Tissue-Protective Effects of NKG2A in Immune-Mediated Clearance of Virus Infection

Virus infection triggers a CD8⁺ T cell response that aids in virus clearance, but also expresses effector functions that may result in tissue injury. CD8⁺ T cells express a variety of activating and inhibiting ligands, though regulation of the expression of inhibitory receptors is not well understood. The ligand for the inhibitory receptor, NKG2A, is the non-classical MHC-I molecule Qa1^b, which may also serve as a putative restricting element for the T cell receptors of purported regulatory CD8⁺ T cells. We have previously shown that Qa1^b-null mice suffer considerably enhanced immunopathologic lung injury in the context of CD8⁺ T cell-mediated clearance of influenza infection, as well as evidence in a non-viral system that failure to ligate NKG2A on CD8⁺ effector T cells may represent an important component of this process. In this report, we examine the requirements for induction of NKG2A expression, and show that NKG2A expression by CD8⁺ T cells occurs as a result of migration from the MLN to the inflammatory lung environment, irrespective of peripheral antigen recognition. Further, we confirmed that NKG2A is a mediator in limiting immunopathology in virus infection using mice with a targeted deletion of NKG2A, and infecting the mutants with two different viruses, influenza and adenovirus. In neither infection is virus clearance altered. In influenza infection, the enhanced lung injury was associated with increased chemoattractant production, increased infiltration of inflammatory cells, and significantly enhanced alveolar hemorrhage. The primary mechanism of enhanced injury was the loss of negative regulation of CD8⁺ T cell effector function. A similar effect was observed in the livers of mutant mice infected intravenously with adenovirus. These results demonstrate the immunoregulatory role of CD8⁺ NKG2A expression in virus infection, which negatively regulates T cell effector functions and contributes to protection of tissue integrity during virus clearance.     

Interictal Neurocognitive Processing of Visual Stimuli in Migraine: Evidence from Event-Related Potentials

Research has established decreased sensory habituation as a defining feature in migraine, while decreased cognitive habituation has only been found with regard to cognitive assessment of the relative probability of the occurrence of a stimulus event. Our study extended the investigation of interictal habituation in migraine to include cognitive processing when viewing of a series of visually-complex images, similar to those we encounter on the internet everyday. We examined interictal neurocognitive function in migraine from a habituation perspective, using a novel paradigm designed to assess how the response to a series of images changes over time. Two groups of participants--migraineurs (N = 25) and non-migraine controls (N = 25)--were asked to view a set of 232 unfamiliar logos in the context of a target identification task as their brain electrical responses were recorded via event-related potentials (ERPs). The set of logos was viewed serially in each of 10 separate trial blocks, with data analysis focusing on how the ERP responses to the logos in frontal electrodes from 200-600 ms changed across time within each group. For the controls, we found that the amplitude of the late positive potential (LPP) ERP component elicited by the logos had no significant change across trial blocks. In contrast, in migraineurs we found that the LPP significantly increased in amplitude across trial blocks, an effect consistent with a lack of habituation to visual stimuli seen in previous research. Our findings provide empirical support abnormal cognitive processing of complex visual images across time in migraineurs that goes beyond the sensory-level habituation found in previous research.     

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.     

CD164 monoclonal antibodies that block hemopoietic progenitor cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor

The novel sialomucin, CD164, functions as both an adhesion receptor on human CD34+ cell subsets in bone marrow and as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation. These diverse effects are mediated by at least two functional epitopes defined by the mAbs, 103B2/9E10 and 105A5. We report here the precise epitope mapping of these mAbs together with that of two other CD164 mAbs, N6B6 and 67D2. Using newly defined CD164 splice variants and a set of soluble recombinant chimeric proteins encoded by exons 1-6 of the CD164 gene, we demonstrate that the 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164. The N6B6 and 67D2 mAbs, in contrast, recognize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3. On the basis of their sensitivities to reducing agents and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by analogy with CD34 epitope classification. The class I 105A5 epitope is sialidase, O-glycosidase, and O-sialoglycoprotease sensitive; the class II 103B2/9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensitive; and the class III N6B6 and 67D2 epitopes are not removed by such enzyme treatments. Collectively, this study indicates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type specific post-translational modifications and suggests a role for epitope-associated carbohydrate structures in CD164 function.     

Effects of antiplatelet agents on platelet aggregation induced by platelet--activating factor (PAF) in human whole blood.

