Detection of a novel microsporidium with intranuclear localization in farmed Penaeus vannamei from Latin America.

Microsporidia are emerging intracellular parasites of most known animal phyla in all ecological niches. In shrimp aquaculture, the microsporidium Enterocytozoon hepatopenaei (EHP) is a major cause of concern inflicting tremendous losses to shrimp producers in southeast Asia. During a histopathological examination of Penaeus vannamei samples originating in a country from Latin America presenting slow growth, we observed abnormal nuclei in the epithelial cells of the hepatopancreas. A PCR screening of the samples using DNA isolated from paraffin embedded tissues for the SSU rRNA gene of EHP provided a 149 bp amplicon. In situ hybridization using the SSU rRNA gene probe provided a positive signal in the nuclei instead of the cytoplasm. Sequence analysis of the SSU rRNA gene product revealed a 91.3 %, 89.2 % and 85.4 % sequence identity to Enterocytozoon bieneusi, E. hepatopenaei and Enterospora canceri respectively. Furthermore, phylogenetic analysis revealed the newly discovered microsporidium clustered with E. bieneusi. Considering the intranuclear location of the novel microsporidium and the differences in the sequence of the SSU rRNA, we tentatively consider this parasite a new member of the genus Enterospora sp. The pathogenicity and distribution of the shrimp Enterospora sp. are currently unknown. Our future efforts are focused on the characterization and development of diagnostic tools for this parasite to understand if it acts as an emergent pathogen that might require surveillance to prevent its spread.

Medicina de Família e Comunidade, Atenção Primaria e Violência: Formação e ação em Iberoamerica / Family Medicine, Primary Care and Violence: Training and action in Iberoamerica / Medicina de Família e Comunidade, Atenção Primaria e Violência: Formação e ação em Iberoamerica

Objetivo: Identificar la percepción de médicos de familia y comunidad, así como de otros profesionales de Atención Primaria, en los 20 países que conforman la Confederación Iberoamericana de Medicina de Familia (CIMF), sobre las formas de violencia más prevalentes en su país y en las comunidades que asisten. Además, identificar la percepción sobre sus propias capacitación y motivación, además aquellas de los médicos de familia en sus países, para abordar la violencia y contribuir a la cultura de la paz. Métodos: Estudio exploratorio, corte-transversal, de carácter descriptivo y enfoque cuantitativo, realizado en los 20 países miembros de CIMF, entre los meses de septiembre de 2017 a marzo de 2018. Se construyó un instrumento tipo encuesta, a partir de revisión bibliográfica del fenómeno de estudio, discusión y validación con diferentes profesionales de la medicina familiar considerados expertos en el tema. Se divulgó con el apoyo de las diferentes sociedades científicas de Medicina Familiar que componen los 20 países de CIMF, alcanzando 242 respuestas. Resultados: Más del 92% de profesionales consideran carecer de formación suficiente para abordar la violencia en su cotidianidad laboral y solo 24% considera haber recibido formación suficiente en la Cultura de Paz. Por otro lado, es alarmante en la región la percepción de prevalencia de los diversos tipos de violencia desde el punto de vista personal, familiar y comunitario. Conclusiones: Es necesario integrar en la formación de los médicos familiares y profesionales de la Atención Primaria, asimismo en los currículos de pregrado de Medicina, contenidos relacionados con el abordaje de la violencia y la contribución a la cultura de paz para superar la violencia. Es visible la brecha de conocimiento en estos temas por parte de los médicos de familia y demás profesionales que actúan en la Atención Primaria. Por otro lado, es notable el potencial beneficio de tener esos profesionales actuando en esto grave problema de salud por su elevada prevalencia y especialmente considerando su contacto frecuente y longitudinal con las personas, familias y comunidades quienes han sido víctimas de violencia.

