Leukocyte Adhesion Deficiency Type Research Articles

Investigation of ITGB2 gene in 12 new cases of leukocyte adhesion deficiency-type I revealed four novel mutations from Iran.

Leukocyte adhesion deficiency type I (LAD-I) is a rare, autosomal recessive inherited immunodeficiency disease. LAD-I is caused by mutations in the ITGB2 gene and characterized by recurrent severe bacterial infections, as well as impaired wound healing with lack of pus formation. In this study, we investigated ITGB2 gene mutations in 12 patients and their parents. Genomic DNA was extracted from whole blood samples. All coding regions of the ITGB2 gene were amplified using PCR and followed by direct sequencing. Genetic analysis revealed 12 different homozygous mutations, including six missense (c.382G>A, c.2146G>C, c.715G>A, c.691G>C, c.1777C and new c.1686C>A), two new nonsense (c.1336G>T and c.1821C>A), three-frame shift (c.1143delc, c.1907delA and new c.474dupC) and a splice site (c.1877+2T>C). Flow cytometry analysis of CD11/CD18 expression on neutrophils revealed defect in CD18 in all twelve cases (1.4% to 42%), CD11a in ten cases (0.1% to 26.7%), CD11b in nine cases (1.2% to 58.8%), and CD11c in all cases (0 % to 18.1%). The patients' parents were both heterozygous carriers. Our findings showed four new mutations in the ITGB2 gene. These results can be used for decisive genetic diagnosis, genetic counseling, as well as prenatal diagnosis for all patients who are suspended to LADI.

Adaptive immune defects in a patient with leukocyte adhesion deficiency type III with a novel mutation in FERMT3

_To the Editor,_ Leukocyte adhesion deficiency (LAD) is a rare primary immunodeficiency disease characterized by impairment of phagocyte adhesion. Three subtypes have been classified by distinct phases of the adhesion cascade. LAD-III is caused by defects in signaling pathways used for integrin activation in all hematopoietic cell types leading to recurrent infections with poor platelet aggregation resembling Glanzmann’s thrombasthenia. Mutations in FERMT3 have been identified to underlie LAD-III. FERMT3 encodes kindlin-3, one of the focal adhesion proteins which contain a FERM domain located at the carboxyl terminus binding to b-integrin cytoplasmic tails. This molecule cooperates with the cytoskeletal protein talin leading to integrin activation. It also stabilizes active conformations of the integrin subunits and the ligand binding. Evidently, integrins are widely expressed in many cell types including T and B lymphocytes. Defects in integrin function therefore could lead to both innate and adaptive immune dysfunctions. However, almost all reported cases of LAD-III only had innate immune defects. Here, we describe a female Thai patient who was diagnosed with LAD-III, yet presenting with a mild atypical phenotype in which a humoral immune defect was detected.

Highlighting the problematic reliance on CD18 for diagnosing leukocyte adhesion deficiency type 1.

Leukocyte adhesion deficiency type 1 (LAD-1) is an autosomal recessive primary immunodeficiency, hallmarked by defective polymorphonuclear transmigration. It is caused by mutations in the gene encoding CD18, which interfere with the CD18/CD11 heterodimerization and expression on leukocyte cell surface. LAD-1 diagnosis rests primarily on the measurement of CD18 expression. However, CD18 measurement entails its pitfalls. Here we present a cohort of ten LAD patients and a review of the relevant literature illustrating the difficulties in sole reliance on CD18 measurement for initial diagnosis. These include normal range expression in some mutations, great variability between patients with the same mutation and subjective interpretation of results. We think there is a need for additional markers as part of the initial LAD diagnostic algorithm. We suggest CD11a expression, which was near absent in all patients in our cohort. The dual use of CD18 and CD11a can increase testing sensitivity and prevent delayed diagnosis of LAD-1.

