Cerebral Expression of the Neuregulin-1 Gene NRG1 during Induced Spatial Memory Impairment and Its Reversal in Aging Mice.

We studied the effects of chronic intranasal administration of amyloidogenic fibrils of the proinflammatory protein S100A9 alone or in combination with glutamate antibodies on the expression of the neuregulin-1 gene (NRG1), a regulator of various physiological processes, in particular, regulation of neurogenesis and apoptosis, in the hippocampus, prefrontal cortex, and cerebellum of aging C57BL/6 mice under conditions of long-term memory disturbances. Under conditions of amnesia induced by S100A9 fibrils, pronounced (>90%) blockade of the expression of the NRG1 gene was found in all cerebral structures. Glutamate antibodies prevented/corrected disturbances in the cerebral expression of the NRG1 gene, thereby maintaining the activity of the NRG1/ErbB molecular signaling system, probably associated with the formation of spatial memory.

Measurement of Immunological Parameters to Assess Human Body Readiness for Physical Load.

We measured the level of natural antibodies (nAb) to glutamate and GABA reflecting the balance of excitation and inhibition systems and involved in the adaptation processes in athletes receiving normalized physical activity in the dynamics of training (figure skaters, football players, and people actively involved in sports). It was found that each subject has an individual immunological profile and its parameters change in accordance with the training load. The measured levels of nAbs to GABA and glutamate correlate the physical activity of a person. The surveyed football players were divided into 3 groups according to the results of the analysis. Subjects of the first group had reliably high immunological indices in comparison with the control and were at the peak of physical form; in the third group, low immunological indices relative to the control indicated exhaustion and fatigue. The indicators of the second group corresponded to normal and demonstrated the resource of adaptation to load. The developed method can be used for assessing person's readiness for physical activity.

Effects of Antibodies to Glutamate on Cerebral Expression of the Tnfrsf1A Gene under Conditions of Spatial Amnesia Induced by Proinflammatory Protein S100A9 Fibrils in Aging Mice.

Proinflammatory S100A9 protein is a promoter of inflammation-linked neurodegeneration and the Tnfrsf1A gene encodes the TNF receptor 1A that binds TNFα to function as a regulator of inflammation. We studied the effects of chronic intranasal administration of in vitro prepared S100A9 fibrils alone or in combination with anti-glutamate antibodies on the expression of the Tnfrsf1A gene in the hippocampus, prefrontal cortex, and cerebellum of aging C57BL/6 mice under conditions of impaired spatial memory. A differential cerebral pattern of Tnfrsf1A gene activity and its modification by S100A9 fibrillar structures were observed: inhibition of Tnfrsf1A gene expression in the hippocampus and cerebellum and its activation in the prefrontal cortex. Anti-glutamate antibodies normalized the expression of the Tnfrsf1A gene in the prefrontal cortex by affecting the TNF signaling pathway and preventing the development of inflammation.

Integration of metabolomics, genomics, and immune phenotypes reveals the causal roles of metabolites in disease.

BACKGROUND: Recent studies highlight the role of metabolites in immune diseases, but it remains unknown how much of this effect is driven by genetic and non-genetic host factors. RESULT: We systematically investigate circulating metabolites in a cohort of 500 healthy subjects (500FG) in whom immune function and activity are deeply measured and whose genetics are profiled. Our data reveal that several major metabolic pathways, including the alanine/glutamate pathway and the arachidonic acid pathway, have a strong impact on cytokine production in response to ex vivo stimulation. We also examine the genetic regulation of metabolites associated with immune phenotypes through genome-wide association analysis and identify 29 significant loci, including eight novel independent loci. Of these, one locus (rs174584-FADS2) associated with arachidonic acid metabolism is causally associated with Crohn's disease, suggesting it is a potential therapeutic target. CONCLUSION: This study provides a comprehensive map of the integration between the blood metabolome and immune phenotypes, reveals novel genetic factors that regulate blood metabolite concentrations, and proposes an integrative approach for identifying new disease treatment targets.

Interactions of neuroimmune signaling and glutamate plasticity in addiction.

