Molecular dynamics simulations of CXCL-8 and its interactions with a receptor peptide, heparin fragments, and sulfated linked cyclitols

CXCL-8 (Interleukin 8) is a CXC chemokine with a central role in the human immune response. We have undertaken extensive in silico analyses to elucidate the interactions of CXCL-8 with its various binding partners, which are crucial for its biological function. Sequence and structure analyses showed that residues in the thirdq β-sheet and basic residues in the heparin binding site are highly variable, while residues in the second β-sheet are highly conserved. Molecular dynamics simulations in aqueous solution of dimeric CXCL-8 have been performed with starting geometries from both X-ray and NMR structures showed shearing movements between the two antiparallel C-terminal helices. Dynamic conservation analyses of these simulations agreed with experimental data indicating that structural differences between the two structures at quaternary level arise from changes in the secondary structure of the N-terminal loop, the 310-helix, the 30s, 40s, and 50s loops and the third β-sheet, resulting in a different interhelical separation. Nevertheless, the observation of these different states indicates that CXCL-8 has the potential to undergo conformational changes, and it seems likely that this feature is relevant to the mode of binding of glycosaminoglycan (GAG) mimetics such as cyclitols. Simulations of the receptor peptide fragment−CXCL-8 complex identified several specific interactions of the receptor peptide with CXCL-8 that could be exploited in the structure-based design of competitive peptides and nonpeptidic molecules targeting CXCL-8 for combating inflammatory diseases. Simulations of the CXCL-8 dimer complexed with a 24-mer heparin fragment and of the CXCL-8−receptor peptide complex revealed that Arg60, Lys64, and Arg68 in the dimer bind to cyclitols in a horseshoe pattern, defining a region which is spatially distinct from the receptor binding site. There appears to be an optimum number of sulfates and an optimum length of alkyl spacers required for the interaction of cyclitol inhibitors with the dimeric form of CXCL-8. Calculation of the binding affinities of cyclitol inhibitors reflected satisfactorily the ranking of experimentally determined inhibitory potencies. The findings of these molecular modeling studies will help in the search for inhibitors which can modulate various CXCL-8 biological activities and serve as an excellent model system to study CXC-inhibitor interactions.

A combination of local inflammation and central memory T cells potentiates immunotherapy in the skin

Adoptive T cell therapy uses the specificity of the adaptive immune system to target cancer and virally infected cells. Yet the mechanism and means by which to enhance T cell function are incompletely described, especially in the skin. In this study, we use a murine model of immunotherapy to optimize cell-mediated immunity in the skin. We show that in vitro - derived central but not effector memory-like T cells bring about rapid regression of skin-expressing cognate Ag as a transgene in keratinocytes. Local inflammation induced by the TLR7 receptor agonist imiquimod subtly yet reproducibly decreases time to skin graft rejection elicited by central but not effector memory T cells in an immunodeficient mouse model. Local CCL4, a chemokine liberated by TLR7 agonism, similarly enhances central memory T cell function. In this model, IL-2 facilitates the development in vivo of effector function from central memory but not effector memory T cells. In a model of T cell tolerogenesis, we further show that adoptively transferred central but not effector memory T cells can give rise to successful cutaneous immunity, which is dependent on a local inflammatory cue in the target tissue at the time of adoptive T cell transfer. Thus, adoptive T cell therapy efficacy can be enhanced if CD8+ T cells with a central memory T cell phenotype are transferred, and IL-2 is present with contemporaneous local inflammation. Copyright © 2012 by The American Association of Immunologists, Inc.

CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver

CD1d-restricted natural killer T (NKT) cells expressing invariant Valpha14Jalpha18 T cell receptor alpha-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver ( approximately 0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-gamma (IFN-gamma) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-gamma in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.

Impeding the adaptive response of prostate cancer to androgen targeted therapies with relaxin receptor antagonist

Androgen deprivation and androgen targeted therapies (ATT) are established treatments for prostate cancer (PCa). Although initially effective, ATT induces an adaptive response that leads to treatment resistance. Increased expression of relaxin-2 (RLN2) is an important alteration in the adaptive response. RLN2 has a well described role in PCa cell proliferation, adhesion and tumour growth. The objectives of this study were to develop cell models for studies of RLN2 signalling and to implement in vitro assays for evaluating the therapeutic properties of the unique RLN2 receptor (RXFP1) antagonist

Mutational analysis of the insulin‑like growth factor 1 receptor tyrosine kinase domain in non-small cell lung cancer patients

The insulin‑like growth factor 1 receptor (IGF1R) pathway plays an important role in the pathogenesis of non‑small cell lung cancer (NSCLC) and also provides a mechanism of resistance to targeted therapies. IGF1R is therefore an ideal therapeutic target and several inhibitors have entered clinical trials. However, thus far the response to these inhibitors has been poor, highlighting the importance of predictive biomarkers to identify patient cohorts who will benefit from these targeted agents. It is well‑documented that mutations and/or deletions in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain predict sensitivity of NSCLC patients to EGFR TK inhibitors. Single‑nucleotide polymorphisms (SNPs) in the IGF pathway have been associated with disease, including breast and prostate cancer. The aim of the present study was to elucidate whether the IGF1R TK domain harbours SNPs, somatic mutations or deletions in NSCLC patients and correlates the mutation status to patient clinicopathological data and prognosis. Initially 100 NSCLC patients were screened for mutations/deletions in the IGF1R TK domain (exons 16‑21) by sequencing analysis. Following the identification of SNP rs2229765, a further 98 NSCLC patients and 866 healthy disease‑free control patients were genotyped using an SNP assay. The synonymous SNP (rs2229765) was the only aberrant base change identified in the IGF1R TK domain of 100 NSCLC patients initially analysed. SNP rs2229765 was detected in exon 16 and was found to have no significant association between IGF1R expression and survival. The GA genotype was identified in 53.5 and 49.4% of NSCLC patients and control individuals, respectively. No significant difference was found in the genotype (P=0.5487) or allele (P=0.9082) frequencies between the case and control group. The present findings indicate that in contrast to the EGFR TK domain, the IGF1R TK domain is not frequently mutated in NSCLC patients. The synonymous SNP (rs2229765) had no significant association between IGF1R expression and survival in the cohort of NSCLC patients.