Role of bacteria in leukocyte adhesion deficiency-associated periodontitis

Leukocyte adhesion deficiency Type I (LAD-I)–associated periodontitis is an aggressive form of inflammatory bone loss that has been historically attributed to lack of neutrophil surveillance of the periodontal infection. However, this form of periodontitis has proven unresponsive to antibiotics and/or mechanical removal of the tooth-associated biofilm. Recent studies in LAD-I patients and relevant animal models have shown that the fundamental cause of LAD-I periodontitis involves dysregulation of a granulopoietic cytokine cascade. This cascade includes interleukin IL-23 (IL-23) and IL-17 that drive inflammatory bone loss in LAD-I patients and animal models and, moreover, foster a nutritionally favorable environment for bacterial growth and development of a compositionally unique microbiome. Although the lack of neutrophil surveillance in the periodontal pockets might be expected to lead to uncontrolled bacterial invasion of the underlying connective tissue, microbiological analyses of gingival biopsies from LAD-I patients did not reveal tissue-invasive infection. However, bacterial lipopolysaccharide was shown to translocate into the lesions of LAD-I periodontitis. It is concluded that the bacteria serve as initial triggers for local immunopathology through translocation of bacterial products into the underlying tissues where they unleash the dysregulated IL-23–IL-17 axis. Subsequently, the IL-23/IL-17 inflammatory response sustains and shapes a unique local microbiome which, in turn, can further exacerbate inflammation and bone loss in the susceptible host.

Kindlin-3-mediated integrin adhesion is dispensable for quiescent but essential for activated hematopoietic stem cells.

Hematopoietic stem cells (HSCs) generate highly dividing hematopoietic progenitor cells (HPCs), which produce all blood cell lineages. HSCs are usually quiescent, retained by integrins in specific niches, and become activated when the pools of HPCs decrease. We report that Kindlin-3-mediated integrin activation controls homing of HSCs to the bone marrow (BM) and the retention of activated HSCs and HPCs but not of quiescent HSCs in their BM niches. Consequently, Kindlin-3-deficient HSCs enter quiescence and remain in the BM when cotransplanted with wild-type hematopoietic stem and progenitor cells (HSPCs), whereas they are hyperactivated and lost in the circulation when wild-type HSPCs are absent, leading to their exhaustion and reduced survival of recipients. The accumulation of HSPCs in the circulation of leukocyte adhesion deficiency type III patients, who lack Kindlin-3, underlines the conserved functions of Kindlin-3 in man and the importance of our findings for human disease.

Novel combination of ITGB2 mutations causing leukocyte adhesion deficiency type 1 (LAD-1)

In this case report, we describe for the first time the identification of a compound heterozygote mutation in the β2 integrin gene (ITGB 2), which is associated with Leukocyte Adhesion Deficiency type 1 (LAD-1). The patient was a 6-year-old male referred for evaluation of multiple recurrent infections. Laboratory evaluation revealed persistent leukocytosis with normal immunoglobulins, complement, mannose-binding lectin, and vaccine antibodies. There was also a complete absence of the integrins - CD18 and CD11a on monocytes, granulocytes and lymphocytes, and CD11b on monocytes and lymphocytes with normal expression on granulocytes. Gene sequencing showed two heterozygous mutations, one being a missense mutation, G284S in exon 7 (c.850G>A), and the second, a single base-pair deletion resulting in a premature stop codon (c.2070delT). Based on the clinical and laboratory findings, a diagnosis of LAD-1 was reached, but it was delayed by an apparent lack of early recognition of the clinical features. LAD-1 should be considered as a potential diagnosis and aggressively pursued in any patient who presents with leukocytosis, delayed umbilical cord separation and non-purulent recurrent infections. Key diagnostic work-up includes flow cytometry for CD18, CD11a and CD11b on white blood cells followed by appropriate confirmatory gene sequencing analysis.

Optimal T Cell Activation and B Cell Antibody Responses In Vivo Require the Interaction between Leukocyte Function-Associated Antigen-1 and Kindlin-3.

