CD40 is essential in the upregulation of TRAF proteins and NF-kappaB-dependent proinflammatory gene expression after arterial injury

Despite extensive investigations, restenosis, which is characterized primarily by neointima formation, remains an unsolved clinical problem after vascular interventions. A recent study has shown that CD40 signaling through TNF receptor associated factor 6 (TRAF6) plays a key role in neointima formation after carotid artery injury; however, underlying mechanisms are not clearly elucidated. Because neointima formation may vary significantly depending on the type of injury, we first assessed the effect of CD40 deficiency on neointima formation in 2 injury models, carotid artery ligation and femoral artery denudation injury. Compared with wild-type mice, CD40 deficiency significantly reduced neointima formation and lumen stenosis in two different models. Further, we investigated the mechanism by which CD40 signaling affects neointima formation after arterial injury. In wild-type mice, the expression levels of CD40, several TRAF proteins, including TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6, as well as total NF-kB p65 and phospho-NF-kB p65, in the carotid artery were markedly upregulated within 3-7 days after carotid ligation. Deficiency of CD40 abolished the injury-induced upregulation of TRAFs including TRAF6 and NF-kB-p65 in the injured vessel wall. Further, CD40(-/-) mice showed a significant decrease in the recruitment of neutrophils (at 3, 7d) and macrophages (at 7, 21d) into injured artery; this effect was most likely attributed to inhibition of NF-kB activation and marked downregulation of NF-kB-related gene expression, including cytokines (TNFα, IL-1β, IL-6), chemokines (MCP-1), and adhesion molecules (ICAM-1, VCAM-1). Moreover, neutrophil recruitment in a model of thioglycollate-induced peritonitis is impaired in CD40-deficient mice. In vitro data revealed that CD40 deficiency blocked CD40L-induced NF-kB p65 nuclear translocation in leukocytes. Altogether, our data identified for the first time that CD40 is essential in the upregulation of TRAF6, NF-kB activation, and NF-kB-dependent proinflammatory genes in vivo. Our findings firmly established the role for CD40 in neointima formation in 2 distinct injury models.     

Location of immunization and interferon-γ are central to induction of salivary gland dysfunction in Ro60 peptide immunized model of Sjögren's syndrome

Introduction

Anti-Ro antibodies can be found in the serum of the majority of patients with Sjögren''s syndrome (SS). Immunization with a 60-kDa Ro peptide has been shown to induce SS-like symptoms in mice. The aim of this study was to investigate factors involved in salivary gland (SG) dysfunction after immunization and to test whether the induction of SS could be improved.

Methods

Ro60 peptide immunization was tested in Balb/c mice, multiple antigenic peptide (MAP)-Ro60 and Pertussis toxin (PTX) were tested in SJL/J mice. In addition, two injection sites were compared in these two strains: the abdominal area and the tailbase. Each group of mice was tested for a loss of SG function, SG lymphocytic infiltration, anti-Ro and anti-La antibody formation, and cytokine production in cultured cells or homogenized SG extracts.

Results

Ro60 peptide immunization in the abdominal area of female Balb/c mice led to impaired SG function, which corresponded with increased Th1 cytokines (IFN-γ and IL-12) systemically and locally in the SG. Moreover, changing the immunization conditions to MAP-Ro60 in the abdominal area, and to lesser extend in the tailbase, also led to impaired SG function in SJL/J mice. As was seen in the Balb/c mice, increased IFN-γ in the SG draining lymph nodes accompanied the SG dysfunction. However, no correlation was observed with anti-MAP-Ro60 antibody titers, and there was no additional effect on disease onset or severity.

Conclusions

Effective induction of salivary gland dysfunction after Ro60 peptide immunization depended on the site of injection. Disease induction was not affected by changing the immunization conditions. However, of interest is that the mechanism of action of Ro60 peptide immunization appears to involve an increase in Th1 cytokines, resulting in the induction of SG dysfunction.     

Genome-Wide Comparative in silico Analysis of Calcium Transporters of Rice and Sorghum

The mechanism of calcium uptake, translocation and accumulation in Poaceae has not yet been fully understood. To address this issue, we conducted genome-wide comparative in silico analysis of the calcium (Ca2+) transporter gene family of two crop species, rice and sorghum. Gene annotation, identification of upstream cis-acting ele- ments, phylogenetic tree construction and syntenic mapping of the gene family were performed using several bio- informatics tools. A total of 31 Ca2+ transporters, distributed on 9 out of 12 chromosomes, were predicted from rice genome, while 28 Ca2+ transporters predicted from sorghum are distributed on all the chromosomes except chromosome 10 (Chr 10). Interestingly, most of the genes on Chr 1 and Chr 3 show an inverse syntenic relation- ship between rice and sorghum. Multiple sequence alignment and motif analysis of these transporter proteins re- vealed high conservation between the two species. Phylogenetic tree could very well identify the subclasses of channels, ATPases and exchangers among the gene family. The in silico cis-regulatory element analysis suggested diverse functions associated with light, stress and hormone responsiveness as well as endosperm- and meris- tem-specific gene expression. Further experiments are warranted to validate the in silico analysis of the predicted transporter gene family and elucidate the functions of Ca2+ transporters in various biological processes.     