Objective: To identify the perception of family and community doctors - as well as other professionals in 20 countries that make up the Ibero-American Confederation of Family Medicine (CIMF) - on the most prevalent forms of violence in their country and in the communities they attend. Also, to identify the perception about their own motivation and ability, as well as that of family physicians from their countries, to address violence and contribute to the culture of peace. Methods: Cross-sectional, exploratory study, descriptive and quantitative approach, carried out in the 20 member countries of CIMF, between the months of September 2017 to March 2018. A survey was designed based on a literature review of the study phenomenon, discussion and validation with different family medicine professionals considered to be experts in the subject. It was disseminated with the support of the different scientific societies of Family Medicine that make up the 20 countries of the CIMF, reaching 242 responses. Results: More than 92% of professionals consider that they lack sufficient training to deal with violence in their daily work and only 24% consider that they have received sufficient training in the Culture of Peace. On the other hand, the perception of prevalence of the different types of violence from the personal, family and community point of view in the region is alarming. Conclusions: It is necessary to integrate in the training of family doctors and primary care professionals, as well as in the undergraduate curricula of Medicine, contents related to the approach to violence and the contribution to the culture of peace to overcome violence. The knowledge gap on these issues is visible by family doctors and other professionals who work in Primary Care. On the other hand, the potential benefit of having these professionals acting in this serious and prevalent health problem is remarkable, especially considering their frequent and longitudinal contact with people, families and communities who have been victims of violence.

Objetivo: Identificar a percepção de médicos de família e comunidade, bem como outros profissionais, em 20 países que compõem a Confederação Ibero-americana de Medicina de Família (CIMF), sobre as formas mais prevalentes de violência em seu país e nas comunidades que atendem. Além disso, identificar a percepção sobre suas próprias motivação e capacitação, além daquelas dos médicos de família de seus países para abordar a violência e contribuir para a cultura da paz. Métodos: Estudo corte-transversal, exploratório, de abordagem descritiva e quantitativa, realizado nos 20 países membros da CIMF entre os meses de setembro 2017 a março de 2018. A pesquisa foi projetada com base em uma revisão da literatura sobre o fenômeno de estudo. Um questionário foi elaborado e validado com diferentes profissionais de medicina de família considerados especialistas no assunto e posteriormente disseminado com o apoio das diferentes sociedades científicas de Medicina de Família que compõem os 20 países do CIMF, alcançando 242 respostas. Resultados: Mais de 92% dos profissionais consideram que não possuem treinamento suficiente para lidar com a violência em seu cotidiano de trabalho e apenas 24% consideram que receberam treinamento suficiente na Cultura de Paz. Por outro lado, a percepção da prevalência, na região, dos diferentes tipos de violência, do ponto de vista pessoal, familiar e comunitário é alarmante. Conclusões: É necessário integrar na formação de médicos de família e os profissionais de cuidados primários, bem como nos currículos de graduação de Medicina, conteúdos relacionados com a abordagem à violência e a contribuição para a cultura da paz para a superação da mesma. A lacuna de conhecimento sobre essas questões é visível pelos médicos de família e outros profissionais que trabalham na Atenção Primára. Por outro lado, é notável, o benefício potencial de ter esses profissionais atuando nesse grave e prevalente problema de saúde, especialmente considerando seu contato frequente e longitudinal com pessoas, famílias e comunidades vítimas de violência.

Expansión de la medicina familiar en América Latina: desafíos y líneas de acción / Expansion of family medicine in Latin America: challenges and lines of action / Expansão da medicina familiar na América Latina: desafios e linhas de ação

RESUMEN Este documento tiene por objetivos resumir los desafíos actuales de la medicina familiar en América Latina y proponer posibles líneas de acción para consolidar su desarrollo. En los últimos 40 años, los sistemas de salud de la Región de las Américas han encarado reformas cuyos resultados fueron negativos en términos de equidad, y la atención primaria de la salud, lejos de ser aquella estrategia destinada a reducirla, se restringió a una política focal y selectiva. En este contexto, las propuestas técnicas de expansión de las plazas de formación en medicina familiar y su inserción en las carreras de Medicina, han carecido de coherencia y de una dirección política clara, por lo que su falta de eficacia puede leerse como un síntoma de estas reformas incompletas. Al respecto, la Confederación Iberoamericana de Medicina Familiar realizó recomendaciones sobre el compromiso político de los gobiernos para asegurar la estructura y el financiamiento necesarios, consolidar el modelo de medicina familiar como mecanismo de instrumentación de la atención primaria de la salud, la jerarquización de los programas de formación, las condiciones laborales de los médicos de familia y la certificación profesional, entre otras. Estas recomendaciones técnicas, sin acción política coherente y oportuna, no serán más exitosas que los intentos previos.

