Receptor-ligand Combinations Research Articles

In Silico Screening of Breadfruit (Artocarpus altilis) Prenylated Flavonoids Identify Potential SARS-CoV Inhibitors

Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global health threat. Traditional herbals and dietary plants with medicinal values have a long antiviral history and, thus, are extensively studied in COVID-19 therapeutics development. Breadfruit (Artocarpus altilis) is a food crop with rich nutrient composition. This study screened selected breadfruit prenylated flavonoids for their potential inhibitory activities against the SARS-CoV family receptors using molecular docking and molecular dynamics (MD) simulation. The A. altilis prenylated flavonoids were selected as target ligands (artocarpin, artoindonesianin V, artonin M, cudraflavone A and cycloartobiloxanthone) and molecular targets from the SARS-CoV family were designated as receptors. Molecular docking was applied with the Lamarckian Genetic algorithm to measure the receptor-ligand orientation using AutoDock Vina software. The structural interactions of the receptor-ligand complexes were visualised using the Biovia Discovery Studio 4.5. Under all possible receptor-ligand combinations, the complexes’ minimum binding affinities (MBA) ranged from -5.5 to -9.1 kcal/mol and held by hydrophobic interactions, hydrogen bonds and electrostatic attractions. Receptor-ligand complexes with the least MBA (<-6.0 kcal/mol) along with strong structural interactions were validated by MD simulation using the GROMACS software. The 5RE4-artocarpin and 5RE4-artoindonesianin V showed the highest hydrophobic interactions at MBA=-6.6 kcal/mol and -6.4 kcal/mol, respectively. The trajectory analysis of 5RE4-artocarpin and 5RE4-artoindonesianin V complexes was fairly stable throughout a 50 ns MD simulation run. The findings conclude that artocarpin and artoindonesianin V are good potential SARS-CoV family receptor inhibitors.

Plant Development: Regular Ovule Spacing Is Controlled by Process-Specific Receptor–Ligand Combinations

Ovules are initiated in a highly regular pattern along the placenta inside the gynoecia of flowering plants. New research reveals that two separate EPFL peptide-mediated pathways functioning through a set of ERECTA-family receptors coordinate ovule patterning with fruit growth.

Higher Accuracy Achieved for Protein-Ligand Binding Pose Prediction by Elastic Network Model-Based Ensemble Docking.

Molecular docking plays an indispensable role in predicting the receptor-ligand interactions in which the protein receptor is usually kept rigid, whereas the ligand is treated as being flexible. Because of the inherent flexibility of proteins, the binding pocket of apo receptors might undergo significant conformational rearrangement upon ligand binding, which limits the prediction accuracy of docking. Here, we present an iterative anisotropic network model (iterANM)-based ensemble docking approach, which generates multiple holo-like receptor structures starting from the apo receptor and incorporates protein flexibility into docking. In a validation data set consisting of 233 chemically diverse cyclin-dependent kinase 2 (CDK2) inhibitors, the iterANM-based ensemble docking achieves higher capacity to reproduce native-like binding poses compared with those using single apo receptor conformation or conformational ensemble from molecular dynamics simulations. The prediction success rate within the top5-ranked binding poses produced by the iterANM can further be improved through reranking with the molecular mechanics-Poisson-Boltzmann surface area method. In a smaller data set with 58 CDK2 inhibitors, the iterANM-based ensemble shows a higher success rate compared with the flexible receptor-based docking procedure AutoDockFR and other receptor conformation generation approaches. Further, an additional docking test consisting of 10 diverse receptor-ligand combinations shows that the iterANM is robustly applicable for different receptor structures. These results suggest the iterANM-based ensemble docking as an accurate, efficient, and practical framework to predict the binding mode of a ligand for receptors with flexibility.

Asymmetrical diversification of the receptor-ligand interaction controlling self-incompatibility in Arabidopsis.

How two-component genetic systems accumulate evolutionary novelty and diversify in the course of evolution is a fundamental problem in evolutionary systems biology. In the Brassicaceae, self-incompatibility (SI) is a spectacular example of a diversified allelic series in which numerous highly diverged receptor-ligand combinations are segregating in natural populations. However, the evolutionary mechanisms by which new SI specificities arise have remained elusive. Using in planta ancestral protein reconstruction, we demonstrate that two allelic variants segregating as distinct receptor-ligand combinations diverged through an asymmetrical process whereby one variant has retained the same recognition specificity as their (now extinct) putative ancestor, while the other has functionally diverged and now represents a novel specificity no longer recognized by the ancestor. Examination of the structural determinants of the shift in binding specificity suggests that qualitative rather than quantitative changes of the interaction are an important source of evolutionary novelty in this highly diversified receptor-ligand system.