Chronic use of drugs of abuse affects neuroimmune signaling; however, there are still many open questions regarding the interactions between neuroimmune mechanisms and substance use disorders (SUDs). Further, chronic use of drugs of abuse can induce glutamatergic changes in the brain, but the relationship between the glutamate system and neuroimmune signaling in addiction is not well understood. Therefore, the purpose of this review is to bring into focus the role of neuroimmune signaling and its interactions with the glutamate system following chronic drug use, and how this may guide pharmacotherapeutic treatment strategies for SUDs. In this review, we first describe neuroimmune mechanisms that may be linked to aberrant glutamate signaling in addiction. We focus specifically on the nuclear factor-kappa B (NF-κB) pathway, a potentially important neuroimmune mechanism that may be a key player in driving drug-seeking behavior. We highlight the importance of astroglial-microglial crosstalk, and how this interacts with known glutamatergic dysregulations in addiction. Then, we describe the importance of studying non-neuronal cells with unprecedented precision because understanding structure-function relationships in these cells is critical in understanding their role in addiction neurobiology. Here we propose a working model of neuroimmune-glutamate interactions that underlie drug use motivation, which we argue may aid strategies for small molecule drug development to treat substance use disorders. Together, the synthesis of this review shows that interactions between glutamate and neuroimmune signaling may play an important and understudied role in addiction processes and may be critical in developing more efficacious pharmacotherapies to treat SUDs.

Autoimmune Epilepsy - Novel Multidisciplinary Analysis, Discoveries and Insights.

Epilepsy affects ~50 million people. In ~30% of patients the etiology is unknown, and ~30% are unresponsive to anti-epileptic drugs. Intractable epilepsy often leads to multiple seizures daily or weekly, lasting for years, and accompanied by cognitive, behavioral, and psychiatric problems. This multidisciplinary scientific (not clinical) 'Perspective' article discusses Autoimmune Epilepsy from immunological, neurological and basic-science angles. The article includes summaries and novel discoveries, ideas, insights and recommendations. We summarize the characteristic features of the respective antigens, and the pathological activity in vitro and in animal models of autoimmune antibodies to: Glutamate/AMPA-GluR3, Glutamate/NMDA-NR1, Glutamate/NMDA-NR2, GAD-65, GABA-R, GLY-R, VGKC, LGI1, CASPR2, and ß2 GP1, found in subpopulations of epilepsy patients. Glutamate receptor antibodies: AMPA-GluR3B peptide antibodies, seem so far as the most exclusive and pathogenic autoimmune antibodies in Autoimmune Epilepsy. They kill neural cells by three mechanisms: excitotoxicity, Reactive-Oxygen-Species, and complement-fixation, and induce and/or facilitate brain damage, seizures, and behavioral impairments. In this article we raise and discuss many more topics and new insights related to Autoimmune Epilepsy. 1. Few autoimmune antibodies tilt the balance between excitatory Glutamate and inhibitory GABA, thereby promoting neuropathology and epilepsy; 2. Many autoantigens are synaptic, and have extracellular domains. These features increase the likelihood of autoimmunity against them, and the ease with which autoimmune antibodies can reach and harm these self-proteins. 3. Several autoantigens have 'frenetic character'- undergoing dynamic changes that can increase their antigenicity; 4. The mRNAs of the autoantigens are widely expressed in multiple organs outside the brain. If translated by default to proteins, broad spectrum detrimental autoimmunity is expected; 5. The autoimmunity can precede seizures, cause them, and be detrimental whether primary or epiphenomenon; 6. Some autoimmune antibodies induce, and associate with, cognitive, behavioral and psychiatric impairments; 7. There are evidences for epitope spreading in Autoimmune Epilepsy; 8. T cells have different 'faces' in the brain, and in Autoimmune Epilepsy: Normal T cells are needed for the healthy brain. Normal T cells are damaged by autoimmune antibodies to Glutamate/AMPA GluR3, which they express, and maybe by additional autoantibodies to: Dopamine-R, GABA-R, Ach-R, Serotonin-R, and Adrenergic-R, present in various neurological diseases (summarized herein), since T cells express all these Neurotransmitter receptors. However, autoimmune and/or cytotoxic T cells damage the brain; 9. The HLA molecules are important for normal brain function. The HLA haplotype can confer susceptibility or protection from Autoimmune Epilepsy; 10. There are several therapeutic strategies for Autoimmune Epilepsy.

Effect of Anti-Glutamate Antibodies in Modeled Parkinsonian Syndrome.