ABSTRACT This article summarizes the current challenges of family medicine in Latin America and proposes possible lines of action to consolidate its development. In the last 40 years, the health systems of the Region of the Americas have faced reforms whose results were negative in terms of equity, and primary health care, far from being a strategy designed to reduce it, was restricted to a selective and focal policy. In this context, the technical proposals for expansion of training positions in family medicine and their insertion in medical careers have lacked consistency and a clear political direction, and thus their lack of effectiveness can be considered a symptom of these incomplete reforms. In this regard, the Ibero-American Confederation of Family Medicine made recommendations on the political commitment of governments to ensure the necessary structure and funding, consolidate the model of family medicine as a mechanism for the implementation of primary health care, the hierarchy of programs of training, the working conditions of family doctors and professional certification, among others. These technical recommendations, without a consistent and timely political action, will not be more successful than previous attempts.

RESUMO Este documento tem como objetivo resumir os desafios atuais da medicina familiar na América Latina e propor possíveis linhas de ação para consolidar seu desenvolvimento. Nos últimos 40 anos, os sistemas de saúde da Região das Américas encararam reformas cujos resultados foram negativos em termos de igualdade, e a atenção primária à saúde, longe de sê-la aquela estratégia destinada a reducir-la, restringiu-se a uma política focal e seletiva. Neste contexto, as propostas técnicas de expansão das praças de formação em medicina familiar e sua inserção nas carreiras de Medicina, não dispuseram de coerência e de uma direção política clara, razão pela qual sua falta de eficácia se pode ler como um sintoma destas reformas incompletas. Neste sentido, a Confederação Ibero-americana de Medicina Familiar realizou recomendações sobre o compromisso político dos governos a fim de assegurar a estrutura e o financiamento necessários, consolidar o modelo de medicina familiar como mecanismo de instrumentação da atenção primária à saúde, a hierarquização dos programas de formação, as condições laborais dos médicos de família e a certificação profissional, entre outras. Estas recomendações técnicas, sem ação política coerente e oportuna, não serão mais exitosos que as tentativas prévias.

Influence of simultaneous liver and peritoneal resection on postoperative morbi-mortality and survival in patients with colon cancer treated with surgical cytoreduction and intraperitoneal hyperthermic chemotherapy. / Influencia de la resección hepática y peritoneal simultánea, en la morbimortalidad y supervivencia de los pacientes con cáncer de colon intervenidos mediante cirugía citorreductora con quimioterapia intraperitoneal hipertérmica.

INTRODUCTION: Cytoreductive surgery plus intraperitoneal hyperthermic chemotherapy (HIPEC) has recently been established as the treatment of choice for selected patients with peritoneal carcinomatosis of colonic origin. Until recently, the simultaneous presence of peritoneal and hepatic dissemination has been considered a contraindication for surgery. The aim of this paper is to analyze the morbidity, mortality and survival of patients with simultaneous peritoneal and hepatic resection with HIPEC for peritoneal carcinomatosis secondary to colon cancer. METHODS: Between January 2010 and January 2015, 61 patients were operated on, 16 had simultaneous peritoneal and hepatic dissemination (group RH+), and 45 presented only peritoneal dissemination (group RH-). RESULTS: There were no differences between the groups in terms of demographic data, length of surgery and extension of peritoneal disease. Postoperative grade III-V complications were significantly higher in the RH+ group (56.3 vs. 26.6%; P=.032). For the whole group, mortality rate was 3.2% (two patients in group RH-, and none in group RH+). Patients with liver resection had a longer postoperative stay (14.4 vs. 23.1 days) (P=.027). Median overall survival was 33 months for RH-, and 36 for RH+ group. Median disease-free survival was 16 months for RH-, and 24 months for RH+ group. CONCLUSIONS: Simultaneous peritoneal cytoreduction and hepatic resection resulted in a significantly higher Clavien grade III-V morbidity and a longer hospital stay, although the results are similar to other major abdominal interventions. The application of multimodal oncological and surgical treatment may obtain similar long-term survival results in both groups.

Targeting of Ly9 (CD229) Disrupts Marginal Zone and B1 B Cell Homeostasis and Antibody Responses.

