Leukocyte Adhesion Deficiency Research Articles

The spectrum of primary immunodeficiencies at a tertiary care hospital in Pakistan

BackgroundPrimary Immunodeficiency Disorders (PIDs) are well-known disorders in the West. but the recognition and diagnosis of these disorders is challenging in developing countries. We present the spectrum of PIDs seen at a tertiary care center in Pakistan, identified using clinical case definitions and molecular methods. MethodsA retrospective chart review of children suspected to have PID was conducted at the Aga Khan University Hospital (AKUH) Karachi, Pakistan from 2010 to 2016. Data on demographics, clinical features, family history of consanguinity, sibling death, details of laboratory workup done for PID and molecular tests targeted panel next generation sequencing (NGS) or whole exome sequencing (WES) performed at the Geha laboratory at Boston Children’s Hospital, USA was collected. The study was exempted from the Ethical Review Committee of AKUH. ResultsA total of 43 children visited the hospital with suspected PID during the study period. Genetic testing was performed in 31/43 (72.1%) children. A confirmed diagnosis of PID was established in 20/43 (46.5%) children. A pathogenic gene variant was identified in 17(85%) of the 20 confirmed cases (Table 1). Twelve (60%) of the confirmed cases of PID were male. The most common presenting symptom was recurrent diarrhea 11/20 (55%). The mean (±S.D) age of the cases at the time of diagnosis was 4.2 (±4.1) years. Chronic granulomatous disease (CGD) was the most common 6/20 (30%) disorder, followed by severe combined immunodeficiency (SCID) 3/20 (15%), leukocyte adhesion deficiency (LAD) 3/20 (15%), agammaglobulinemia/hypogammaglobulinemia 3/20 (15%), and Hermansky-Pudlak Syndrome (HPS) 2/20 (10%). Wiskott-Aldrich Syndrome, Immunodeficiency Centromeric Instability and Facial Anomalies Syndrome (ICF 2), Trichohepatoenteric syndrome (TRES), and C3 deficiency were each diagnosed once {1/20 (4.3%) each} (Table 1). Of these 20 confirmed cases, almost all 19/20 (95%) had a family history of consanguinity. Sibling death was reported in 5/20 (25%) of these cases. Five out of the 20 (25%) children died over the 7-year period for various reasons. ConclusionPIDs are not uncommon in Pakistan; their diagnosis may be missed or delayed due to the overlapping of clinical features of PID with other diseases and a lack of diagnostic facilities. There is a need to build capacity for early recognition and diagnosis of PIDs to decrease morbidity and mortality.

Leukocyte adhesion deficiency type1 in Algeria

Leukocyte adhesion deficiency type I (LAD-I) is an autosomal recessive disease characterized by impaired leukocyte diapedesis to sites of infection. The disorder is caused by mutations in the ITGB2 (integrin beta-2) gene. Herein, we report the first series of Algerian patients with LAD-I. In this study, we enrolled twelve patients. All suffered from the severe form of the disease, with no significant delay in umbilical cord separation, except for two patients, however omphalitis was present in most cases. The analysis of the spectrum of mutations showed three different homozygous mutations, two were known mutations (missense mutation: c.533C˃T and nonsense mutation: c.562C > T) and one was novel nonsense mutation (c.1401C > A). Thus, the three mutations affect CD18 and are responsible for the severe form of the disease.

Frontline Science: Activation of metabolic nuclear receptors restores periodontal tissue homeostasis in mice with leukocyte adhesion deficiency-1.