Kindlin-3 is an important integrin regulator that is mutated in the rare genetic disorder, leukocyte adhesion deficiency type III, a disorder characterized by defective neutrophil trafficking and platelet function, leading to recurrent bacterial infections and bleeding. Kindlin-3 is also known to regulate T cell adhesion in vitro and trafficking in vivo, but whether the integrin/kindlin interaction regulates T or B cell activation in vivo is unclear. In this study, we used TTT/AAA β2-integrin knock-in (KI) mice and TCR-transgenic (OT-II) KI mice, in which the integrin/kindlin connection is disrupted, to investigate the role of the integrin/kindlin interaction in T cell activation. We show that basal T cell activation status in these animals in vivo is normal, but they display reduced T cell activation by wild-type Ag-loaded dendritic cells in vitro. In addition, T cell activation in vivo is reduced. We also show that basal Ab levels are normal in TTT/AAA β2-integrin KI mice, but B cell numbers in lymph nodes and IgG and IgM production after immunization are reduced. In conclusion, we show that the integrin/kindlin interaction is required for trafficking of immune cells, as well as for T cell activation and B cell Ab responses in vivo. These results imply that the immunodeficiency found in leukocyte adhesion deficiency type III patients, in addition to being caused by defects in neutrophil function, may be due, in part, to defects in lymphocyte trafficking and activation.

Leukocyte adhesion deficiency type III: clinical features and treatment with stem cell transplantation.

Leukocyte adhesion deficiency type III (LADIII) is an autosomal recessive disorder that presents with a severe leukocyte adhesion defect and a Glanzmann-type thrombocytopathy. Hematopoietic stem cell transplantation (HSCT)--the only definitive treatment for LADIII--appears to have a high rate of complications. In this study, we describe a new group of patients with LADIII, highlighting further clinical and immunologic aspects of this disease, and reevaluating the effectiveness of HSCT for its treatment. The patients had clinical and laboratory findings consistent with LADIII. Molecular analysis confirmed the presence of a mutation in the kindlin-3 gene. HSCT was carried out in 3 patients and was successful in 2. The diagnosis of LADIII should be considered in all patients who present with recurrent infections and a bleeding diathesis, regardless of the leukocyte count. LADIII is a primary immune deficiency, which can be successfully corrected by bone marrow transplantation if applied early in the course of the disease using appropriate conditioning.

Leukocyte Adhesion Deficiency Type I: The Outcome of Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation

Leukocyte Adhesion Deficiency Type I: The Outcome of Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation

In this issue – August 2014

Bovine viral diarrhoea virus (BVDV) type 1 is endemic in Australian cattle populations, costing the national industry an estimated AUD57.9 million annually.1 BVDV control programs based on identifying and removing persistently infected cattle have been efficiently and effectively implemented in Europe and are gaining pace in New Zealand. This review discusses the challenges to BVDV control in Australia, including availability of infrastructure, reliability and availability of diagnostic tests, stakeholder compliance and the potential of other species, particularly sheep, to act as a potential reservoir host for the virus. Network analysis of the movements and connections of Australian beekeepers showed the nomadic beekeeper network was fragmented into 34 disconnected components.2 The largest component network linked 288 locations from Duaringa in central Queensland to Nelson in the far south-west of Victoria. Almond orchards and lucerne seed production were locations of major hive congregations driven by the opportunity to provide paid pollination services. In the 3 months after completion of almond pollination, movement of hives occurred to 49 locations spread over >2000 km, ranging from south-east Queensland to southern Victoria. This highlights how rapidly pests or diseases could spread if introduced to major congregation areas. The authors discuss the implications of these findings for improving surveillance and disease control policy for the honeybee industry. A study conducted at Charles Sturt University reports that Echinococcus granulosus is still present in Tasmania and being transmitted at low levels between dogs and cattle, although provisional eradication of E. granulosus from Tasmania was declared in 1996.3 Faecal samples from 1425 rural domestic dogs from Tasmania to south-eastern Queensland were tested for the presence of E. granulosus and other intestinal helminth infections. A total of 45 dogs (3.2%) were found to be E. granulosus coproantigen-positive, with 24 of these dogs originating from Tasmania and 16 from NSW. Three of the Tasmanian dogs were also positive for E. granulosus on coproPCR. The study also found that owner behaviour perpetuates the transmission of E. granulosus. Most owners did not deworm their dogs frequently enough to ensure they could never have a patent infection of E. granulosus. In addition, approximately 45% of the dog owners surveyed reported feeding offal of lambs or mutton and whole rabbits to their dogs, and >74% of owners admitted their dogs had opportunities to access carcases of wildlife or livestock. This month's case report is the first reported case of leucocyte adhesion deficiency type III (LAD III) in a dog in Australia and the first reported case of LAD III in a mixed-breed dog worldwide.4 This condition is attributable to a mutation in the KINLIN-3 gene that impairs activation of β-integrins, resulting in absence of neutrophil chemotaxis and adhesion and platelet adhesion and aggregation. Affected patients exhibit recurrent infections, marked leucocytosis and a severe bleeding tendency causing increased mucosal haemorrhage and prolonged postoperative haemorrhage. The authors describe the clinical signs, diagnosis and management of LAD III in a 16-month-old male German Shepherd × Rottweiler dog. A survey of Thoroughbred and Standardbred trainers investigated the magnitude of and risk factors associated with horse wastage in the Australian racing industry during the 2002–03 race year.5 The results showed an overall exit rate of around 40% in Thoroughbred and Standardbred stables. Poor performance was the most commonly reported reason for leaving, followed by illness/injury. Unsuitable temperament/behaviour was less frequently reported. A larger percentage of Standardbreds than Thoroughbreds went to different trainers or directly to knackeries, while a higher percentage of Thoroughbreds went to stud, auctions or entered ridden equestrian pursuits. Statistically significant risk factors influencing the destinations of both Thoroughbred and Standardbred racing horses included whether the trainer owned the horses, horse sex and age and reasons for leaving. A study conducted at Oregon and Colorado State Universities reviewed the clinical features and factors associated with short- and long-term survival of New World camelids with acquired urethral obstruction caused by urolithiasis.6 The most common clinical signs included straining to urinate, dribbling urine and anorexia, which are also frequently observed in small ruminants with obstructive urolithiasis. Prognosis appeared to be worse and occurrence of complications appeared to be more frequent than in small ruminants, particularly after tube cystostomy and bladder marsupialisation. Incomplete urethral obstruction at admission and surgical treatment were associated with increased odds of short-term survival. Recurrence of urethral obstruction had a poor prognosis for long-term survival.

Defective Neutrophil Recruitment in Leukocyte Adhesion Deficiency Type I Disease Causes Local IL-17–Driven Inflammatory Bone Loss

Leukocyte adhesion deficiency type I (LAD-I), a disease syndrome associated with frequent microbial infections, is caused by mutations on the CD18 subunit of β₂ integrins. LAD-I is invariably associated with severe periodontal bone loss, which historically has been attributed to the lack of neutrophil surveillance of the periodontal infection. We provide an alternative mechanism by showing that the cytokine interleukin-17 (IL-17) plays a major role in the oral pathology of LAD-I. Defective neutrophil recruitment in LAD-I patients or in LFA-1 (CD11a/CD18)-deficient mice--which exhibit the LAD-I periodontal phenotype--was associated with excessive production of predominantly T cell-derived IL-17 in the periodontal tissue, although innate lymphoid cells also contributed to pathological IL-17 elevation in the LFA-1-deficient mice. Local treatment with antibodies to IL-17 or IL-23 in LFA-1-deficient mice not only blocked inflammatory periodontal bone loss but also caused a reduction in the total bacterial burden, suggesting that the IL-17-driven pathogenesis of LAD-I periodontitis leads to dysbiosis. Therefore, our findings support an IL-17-targeted therapy for periodontitis in LAD-I patients.