Marginal zone (MZ) and B1 B cells have the capacity to respond to foreign Ags more rapidly than conventional B cells, providing early immune responses to blood-borne pathogens. Ly9 (CD229, SLAMF3), a member of the signaling lymphocytic activation molecule family receptors, has been implicated in the development and function of innate T lymphocytes. In this article, we provide evidence that in Ly9-deficient mice splenic transitional 1, MZ, and B1a B cells are markedly expanded, whereas development of B lymphocytes in bone marrow is unaltered. Consistent with an increased number of these B cell subsets, we detected elevated levels of IgG3 natural Abs and a striking increase of T-independent type II Abs after immunization with 2,4,6-trinitrophenyl-Ficoll in the serum of Ly9-deficient mice. The notion that Ly9 could be a negative regulator of innate-like B cell responses was supported by the observation that administering an mAb directed against Ly9 to wild-type mice selectively eliminated splenic MZ B cells and significantly reduced the numbers of B1 and transitional 1 B cells. In addition, Ly9 mAb dramatically diminished in vivo humoral responses and caused a selective downregulation of the CD19/CD21/CD81 complex on B cells and concomitantly an impaired B cell survival and activation in an Fc-independent manner. We conclude that altered signaling caused by the absence of Ly9 or induced by anti-Ly9 may negatively regulate development and function of innate-like B cells by modulating B cell activation thresholds. The results suggest that Ly9 could serve as a novel target for the treatment of B cell-related diseases.

Outcomes of an enhanced recovery after surgery programme for pancreaticoduodenectomy.

BACKGROUND: Enhanced recovery after surgery (ERAS) has demonstrated in colorectal surgery a reduction in morbidity and length of stay without compromising security. Experience with ERAS programs in pancreatoduodenectomy (PD) is still limited. The aims of this study were first to evaluate the applicability of an ERAS program for PD patients in our hospital, and second to analyze the postoperative results. METHODS: A retrospective study using a prospectively maintained database identified 41 consecutive PD included in an ERAS program. Key elements studied were early removal of tubes and drainages, early oral feeding and early mobilization. Variables studied were mortality, morbidity, perioperative data, length of stay, re-interventions and inpatient readmission. This group of patients was compared with an historic control group of 44 PD patients with a standard postoperative management. RESULTS: A total of 85 pancreatoduodenectomies were analyzed (41 patients in the ERAS group, and 44 patients in the control group. General mortality was 2.4% (2 patients) belonging to the control group. There were no statistical differences in mortality, length of stay in intensive care, reoperationss, and readmissions. ERAS group had a lower morbidity rate than the control group (32 vs. 48%; P=.072), as well as a lower length of stay (14.2 vs. 18.7 days). All the key ERAS proposed elements were achieved. CONCLUSIONS: ERAS programs may be implemented safely in pancreaticoduodenectomy. They may reduce the length of stay, unifying perioperative care and diminishing clinical variability and hospital costs.

Determination of soluble tumor necrosis factor receptor 2 produced by alternative splicing.

Soluble cytokine receptors have proven to be very useful biomarkers in a large variety of diseases, including cancer, infections, and chronic inflammatory diseases. These soluble receptors are produced by proteolytic cleavage or alternative splicing. Several cytokine receptors including tumor necrosis factor receptor 2 (TNFR2) can be generated by both mechanisms. However, the conventional ELISA systems do not differentiate between these two types of soluble receptors. We describe a sandwich ELISA to specifically quantify soluble TNFR2 protein generated by alternative splicing. This method requires the use of a capturing monoclonal antibody (mAb) specific of an epitope present in the soluble TNFR2 generated by alternatively splicing but absent in the proteolytically generated isoform. Here we present a detailed protocol for the production and validation of such a mAb. This method has the potential to be applied for measuring other soluble cell surface molecules generated by alternative splicing.

Cutting edge: Ly9 (CD229), a SLAM family receptor, negatively regulates the development of thymic innate memory-like CD8+ T and invariant NKT cells.

Signaling lymphocytic activation molecule family receptors and the specific adapter signaling lymphocytic activation molecule-associated protein modulate the development of innate-like lymphocytes. In this study, we show that the thymus of Ly9-deficient mice contains an expanded population of CD8 single-positive cells with the characteristic phenotype of innate memory-like CD8(+) T cells. Moreover, the proportion of these innate CD8(+) T cells increased dramatically postinfection with mouse CMV. Gene expression profiling of Ly9-deficient mice thymi showed a significant upregulation of IL-4 and promyelocytic leukemia zinc finger. Analyses of Ly9(-/-)IL4ra(-/-) double-deficient mice revealed that IL-4 was needed to generate the thymic innate CD8(+) T cell subset. Furthermore, increased numbers of invariant NKT cells were detected in Ly9-deficient thymi. In wild-type mice, IL-4 levels induced by α-galactosylceramide injection could be inhibited by a mAb against Ly9. Thus, Ly9 plays a unique role as an inhibitory cell surface receptor regulating the size of the thymic innate CD8(+) T cell pool and the development of invariant NKT cells.

