A vesicle surface tyrosine kinase regulates phagosome maturation.

Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered via a complex route from phagosomes to lysosomes for degradation, but the molecular mechanisms involved in the phagosome maturation process are not well understood. Here, we identify a novel vesicle-associated receptor tyrosine kinase-like protein, VSK3, in D. discoideum. We demonstrate how VSK3 is involved in phagosome maturation. VSK3 resides on the membrane of late endosomes/lysosomes with its C-terminal kinase domain facing the cytoplasm. Inactivation of VSK3 by gene disruption reduces the rate of phagocytosis in cells, which is rescued by re-expression of VSK3. We found that the in vivo function of VSK3 depends on the presence of the kinase domain and vesicle localization. Furthermore, VSK3 is not essential for engulfment, but instead, is required for the fusion of phagosomes with late endosomes/lysosomes. Our findings suggest that localized tyrosine kinase signaling on the surface of endosome/lysosomes represents a control mechanism for phagosome maturation.

How human leukocytes track down and destroy pathogens: lessons learned from the model organism Dictyostelium discoideum.

Human leukocytes, including macrophages and neutrophils, are phagocytic immune cells that capture and engulf pathogens and subsequently destroy them in intracellular vesicles. To accomplish this vital task, these leukocytes utilize two basic cell behaviors-chemotaxis for chasing down infectious pathogens and phagocytosis for destroying them. The molecular mechanisms controlling these behaviors are not well understood for immune cells. Interestingly, a soil amoeba, Dictyostelium discoideum, uses these same behaviors to pursue and injest its bacterial food source and to organize its multi-cellular development. Consequently, studies of this model system have provided and will continue to provide us with mechanistic insights into the chemotaxis and phagocytosis of immune cells. Here, we review recent research in these areas that have been conducted in the Chemotaxis Signal Section of NIAID's Laboratory of Immunogenetics.

Seroconversion after measles vaccination at nine and fifteen months of age.

BACKGROUND: Despite high vaccination coverage, single dose measles immunization programs have been unsuccessful in eliminating the disease. Because seroconversion rates are lower in infants vaccinated before 12 months of age, a second dose of measles vaccine is recommended at 15 months. The aim of this study was to determine the seroconversion rates in children after the first and second doses of measles vaccinations at 9 and 15 months of age. METHODS: Study population comprised 116 infants attending the Well Baby Clinic of Istanbul University, Faculty of Medicine. Serum specimens were obtained from children before and 1 month after the first measles (Rouvax, Schwarz strain 1000 TCID(50)) vaccine given at 9 months. A second dose was given to 72 children at 15 months of age as measles-mumps-rubella (Trimovax, Schwarz measles strain, 1000 TCID(50); Urabe Am 9 mumps strain, 5000 TCID(50); Wister RA 27/3 rubella strain, 1000 TCID(50)). Third blood samples were collected 20 months after the second vaccine. RESULTS: Passive antibody positivity rate was 5.2% at the age of 9 months. Seroconversion rate was 77.6% after the first dose and 81.9% after the second dose of measles vaccine. Of 15 children who were seronegative, 13 (86.7%) became seropositive after the immunization at 15 months. Eleven children (19.2%) seroconverted from positive to negative after the second vaccine. CONCLUSION: The two dose schedule seems to increase the seropositivity rate. Our findings also indicate that increasing vaccination coverage and revaccination at 6 years of age are important even with the early two dose schedule.

A simple PCR/RFLP analysis can differentiate between Candida albicans, Aspergillus niger, and Aspergillus fumigatus.

The clinical management of immunocompromised patients depends on the rapid identification of infectious agents such as fungal pathogens. The procedure described here for accomplishing this uses a sensitive polymerase chain reaction method, previously reported, combined with restriction-enzyme digestion to distinguish between Candida and Aspergillus species and to classify Aspergillus strains.

[The existence of contamination in enzymes used for the isolation of Aspergillus and Candida species DNA]. / Aspergillus ve Candida cinsi DNA'sinin izolasyonunda kullanilan enzimlerde kontaminasyon varliginin arastirilmasi.

Molecular biological methods which are widely used in different fields, have been replaced with conventional diagnostic tests for the early diagnosis of invasive fungal infections, recently. Polymerase chain reaction (PCR) is one of these methods, with high specificity and sensitivity, which is accepted throughout the world. However, the enzymes that are used for the isolation of target DNA, may be contaminated with the gene sequences of some other fungal species in the preparation steps and may affect the PCR results. The studies showed that, the contamination of these enzymes with any type of fungi during their production steps, leads false positive results in PCR tests. So, the additional studies are recommended for minimizing the contamination. The aim of this study was to search whether the enzymes necessary for the isolation of fungal target DNA, used in PCR, are contaminated or not. For this purpose, five different enzymes namely, Zymolase 20T (from Arthrobacter luteus, two examples from different companies), lyticase (from Arthrobacter luteus), lysing enzyme (from Trichoderma harzianum) and proteinase K (from Tritrachium album) have been investigated for the presence of contamination. As a result, both of zymolase 20T enzymes were found to be contaminated from some fungal species with the demonstration of 18S rRNA gene sequences, while the other enzymes were found non-contaminated. By the help of this method, the most suitable enzyme for PCR was chosen and fungal contamination was prevented.