Effect of modular conjugation strategy for N-acetylgalactosamine-targeted antisense oligonucleotides

The asialoglycoprotein receptor (ASGPr) and N-acetylgalactosamine (GalNAc) is one of the most reliable receptor-ligand combinations for delivering antisense oligonucleotides (ASOs) to the liver. Here, we show that a modular GalNAc conjugation strategy allows us to reinforce the activity of the parent, naked 2′,4′-BNA/LNA gapmer targeting apolipoprotein B. The conjugation partly reduced a possible hepatotoxicity of the parent ASO. The structure-activity study revealed the significance of the metabolic susceptibility of the GalNAc moiety to nucleolytic cleavage that results in exposure of the parent gapmer. The broad usefulness of our delivery strategy of ASOs to the liver has been demonstrated.

A protective effect conferred by KIR3DL1 and its cognate ligand against cervical cancer among ethnic Han Chinese population and its potential mechanism

To explore the role of inhibitory KIR (iKIR) and its cognate HLA ligand in the occurrence and development of cervical cancer among ethnic Han Chinese and its potential mechanism. Peripheral blood samples from 265 Han Chinese patients with cervical intraepithelial neoplasia (CIN)/cervical cancer and 200 ethnically matched healthy controls were collected. The results of KIR PCR-SSP, HLA PCR-rSSO and KIR3DL1 PCR-SBT, together with cervical cancer data from the TCGA database, were used to assess the association of iKIR genes, receptor-ligand gene combinations, iKIR transcription level in the tumor tissue and the KIR3DL1 alleles with the occurrence and development of cervical cancer. Among the four iKIR genes (KIR2DL1, 2DL2/3, 3DL1 and 3DL2), the frequencies of KIR3DL1 and KIR3DL1-HLA-Bw4 genes among controls were significantly higher than those of the cervical cancer group (96.5% vs. 87.0%, P = 0.018; 81.5% vs. 64.8%, P=0.009). The survival rate of cervical cancer patients with a high transcription level of KIR3DL1 in tumor tissues was significantly higher than those with a low/medium transcription level (P=0.028). The frequency of strong-inhibitory and high-expression KIR3DL1*01502 allele among the healthy population was significantly higher than that of the cervical cancer group (76.0% vs. 59.3%, P =0.015). Combined KIR3DL1 and KIR3DL1-HLA-Bw4 can confer a protective effect against the development of cervical cancer, which may be attributed to the strong-inhibitory and high-expression allele of KIR3DL1*01502.

Association between KIR-HLA combination and ulcerative colitis and Crohn's disease in a Japanese population.

Inflammatory bowel disease (IBD) consists of ulcerative colitis (UC) and Crohn’s disease (CD). Natural killer cell responses play a crucial role in autoimmune disease through innate immunity, in which killer cell immunoglobulin-like receptors (KIRs) are closely involved. Although the genetic combination of KIRs with their specific HLA class I ligands has been associated with IBD in Caucasians, such KIR-HLA receptor-ligand combinations are not fully understood in the Japanese. We investigated 14 KIR genes along with HLA-Bw and -C ligands in 90 patients with UC and 50 patients with CD and compared them with the characteristics of 325 healthy control subjects. The frequency of HLA-Bw4 was significantly increased in patients with UC (P = 1.3 × 10−6; odds ratio [OR] = 3.39) and CD (P = 0.0065; OR = 2.32) versus controls. The UC group had a significantly higher frequency of KIR2DS3 (P = 0.024; OR = 1.94) and lower frequency of KIR2DS4 (P = 0.019; OR = 0.40) and KIR2DL1-HLA-C2 (P = 0.035; OR = 0.47). The Tel-A/B haplotype was significantly decreased in UC patients (P = 0.0056; OR = 0.49). The frequency of KIR3DL1-HLA-Bw4 was significantly higher in patients with UC (P = 4.3 × 10−6; OR = 3.12) and CD (P = 0.0067; OR = 2.30). In conclusion, HLA-Bw4 and KIR-HLA pairs may play an important role in the genetic susceptibility to IBD in the Japanese.