Signaling lymphocyte activation molecule regulates development of colitis in mice.

BACKGROUND & AIMS: Signaling lymphocyte activation molecule (Slamf)1 is a co-stimulatory receptor on T cells and regulates cytokine production by macrophages and dendritic cells. Slamf1 regulates microbicidal mechanisms in macrophages, therefore we investigated whether the receptor affects development of colitis in mice. METHODS: We transferred CD45RB(hi) CD4(+) T cells into Rag(-/-) or Slamf1(-/-)Rag(-/-) mice to induce colitis. We also induced colitis by injecting mice with an antibody that activates CD40. We determined the severity of enterocolitis based on disease activity index, histology scores, and levels of cytokine production, and assessed the effects of antibodies against Slamf1 on colitis induction. We quantified migration of monocytes and macrophage to inflamed tissues upon induction of colitis or thioglycollate-induced peritonitis and in response to tumor necrosis factor-α in an air-pouch model of leukocyte migration. RESULTS: Colitis was reduced in Slamf1(-/-)Rag(-/-) mice, compared with Rag(-/-) mice, after transfer of CD45RB(hi) CD4(+) T cells or administration of the CD40 agonist. The numbers of monocytes and macrophages were reduced in inflamed tissues of Slamf1(-/-)Rag(-/-) mice, compared with Rag(-/-) mice, after induction of colitis and other inflammatory disorders. An antibody that inhibited Slamf1 reduced the level of enterocolitis in Rag(-/-) mice. CONCLUSIONS: Slamf1 contributes to the development of colitis in mice. It appears to indirectly regulate the appearance of monocytes and macrophages in inflamed intestinal tissues. Antibodies that inhibit Slamf1 reduce colitis in mice, so human SLAMF1 might be a therapeutic target for inflammatory bowel disease.

Cutting edge: Slamf8 is a negative regulator of Nox2 activity in macrophages.

Slamf8 (CD353) is a cell surface receptor that is expressed upon activation of macrophages (MΦs) by IFN-γ or bacteria. In this article, we report that a very high NADPH oxidase (Nox2) enzyme activity was found in Slamf8(-/-) MΦs in response to Escherichia coli or Staphylococcus aureus, as well as to PMA. The elevated Nox2 activity in Slamf8(-/-) MΦs was also demonstrated in E. coli or S. aureus phagosomes by using a pH indicator system and was further confirmed by a reduction in the enzyme activity after transfection of the receptor into Slamf8-deficient primary MΦs or RAW 264.7 cells. Upon exposure to bacteria or PMA, protein kinase C activity in Slamf8(-/-) MΦs is increased. This results in an enhanced phosphorylation of p40phox, one key component of the Nox2 enzyme complex, which, in turn, leads to greater Nox2 activity. Taken together, the data show that, in response to inflammation-associated stimuli, the inducible receptor Slamf8 negatively regulates inflammatory responses.

SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages.

Phagocytosis is a pivotal process by which macrophages eliminate microorganisms after recognition by pathogen sensors. Here we unexpectedly found that the self ligand and cell surface receptor SLAM functioned not only as a costimulatory molecule but also as a microbial sensor that controlled the killing of gram-negative bacteria by macrophages. SLAM regulated activity of the NADPH oxidase NOX2 complex and phagolysosomal maturation after entering the phagosome, following interaction with the bacterial outer membrane proteins OmpC and OmpF. SLAM recruited a complex containing the intracellular class III phosphatidylinositol kinase Vps34, its regulatory protein kinase Vps15 and the autophagy-associated molecule beclin-1 to the phagosome, which was responsible for inducing the accumulation of phosphatidylinositol-3-phosphate, a regulator of both NOX2 function and phagosomal or endosomal fusion. Thus, SLAM connects the gram-negative bacterial phagosome to ubiquitous cellular machinery responsible for the control of bacterial killing.

