Mast cell signaling: The role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants

The aggregation by antigen of the IgE bound to its high affinity receptor on mast cells initiates a complex series of biochemical events that result in the release of inflammatory mediators. The essential role of the protein tyrosine kinase Syk has been appreciated for some time, and newer results have defined the mechanism of its activation. The use of siRNA has defined the relative contribution of Syk, Fyn and Gab2 to signaling and has made possible a screening study to identify previously unrecognized molecules that are involved in these pathways.     

Effect of Killer Immunoglobulin-Like Receptors in the Response to Combined Treatment in Patients with Chronic Hepatitis C Virus Infection

Killer immunoglobulin-like receptors (KIRs) are related to the activation and inhibition of NK cells and may play an important role in the innate response against infection with viruses such as hepatitis C virus (HCV). We examined whether the different combinations of KIRs with their HLA class I ligands influenced the response to combined treatment (pegylated alpha interferon and ribavirin) of patients infected by HCV. A total of 186 consecutive patients diagnosed with chronic HCV infection were analyzed. Seventy-seven patients exhibited HCV RNA levels at 6 months posttreatment and were called nonresponders (NR), while 109 cleared viral RNA and were named sustained viral responders (SVR). Patients were typed for HLA-B, HLA-Cw, KIR genes, and HCV genotype. In our study, the frequency of the KIR2DL2 allele was significantly increased in NR (P < 0.001; odds ratio [OR] = 1.95), as was the frequency of the KIR2DL2/KIR2DL2 genotype (P < 0.005; OR = 2.52). In contrast, the frequencies of the KIR2DL3 genotype (P < 0.001) and KIR2DL3/KIR2DL3 genotype (P < 0.05; OR = 0.54) were significantly increased in the SVR. Different combinations of KIR2DL2 and KIR2DL3 alleles with their ligands were analyzed. The frequency of the KIR2DL2/KIR2DL2-HLA-C1C2 genotype was significantly increased in the NR (P < 0.01; OR = 3.15). Additionally, we found a higher frequency of the KIR2DL3/KIR2DL3-HLA-C1C1 genotype in the SVR group (P < 0.05; OR = 0.33). These results were not affected by the HCV genotype. In conclusion, patients who carried the KIR2DL2/KIR2DL2-HLA-C1C2 genotype were less prone to respond to treatment. However, the KIR2DL3/KIR2DL3-HLA-C1C1 genotype clearly correlated with a satisfactory response to treatment, defined by the clearance of HCV RNA.Hepatitis C virus (HCV) infection is a common chronic disease affecting over 170 million people worldwide (48). Around 80% of these individuals evolve to chronic infection, and 10 to 20% of patients develop cirrhosis over a 20-year period. A minority (2%) progresses to hepatocellular carcinoma annually (18). Several host factors including age, body mass index (BMI), gender, fibrosis, cirrhosis, or the absence of cirrhosis and several viral factors including viral genotype and viral load can influence the response to treatment (6, 32, 42). Pegylated alpha interferon (Peg-IFN-α) plus ribavirin (combined therapy) constitute the most effective therapy for the treatment of chronic HCV infection (13). Since this treatment carries serious side effects, it is necessary to identify those patients who can clear HCV infection in order to reduce the period of this aggressive therapy.Natural killer (NK) cells are lymphocytes that play an important role in the host defense against HCV infection (14). NK cell activity is determined by the balance of different signals received and the equilibrium between inhibitory and activating receptors (3). Some receptors are specific for human leukocyte antigen (HLA) class I molecules (4). NK cells check the surface of the surrounding cells, detect the presence of their HLA class I molecules, and then discriminate between healthy, infected, or transformed cells (10). When NK cells contact target cells, the resulting interactions of their receptors produce either activating or inhibitory signals. If the expression of HLA class I molecules on the target cell is absent or reduced, the inhibitory signal is not generated (25).Killer cell immunoglobulin-like receptors (KIRs) are members of a group of regulatory molecules expressed on NK cells and a subset of T cells (30). This family of polymorphic genes is located on chromosome 19 (19q13.4), within the leukocyte receptor complex. The leukocyte receptor complex also encodes a number of genetically and functionally related genes. KIRs with long cytoplasmic tails are inhibitors, based on the presence of immunoreceptor tyrosine-based inhibition motifs in their cytoplasmic domains. KIRs with short tails interact with adaptor molecules such as DAP-12 (DNAX activation protein), which contain immunoreceptor tyrosine-based activation motifs and transmit activating signals (24). Several inhibitory KIRs have been well defined. KIR2DL1 binds the subset of HLA-Cw molecules with lysine at position 80 of the heavy chain (HLA-C2 group). KIR2DL2 and KIR2DL3 bind the subset of HLA-Cw molecules with asparagine at position 80 (HLA-C1 group) (34).Studies that have associated KIR genotypes with diseases have identified mainly viral infections and autoimmune diseases (22, 45). The importance of NK cells in the resolution of viral infections has prompted studies that correlate KIRs and their ligands with outcomes (12). Some studies identified a relationship between KIR genotypes and outcomes with several infectious agents such as human immunodeficiency virus (27, 29), cytomegalovirus (7), hepatitis B virus (28), and HCV (21, 26).Recently, a protective association of the inhibitory receptor KIR2DL3 with HLA-C^Asn80 (HLA-C1) and its effect on the course of HCV infection were described (21). The prevalence of KIR2DL3 and its ligand HLA-C1 is increased in individuals who eliminate HCV spontaneously, in contrast to those who remain chronically infected. The protective effect of KIR2DL3/HLA-C^Asn80 was observed only among individuals who carried both homozygous genes and had received a low HCV exposure dose. Recently, we found that the frequency of HLA-Bw4^I80 ligand and the activating receptor KIR3DS1 was increased healthy in HCV carriers compared to patients who had developed hepatocellular carcinoma (26).The aim of this study was to investigate the influence of KIR genes and KIR-HLA combinations on the response to combined therapy with Peg-IFN-α-2b and ribavirin in a group of patients with HCV infection.     