Congenital disorder of fucosylation type 2c (LADII) presenting with short stature and developmental delay with minimal adhesion defect

Leukocyte adhesion deficiency type II is a hereditary disorder of neutrophil migration caused by mutations in the guanosine diphosphate-fucose transporter gene (SLC35C1). In these patients, inability to generate key fucosylated molecules including sialyl Lewis X leads to leukocytosis and recurrent infections, in addition to short stature and developmental delay. We report two brothers with short stature and developmental delay who are compound heterozygotes for novel mutations in SLC35C1 resulting in partial in vivo defects in fucosylation. Specifically, plasma glycoproteins including immunoglobulin G demonstrated marked changes in glycoform distribution. While neutrophil rolling on endothelial selectins was partially impeded, residual adhesion proved sufficient to avoid leukocytosis or recurrent infection. These findings demonstrate a surprising degree of immune redundancy in the face of substantial alterations in adhesion molecule expression, and show that short stature and developmental delay may be the sole presenting signs in this disorder.

Kindlin-3–independent adhesion of neutrophils from patients with leukocyte adhesion deficiency type III

Kindlin-3–independent adhesion of neutrophils from patients with leukocyte adhesion deficiency type III

Primary immunodeficiencies underlying fungal infections

We review the primary immunodeficiencies (PIDs) underlying an increasing variety of superficial and invasive fungal infections. We also stress that the occurrence of such fungal infections should lead physicians to search for the corresponding single-gene inborn errors of immunity. Finally, we suggest that other fungal infections may also result from hitherto unknown inborn errors of immunity, at least in some patients with no known risk factors. An increasing number of PIDs are being shown to underlie fungal infectious diseases in children and young adults. Inborn errors of the phagocyte NADPH oxidase complex (chronic granulomatous disease), severe congenital neutropenia (SCN) and leukocyte adhesion deficiency type I confer a predisposition to invasive aspergillosis and candidiasis. More rarely, inborn errors of interferon-γ immunity underlie endemic mycoses. Inborn errors of interleukin-17 immunity have recently been shown to underlie chronic mucocutaneous candidiasis (CMC), while inborn errors of caspase recruitment domain-containing protein 9 (CARD9) immunity underlie deep dermatophytosis and invasive candidiasis. CMC, invasive candidiasis, invasive aspergillosis, deep dermatophytosis, pneumocystosis, and endemic mycoses can all be caused by PIDs. Each type of infection is highly suggestive of a specific type of PID. In the absence of overt risk factors, single-gene inborn errors of immunity should be sought in children and young adults with these and other fungal diseases.

Spectrum of primary immunodeficiency disorders in Sri Lanka

BackgroundWhile primary immunodeficiencies (PID has been recognized in the west for decades, recognition has been delayed in the third world. This study attempts to detail the spectrum of PID, the therapy provided, and constraints in the diagnosis and treatment in a middle income country such as Sri Lanka.MethodsNine hundred and forty two patients with recurrent infections and features suggestive of immune deficiency, referred from the entire country in a 4 year period, to the sole immunology unit in Sri Lanka were included. The following tests were performed. Full blood counts, serum Immunoglobulin and complement C3 and C4 levels, functional antibody levels, enumeration of lymphocyte subsets, in vitro and in vivo T cell functional assays,, nitroblue tetrazolium assay to diagnose chronic granulomatous disease, hair shaft assay to diagnose Griscelli syndrome. Sequencing of the common gamma chain to identify x linked severe combined immune deficiency, and X linked agammaglobulinemia was confirmed by assaying for Btk mutations by single sequence conformation polymorphism. HIV/AIDS was excluded in all patients.ResultsSeventy three patients were diagnosed with a primary immune deficiency. The majority (60.27%) had antibody deficiency. Common variable immune deficiency was the commonest (28.76%), followed by X linked agammaglobulinemia (XLA) (20.54%). Five patients had possible hyper IgM syndrome.Ten patients had severe combined immune deficiency (SCID), including 2 with x linked SCID, in addition to DiGeorge syndrome (2), ataxia telangiectasia (6), autosomal dominant hyper IgE syndrome (2), chronic granulomatous disease (4), leucocyte adhesion deficiency type 1 (2) and Griscelli syndrome (3).Patients with autoinflammatory, innate immune and complement defects could not be identified due to lack of facilities.ConclusionsAntibody deficiency is the commonest PID, as in the west.IgA deficiency is rare. Autoinflammatory diseases, innate immune and complement deficiencies could not be identified due to lack of diagnostic facilities. Lack of awareness of PID among adult physicians result in delay in treatment of adult patients. While treatment of antibody deficiencies provided in state hospitals has extended life expectancy, there is no treatment available for severe T cell defects.