β2 Integrins mediate neutrophil-endothelial adhesion and recruitment of neutrophils to sites of inflammation. The diminished expression of β2 integrins in patients with mutations in the ITGB2 (CD18) gene (leukocyte adhesion deficiency-Type 1; LAD1) results in few or no neutrophils in peripheral tissues. In the periodontium, neutrophil paucity is associated with up-regulation of IL-23 and IL-17, which drive inflammatory bone loss. Using a relevant mouse model, we investigated whether diminished efferocytosis (owing to neutrophil scarcity) is associated with LAD1 periodontitis pathogenesis and aimed to develop approaches to restore the missing efferocytosis signals. We first showed that CD18-/- mice phenocopied human LAD1 in terms of IL-23/IL-17-driven inflammatory bone loss. Ab-mediated blockade of c-Mer tyrosine kinase (Mer), a major efferocytic receptor, mimicked LAD1-associated up-regulation of gingival IL-23 and IL-17 mRNA expression in wild-type (WT) mice. Consistently, soluble Mer-Fc reversed the inhibitory effect of efferocytosis on IL-23 expression in LPS-activated Mϕs. Adoptive transfer of WT neutrophils to CD18-/- mice down-regulated IL-23 and IL-17 expression to normal levels, but not when CD18-/- mice were treated with blocking anti-Mer Ab. Synthetic agonist-induced activation of liver X receptors (LXR) and peroxisome proliferator-activated receptors (PPAR), which link efferocytosis to generation of homeostatic signals, inhibited the expression of IL-23 and IL-17 and favorably affected the bone levels of CD18-/- mice. Therefore, our data link diminished efferocytosis-associated signaling due to impaired neutrophil recruitment to dysregulation of the IL-23-IL-17 axis and, moreover, suggest LXR and PPAR as potential therapeutic targets for treating LAD1 periodontitis.

Diapedesis and LFA-1 mediate tissue immune cell effector activity via intrinsic complement C3 licensing

Abstract Intracellularly generated and autocrine-functioning complement component C3 is a critical integral part of normal human CD4+ Th1 and cytotoxic CD8+ T cells responses. Increased or decreased intracellular C3 result in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression are, however, undefined. By comparing transcriptomes from blood and tissue, we identified the complement system, including C3, as one of the most significantly enriched biological pathways in tissue-occupying cells of human macrophages, CD4+ and CD8+ T cells. By leveraging a novel C3 reporter mouse, we confirmed that C3 gene transcription is a feature of immune cells in tissues, is induced during trans-endothelial diapedesis, and is dependent on the integrin intercellular adhesion molecule (ICAM)-1 liganding lymphocyte function-associated antigen (LFA)-1. Consequently, monocytes and T cells from patients with leukocyte adhesion deficiency (LAD)-1 had reduced C3 and diminished effector activities, which could be rescued proportionally by normalization of intracellular C3. In synovia of patients with rheumatoid arthritis (RA), C3 transcript expression by CD4+ T cells was linearly associated with disease severity and acted as a biomarker distinguishing inflamed versus uninflamed RA. Our study defines the integrin network as a novel and key controller of intracellular complement, demonstrates that perturbations in the LFA-1–C3 axis contribute to primary human immunodeficiency and identifies T cell C3 production as a biomarker of the severity of autoimmunity.

Recruitment of kindlin-3 to plasma membrane through its PH domain precedes high affinity β2 integrin activation and neutrophil arrest

Abstract Leukocytes including neutrophils must adhere to the vascular endothelium in order to respond to injury or infection. Integrin-mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin-3 is required for leukocyte adhesion; mutations of kindlin-3 cause leukocyte adhesion deficiency type III syndrome. However, the molecular mechanism by which kindlin-3 regulates β2 integrin activation is unknown. Here we measured the spatiotemporal dynamics of kindlin-3 and β2 integrin conformation changes during neutrophil and HL-60 cell rolling and arrest under flow. Using high-resolution quantitative dynamic footprinting (qDF) microscopy and kindlin-3-fluorescent protein (FP) fusion proteins, we find that kindlin-3 is recruited to the plasma membrane prior to induction of the H+ β2 integrin conformation. Deletion of kindlin-3 or its lipid-binding pleckstrin homology (PH) domain completely abolished H+ β2 integrin induction. Intravital imaging of bone marrow chimeric mice revealed that EGFP-tagged kindlin-3-expressing neutrophils, but not control kindlin-3-deficient neutrophils, arrested in cremaster venules in vivo in response to CXCL1. Our data show that kindlin-3 is indispensable for the H+ β2 integrin conformation and crucial for integrin-mediated leukocyte adhesion during inflammation.