We studied the effect of single and repeated intranasal administration of antibodies to glutamate in experimental parkinsonian syndrome induced by injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to C57BL/6J mice. Intranasal administration of anti-glutamate antibodies to mice in parallel with administration of MPTP over 10 days alleviated parkinsonian symptoms (oligokinesia and rigidity). In the serum of mice injected with antibodies to glutamate and/or MPTP, the titers of autoantibodies to glutamate and dopamine were higher than in control animals receiving saline. Single intranasal administration of anti-glutamate antibodies to mice with established parkinsonian syndrome did not affect the severity of parkinsonian symptoms.

Antibodies to Glutamate Facilitate Spatial Memory Formation in the Morris Water Maze in Aging C57BL/6 mice.

Intranasal administration of antibodies to glutamate in a dose of 250 µg/kg for two weeks facilitated spatial learning and memory formation in the Morris water maze in aging C57BL/6 mice. In animals treated with glutamate antibodies, the content of serotonin and dopamine metabolites 3-MT and HVA in the hippocampus decreased, but no changes in the metabolism of neurotransmitter acids were revealed. In the prefrontal cortex, dopamine level decreased and the content of its metabolite DOPAC increased; in parallel, an increase in excitatory and inhibitory amino acids (aspartic acid, glutamate, glycine, taurine, and GABA) was observed.

Natural variants of α-gliadin peptides within wheat proteins with reduced toxicity in coeliac disease.

The only generally accepted treatment of coeliac disease (CD) is a lifelong gluten-free diet. Wheat gluten proteins include gliadins, low and high molecular weight glutenins. However, we have found significant structural variations within these protein families among different cultivars. To determine which structural motifs might be less toxic than others, we assessed five variants of α-gliadin immunodominant CD-toxic peptides synthesised as 16mers in CD T cell stimulation assays with gluten-sensitive T cell lines generated from duodenal biopsies from CD-affected individuals. The peptides harboured the overlapping T cell epitopes DQ 2.5-glia-α-2 and naturally occurring variants that differed in certain amino acids (AA). The results revealed that introduction of two selected AA substitutions in α-gliadin peptides reduced immunogenicity. A peptide with three AA substitutions involving two glutamic acids (E) and one glutamine residue (G) revealed the peptide was negative in 5:5 samples. We used CD small-intestinal organ culture to assess CD toxicity that revealed two peptides with selected substitution of both glutamic acid (E) and proline (P) residues abrogated evidence of CD toxicity.

Disorder-to-order transition in PE-PPE proteins of Mycobacterium tuberculosis augments the pro-pathogen immune response.

A growing body of evidence supports the hypothesis that intrinsically disordered proteins often mediate host-pathogen interactions and modulate host functions for pathogen survival and virulence. Mycobacterium tuberculosis (M.tb) has evolved largely through reductive evolution, with a few exceptions such as the glycine-alanine-rich PE-PPE/PGRS protein family, which has been expanding in pathogenic mycobacteria. Here, our analyses of the M.tb proteome and secretome revealed that the PE-PGRS subfamily is enriched for disordered regions and disordered binding sites, pointing to their importance in host-pathogen interactions. As a case study, the secondary structure of PE35-PPE68 and PE32-PPE65 of the pathogenesis-related RD1 and RD8 regions was analyzed through Fourier-transform infrared spectroscopy. These disordered proteins displayed a considerable structural shift from disordered to ordered while engaged in the formation of complexes. While these proteins are immunogenic individually and enhance the pro-pathogen response, their corresponding complexes enhanced the responses manifold as displayed here by PE35 and PPE68. It is likely that M.tb exploits such disorder-order structural dynamics as a strategy to mount a pro-pathogen response and subvert host defense for productive infection. This functional gain also serves as a means to compensate genomic content loss due to reductive evolution.

ß1-Integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells.

Although the impact of Th17 cells on autoimmunity is undisputable, their pathogenic effector mechanism is still enigmatic. We discovered soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex proteins in Th17 cells that enable a vesicular glutamate release pathway that induces local intracytoplasmic calcium release and subsequent damage in neurons. This pathway is glutamine dependent and triggered by binding of ß1-integrin to vascular cell adhesion molecule 1 (VCAM-1) on neurons in the inflammatory context. Glutamate secretion could be blocked by inhibiting either glutaminase or KV1.3 channels, which are known to be linked to integrin expression and highly expressed on stimulated T cells. Although KV1.3 is not expressed in CNS tissue, intrathecal administration of a KV1.3 channel blocker or a glutaminase inhibitor ameliorated disability in experimental neuroinflammation. In humans, T cells from patients with multiple sclerosis secreted higher levels of glutamate, and cerebrospinal fluid glutamine levels were increased. Altogether, our findings demonstrate that ß1-integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells upon direct cell-cell contact between Th17 cells and neurons.