OR9 Certain KIR-HLA genotypes show improved progression-free survival in patients with multiple myeloma treated with Isatuximab (SAR650984, Anti-CD38 MAB), lenalidomide and dexamethasone

Aim The functional competency of NK cell is determined by the interactions of germline-encoded killer cell immunoglobulin-like receptors (KIR) and their cognate HLA class I ligands, which are substantially variable between individuals. To determine if certain KIR-HLA interactions modulate the efficacy of Isatuximab (Isa), a chimeric anti-CD38 monoclonal antibody used in clinical trials treating multiple myeloma. Methods We characterized KIR and HLA class I types using luminex rSSO methods on a cohort of 57 patients with relapsed and/or refractory multiple myeloma (RRMM) enrolled on the Isa/Len/Dex trial (NCT01749969). We used Kaplan-Meier methods to estimate progression-free survival (PFS) between patients carrying different KIR genotypes, and between patients carrying or missing distinct KIR + HLA pairs. Results Patients with AA KIR genotypes (carrying more inhibitory KIRs and 0–1 activating KIR) have higher median PFS compared to those carrying Bx (AB or BB) genotypes (carrying less inhibitory KIRs and 2–6 activating KIRs) (Fig. A). Moreover, patients carrying KIR2DL3+ and HLA-C1+, KIR3DL1+ and HLA-B Bw4I80+, and/or KIR3DL2+ and HLA-A3/A11+ have higher median PFS compared to those that were missing these receptor-ligand combinations (Fig.D, E, F). In contrast, patients carrying KIR3DL1+ and HLA-B Bw4T80+, KIR2DL1+ and HLA-C2+ and/or KIR2DL2+ and HLA-C1+ showed worst PFS compared to those missing these KIR-HLA pairs (Fig.E, B, C). Carriers of KIR2DS2 and/or KIR2DS3 showed worst PFS compared to those missing these activating KIRs. Presence/absence of KIR2DS1, 2DS4, 2DS5, and/or 3DS1 did not make any difference in PFS. Conclusions Our results demonstrate that certain KIR-HLA interactions are associated with improved progression free survival in patients with RRMM treated with Isa/Len/Dex. The beneficial impact of individual KIR-HLA combinations is likely due to differences in binding strength or specificity, which influence NK licensing and/or inhibition, and effects the ability to induce ADCC.

Killer Immunoglobulin-Like Receptor Profiles Are not Associated with Risk of Amoxicillin-Clavulanate-Induced Liver Injury in Spanish Patients.

Natural killer cells are an integral part of the immune system and represent a large proportion of the lymphocyte population in the liver. The activity of these cells is regulated by various cell surface receptors, such as killer Ig-like receptors (KIR) that bind to human leukocyte antigen (HLA) class I ligands on the target cell. The composition of KIR receptors has been suggested to influence the development of specific diseases, in particularly autoimmune diseases, cancer and reproductive diseases. The role played in idiosyncratic drug-induced liver injury (DILI) is currently unknown. In this study, we examined KIR gene profiles and HLA class I polymorphisms in amoxicillin-clavulanate (AC) DILI patients in search for potential risk associations. One hundred and two AC DILI patients and 226 controls were genotyped for the presence or absence of 16 KIR loci, including the two pseudogenes 2DP1 and 3DP1. No significant differences were found in the distribution of individual KIRs between patients and controls, which were comparable to previously reported KIR data from ethnically similar cohorts. The 21.6 and 21.2% of the patients and controls, respectively, were homozygous haplotype A carriers, while 78.4 and 78.8%, respectively, contained at least one B haplotype (Bx). The genotypes translated into 27 (AC DILI) and 46 (controls) different gene profiles, with 19 being present in both groups. The most frequent Bx gene profile containing KIRs 2DS2, 2DL2, 2DL3, 2DP1, 2DL1, 3DL1, 2DS4, 3DL2, 3DL3, 2DL4, and 3PD1 was present in 16% of the DILI patients and 14% of the controls. The distribution of HLA class I epitopes did not differ significantly between AC DILI patients and controls. The most frequent receptor-ligand combinations in the DILI patients were 2DL3 + epitope C1 (67%) and 3DL1 + Bw4 motif (67%), while 2DL1 + epitope C2 (69%) and 3DL1 + Bw4 motif (69%) predominated in the controls. This is to our knowledge the first analysis of KIR receptor-HLA ligand associations in DILI, although our findings do not support evidence of these genetic variations playing a major role in AC DILI development.