Distinct Contributions of CD18 Integrins for Binding and Phagocytic Internalization of Pseudomonas aeruginosa

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.

Recruitment of kindlin‐3 to plasma membrane through its PH domain precedes high affinity β2 integrin activation and neutrophil arrest

Leukocytes including neutrophils must adhere to the vascular endothelium in order to respond to injury or infection. Integrin‐mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin‐3 is required for leukocyte adhesion; mutations of kindlin‐3 cause leukocyte adhesion deficiency type III syndrome. However, the molecular mechanism by which kindlin‐3 regulates β2 integrin activation is unknown. Here we measured the spatiotemporal dynamics of kindlin‐3 and β2 integrin conformation changes during neutrophil and HL‐60 cell rolling and arrest under flow. Using high‐resolution quantitative dynamic footprinting (qDF) microscopy and kindlin‐3‐fluorescent protein (FP) fusion proteins, we find that kindlin‐3 is recruited to the plasma membrane prior to induction of the H+ β2 integrin conformation. Deletion of kindlin‐3 or its lipid‐binding pleckstrin homology (PH) domain completely abolished H+ β2 integrin induction. Intravital imaging of bone marrow chimeric mice revealed that EGFP‐tagged kindlin‐3‐expressing neutrophils, but not control kindlin‐3‐deficient neutrophils, arrested in cremaster venules in vivo in response to CXCL1. Our data show that kindlin‐3 is indispensable for the H+ β2 integrin conformation and crucial for integrin‐mediated leukocyte adhesion during inflammation.Support or Funding InformationThis work was supported by grants from the National Institutes of Health, USA (HL078784 to K.L., R01HL145454 to Z.F.), a Postdoctoral Fellowship (19POST34450228 to L.W.) and a Career Development Award (18CDA34110426 to Z.F.) from the American Heart Association, USA.

Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic Tcell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immunecells, including Tcells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by Tcells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

MSCs rescue impaired wound healing in a murine LAD1 model by adaptive responses to low TGF‐β1 levels

Mutations in the CD18 gene encoding the common β‐chain of β2 integrins result in impaired wound healing in humans and mice suffering from leukocyte adhesion deficiency syndrome type 1 (LAD1). Transplantation of adipose tissue‐derived mesenchymal stem cells (MSCs) restores normal healing of CD18−/− wounds by restoring the decreased TGF‐β1 concentrations. TGF‐β1 released from MSCs leads to enhanced myofibroblast differentiation, wound contraction, and vessel formation. We uncover that MSCs are equipped with a sensing mechanism for TGF‐β1 concentrations at wound sites. Low TGF‐β1 concentrations as occurring in CD18−/− wounds induce TGF‐β1 release from MSCs, whereas high TGF‐β1 concentrations suppress TGF‐β1 production. This regulation depends on TGF‐β receptor sensing and is relayed to microRNA‐21 (miR‐21), which subsequently suppresses the translation of Smad7, the negative regulator of TGF‐β1 signaling. Inactivation of TGF‐β receptor, or overexpression or silencing of miR‐21 or Smad7, abrogates TGF‐β1 sensing, and thus prevents the adaptive MSC responses required for tissue repair.

Successful hematopoietic stem cell transplant in leukocyte adhesion deficiency type III presenting primarily as malignant infantile osteopetrosis

Leukocyte adhesion deficiency type III (LAD-III) is caused by mutations in FERMT3 that encodes Kindlin-3 which regulates integrins activation. LAD-III predisposes to infections and bleeding. Osteopetrosis was reported in some cases. We report three patients who presented as malignant infantile osteopetrosis. One had recurrent infections and none had bleeding. Exome sequencing revealed a novel homozygous mutation in FERMT3 c.1555C > T (p.Gln519Ter). Two patients underwent successful hematopoietic stem cell transplant (HSCT) from matched siblings with resolution of osteopetrosis. The third patient died secondary to sepsis prior to HSCT. Our results support early HSCT in LAD-III prior to the occurrence of life-threatening complications.