Neurotoxic potential of reactive astrocytes in canine distemper demyelinating leukoencephalitis.

Canine distemper virus (CDV) causes a fatal demyelinating leukoencephalitis in young dogs resembling human multiple sclerosis. Astrocytes are the main cellular target of CDV and undergo reactive changes already in pre-demyelinating brain lesions. Based on their broad range of beneficial and detrimental effects in the injured brain reactive astrogliosis is in need of intensive investigation. The aim of the study was to characterize astrocyte plasticity during the course of CDV-induced demyelinating leukoencephalitis by the aid of immunohistochemistry, immunofluorescence and gene expression analysis. Immunohistochemistry revealed the presence of reactive glial fibrillary acidic protein (GFAP)+ astrocytes with increased survivin and reduced aquaporin 4, and glutamine synthetase protein levels, indicating disturbed blood brain barrier function, glutamate homeostasis and astrocyte maladaptation, respectively. Gene expression analysis revealed 81 differentially expressed astrocyte-related genes with a dominance of genes associated with neurotoxic A1-polarized astrocytes. Accordingly, acyl-coA synthetase long-chain family member 5+/GFAP+, and serglycin+/GFAP+ cells, characteristic of A1-astrocytes, were found in demyelinating lesions by immunofluorescence. In addition, gene expression revealed a dysregulation of astrocytic function including disturbed glutamate homeostasis and altered immune function. Observed findings indicate an astrocyte polarization towards a neurotoxic phenotype likely contributing to lesion initiation and progression in canine distemper leukoencephalitis.

Evidence for a dynorphin-mediated inner ear immune/inflammatory response and glutamate-induced neural excitotoxicity: an updated analysis.

Acoustic overstimulation (AOS) is defined as the stressful overexposure to high-intensity sounds. AOS is a precipitating factor that leads to a glutamate (GLU)-induced Type I auditory neural excitotoxicity and an activation of an immune/inflammatory/oxidative stress response within the inner ear, often resulting in cochlear hearing loss. The dendrites of the Type I auditory neural neurons that innervate the inner hair cells (IHCs), and respond to the IHC release of the excitatory neurotransmitter GLU, are themselves directly innervated by the dynorphin (DYN)-bearing axon terminals of the descending brain stem lateral olivocochlear (LOC) system. DYNs are known to increase GLU availability, potentiate GLU excitotoxicity, and induce superoxide production. DYNs also increase the production of proinflammatory cytokines by modulating immune/inflammatory signal transduction pathways. Evidence is provided supporting the possibility that the GLU-mediated Type I auditory neural dendritic swelling, inflammation, excitotoxicity, and cochlear hearing loss that follow AOS may be part of a brain stem-activated, DYN-mediated cascade of inflammatory events subsequent to a LOC release of DYNs into the cochlea. In support of a DYN-mediated cascade of events are established investigations linking DYNs to the immune/inflammatory/excitotoxic response in other neural systems.

Reduced Microglial Activity and Enhanced Glutamate Transmission in the Basolateral Amygdala in Early CNS Autoimmunity.

Emotional dysfunction is common in multiple sclerosis (MS) patients and in mouse models of MS, including experimental autoimmune encephalomyelitis (EAE); however, the etiology of these behaviors is poorly understood. To identify CNS changes associated with these behaviors, we focused on the basolateral amygdala (BLA) because of its central role in the regulation of emotional behavior. Whole-cell recordings were performed in the principal neurons of the BLA in early EAE, before demyelination, T-cell invasion, and motor dysfunction. EAE female mice displayed increased frequency of mEPSCs, with no alteration in amplitude or evoked EPSC paired-pulse ratio compared with controls. We found an increase in the AMPA-NMDA ratio and dendritic spine density, indicating increased numbers of glutamatergic synapses. We saw similar electrophysiological changes in BLA principal neurons after microglia were either inactivated (minocycline) or depleted (Mac1-Saporin) in the BLA. Microglia regulate synapses through pruning, directed by complement protein 3 (C3) expression. C3 was downregulated in the BLA in EAE. Ultrastructural analysis of microglia revealed more complex ramifications and reduced extracellular digestion of cellular elements. We also observed reduced IBA-1 and CD68 staining and lack of proinflammatory cytokine expression in the amygdala. Thus, early EAE is a state of microglial "deactivation" associated with reduced synaptic pruning. This contrasts with the prototypic microglial activation commonly associated with inflammatory CNS disease. Additionally, these data support a role for the acquired immune system to influence both neuronal and microglial function in early CNS autoimmunity.SIGNIFICANCE STATEMENT Microglia help regulate synaptic homeostasis, but there has been little evidence for how this might be important in neuroinflammatory diseases. The data from this study reveal increased synaptic activity and spine density in early stages of experimental autoimmune encephalomyelitis (an animal model of multiple sclerosis) in the basolateral amygdala, a nucleus important in the types of behavioral changes we have previously described. These electrophysiological and morphological effects occurred without significant elevation of local inflammatory cytokines or local demyelination. Unexpectedly, in the context of inflammatory state, we found that microglia were "deactivated." This study provides strong evidence for a link between microglial activity and synaptic function; the conclusions contrast with the generally accepted view that microglia are activated in inflammatory disease.