The β2 integrin–kindlin-3 interaction is essential for T-cell homing but dispensable for T-cell activation in vivo

AbstractKindlin-3 is mutated in the rare genetic disorder, leukocyte adhesion deficiency type III, which is characterized by deficient integrin-mediated adhesion of leukocytes and platelets. However, the specific roles of kindlin-3–β2-integrin interactions in T-cell adhesion and homing and immune responses in vivo remain unclear. Here, we show that the TTT motif in β2 integrins controls kindlin-3 binding. Mutation of the kindlin-3 binding site in β2 integrins caused a loss of firm adhesion of T cells under both static and shear flow conditions and a reduction of T-cell homing to lymph nodes in vivo. However, atomic force microscopy studies of integrin-ligand bonds revealed that initial ligand binding could still occur, and 2-dimensional T-cell migration was reduced but not abolished by the TTT/AAA mutation in the β2 integrin. Importantly, dendritic cell–mediated T-cell activation in vivo was normal in TTT/AAA β2 integrin knock-in mice. Our results reveal a selective role of the kindlin-3–integrin association for lymphocyte functions in vivo; the integrin–kindlin-3 interaction is particularly important in adhesion strengthening under shear flow, and for T-cell homing to lymph nodes, but dispensable for T cell activation which occurs in a shear-free environment.

Clinical analysis of a Chinese child with leukocyte adhesion deficiency type 1

Leukocyte adhesion deficiency type 1 (LAD-I) is rare. We present 1 case of LAD-I patient diagnosed by gene analysis. His clinical manifestations and genetic mutation features are analyzed in this article. The clinical material of the LAD-I patient who was diagnosed by gene analysis was retrospectively analyzed. The patient was a 2-month-old boy. He had a complaint of recurrent fever and cough for 30 days. Pulmonary CT indicated a small to moderate quantity pleural effusion on the right side. His peripheral blood leukocyte and C-reactive protein (CRP) was always significantly higher than normal. After hospitalization he had diarrheal diseases, routine stool test showed 2 RBC cells/high power (HP), WBC 30 cells/HP, stool cultures were negative, digestive tract ultrasonography showed an array of defects, in the sigmoid colon and rectal mucosa suggestive of ulcerative colitis. He was treated with cefoperazone and sulbactam and vancomycin. He had a history of impetigo in his neonatal period and without delayed umbilical cord exfoliation. His family history was normal. ITGB2 genetic mutation analysis revealed a homozygous mutation (1062A > T). His parents did not participate in this study. He had no fever but had diarrheal disease after 1 month of follow up. This patient had suffered from impetigo, pleural effusion, diarrheal diseases, markedly increased peripheral white blood cell and ITGB2 genetic mutation analysis showed that homozygous mutation (1062A > T). He received a diagnosis of LAD-I.

Long-Term Follow-up of Foamy Viral Vector-Mediated Gene Therapy for Canine Leukocyte Adhesion Deficiency

The development of leukemia following gammaretroviral vector-mediated gene therapy for X-linked severe combined immunodeficiency disease and chronic granulomatous disease (CGD) has emphasized the need for long-term follow-up in animals treated with hematopoietic stem cell gene therapy. In this study, we report the long-term follow-up (4-7 years) of four dogs with canine leukocyte adhesion deficiency (CLAD) treated with foamy viral (FV) vector-mediated gene therapy. All four CLAD dogs previously received nonmyeloablative conditioning with 200 cGy total body irradiation followed by infusion of autologous, CD34(+) hematopoietic stem cells transduced by a FV vector expressing canine CD18 from an internal Murine Stem Cell Virus (MSCV) promoter. CD18(+) leukocyte levels were >2% following infusion of vector-transduced cells leading to ongoing reversal of the CLAD phenotype for >4 years. There was no clinical development of lymphoid or myeloid leukemia in any of the four dogs and integration site analysis did not reveal insertional oncogenesis. These results showing disease correction/amelioration of disease in CLAD without significant adverse events provide support for the use of a FV vector to treat children with leukocyte adhesion deficiency type 1 (LAD-1) in a human gene therapy clinical trial.