Impedance aggregometry was used to evaluate the potency of anti-platelet agents on Platelet Activating Factor (PAF)--induced platelet aggregation in citrated human whole blood. Drugs were tested for ability to inhibit maximum aggregation to PAF. Dose response curves were obtained and the concentration of drug producing 50% inhibition of maximum aggregation (ED50) determined. ED50's (microM) for specific PAF antagonists WEB 2086, Ro 19-3704, FR-900452, BN 52021, L-652,731, CV 3988, WEB 2118 and 48740 RP are: 0.39, 2.4, 4.7, 19.5, 21.0, 5.32, 161.0, 924.0, respectively. ED50's for non-specific PAF antagonists, diltiazem, propranolol, ketotifen, procaine HCL, and lidocaine HCL are: 38.0, 56.0, 250.0, 513.0 and 768.0, respectively. Ibuprofen was inactive at 2300 microM. Results are consistent with concept that there are specific receptors on platelets mediating PAF-induced aggregation in whole blood. Aggregation is inhibited potently by specific and competitive PAF receptor antagonists. Whole blood aggregometry may be a valid method for predicting in vivo activity of PAF antagonists.     

Exploring how extracellular electric field modulates neuron activity through dynamical analysis of a two-compartment neuron model

To investigate how extracellular electric field modulates neuron activity, a reduced two-compartment neuron model in the presence of electric field is introduced in this study. Depending on neuronal geometric and internal coupling parameters, the behaviors of the model have been studied extensively. The neuron model can exist in quiescent state or repetitive spiking state in response to electric field stimulus. Negative electric field mainly acts as inhibitory stimulus to the neuron, positive weak electric field could modulate spiking frequency and spike timing when the neuron is already active, and positive electric fields with sufficient intensity could directly trigger neuronal spiking in the absence of other stimulations. By bifurcation analysis, it is observed that there is saddle-node on invariant circle bifurcation, supercritical Hopf bifurcation and subcritical Hopf bifurcation appearing in the obtained two parameter bifurcation diagrams. The bifurcation structures and electric field thresholds for triggering neuron firing are determined by neuronal geometric and coupling parameters. The model predicts that the neurons with a nonsymmetric morphology between soma and dendrite, are more sensitive to electric field stimulus than those with the spherical structure. These findings suggest that neuronal geometric features play a crucial role in electric field effects on the polarization of neuronal compartments. Moreover, by determining the electric field threshold of our biophysical model, we could accurately distinguish between suprathreshold and subthreshold electric fields. Our study highlights the effects of extracellular electric field on neuronal activity from the biophysical modeling point of view. These insights into the dynamical mechanism of electric field may contribute to the investigation and development of electromagnetic therapies, and the model in our study could be further extended to a neuronal network in which the effects of electric fields on network activity may be investigated.     

Dipsticks for rapid detection of Plasmodium in vectoring Anopheles mosquitoes

Malaria remains the most serious vector-borne disease, affecting some 300-500 million people annually, transmitted by many species of Anopheles mosquitoes (Diptera: Culicidae). Monoclonal antibodies developed against specific circumsporozoite (CS) proteins of the main malaria parasites Plasmodium falciparum and P. vivax have been used previously for enzyme-linked immunosorbent assays (ELISA), widely employed for detection of malaria sporozoites in vector Anopheles for local risk assessment, epidemiological studies and targeting vector control. However, ELISA procedures are relatively slow and impractical for field use. To circumvent this, we developed rapid wicking assays that identify the presence or absence of specific peptide epitopes of CS protein of the most important P. falciparum and two strains (variants 210 and 247) of the more widespread P. vivax. The resulting assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. In laboratory assessment, dipsticks identified 1 ng/ mL of any of these three CS protein antigens, with sensitivity nearly equal to the CS standard ELISA. We have developed and are evaluating a combined panel assay that will be both qualitative and quantitative. This quick and easy dipstick test (VecTest Malaria) offers practical advantages for field workers needing to make rapid surveys of malaria vectors.     

Effect of fructose-1,6-bisphosphate on glutamate uptake and glutamine synthetase activity in hypotix astrocyte cultures

Astrocytes are important in regulating the microencironment of neurons both by catabolic and synthetic pathways. The glutamine synthetase (GS) activity observed in astrocytes affects neurons by removing toxic substances, NH₃ and glutamate; and by providing an important neuronal substrate, glutamine. This glutamate cycle might play a critical role during periods of hypoxia and ischemia, when an increase in extracellular excitatory amino acids is observed. It was previously shown in our laboratory that fructose-1,6-bisphosphate (FBP) protected cortical astrocyte cultures from hypoxic insult and reduced ATP loss following a prolonged (18–30 hrs) hypoxia. In the present study we established the effects of FBP on the level of glutamate uptake and GS activity under normoxic and hypoxic conditions. Under normoxic conditions, [U-¹⁴C]glutamate uptake and glutamine production were independent of FBP treatment; whereas under hypoxic conditions, the initial increase in glutamate uptake and an overall increase in glutamine production in astrocytes were FBP-dependent. Glutamine synthetase activity was dependent on FBP added during the 22 hours of either normoxic- or hypoxic-treatment, hence significant increases in activity were observed due to FBP regardless of the oxygen/ATP levels in situ. These studies suggest that activation of GS by FBP may provide astrocytic protection against hypoxic injury.