MAGP2 controls Notch via interactions with RGD binding integrins: Identification of a novel ECM–integrin–Notch signaling axis

Canonical Notch signaling involves Notch receptor activation via interaction with cell surface bound Notch ligand. Recent findings also indicate that Notch signaling may be modulated by cross-talk with other signaling mechanisms. The ECM protein MAGP2 was previously shown to regulate Notch in a cell type dependent manner, although the molecular details of this interaction have not been dissected. Here, we report that MAGP2 cell type specific control of Notch is independent of individual Notch receptor-ligand combinations but dependent on interaction with RGD binding integrins. Overexpressed MAGP2 was found to suppress transcriptional activity from the Notch responsive Hes1 promoter activity in endothelial cells, while overexpression of a RGD→RGE MAGP2 mutant increased Notch signaling in the same cell type. This effect was not unique to MAGP2 since the RGD domain of the ECM protein EGFL7 was also found to be an important modulator of Hes1 promoter activity. Independently of MAGP2 or EGFL7, inhibition of RGD-binding integrins with soluble RGD peptides also increased accumulation of active N1ICD fragments and Notch responsive promoter activity independently of changes in Notch1, Jag1, or Dll4 expression. Finally, β1 or β3 integrin blocking antibodies also enhanced Notch signaling. Collectively, these results answer the question of how MAGP2 controls cell type dependent Notch signaling, but more importantly uncover a new mechanism to understand how extracellular matrices and cellular environments impact Notch signaling.

The effect of KIR2D-HLA-C receptor-ligand interactions on clinical outcome in a HIV-1 CRF01_AE-infected Thai population.

Class I human leukocyte antigen (HLA) alleles interact with both cytotoxic T lymphocytes through their T-cell receptors, and natural killer cells through their killer immunoglobulin-like receptors (KIRs). Compared with the reported protective effect of KIR3DL1/S1-HLA-Bw4 interactions in HIV-infected patients, the effect of KIR2D-HLA-C combinations on HIV control remains unclear. Here, we investigate the effect of KIR2D-HLA-C combinations on HIV disease progression. We performed a cross-sectional and longitudinal analysis of a Thai HIV cohort. Two hundred and nine HIV-1 CRF01_AE-infected, treatment-naive Thai patients (CD4 T-cell counts of >200/μl) and 104 exposed seronegatives were studied. The effect of KIR-HLA receptor-ligand combinations on viral transmission and survival rate was statistically analyzed. We found the following results: higher frequency of patients expressing both KIR2DL3 and HLA-C1 among infected patients compared with exposed seronegative (odds ratio 4.8, P = 0.004), higher viral load in patients expressing HLA-C1 with KIR2DL3 compared with those without this receptor-ligand combination (median 4.8 vs. 4.2 log copies/ml, P = 0.033), higher numbers of KIR2DL3-HLA-C1 interactions was associated with a higher viral load (β = 0.13, P = 0.039 by linear regression model), and higher mortality rate in carriers of the KIR2DL3-HLA-C1 combination (adjusted hazard ratio 1.9, P = 0.012 by Cox hazard model). We have identified a deleterious effect of the KIR2DL3-HLA-C1 receptor-ligand combination on HIV clinical outcomes in a Thai cohort. Further investigation into mechanisms underlying this susceptibility may aid the understanding of the role of natural killer cells in HIV disease control and pathogenesis.

Investigating the mechanism of TLR signaling crosstalk in mouse macrophages (IRM5P.702)

Abstract It is well established that the TRIF- and MyD88-dependent signaling branches in the TLR4 pathway cooperate to induce a strong innate immune response. It is also known that certain toll-like receptor (TLR) ligand combinations can induce synergistic production of some cytokines. However, the molecular mechanisms underlying this signaling pathway cooperation remain elusive. We conducted a microarray analysis of mouse bone marrow-derived macrophages stimulated with all pairwise combinations of the TLR ligands LPS, Pam3CSK4, R848 and poly I:C to characterize synergy in transcript induction. We find that the crosstalk between the different TLR pathways is most prevalent in combinations which induce both the TRIF and the MyD88 pathways, which is essentially a mimic of the TLR4 pathway. Genes synergistically induced under dual TLR stimulation are mainly cytokines and immune effectors, including IL-6, IL-10, IL-1α, IL-1β, haptoglobin, lipocalin 2, and SOCS3. Based on these data, over 200 synergy factor candidates were picked and targeted using RNAi screening, with IL-6 and IL-12p40 secretion as read outs. A total of 32 hits were identified in the primary screening, including well-known regulators of secondary response gene transcription such as Nfkbiz, Irak2, Tnfaip3 and Tnip1. Secondary screening is ongoing to verify what role the 32 identified genes play in the MyD88/TRIF-dependent synergistic induction of immune regulators.

