*0501 is significantly increased in this group (pc

HLA-DR and DQ alleles have been detected by DNA typing in Ashkenazi and non-Ashkenazi Jews from Israel. Allele frequencies, characteristic DR/DQ linkage disequilibria, population distances and their corresponding dendrogram by using the Neighbor-Joining method were used to study relatedness between Jewish and other Mediterranean and non Mediterranean populations. Closest relatedness is observed between Ashkenazi and non-Ashkenazi Jews, and, in decreasing order, also with Algerians, Spaniards (including Spanish-Basques), French and Italians. Also, particular characteristic Central European alleles are observed in Ashkenazi Jews and Mediterranean/African alleles in non-Ashkenazi Jews. This is consistent with historical data, Jews being an ancient Mediterranean population, who have had a certain degree of admixture with their 2000–3000 years old neighbors in spite of cultural and religious traditions which have preserved identity outside Israel.