β2 Integrins-Multi-Functional Leukocyte Receptors in Health and Disease.

β2 integrins are heterodimeric surface receptors composed of a variable α (CD11a-CD11d) and a constant β (CD18) subunit and are specifically expressed by leukocytes. The α subunit defines the individual functional properties of the corresponding β2 integrin, but all β2 integrins show functional overlap. They mediate adhesion to other cells and to components of the extracellular matrix (ECM), orchestrate uptake of extracellular material like complement-opsonized pathogens, control cytoskeletal organization, and modulate cell signaling. This review aims to delineate the tremendous role of β2 integrins for immune functions as exemplified by the phenotype of LAD-I (leukocyte adhesion deficiency 1) patients that suffer from strong recurrent infections. These immune defects have been largely attributed to impaired migratory and phagocytic properties of polymorphonuclear granulocytes. The molecular base for this inherited disease is a functional impairment of β2 integrins due to mutations within the CD18 gene. LAD-I patients are also predisposed for autoimmune diseases. In agreement, polymorphisms within the CD11b gene have been associated with autoimmunity. Consequently, β2 integrins have received growing interest as targets in the treatment of autoimmune diseases. Moreover, β2 integrin activity on leukocytes has been implicated in tumor development.

Successful umbilical cord blood transplantation in children with leukocyte adhesion deficiency type I.

This study aims to investigate the efficacy and safety of umbilical cord blood transplantation (UCBT) without serotherapy for treating children with leukocyte adhesion deficiency type I (LAD-I). Clinical characteristics and data of five children with LAD-I who underwent UCBT at our hospital between September 2016 and September 2018 were retrospectively analyzed. Five (two boys and three girls) patients with LAD-I were included. The median age at UCBT was 9 months (range, 8 to 32 months). The same myeloablative conditioning regimen was administered for each patient and included busulfan, fludarabine, and cyclophosphamide. HLA matching of patients and umbilical cord blood was 8/10 to 10/10. The median dose of total nucleated cells (TNC) infused was 10.2×107/kg (range, 4.5×107 to 20.6×107/kg) and the median dose of CD34+ cells was 3.2×105/kg (range, 1.9×105 to 5.7×105/kg). The median time of neutrophil engraftment was 20 days (range, 13 to 28 days). The median time of platelet engraftment was 36 days (range, 32 to 56 days). All patients received complete donor chimerism (CDC). Four of the five patients developed grade II-IV acute graft-versus-host disease (GvHD). The median follow-up time after transplantation was 19 months (range, 8 to 38 months). Four of the patients survived and achieved complete clinical remission. The other patient died of bronchiolitis obliterans 8 months after UCBT. UCBT is an effective treatment method for LAD-I patients. Also, severe LAD-I patients should undergo stem cell transplantation as early as possible.

Parvovirus B19-Associated Graft Failure after Allogeneic Hematopoietic Stem Cell Transplantation