Polymorphisms of killer immunoglobulin-like receptors (KIRs) and HLA ligands in northeastern Thais

Killer cell immunoglobulin-like receptors (KIRs) are cell surface receptors on natural killer (NK) cells and subsets of T cells. The functions of NK cells are partly regulated by interactions between KIRs and HLA ligands on target cells. In this study, the presence or absence of 17 KIR genes and their known HLA ligands have been investigated in 235 unrelated individuals living in northeastern Thailand (NET). Subtypes of KIR2DS4 including full length (KIR2DS4F) and deleted forms (KIR2DS4D) have also been determined. Framework genes (KIR2DL4, 3DL2, 3DL3, and 3DP1) were found in all individuals and KIR genes belonging to the A haplotype (KIR2DL1, 2DL3, 3DL1, and 2DS4) were present in more than 90% of NET. KIR2DS4D (61.7%) was more common than KIR2DS4F (52.8%). A total of 33 different KIR genotypes were observed. Of these, three new genotypes were identified. The most common genotype (AA) was observed in 35.7% of NET, and HLA-C alleles bearing the C1 epitope (HLA-C1) had the highest frequency (97%). All individuals had at least one inhibitory KIR and its corresponding HLA ligand; 40.9% of NET had three pairs of receptor-ligand combinations, and 18.3% had all three receptor-ligand combinations of KIR2DL3+C1, 3DL1+Bw4, and 3DL2+A11. Surprisingly, the patterns of KIR gene frequencies in NET are more similar to those of Caucasians than Japanese, Korean, and Chinese. This is the first report on complete analysis of KIR and known HLA ligands in Thais. These data provide basic knowledge on KIR for further studies on disease associations and transplantation in northeastern Thais.

Abstract LB-332: Purified ex vivo expanded and activated human Natural Killer cells efficiently kill glioblastoma cells in a dose- and donor-dependent manner.

Abstract Glioblastoma (GBM) is the most malignant and frequent brain tumor. Despite combined surgery, chemo- and radiotherapy, GBM patients' survival remains very low, emphasizing the need for further development of novel therapies. The promising strategy is adoptive cellular immunotherapy. Studies investigating purified Natural Killer (NK) cells in adoptive transfer therapies for solid tumors are sparse. Most clinical trials in GBM patients have used Lymphokine Activated Killer (LAK) cells that are composed of T-lymphocytes and NK cells, where the former can contribute to Graft vs Host Disease (GvHD) and inhibit NK cytotoxicity. We hypothesized that purified allogeneic NK cells may be potent effectors for adoptive transfer in GBM patients due to their cytotoxicity, cytokine production and immunomodulatory properties. We performed in vitro cytotoxicity experiments using patient-derived GBM cells co-cultured with resting or ex vivo expanded and activated NK cells isolated from healthy donors or LAK cells derived from the same donors. We also investigated the role of Killer Immunoglobulin-like Receptor (KIR) versus Human Leucocyte Antigen (HLA) ligand mismatch in determining potency of NK cell-mediated cytotoxicity in vitro and in vivo in human GBM-bearing Nod-Scid mice, treated with activated human NK cells isolated from 2 different donors with or without KIR-HLA ligand mismatch. The primary end points were animals' survival, NK persistence in vivo and mechanisms of therapeutic efficacy. All GBM tumors investigated for cytotoxicity expressed variable levels of MHC class I, MICA/B, ULBP1-3 as evaluated by flow cytometry. High resolution HLA-typing of the GBM cells and KIR-typing of donors was performed at genomic level. Resting and activated NK cells were also characterized for expression levels of NKp46, NKG2D/A and KIRs by flow cytometric phenotyping. Activated NK cells killed GBM cells more efficiently than resting NK cells and LAK cells in a dose- and donor-dependent manner. By examining tumor-donor pairs for KIR-HLA ligand mismatch we could identify the receptor-ligand combinations that determine greater cytotoxic potential in vitro in suspension cultures vs. in vivo in solid tumors in mice. We demonstrate that KIR-HLA ligand mismatch was important in killing the GBM cells by resting NK cells, however, the cytotoxicity mediated by activated NK cells was less dependent on KIR-HLA mismatch. Activated NK cells upregulated expression of particular KIRs when compared to resting NK cells from the same donor and this partially explains why activated NK cell - mediated cytotoxicity is less dependent on KIR-HLA ligand mismatch. We conclude that purified and activated NK cells are better effectors against gliomas than LAK cells. However, the KIR-HLA mismatch in solid tumors may not be the primary determinant of activated NK potency in vitro but may have relevance in vivo. Citation Format: Justyna Kmiecik, Andrea Gras Navarro, Per Ø. Enger, Mateusz Zelkowski, Jacques Zimmer, Martha Chekenya Enger. Purified ex vivo expanded and activated human Natural Killer cells efficiently kill glioblastoma cells in a dose- and donor-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-332. doi:10.1158/1538-7445.AM2013-LB-332 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Activating KIR/HLA complexes in classic Kaposi's Sarcoma.