Mouse CD84 is a pan-leukocyte cell-surface molecule that modulates LPS-induced cytokine secretion by macrophages.

CD84 is 1 of the 9 SLAM family cell-surface receptors involved in leukocyte activation. The CD84 ectodomain is highly glycosylated, and its cytoplasmic tail contains 2 copies of an ITSM, which can be phosphorylated. Here, we report that although mouse CD84 was present on all BM HSCs, its expression declined in developing thymic and BM lymphocytes. However, CD84 expression levels did increase significantly during the later maturation stages and were expressed abundantly on mature B and T cells. Among lymphocyte subsets, the highest expression was found on innate-like lymphocytes; specifically, on NKT and marginal zone B cells. Splenic CD4+ T(FH) cells exhibited higher levels of CD84 compared with the other CD4+ T cell subsets. CD84 was expressed abundantly on monocytes, macrophages, granulocytes, and DCs. Moreover, as the function of CD84 in myeloid cells remains unknown, we focused on the role this receptor plays in mouse macrophage activation. Transfection of CD84 in RAW-264.7 macrophages led to an increase in MAPK phosphorylation and NF-κB activation upon LPS stimulation. Concomitantly, the presence of CD84 increased the LPS-induced secretion of TNF-α and MCP-1 but lowered IL-10 and IL-6 production significantly. This modulatory effect was mediated by Y(300) within the second ITSM of CD84. Additionally, CD84 knock-down decreased TNF-α and IL-6 production in LPS-activated BMDMs. Taken together, these results show that mouse CD84 is a pan-leukocyte receptor, able to modulate signaling pathways downstream of TLR4, and regulates macrophage cell-fate decisions and effector functions.

GITR engagement preferentially enhances proliferation of functionally competent CD4+CD25+FoxP3+ regulatory T cells.

Naturally occurring regulatory T cells (Treg) express high levels of glucocorticoid-induced tumour necrosis factor receptor (GITR). However, studies of the role of GITR in Treg biology has been complicated by the observation that upon activation effector CD4(+) T (Teff) cells also express the receptor. Here, we dissect the contribution of GITR-induced signaling networks in the expansion and function of FoxP3(+) Treg. We demonstrate that a high-affinity soluble Fc-GITR-L dimer, in conjugation with alphaCD3, specifically enhances in vitro proliferation of Treg, which retain their phenotypic markers (CD25 and FoxP3) and their suppressor function, while minimally affecting Teff cells. Furthermore, Fc-GITR-L does not impair Teff susceptibility to suppression, as judged by cocultures employing GITR-deficient and GITR-sufficient CD4(+) T-cell subsets. Notably, this expansion of Treg could also be seen in vivo, by injecting FoxP3-IRES-GFP mice with Fc-GITR-L even in the absence of antigenic stimulation. In order to test the efficacy of these findings therapeutically, we made use of a C3H/HeJ hemophilia B-prone mouse model. The use of liver-targeted human coagulation factor IX (hF.IX) gene therapy in this model has been shown to induce liver toxicity and the subsequent failure of hF.IX expression. Interestingly, injection of Fc-GITR-L into the hemophilia-prone mice that were undergoing liver-targeted hF.IX gene therapy increased the expression of F.IX and reduced the anticoagulation factors. We conclude that GITR engagement enhances Treg proliferation both in vitro and in vivo and that Fc-GITR-L may be a useful tool for in vivo tolerance induction.

SLAM family receptors and the SLAM-associated protein (SAP) modulate T cell functions.

One or more of the signaling lymphocytic activation molecule (SLAM) family (SLAMF) of cell surface receptors, which consists of nine transmembrane proteins, i.e., SLAMF1-9, are expressed on most hematopoietic cells. While most SLAMF receptors serve as self-ligands, SLAMF2 and SLAMF4 use each other as counter structures. Six of the receptors carry one or more copies of a unique intracellular tyrosine-based switch motif, which has high affinity for the single SH2-domain signaling molecules SLAM-associated protein and EAT-2. Whereas SLAMF receptors are costimulatory molecules on the surface of CD4+, CD8+, and natural killer (NK) T cells, they also involved in early phases of lineage commitment during hematopoiesis. SLAMF receptors regulate T lymphocyte development and function and modulate lytic activity, cytokine production, and major histocompatibility complex-independent cell inhibition of NK cells. Furthermore, they modulate B cell activation and memory generation, neutrophil, dendritic cell, macrophage and eosinophil function, and platelet aggregation. In this review, we will discuss the role of SLAM receptors and their adapters in T cell function, and we will examine the role of these receptors and their adapters in X-linked lymphoproliferative disease and their contribution to disease susceptibility in systemic lupus erythematosus.