[The use of real-time polymerase chain reaction and enzyme immunoassay for the diagnosis of acute parvovirus B19 infections in immunosuppressed patients]. / Immünsüpresif hastalarda akut parvovirus B19 enfeksiyonunun tanisinda enzim immún yóntemi ile gerçek zamanli polimeraz zincir reaksiyonunun kullanimi.

Human parvovirus B19 (PV-B19) infection may lead to very serious clinical situations such as transient aplastic crisis in patients with hemolytic anemia, thrombocytopenia, neutropenia and transient arthritis accompanied with erythema infectiosum, especially in immunosuppressed patients. Early diagnosis of PV-B19 infection is of critical importance especially in immunosuppressed patients since the necessary precautions can be undertaken accordingly. In this study, PV-B19 IgM and IgG antibodies and viral DNA have been searched by enzyme immunoassay (ELISA) and real-time polymerase chain reaction (PCR), respectively, in 50 PV-B19 suspected immunosuppressed patients. Viral IgM, IgG and DNA positivities were detected in 7 (14%), 20 (40%) and 7 (14%) of the patients, respectively. During the first week three patients were found DNA and IgM positive but IgG negative, while four patients were found positive for the viral DNA, IgM and IgGs. The DNA copy numbers were high in all of the patients during the first week, with a gradual decrease during a seven-week follow-up period. IgM antibodies have disappeared in the sixth week in three of the patients and at the end of the seventh week in four of the patients. Although the IgG antibodies were negative in three patients in the first week, they became positive in the second week and the titers gradually increased during the following weeks. According to the results of this study, it can be concluded that, in high risk groups such as immunosuppressed patients, in addition to ELISA, real-time PCR method would be helpful for the early diagnosis of PV-B19 infections.

Using PCR and Real-Time PCR (LightCycler) for Diagnosis and Follow up of Invasive Fungal Infections in Patients with Hematological Malignancies and Transplantation.

In modern medicine, for early diagnosis of infections, tests that have high specificity and sensitivity should be preferred. For this reason, especially for patients with hematological malignancies and transplantation, that have high mortality and morbidity ratios, some molecular biological techniques are coming into use today for differential diagnosis and follow up of invasive fungal infections. In the coming years it will become easier to early diagnose and also to plan optimal treatments, by using these techniques for diagnosis of fungal infections. In this study, we made fungal DNA isolation from blood samples, which were taken from patients, in immunodeficiency state, with hematological malignancies and transplantation. For polymerase chain reaction (PCR), we used universal fungal primers. By using real-time PCR samples from 20 patients, which were found positive by PCR, the amount of DNA was measured. In real-time PCR, we used Aspergillus colonies as our standard, and for this purpose, SDA petri dishes were incubated for 72 hours at 30°C and; 101-106 cfu/mL serial dilutions were made by using hemocytometry. We performed DNA isolation and PCR from these dilutions. Fungal species specific products giving one band of 500 bp, were fixed in 2% agarose gel. We measured the DNA amounts by real-time PCR by using Sybr Green I. Standard dilution series and extraction samples were studied at the same time by real-time PCR. Measurement of the DNA quantities in the separate samples, one at the time of first day and the other after 15 days were interpreted by the LightCycler system. Results of real-time PCR in these two different samples were compared; it was noted that fungal DNA was increased in 5, decreased in 6 and equivalent in 9 patients. These findings showed that real-time PCR is a new, specific and a sensitive method among the other quantitative PCR systems. This method was also quicker than the other quantitative PCR systems. Currently, the investigators prefer this method because, it gives early idea about patient condition, for early diagnosis of infections and treatment patients who are under risk of some kind of infection, and for follow up treatment results. Additionally for diagnosis of fungal infections serial samples in PCR, together with other conventional diagnostic methods, should used.

The Elmo family forms an ancient group of actin-regulating proteins.

The Elmo protein family members are important mediators of small G protein activity, regulating actin-mediated processes such as chemotaxis and engulfment. Until recently,1 Elmo function has not been explored in professional phagocytes such as Dictyostelium discoideum. We discuss the significance of this family with respect to pathways that regulate Rac signaling, we present a comparison of Elmo proteins between representative taxa, and discuss our findings on ElmoA, one of six Elmo proteins found in D. discoideum.