Background Parvovirus B19 (PVB19) is a single-stranded DNA virus that infects erythroid progenitor cells, classically causing a pure red cell aplasia, but sometimes causing fulminant pancytopenia. PVB19 has been implicated in solid organ allograft failure, but no such literature exists for allogeneic hematopoietic stem cell transplantation (allo-HSCT). PVB19 may be an underappreciated complication in allo-HSCT. Methods Retrospective report of two pediatric patients diagnosed with PVB19 infection and allo-HSCT graft failure. Results Case #1: 1 year-old male with leukocyte adhesion deficiency (LAD) received reduced intensity conditioning with alemtuzumab, fludarabine, thiotepa, and melphalan, followed by a 10/10 HLA- matched, ABO-compatible, sibling donor bone marrow graft (4.85 × 108 TNC/kg and 9.74 × 106 CD34+ cells/kg). He achieved neutrophil and platelet engraftment by days +23 and +25, respectively. Day +29 chimerism was 100% donor unseparated. Patient had biopsy-proven acute GI GVHD at day +77, responsive to systemic steroids. Evaluation for unexplained transfusion-dependent normocytic normochromic anemia at day +88 revealed PVB19 by PCR (3900 copies/mL). Patient had history of intermittent, non-palpable, and non-specific blanching erythematous rash. At day +105, donor chimerism decreased to 9% unseparated and 5% myeloid engraftment. Flow cytometry revealed repopulation of original LAD phenotype, confirming secondary graft failure. The patient received a second allo-HSCT using the same donor at day +225. Case #2: 13 year-old male with acute myeloid leukemia received myeloablative conditioning with fludarabine and busulfan followed by a T cell-replete, 5/10 HLA-mismatched, ABO-compatible, sibling bone marrow graft (3.60 × 108 TNC/kg and 4.51 × 106 CD34+ cells/kg). GVHD prophylaxis included post-transplant cyclophosphamide, tacrolimus, and mycophenolate mofetil. He achieved neutrophil engraftment by day +16. Patient had non-specific blanching rash with bone pain during the post-HSCT recovery period. Patient had low level CMV viremia treated with ganciclovir and valganciclovir early post-HSCT. Day +26 donor chimerism showed 89% unseparated and 100% myeloid engraftment. Patient developed pancytopenia unresponsive to G-CSF. Bone marrow exam on day +40 showed mixed chimerism, no recurrent AML, but detectable PVB19 by PCR. Blood PVB19 PCR was positive (312 copies/mL). The patient was diagnosed with secondary graft failure with persistent severe pancytopenia. He received a rescue allo-HSCT from the same donor at day + 69. Conclusions There are many risk factors and potential confounders in determining the exact etiology of graft failure after allo-HSCT. These two cases highlight the importance of including PVB19 PCR in the diagnostic evaluation for graft failure. PVB19 infection may be an important risk factor for allo-HSCT graft failure.

Flow Cytometry for the Diagnosis of Primary Immunodeficiency Diseases: A Single Center Experience

PurposeWhile there is an urgent need for diagnosis and therapeutic intervention in patients with primary immunodeficiency diseases (PIDs), current genetic tests have drawbacks. We retrospectively reviewed the usefulness of flow cytometry (FCM) as a quick tool for immunophenotyping and functional assays in patients suspected to have PIDs at a single tertiary care institute.MethodsBetween January 2001 and June 2018, patients suspected of having PIDs were subjected to FCM tests, including lymphocyte subset analysis, detection of surface- or intracellular-target proteins, and functional analysis of immune cells, at Samsung Medical Center, Seoul, Korea. The genetic diagnosis was performed using Sanger or diagnostic exome sequencing.ResultsOf 60 patients diagnosed with definite or probable PID according to the European Society of Immune Deficiencies criteria, 24 patients were provided with useful information about immunological dysfunction after initial FCM testing. In 10 patients, the PID diagnosis was based on abnormal findings in FCM testing without genetic tests. The FCM findings provided strong evidence for the diagnosis of severe combined immunodeficiency (n = 6), X-linked chronic granulomatous diseases (CGD) (n = 6), leukocyte adhesion deficiency type 1 (n = 3), X-linked agammaglobulinemia (n = 11), autoimmune lymphoproliferative syndrome-FASLG (n = 1), and familial hemophagocytic lymphohistiocytosis type 2 (n = 1), and probable evidence for autosomal recessive-CGD (n = 2), autosomal dominant-hyper-immunoglobulin E (IgE)-syndrome (n = 1), and STAT1 gain-of-function mutation (n = 1). In PIDs derived from PIK3CD (n = 2), LRBA (n = 2), and CTLA4 mutations (n = 3), the FCM test provided useful evidence of immune abnormalities and a tool for treatment monitoring.ConclusionsThe initial application of FCM, particularly with known protein targets on immune cells, would facilitate the timely diagnosis of PIDs and thus would support clinical decisions and improve the clinical outcome.