The SLAM and SAP gene families control innate and adaptive immune responses.

The nine SLAM-family genes, SLAMF1-9, a subfamily of the immunoglobulin superfamily, encode differentially expressed cell-surface receptors of hematopoietic cells. Engagement with their ligands, which are predominantly homotypic, leads to distinct signal transduction events, for instance those that occur in the T or NK cell immune synapse. Upon phosphorylation of one or more copies of a unique tyrosine-based signaling motif in their cytoplasmic tails, six of the SLAM receptors recruit the highly specific single SH2-domain adapters SLAM-associated protein (SAP), EAT-2A, and/or EAT-2B. These adapters in turn bind to the tyrosine kinase Fyn and/or other protein tyrosine kinases connecting the receptors to signal transduction networks. Individuals deficient in the SAP gene, SH2D1A, develop an immunodeficiency syndrome: X-linked lympho-proliferative disease. In addition to operating in the immune synapse, SLAM receptors initiate or partake in multiple effector functions of hematopoietic cells, for example, neutrophil and macrophage killing and platelet aggregation. Here we discuss the current understanding of the structure and function of these recently discovered receptors and adapter molecules in the regulation of adaptive and innate immune responses.

Differential expression of CD150 (SLAM) family receptors by human hematopoietic stem and progenitor cells.

OBJECTIVES: Human hematopoietic stem cell (HSC)-containing grafts are most commonly used to treat various blood diseases, including leukemias and autoimmune disorders. CD150 (SLAM) family receptors have recently been shown to be differentially expressed by mouse HSC and progenitor cells. Members of the CD150 family are key regulators of leukocyte activation and differentiation. The goal of the present study is to analyze the expression patterns of the CD150 receptors CD48, CD84, CD150 (SLAM), CD229 (Ly9), and CD244 (2B4) on the different sources of human hematopoietic stem and progenitor cells. MATERIALS AND METHODS: Expression of CD150 receptors was analyzed on human mobilized peripheral blood CD133(+)-isolated cells and CD34(+) bone marrow (BM) and umbilical cord blood (CB) cells using multicolor flow cytometry. RESULTS: CD244 was present on most CD133(+)Lin(-)-mobilized cells and CD34(+)Lin(-) BM and CB cells, including virtually all CD38(-)Lin(-) primitive progenitor cells. CD48 had a restricted expression pattern on CD133(+)Lin(-)CD38(-) cells, while its levels were significantly higher in CD34(+)Lin(-) BM and CB cells. In addition, CD84 was present on a significant number of CD133(+)Lin(-) cells, but only on a small fraction of CD133(+)Lin(-)CD38(-) peripheral blood mobilized cells. In contrast, CD84 was expressed on practically all CD34(+)Lin(-) BM cells. No CD150 expression was observed in mobilized peripheral blood CD133(+)Lin(-) or CD34(+)Lin(-) BM and CB cells. Furthermore, only a small fraction of CD34(+)Lin(-) BM and CB cells expressed CD229. CONCLUSIONS: Our results show that CD150 family molecules are present on human hematopoietic stem and progenitor cells and that their expression patterns differ between humans and mice.

Cutting edge: MyD88 controls phagocyte NADPH oxidase function and killing of gram-negative bacteria.

MyD88 is an adaptor protein for the TLR family of proteins that has been implicated as a critical mediator of innate immune responses to pathogen detection. In this study, we report that MyD88 plays a crucial role in killing Gram-negative bacteria by primary macrophages via influencing NADPH oxidase function. Peritoneal macrophages from MyD88-/- mice exhibited a marked inability to kill Escherichia coli (F18) or an attenuated strain of Salmonella typhimurium (sseB) in vitro. This defect in killing was due to diminished NADPH oxidase-mediated production of superoxide anion in response to bacteria by MyD88-/- phagocytes as a consequence of defective NADPH oxidase assembly. Defective oxidase assembly in MyD88-deficient macrophages resulted from impaired p38 MAPK activation and subsequent phosphorylation of p47phox. Together these data demonstrate a pivotal role for MyD88 in killing Gram-negative bacteria via modulation of NADPH oxidase activity in phagocytic cells.