Associations between HLA Class I Alleles and Escape Mutations in the Hepatitis B Virus Core Gene in New Zealand-Resident Tongans

The full repertoire of hepatitis B virus (HBV) peptides that bind to the common HLA class I molecules found in areas with a high prevalence of chronic HBV infection has not been determined. This information may be useful for designing immunotherapies for chronic hepatitis B. We identified amino acid residues under positive selection pressure in the HBV core gene by phylogenetic analysis of cloned DNA sequences obtained from HBV DNA extracted from the sera of Tongan subjects with inactive, HBeAg-negative chronic HBV infections. The repertoires of positively selected sites in groups of subjects who were homozygous for either HLA-B*4001 (n = 10) or HLA-B*5602 (n = 7) were compared. We identified 13 amino acid sites under positive selection pressure. A significant association between an HLA class I allele and the presence of nonsynonymous mutations was found at five of these sites. HLA-B*4001 was associated with mutations at E77 (P = 0.05) and E113 (P = 0.002), and HLA-B*5602 was associated with mutations at S21 (P = 0.02). In addition, amino acid mutations at V13 (P = 0.03) and E14 (P = 0.01) were more common in the seven subjects with an HLA-A*02 allele. In summary, we have developed an assay that can identify associations between HLA class I alleles and HBV core gene amino acids that mutate in response to selection pressure. This is consistent with published evidence that CD8⁺ T cells have a role in suppressing viral replication in inactive, HBeAg-negative chronic HBV infection. This assay may be useful for identifying the clinically significant HBV peptides that bind to common HLA class I molecules.The most potent nucleoside/nucleotide analogue drugs used to treat chronic hepatitis B reduce serum hepatitis B virus (HBV) DNA to undetectable levels in over 90% of subjects (5, 10). It was originally hoped that such a substantial reduction in viral titers would reverse T-cell tolerance for HBV antigens (17, 30) and lead to an immune response that permanently suppressed the virus, thus removing the need for expensive, lifelong drug therapy. However, HBeAg seroconversion rates of under 30% suggest that suppression of HBV replication is not sufficient to reverse the defects (4, 15) in the HBV peptide-specific CD8⁺ T-cell compartment that occur in these patients. A therapeutic vaccine that stimulated a diverse repertoire of functional CD8⁺ T cells could make a valuable contribution to management of chronic hepatitis B.The first step in designing a therapeutic vaccine that will suppress viral replication without exacerbating chronic hepatitis B (15) is to identify the HBV peptides that stimulate functional CD8⁺ T cells by binding to the most common HLA class I alleles. These peptides may contribute to the antigen component of a vaccine and to the design of assays for use as correlates of immunity in trials of antiviral therapies. Although some of the HBV peptides that bind to four HLA-A alleles have been published (3, 19, 25, 28), a much wider repertoire of peptide-HLA interactions needs to be identified. There is no established method for finding them (32). Adding pools of peptides to peripheral blood mononuclear cells in enzyme-linked immunospot assays is the most commonly used technique (4), but it has disadvantages. Pools of peptides contain epitopes that are not produced by in vivo antigen-processing mechanisms (32), and the influence of these epitopes on complex mixtures of T cells with degenerate antigen receptors is unknown. False-positive and false-negative results are possible. In addition, it cannot be assumed that the ability of a T cell to secrete gamma interferon in an enzyme-linked immunospot assay correlates with its ability to place clinically significant selection pressure on the virus in vivo.We are proposing an alternative approach, which should lead to the identification of the most clinically significant wild-type peptide antigens. This is to assess the influence of HLA class I alleles on the repertoire of escape mutations (3, 18) encoded in the HBV DNA extracted from the sera of HBeAg-negative subjects with an inactive chronic HBV infection. A functional CD8⁺ T-cell repertoire (15, 22) develops in these subjects at the same time the virus in their sera accumulates amino acid mutations (2). Phylogenetic analysis can distinguish those amino acid mutations that have arisen as a result of positive selection pressure from those that have arisen as a result of random processes (31). CD8⁺ T cells are likely to have placed selection pressure on any of the nonrandom amino acid mutations that preferentially occur in patients with a specific HLA class I allele. It should be possible to obtain the precise amino acid sequences of the peptides that contain these amino acids using immunological assays.This study was carried out with Tongan subjects who are homozygous for one of two common HLA-B alleles. Since there is significant linkage disequilibrium within the HLA class I locus in Tongan people (1), this has allowed two groups of subjects with distinct HLA class I haplotypes to be studied. In addition, we restricted the study to subjects infected with a genotype C3 HBV.     