BackgroundClassic Kaposi's Sarcoma (cKS) is a rare vascular tumor associated with Human Herpesvirus 8 (KSHV) infection, nevertheless not all KSHV-infected individuals have cKS.ObjectiveWe investigated whether particular KIR/HLA receptor/ligand genotypes would be preferentially present in KSHV-infected and uninfected individuals who have or have not developed cKS.MethodsKIR/HLA genotypes were analyzed by molecular genotyping in 50 KSHV-infected individuals who did or did not have cKS and in 33 age-and sex-matched KSHV seronegative individuals.ResultsThere was no association of individual KIR, HLA or receptor ligand combinations with KSHV infection. However, activating KIR and KIR/HLA genotypes were significantly more frequent in cKS cases, specifically KIR3DS1, KIR2DS1, and KIR2DS1 with its HLA-C2 ligand.ConclusionA nonspecific inflammatory response triggered by activation of NK cells upon KIR-HLA interaction could be associated with the pathogenesis of KS.

Lineage-specific co-evolution of the Egf receptor/ligand signaling system

BackgroundThe epidermal growth factor receptor (Egfr) with its numerous ligands has fundamental roles in development, cell differentiation and physiology. Dysfunction of the receptor-ligand system contributes to many human malignancies. Consistent with such various tasks, the Egfr gene family has expanded during vertebrate evolution as a consequence of several rounds of whole genome duplication. Of particular interest is the effect of the fish-specific whole genome duplication (FSGD) on the ligand-receptor system, as it has supplied this largest group of vertebrates with additional opportunities for sub- and/or neofunctionalization in this signaling system.ResultsWe identified the predicted components of the Egf receptor-ligand signaling system in teleost fishes (medaka, platyfish, stickleback, pufferfishes and zebrafish). We found two duplicated egfr genes, egfra and egfrb, in all available teleost genomes. Surprisingly only one copy for each of the seven Egfr ligands could be identified in most fishes, with zebrafish hbegf being the only exception. Special focus was put on medaka, for which we more closely investigated all Egf receptors and Egfr ligands. The different expression patterns of egfra, egfrb and their ligands in medaka tissues and embryo stages suggest differences in role and function. Preferential co-expression of different subsets of Egfr ligands corroborates the possible subfunctionalization and specialization of the two receptors in adult tissues. Bioinformatic analyses of the ligand-receptor interface between Egfr and its ligands show a very weak evolutionary conservation within this region. Using in vitro analyses of medaka Egfra, we could show that this receptor is only activated by medaka ligands, but not by human EGF. Altogether, our data suggest a lineage-specific Egfr/Egfr ligand co-evolution.ConclusionsOur data indicate that medaka Egfr signaling occurs via its two copies, Egfra and Egfrb, each of them being preferentially coexpressed with different subsets of Egfr ligands. This fish-specific occurrence of Egf receptor specialization offers unique opportunities to study the functions of different Egf receptor-ligand combinations and their biological outputs in vertebrates. Furthermore, our results strongly support the use of homologous ligands in future studies, as sufficient cross-specificity is very unlikely for this ligand/receptor system.