The adaptor protein 3BP2 binds human CD244 and links this receptor to Vav signaling, ERK activation, and NK cell killing.

Adaptor proteins, molecules that mediate intermolecular interactions, are crucial for cellular activation. The adaptor 3BP2 has been shown to positively regulate NK cell-mediated cytotoxicity. In this study we present evidence for a physical interaction between 3BP2 and the CD244 receptor. CD244, a member of the CD150 family, is a cell surface protein expressed on NK, CD8+ T, and myeloid cells. CD244 interacts via its Src homology 2 domain with the X-linked lymphoproliferative disease gene product signaling lymphocytic activation molecule-associated protein (SAP)/SH2 domain protein 1A. 3BP2 interacts with human but not murine CD244. CD244-3BP2 interaction was direct and regulated by phosphorylation, as shown by a three-hybrid analysis in yeast and NK cells. Tyr337 on CD244, part of a consensus motif for SAP/SH2 domain protein 1A binding, was critical for the 3BP2 interaction. Although mutation of Tyr337 to phenylalanine abrogated human 3BP2 binding, we still observed SAP association, indicating that this motif is not essential for SAP recruitment. CD244 ligation induced 3BP2 phosphorylation and Vav-1 recruitment. Overexpression of 3BP2 led to an increase in the magnitude and duration of ERK activation, after CD244 triggering. This enhancement was concomitant with an increase in cytotoxicity due to CD244 ligation. However, no differences in IFN-gamma secretion were found when normal and 3BP2-transfected cells were compared. These results indicate that CD244-3BP2 association regulates cytolytic function but not IFN-gamma release, reinforcing the hypothesis that, in humans, CD244-mediated cytotoxicity and IFN-gamma release involve distinct NK pathways.

Characterization of antibodies submitted to the B cell section of the 8th Human Leukocyte Differentiation Antigens Workshop by flow cytometry and immunohistochemistry.

The aim of this study was to characterize the reactivity of monoclonal antibodies (mAbs) that had been submitted to the HLDA8 Workshop. The lineage specificity of target molecules was tested by analyzing their expression patterns on blood cells, leukocytes, and lymphocyte subsets. The expression of target molecules during B cell development, ranging from early precursors to plasma cells, was analyzed using a large panel of B cell lines. Our results have permitted us to characterize the expression of 10 new CD molecules: CD316 (HM1.24, BST2), CD268 (BAFF-R, TNFRSF13C), CD269 (BCMA, TNFRF17), CD267 (TACI, TNFRSF13B), CD275 (ICOSL, B7H2), CD254 (TRANCE, TNFSF11), CD252 (OX40L TNFSF4), CD315 (CD9-P), CD316 (EWI-2, PGRL), and CD307 (IRTA-2 or FcRH5). Three of these new CDs, CD267, CD269, and CD307 presented a B cell-restricted expression pattern. MAbs against these novel cell-surface molecules may offer new tools for research, diagnosis, and therapy.

CD229 (Ly9) lymphocyte cell surface receptor interacts homophilically through its N-terminal domain and relocalizes to the immunological synapse.

CD229 is a member of the CD150 family of the Ig superfamily expressed on T and B cells. Receptors of this family regulate cytokine production and cytotoxicity of lymphocytes and NK cells. The cytoplasmic tail of CD229 binds to SAP, a protein that is defective in X-linked lymphoproliferative syndrome. To identify the CD229 ligand, we generated a soluble Ig fusion protein containing the two N-terminal extracellular domains of human CD229 (CD229-Ig). CD229-Ig bound to CD229-transfected cells, whereas no binding was detected on cells expressing other CD150 family receptors, showing that CD229 binds homophilically. Both human and mouse CD229 interacted with itself. Domain deletion mutants showed that the N-terminal Ig-domain mediates homophilic adhesion. CD229-CD229 binding was severely compromised when the charged amino acids E27 and E29 on the predicted B-C loop and R89 on the F-G loop of the N-terminal domain were mutated to alanine. In contrast, one mutation, R44A, enhanced the homophilic interaction. Confocal microscopy image analysis revealed relocalization of CD229 to the contact area of T and B cells during Ag-dependent immune synapse formation. Thus, CD229 is its own ligand and participates in the immunological synapse.