Bombay and Lewis phenotype testing in correlation with leukocyte adhesion deficiency type II: The blood bank's role in diagnosis

Leukocyte adhesion deficiency type II (LAD II) is a rare hereditary disorder caused by the mutation in the guanosine diphosphate-fucose transporter gene (SLC35C1). LAD II is characterized by inexpression of ABO antigens (i.e., Bombay phenotype) and the lack of Lewis red blood cell (RBC) antigens. In addition to high neutrophil counts, patients also manifest recurrent infections with developmental delays and stunted growth. Blood bank laboratory tests such as blood grouping, RBC phenotyping, antibody screening and identification of the nature of the antibody were conducted in order to confirm the consequences associated with this gene anomaly in this case report. Type and screen results initially identified the patient as group “O” Rh positive with pan-reactive antibody screening cells and identification panel cells. The patient plasma showed significant incompatibility to all O-positive donors' RBCs. The patient's RBCs were tested against anti-H lectin and Lewis (Lea, Leb) typing antisera and showed no reaction, which meant that the patient does not express neither Lewis antigens nor H antigen on his red cell. The presence of anti-H (in a Bombay phenotype individual) explained the reactivity to reagent red cells and crossmatched incompatibility of Group O units as these cells naturally contained the H antigen.

PYROSEQUENCING: ITS POTENTIAL AND LIMITATIONS IN DIAGNOSIS OF INHERITED DISEASES IN CATTLE

A large number of diff erent groups of livestock diseases in which heredity plays a signifi cant role are currently reported. In particular, more than 500 genetically determined morphological and functional disorders have been detected in cattle; for 150 of them, specifi c mutations are known. The sale of bovine genetic materials is associated with the spread of various diseases caused by mutations that occur in the prominent representatives of breeds. The abundance of lethal mutations in the populations requires a broader application of molecular diagnostic methods for detection of monogenic hereditary diseases. DNA pyrosequencing, being the most convenient technique for the rapid diagnosis of single nucleotide mutations in the bovine genome that are located in the regions with known nucleotide sequence, has a potential for meeting this need. Pyrosequencing-based methods for identifi cation of the most common signifi cant mutations in Holstein, Simmental, Brown Swiss and Aberdeen Angus cattle were developed, validated and approved at the FGBU “VGNKI”. Such mutations are associated with leukocyte adhesion defi ciency, complex vertebral malformation, uridine monophosphate synthetase defi ciency, citrullinemia, spinal muscular atrophy, spinal cord demyelination, Brown Swiss haplotype 2, Weaver syndrome, developmental duplications, α-mannosidosis, dwarfi sm, bovine male subfertility, trombocytopathya, arachnomelia syndrome, hypozincemia-like syndrome. These methods are intended to test semen in the breeding centres, scientifi c laboratories working in the fi eld of biotechnology and animal reproduction, livestock reproduction centres. The use of the proposed genetic tests to detect mutant alleles, as well as reduced use of mutation-bearing animals in stock breeding will allow minimizing the occurrence of inherited diseases and thus improving the gene pool of cattle in the country.

Neutrophil Defects and Diagnosis Disorders of Neutrophil Function: An Overview.

Primary disorders of neutrophil function result from impairment in neutrophil responses that are critical for host defense. This chapter summarizes inherited disorders of neutrophils that cause defects in neutrophil adhesion, migration, and oxidative killing. These include the leukocyte adhesion deficiencies, actin defects and other disorders of chemotaxis, hyperimmunoglobulin E syndrome, Chédiak-Higashi Syndrome, neutrophil specific granule deficiency, chronic granulomatous disease, and myeloperoxidase deficiency. Diagnostic tests and treatment approaches are also summarized for each neutrophil disorder.