Use of chemometric methodology in optimizing conditions for competitive binding partial filling affinity capillary electrophoresis

This work expands the knowledge of the use of chemometric response surface methodology (RSM) in optimizing conditions for competitive binding partial filling ACE (PFACE). Specifically, RSM in the form of a Box-Behnken design was implemented in flow-through PFACE (FTPFACE) to effectively predict the significance of injection time, voltage, and neutral ligand (neutral arylsulfonamide) concentration, [L(o)], on protein-neutral ligand binding. Statistical analysis results were used to create a model for response surface prediction via contour and surface plots at a given maximum response (DeltaRMTR) to reach a targeted K(b) = 2.50 x 10(6) M(-1). The adequacy of the model was then validated by experimental runs at the optimal predicted solution (injection time = 2.3 min, voltage = 11.6 kV, [L(o)] = 1.4 microM). The achieved results greatly extend the usefulness of chemometrics in ACE and provide a valuable statistical tool for the study of other receptor-ligand combinations.

KIR/HLA Ligand Incompatibility in Kidney Transplantation

The polymorphic family of killer-cell immunoglobulin-like receptors (KIRs) consists of activating and inhibitory receptors expressed by natural killer (NK) cells and effector T cells that recognize human leukocyte antigen (HLA) class I ligands. It has been suggested that KIR/HLA incompatibility exerts beneficial effects in hematopoietic stem cell transplantation. To elucidate whether certain receptor-ligand combinations between recipient KIR and donor HLA antigens lead to enhanced alloreactivity of NK cells associated with acute rejection (aRx) after kidney transplantation, we analyzed the entirety of matches/mismatches between KIR genes and known HLA ligands for aRx patients (n=105) compared to patients with stable renal function (n=119). Whereas HLA-C ligand incompatibility between donor and recipient has no influence on aRx, grafts derived from donors homozygous for HLA-C group 2 alleles seem to demonstrate a better outcome (P=0.052). Additionally, a higher number of inhibitory receptors in the recipient's genotype (P=0.042), a significant higher number of matches for the receptors KIR2DL2/DS2 (P=0.004), as well as a higher number of mismatches for KIR2DL3 (P=0.014) could be observed for patients with stable renal function. Our data illustrate that certain KIR/HLA class I ligand combinations between donor and recipient might influence graft short-term outcome after renal transplantation.

Association of MCP‐1 and CCR2 polymorphisms with the risk of late acute rejection after renal transplantation in Korean patients

Among the factors modulating transplant rejection, chemokines and their respective receptors deserve special attention. Increased expression of monocyte chemoattractant protein-1 (MCP-1) and its corresponding receptor (chemokine receptor-2, CCR2) has been implicated in renal transplant rejection. To determine the impact of the MCP-1-2518G and CCR2-64I genotypes on renal allograft function, 167 Korean patients who underwent transplantation over a 25-year period were evaluated. Genomic DNA was genotyped using polymerase chain reaction followed by restriction fragment length polymorphism analysis. Fifty-five (32.9%) patients were homozygous for the MCP-1-2518G polymorphism. Nine (5.4%) patients were homozygous for the CCR2-64I polymorphism. None of the investigated polymorphism showed a significant shift in long-term allograft survival. However, a significant increase was noted for the risk of late acute rejection in recipients who were homozygous for the MCP-1-2518G polymorphism (OR, 2.600; 95% CI, 1.125–6.012; P = 0.022). There was also an association between the MCP-1-2518G/G genotype and the number of late acute rejection episodes (P = 0.024). Although there was no difference in the incidence of rejection among recipients stratified by the CCR2-V64I genotype, recipients with the CCR2-V64I GG genotype in combination with the MCP-1-2518G/G genotype had a significantly higher risk of acute or late acute rejection among the receptor-ligand combinations (P = 0.006, P = 0.008, respectively). The MCP-1 variant may be a marker for risk of late acute rejection in Korean patients.

Expression of fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3) in the developing head and face.

Fibroblast growth factors may play an important role in the differential growth of the skull, brain, and facial prominences. In order to understand the role of FGFs in vivo, we have analyzed the competency of head mesenchyme to respond to FGFs via expression of the high affinity receptors FGFR1, 2, and 3. Receptor transcripts, especially those of FGFR2 and FGFR3, were localized to specific regions of the head. We raise the possibilities of particular receptor-ligand combinations and the possible functions of these interactions in the morphogenesis of the head, face, and brain. Finally, we discuss the relationship between FGF receptor expression in the chicken and the phenotypes of FGF receptor mutations in humans.