The effect of Gratiana boliviana (Coleoptera: Chrysomelidae) herbivory on growth and population density of tropical soda apple (Solanum viarum) in Florida

The effect of herbivory by Gratiana boliviana Spaeth (Coleoptera: Chrysomelidae) on the invasive, tropical soda apple (TSA) (Solanum viarum Dunal, Solanaceae), was investigated using exclusion methods and by monitoring the density of G. boliviana and the weed at four locations over a period of 40 months. TSA plants protected by insecticide were taller, wider, and had greater canopy cover that unprotected plants, and plants in closed cages were taller and wider than those in open cages. Survival of plants was higher in plots protected with insecticide than in unprotected plots in both years of a 2-year study. In the population dynamics study, the initial density of TSA was 4–5 times higher at one of the locations than at the other three sites, but within 3 years, TSA density at the high density site had declined by 90%. At the three sites which initially had a low abundance of TSA, density remained low throughout the study. The intrinsic rate of increase of G. boliviana varied between –3.9 and 4.5, but over the 3-year study, was not different from zero, indicating a stable population. The intrinsic rate of increase was lower than zero for the period from October to January, and greater than zero during the January to April period. In the periods from April to July and July to October, the rate of increase was not different from zero. The implications of these results for biological control of TSA in Florida are discussed.     

Crystal structure and molecular dynamics studies of human purine nucleoside phosphorylase complexed with 7-deazaguanine

In humans, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. HsPNP is a target for inhibitor development aiming at T-cell immune response modulation. Here we report the crystal structure of HsPNP in complex with 7-deazaguanine (HsPNP:7DG) at 2.75 Å. Molecular dynamics simulations were employed to assess the structural features of HsPNP in both free form and in complex with 7DG. Our results show that some regions, responsible for entrance and exit of substrate, present a conformational variability, which is dissected by dynamics simulation analysis. Enzymatic assays were also carried out and revealed that 7-deazaguanine presents a lower inhibitory activity against HsPNP (K_i = 200 μM). The present structure may be employed in both structure-based design of PNP inhibitors and in development of specific empirical scoring functions.     

Cloning and purification of recombinant proteins of Mycoplasma hyopneumoniae expressed in Escherichia coli

Mycoplasma hyopneumoniae, the etiological agent of swine enzootic pneumonia, is an important pathogen in the swine industry worldwide. Vaccination is the most cost-effective strategy for controlling and prevention of this disease. However, investigations on pathogenicity mechanisms as well as current serological detection methods and the development of new recombinant subunit vaccines are hampered by the lack of known and well characterized species-specific M. hyopneumoniae antigens. In this work, 54 predicted genes encoding proteins with potential to be used as subunit vaccine or antigens in diagnostic tests were selected, amplified by PCR and cloned into Escherichia coli expression vectors. Recombinant protein expression, solubility and yields were analyzed. The majority of the recombinant proteins were expressed in inclusion bodies. After solubilization with urea or N-lauroyl sarcosine, recombinant proteins were purified by Ni²⁺ affinity chromatography. This approach allowed purification of thirty recombinant M. hyopneumoniae proteins which will be evaluated as vaccine candidates and/or as antigens to be used in diagnostic tests.     

Functional elucidation of a key contact between tRNA and the large ribosomal subunit rRNA during decoding

The selection of cognate tRNAs during translation is specified by a kinetic discrimination mechanism driven by distinct structural states of the ribosome. While the biochemical steps that drive the tRNA selection process have been carefully documented, it remains unclear how recognition of matched codon:anticodon helices in the small subunit facilitate global rearrangements in the ribosome complex that efficiently promote tRNA decoding. Here we use an in vitro selection approach to isolate tRNA^Trp miscoding variants that exhibit a globally perturbed tRNA tertiary structure. Interestingly, the most substantial distortions are positioned in the elbow region of the tRNA that closely approaches helix 69 (H69) of the large ribosomal subunit. The importance of these specific interactions to tRNA selection is underscored by our kinetic analysis of both tRNA and rRNA variants that perturb the integrity of this interaction.