A Role of Kindlin-3 in Integrin αMβ2 Outside-In Signaling and the Syk-Vav1-Rac1/Cdc42 Signaling Axis

Integrins mediate cell-cell and cell-extracellular matrix attachments. Integrins are signaling receptors because their cytoplasmic tails are docking sites for cytoskeletal and signaling proteins. Kindlins are a family of band 4.1-ezrin-radixin-moesin-containing intracellular proteins. Apart from regulating integrin ligand-binding affinity, recent evidence suggests that kindlins are involved in integrin outside-in signaling. Kindlin-3 is expressed in platelets, hematopoietic cells and endothelial cells. In humans, loss of kindlin-3 expression accounts for the rare autosomal disease leukocyte adhesion deficiency (LAD) type III that is characterized by bleeding disorders and defective recruitment of leukocytes into sites of infection. Studies have shown that the loss of kindlin-3 expression leads to poor ligand-binding properties of β1, β2 and β3 integrin subfamilies. The leukocyte-restricted β2 integrin subfamily comprises four members, namely αLβ2, αMβ2, αXβ2 and αDβ2. Integrin αMβ2 mediates leukocyte adhesion, phagocytosis, degranulation and it is involved in the maintenance of immune tolerance. Here we provide further evidence that kindlin-3 is required for integrin αMβ2-mediated cell adhesion and spreading using transfected K562 cells that expressed endogenous kindlin-3 but not β2 integrins. K562 stable cell line expressing si-RNA targeting kindlin-3, but not control-si-RNA, and transfected with constitutively activated integrin αMβ2N329S adhered and spread poorly on iC3b. We also show that kindlin-3 is required for the integrin αMβ2-Syk-Vav1 signaling axis that regulates Rac1 and Cdc42 activities. These findings reinforce a role for kindlin-3 in integrin outside-in signaling.

Leukocyte adhesion deficiency type I in a mixed-breed dog

A 6-month-old, neutered male, mixed-breed dog was examined for a 2-month persistent fever, nonhealing dermal metacarpal area wound, and leukocytosis (47.0-198.0 × 10(3)/μl). Serum chemistry findings included hypoalbuminemia, hyperglobulinemia, hyperphosphatemia, and hyperphosphatasemia. Complete blood cell count results revealed a moderate microcytic, hypochromic nonregenerative anemia with a profound leukocytosis (198.5 × 10(3)/μl), characterized by neutrophilia with toxicity and hypersegmentation, and significant band cells. Tick-borne disease titers (genera Anaplasma, Ehrlichia, and Borrelia) were negative, as were polymerase chain reaction for other infectious agents (genera Hepatozoon, Mycobacterium, Mycoplasma; and Canine distemper virus). No agents were identified in a deep dermal biopsy (conventional and special histochemical stains) of the chronic draining, metacarpal region lesion. Cytology of the draining tract revealed numerous mixed bacteria and a surprising lack of neutrophils. Chronic occult blood loss with iron deficiency was considered a possible cause of the anemia. Differentials for the leukon were chronic established inflammation (occult infectious agent), chronic neutrophilic leukemia, paraneoplastic leukocytosis (neoplastic source of granulocyte colony-stimulating factor [CSF] or granulocyte-macrophage CSF), and leukocyte adhesion deficiency (LAD). The possibility of a LAD disorder was further investigated because of the noted hypersegmented neutrophils, absence of neutrophils in the cytology sample, the animal's young age, and persistence of clinical and laboratory signs. Flow cytometry of blood neutrophils showed a 60% reduction in surface expression of the β2-integrin (CD18) subunit, whereas neutrophil function tests (oxidative burst and phagocytosis) were normal. Genetic testing revealed a homozygous missense mutation in the β2-integrin subunit gene, previously recognized only in purebred Irish Setters, leading to a diagnosis of LAD type 1 disorder in this mixed-breed dog.