Gene Therapy for Lad-I Immunodeficiency: Preclinical Evaluation of HSC Transduction Under Optimized GMP-Conditions

The efficacy of lentiviral gene therapy for the treatment of primary immunodeficiencies (PIDs) has been extensively demonstrated in diseases such as X1-SCID, ADA-SCID, X-CGD and WAS. Leukocyte Adhesion Deficiency type I (LAD-I) has also emerged as a PID that is potentially correctable by gene therapy. To this aim during the recent years we have completed all the pre-clinical work required for the initiation of a gene therapy trial for patients with severe LAD-I. Mutations in the ITGB2 gene (encoding for the β2 subunit of integrins, also known as CD18) impair leukocyte extravasation to inflamed areas. LAD-I patients thus suffer from recurrent and life-threatening bacterial and fungal infections. We have previously demonstrated that the ectopic expression of CD18 driven by a myeloid chimeric promoter in LAD-I leukocytes restores their ability to migrate to inflamed sites. Because the efficacy of hematopoietic gene therapy relies on the stable engraftment of gene-corrected HSCs in treated patients, we have implemented the transduction conditions of human HSCs with this therapeutic lentiviral vector in healthy donor CD34+ cells, either from cord blood or mobilized peripheral blood sources. CD34+ cells were transduced at relatively low multiplicities of infection (MOIs) of 50 i.u./cell in the presence of transduction enhancers. Using an optimized transduction protocol, high transduction efficacies were obtained with minimal loss in hematopoietic progenitors. On average, transduction efficiencies of 70% and mean vector copy numbers of 2-5 copies/cell were identified in hematopoietic progenitor cells. Similar results were obtained when large-scale GMP conditions were used. To assess the transduction efficacy of human HSCs, transduced CD34+ cells were transplanted into immunodeficient NSG mice. In these studies, stable repopulation levels of human cells harboring 1-3 copies of the therapeutic vector per cell were observed up to three months post transplantation. In some cases, secondary transplants were also performed, confirming long-term and stable engraftment of transduced cells. These results have allowed us to obtain the approval for the initiation of the first lentiviral-mediated gene therapy clinical trial for the treatment of LAD-I patients. Disclosures Almarza: Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Rio:Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Law:Rocket Pharmaceuticals: Employment, Equity Ownership. Beard:Rocket Pharmaceuticals: Employment, Equity Ownership. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

Technical note: Development and application of KASP assays for rapid screening of 8 genetic defects in Holstein cattle

Specific DNA mutations underlying several genetic defects associated with embryo loss or reduced calf survivability have been identified in dairy cattle, and a convenient and cost-effective platform is required for their routine screening. We developed Kompetitive allele-specific PCR (KASP) assays for discrimination of the wild-type alleles from the associated defective alleles at each of 8 common genetic defects in Holstein cattle, involving 5 SNP [HH1, HH3, HH4, bovine leukocyte adhesion deficiency (BLAD), and complex vertebral malformation (CVM)] and 3 insertion or deletion mutations [HH5, haplotype for cholesterol deficiency (HCD), and brachyspina (BS)]. A total of 390 cows from a Chinese Holstein herd were genotyped and the carriers identified at 7 of these 8 loci (except HH4), with the highest carrier frequencies found for CVM (10.5%) and HH1 (10.0%), followed by HH3 (2.6%), BS (2.1%), HCD (1.3%), HH5 (0.8%), and BLAD (0.5%). Surprisingly, 102 cows (26.2%) carried at least 1 of the 7 defective alleles. Our results demonstrate that these KASP assays are simple, rapid, and reliable for the detection of multiple genetic defects. The high carrier frequency of these genetic defects indicates an urgent need for routine molecular testing to eliminate the deleterious alleles from Chinese Holstein cattle.

M282 VERY EARLY-ONSET INFLAMMATORY BOWEL DISEASE IN A PATIENT WITH LEUKOCYTE ADHESION DEFICIENCY TYPE 1

We present a patient with leukocyte adhesion deficiency type 1 (LAD1) for which the presenting manifestation was very early-onset inflammatory bowel disease (VEO-IBD).