Macavirus latency-associated protein evades immune detection through regulation of protein synthesis in cis depending upon its glycin/glutamate-rich domain.

Alcelaphine herpesvirus 1 (AlHV-1) is a γ-herpesvirus (γ-HV) belonging to the macavirus genus that persistently infects its natural host, the wildebeest, without inducing any clinical sign. However, cross-transmission to other ruminant species causes a deadly lymphoproliferative disease named malignant catarrhal fever (MCF). AlHV-1 ORF73 encodes the latency-associated nuclear antigen (LANA)-homolog protein (aLANA). Recently, aLANA has been shown to be essential for viral persistence in vivo and induction of MCF, suggesting that aLANA shares key properties of other γ-HV genome maintenance proteins. Here we have investigated the evasion of the immune response by aLANA. We found that a glycin/glutamate (GE)-rich repeat domain was sufficient to inhibit in cis the presentation of an epitope linked to aLANA. Although antigen presentation in absence of GE was dependent upon proteasomal degradation of aLANA, a lack of GE did not affect protein turnover. However, protein self-synthesis de novo was downregulated by aLANA GE, a mechanism directly associated with reduced antigen presentation in vitro. Importantly, codon-modification of aLANA GE resulted in increased antigen presentation in vitro and enhanced induction of antigen-specific CD8+ T cell responses in vivo, indicating that mRNA constraints in GE rather than peptidic sequence are responsible for cis-limitation of antigen presentation. Nonetheless, GE-mediated limitation of antigen presentation in cis of aLANA was dispensable during MCF as rabbits developed the disease after virus infection irrespective of the expression of full-length or GE-deficient aLANA. Altogether, we provide evidence that inhibition in cis of protein synthesis through GE is likely involved in long-term immune evasion of AlHV-1 latent persistence in the wildebeest natural host, but dispensable in MCF pathogenesis.

Alterations in nitric oxide homeostasis during traumatic brain injury.

Changes in nitric oxide (NO) levels have been often associated with various forms of trauma, including secondary damage after traumatic brain injury (TBI). Several studies demonstrate the upregulation of NO synthase (NOS) enzymes, and concomitant increases in brain NO levels, which contribute to the TBI-associated glutamate cytotoxicity, including the pathogenesis of mitochondrial dysfunction. TBI is also associated with elevated NO levels in remote organs, indicating that TBI can induce systemic changes in NO regulation, which can be either beneficial or detrimental. Here we review the possible mechanisms responsible for changes in NO metabolism during TBI. Better understanding of the changes in NO homeostasis in TBI will be necessary to design rational therapeutic approaches for TBI. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Generation of specific antisera directed against D-amino acids: focus on the neuroanatomical distribution of D-glutamate and other D-amino acids.

This review updates the findings about the anatomical distribution (using immunohistochemical techniques) and possible functions of D-glutamate in the central nervous system of mammals, as well as compares the distribution of D-glutamate with the distribution of the most studied D-amino acids: D-serine and D-aspartate. The protocol used to obtain highly specific antisera directed against D-amino acids is also reported. Immunoreactivity for D-glutamate was found in dendrites and cell bodies, but not in nerve fibers. Perikarya containing D-glutamate were found in the mesencephalon and thalamus. The highest density of cell bodies was found in the dorsal raphe nucleus, the mesencephalic central grey matter, the superior colliculus, and in the subparafascicular thalamic nucleus. In comparison with the distribution of immunoreactive cell bodies containing D-serine or D-aspartate, the distribution of D-glutamate-immunoreactive perikarya is less widespread. Currently, the physiological actions mediated by D-glutamate in the brain are unknown but the restricted neuroanatomical distribution of this D-amino acid suggests that D-glutamate could be involved in very specific physiological mechanisms. In this sense, the possible functional roles of D-glutamate are discussed.