An Elmo-like protein associated with myosin II restricts spurious F-actin events to coordinate phagocytosis and chemotaxis.

Elmo proteins positively regulate actin polymerization during cell migration and phagocytosis through activation of the small G protein Rac. We identified an Elmo-like protein, ElmoA, in Dictyostelium discoideum that unexpectedly functions as a negative regulator of actin polymerization. Cells lacking ElmoA display an elevated rate of phagocytosis, increased pseudopod formation, and excessive F-actin localization within pseudopods. ElmoA associates with cortical actin and myosin II. TIRF microscopic observations of functional ElmoA-GFP reveal that a fraction of ElmoA localizes near the presumptive actin/myosin II cortex and the levels of ElmoA and myosin II negatively correlate with that of polymerizing F-actin. F-actin-regulated dynamic dispersions of ElmoA and myosin II are interdependent. Taken together, our data suggest that ElmoA modulates actin/myosin II at the cortex to prevent excessive F-actin polymerization around the cell periphery, thereby maintaining proper cell shape during phagocytosis and chemotaxis.

Fluorescence resonance energy transfer imaging reveals that chemokine-binding modulates heterodimers of CXCR4 and CCR5 receptors.

BACKGROUND: Dimerization has emerged as an important feature of chemokine G-protein-coupled receptors. CXCR4 and CCR5 regulate leukocyte chemotaxis and also serve as a co-receptor for HIV entry. Both receptors are recruited to the immunological synapse during T-cell activation. However, it is not clear whether they form heterodimers and whether ligand binding modulates the dimer formation. METHODOLOGY/PRINCIPAL FINDINGS: Using a sensitive Fluorescence Resonance Energy Transfer (FRET) imaging method, we investigated the formation of CCR5 and CXCR4 heterodimers on the plasma membrane of live cells. We found that CCR5 and CXCR4 exist as constitutive heterodimers and ligands of CCR5 and CXCR4 promote different conformational changes within these preexisting heterodimers. Ligands of CCR5, in contrast to a ligand of CXCR4, induced a clear increase in FRET efficiency, indicating that selective ligands promote and stabilize a distinct conformation of the heterodimers. We also found that mutations at C-terminus of CCR5 reduced its ability to form heterodimers with CXCR4. In addition, ligands induce different conformational transitions of heterodimers of CXCR4 and CCR5 or CCR5(STA) and CCR5(Delta4). CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest a model in which CXCR4 and CCR5 spontaneously form heterodimers and ligand-binding to CXCR4 or CCR5 causes different conformational changes affecting heterodimerization, indicating the complexity of regulation of dimerization/function of these chemokine receptors by ligand binding.

Cutting edge: identification of a pre-ligand assembly domain (PLAD) and ligand binding site in the IL-17 receptor.

IL-17 is the hallmark cytokine of the newly described "Th17" lymphocyte population. The composition, subunit dynamics, and ligand contacts of the IL-17 receptor are poorly defined. We previously demonstrated that the IL-17RA subunit oligomerizes in the membrane without a ligand. In this study, computational modeling identified two fibronectin-III-like (FN) domains in IL-17RA connected by a nonstructured linker, which we predicted to mediate homotypic interactions. In yeast two-hybrid, the membrane-proximal FN domain (FN2), but not the membrane-distal domain (FN1), formed homomeric interactions. The ability of FN2 to drive ligand-independent multimerization was verified by coimmunoprecipitation and fluorescence resonance energy transfer microscopy. Thus, FN2 constitutes a "pre-ligand assembly domain" (PLAD). Further studies indicated that the FN2 linker domain contains the IL-17 binding site, which was never mapped. However, the FN1 domain is also required for high affinity interactions with IL-17. Therefore, although the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker and FN1.

HIV gp120-induced interaction between CD4 and CCR5 requires cholesterol-rich microenvironments revealed by live cell fluorescence resonance energy transfer imaging.

Binding of the human immunodeficiency virus (HIV) envelope gp120 glycoprotein to CD4 and CCR5 receptors on the plasma membrane initiates the viral entry process. Although plasma membrane cholesterol plays an important role in HIV entry, its modulating effect on the viral entry process is unclear. Using fluorescence resonance energy transfer imaging, we have provided evidence here that CD4 and CCR5 localize in different microenvironments on the surface of resting cells. Binding of the third variable region V3-containing gp120 core to CD4 and CCR5 induced association between these receptors, which could be directly monitored by fluorescence resonance energy transfer on the plasma membrane of live cells. Depletion of cholesterol from the plasma membrane abolished the gp120 core-induced associations between CD4 and CCR5, and reloading cholesterol restored the associations in live cells. Our studies suggest that, during the first step of the HIV entry process, gp120 binding alters the microenvironments of unbound CD4 and CCR5, with plasma membrane cholesterol required for the formation of the HIV entry complex.