A novel dimer-tetramer transition captured by the crystal structure of the HIV-1 Nef

HIV-1 Nef modulates disease progression through interactions with over 30 host proteins. Individual chains fold into membrane-interacting N-terminal and C-terminal core (Nef_core) domains respectively. Nef exists as small oligomers near membranes and associates into higher oligomers such as tetramers or hexadecamers in the cytoplasm. Earlier structures of the Nef_core in apo and complexed forms with the Fyn-kinase SH3 domain revealed dimeric association details and the role of the conserved PXXP recognition motif (residues 72–78) of Nef in SH3-domain interactions. The crystal structure of the tetrameric Nef reported here corresponds to the elusive cytoplasmic stage. Comparative analyses show that subunits of Nef_core dimers (open conformation) swing out with a relative displacement of ∼22 Å and rotation of ∼174° to form the ‘closed’ tetrameric structure. The changes to the association are around Asp125, a conserved residue important for viral replication and the important XR motif (residues 107–108). The tetramer associates through C4 symmetry instead of the 222 symmetry expected when two dimers associate together. This novel dimer-tetramer transition agrees with earlier solution studies including small angle X-ray scattering, analytical ultracentrifugation, dynamic laser light scattering and our glutaraldehyde cross-linking experiments. Comparisons with the Nef_core—Fyn-SH3 domain complexes reveal that the PXXP motif that interacts with the SH3-domain in the dimeric form is sterically occluded in the tetramer. However the 151–180 loop that is distal to the PXXP motif and contains several protein interaction motifs remains accessible. The results suggest how changes to the oligomeric state of Nef can help it distinguish between protein partners.     

Identifying hosts of families of viruses: a machine learning approach

Identifying emerging viral pathogens and characterizing their transmission is essential to developing effective public health measures in response to an epidemic. Phylogenetics, though currently the most popular tool used to characterize the likely host of a virus, can be ambiguous when studying species very distant to known species and when there is very little reliable sequence information available in the early stages of the outbreak of disease. Motivated by an existing framework for representing biological sequence information, we learn sparse, tree-structured models, built from decision rules based on subsequences, to predict viral hosts from protein sequence data using popular discriminative machine learning tools. Furthermore, the predictive motifs robustly selected by the learning algorithm are found to show strong host-specificity and occur in highly conserved regions of the viral proteome.     

A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis

Background

In spite of a consistent protection against tuberculosis (TB) in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG) fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB.

Methods/Principal Findings

In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin- a key latency antigen of M. tuberculosis to boost the BCG induced immunity. ‘BCG prime – DNA boost’ regimen (B/D) confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log₁₀ and 1.96 log₁₀ fewer bacilli in lungs and spleen, respectively; p<0.01). In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3⁺) simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)2.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3⁺ Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.     

Tryptophan residue is essential for immunoreactivity of a diagnostically relevant peptide epitope of A. fumigatus

The role of tryptophan (Trp¹⁷) in immunoreactivity of P1, the diagnostically relevant peptide from a major allergen/antigen of Aspergillus fumigatus, was evaluated by chemically modifying tryptophanyl residue of P1. In BIAcore kinetic studies, unmodified P1 showed a 100-fold higher binding with ABPA (Allergic Bronchopulmonary Aspergillosis) patients’ IgG [K_D (equilibrium dissociation constant) = 2.74 e⁻⁸ ± 0.13 M] than the controls’ IgG (K_D = 2.97 e⁻⁶± 0.14 M), whereas chemically-modified P1 showed similar binding [K_D patients’ IgG = 3.25 e⁻⁷± 0.16 M, K_D controls’ IgG = 3.86 e⁻⁷± 0.19 M] indicating loss of specific immunoreactivity of P1 on tryptophan modification. Modified P1 showed loss of specific binding to IgE and IgG antibodies of ABPA patients in ELISA (Enzyme-Linked Immunosorbent Assay). The study infers that tryptophan residue (Trp¹⁷) is essential for immunoreactivity of P1.     

Fine structure analysis of a protein folding transition state; distinguishing between hydrophobic stabilization and specific packing

Developing a detailed understanding of the structure and energetics of protein folding transition states is a key step in describing the folding process. The phi-value analysis approach allows the energetic contribution of side-chains to be mapped out by comparing wild-type with individual mutants where conservative changes are introduced. Studies where multiple substitutions are made at individual sites are much rarer but are potentially very useful for understanding the contribution of each element of a side-chain to transition state formation, and for distinguishing the relative importance of specific packing versus hydrophobic interactions. We have made a series of conservative mutations at multiple buried sites in the N-terminal domain of L9 in order to assess the relative importance of specific side-chain packing versus less specific hydrophobic stabilization of the transition state. A total of 28 variants were prepared using both naturally occurring and non-naturally occurring amino acids at six sites. Analysis of the mutants by NMR and CD showed no perturbation of the structure. There is no correlation between changes in hydrophobicity and changes in stability. In contrast, there is excellent linear correlation between the hydrophobicity of a side-chain and the log of the folding rate, ln(k(f)). The correlation between ln(k(f)) and the change in hydrophobicity holds even for substitutions that change the shape and/or size of a side-chain significantly. For most sites, the correlation with the logarithm of the unfolding rate, ln(k(u)), is much worse. Mutants with more hydrophobic amino acid substitutions fold faster, and those with less hydrophobic amino acid substitutions fold slower. The results show that hydrophobic interactions amongst core residues are an important driving force for forming the transition state, and are more important than specific tight packing interactions. Finally, a number of substitutions lead to negative phi-values and the origin of these effects are described.