Construction and evaluation of pH-sensitive immunoliposomes for enhanced delivery of anticancer drug to ErbB2 over-expressing breast cancer cells.

1,5-Dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (GGLG) liposomes were previously developed to enhance drug delivery efficiency in tumor cells owing to its pH-responsive properties. Herein, we report the modification of GGLG liposomes by conjugating a Fab' fragment of an ErbB2 antibody to the terminus of PEG (polyethylene glycol)-lipid (Fab'-GGLG liposomes). The conjugation of Fab' fragments did not affect the antibody activity, drug (doxorubicin, DOX) encapsulation efficiency, stability during storage or pH-sensitivity. However, the binding affinity of Fab'-GGLG liposomes was enhanced to ErbB2-overexpressing HCC1954 cells specifically, and the cell association increased 10-fold in comparison to GGLG liposomes. Consequently, intracellular DOX delivery was enhanced, with an increased cytotoxicity in HCC1954 cells (i.e., IC50 of 1.17 and 3.08 µg/mL for Fab'-GGLG-DOX and GGLG-DOX liposomes, respectively). Further, a significantly enhanced tumor growth inhibition was obtained in an ErbB2-overexpressing breast cancer-bearing mouse model. Therefore, a potent anticancer drug delivery system was constructed by the immunological modification of pH-sensitive liposomes.

Glufosinate aerogenic exposure induces glutamate and IL-1 receptor dependent lung inflammation.

Glufosinate-ammonium (GLA), the active component of an herbicide, is known to cause neurotoxicity. GLA shares structural analogy with glutamate. It is a powerful inhibitor of glutamine synthetase (GS) and may bind to glutamate receptors. Since these potentials targets of GLA are present in lung and immune cells, we asked whether airway exposure to GLA may cause lung inflammation in mice. A single GLA exposure (1 mg/kg) induced seizures and inflammatory cell recruitment in the broncho-alveolar space, and increased myeloperoxidase (MPO), inducible NO synthase (iNOS), interstitial inflammation and disruption of alveolar septae within 6-24 h. Interleukin 1ß (IL-1ß) was increased and lung inflammation depended on IL-1 receptor 1 (IL-1R1). We demonstrate that glutamate receptor pathway is central, since the N-methyl-D-aspartate (NMDA) receptor inhibitor MK-801 prevented GLA-induced lung inflammation. Chronic exposure (0.2 mg/kg 3× per week for 4 weeks) caused moderate lung inflammation and enhanced airway hyperreactivity with significant increased airway resistance. In conclusion, GLA aerosol exposure causes glutamate signalling and IL-1R-dependent pulmonary inflammation with airway hyperreactivity in mice.

KIR2DL2/2DL3-E(35) alleles are functionally stronger than -Q(35) alleles.

KIR2DL2 and KIR2DL3 segregate as alleles of a single locus in the centromeric motif of the killer cell immunoglobulin-like receptor (KIR) gene family. Although KIR2DL2/L3 polymorphism is known to be associated with many human diseases and is an important factor for donor selection in allogeneic hematopoietic stem cell transplantation, the molecular determinant of functional diversity among various alleles is unclear. In this study we found that KIR2DL2/L3 with glutamic acid at position 35 (E(35)) are functionally stronger than those with glutamine at the same position (Q(35)). Cytotoxicity assay showed that NK cells from HLA-C1 positive donors with KIR2DL2/L3-E(35) could kill more target cells lacking their ligands than NK cells with the weaker -Q(35) alleles, indicating better licensing of KIR2DL2/L3(+) NK cells with the stronger alleles. Molecular modeling analysis reveals that the glutamic acid, which is negatively charged, interacts with positively charged histidine located at position 55, thereby stabilizing KIR2DL2/L3 dimer and reducing entropy loss when KIR2DL2/3 binds to HLA-C ligand. The results of this study will be important for future studies of KIR2DL2/L3-associated diseases as well as for donor selection in allogeneic stem cell transplantation.