Administration of DHA Reduces Endoplasmic Reticulum Stress-Associated Inflammation and Alters Microglial or Macrophage Activation in Traumatic Brain Injury

We investigated the effects of the administration of docosahexaenoic acid (DHA) post-traumatic brain injury (TBI) on reducing neuroinflammation. TBI was induced by cortical contusion injury in Sprague Dawley rats. Either DHA (16 mg/kg in dimethyl sulfoxide) or vehicle dimethyl sulfoxide (1 ml/kg) was administered intraperitonially at 5 min after TBI, followed by a daily dose for 3 to 21 days. TBI triggered activation of microglia or macrophages, detected by an increase of Iba1 positively stained microglia or macrophages in peri-lesion cortical tissues at 3, 7, and 21 days post-TBI. The inflammatory response was further characterized by expression of the proinflammatory marker CD16/32 and the anti-inflammatory marker CD206 in Iba1⁺ microglia or macrophages. DHA-treated brains showed significantly fewer CD16/32⁺ microglia or macrophages, but an increased CD206⁺ phagocytic microglial or macrophage population. Additionally, DHA treatment revealed a shift in microglial or macrophage morphology from the activated, amoeboid-like state into the more permissive, surveillant state. Furthermore, activated Iba1⁺ microglial or macrophages were associated with neurons expressing the endoplasmic reticulum (ER) stress marker CHOP at 3 days post-TBI, and the administration of DHA post-TBI concurrently reduced ER stress and the associated activation of Iba1⁺ microglial or macrophages. There was a decrease in nuclear translocation of activated nuclear factor kappa-light-chain-enhancer of activated B cells protein at 3 days in DHA-treated tissue and reduced neuronal degeneration in DHA-treated brains at 3, 7, and 21 days after TBI. In summary, our study demonstrated that TBI mediated inflammatory responses are associated with increased neuronal ER stress and subsequent activation of microglia or macrophages. DHA administration reduced neuronal ER stress and subsequent association with microglial or macrophage polarization after TBI, demonstrating its therapeutic potential to ameliorate TBI-induced cellular pathology.     

A mass spectrometry assay for detection of endogenous and lentiviral engineered hemoglobin in cultured cells and sickle cell disease mice

Sickle cell disease (SCD) results from a sequence defect in the β-globin chain of adult hemoglobin (HbA) leading to expression of sickle hemoglobin (HbS). It is traditionally diagnosed by cellulose-acetate hemoglobin electrophoresis or high-performance liquid chromatography. While clinically useful, these methods have both sensitivity and specificity limitations. We developed a novel mass spectrometry (MS) method for the rapid, sensitive and highly quantitative detection of endogenous human β-globin and sickle hβ-globin, as well as lentiviral-encoded therapeutic hβAS3-globin in cultured cells and small quantities of mouse peripheral blood. The MS methods were used to phenotype homozygous HbA (AA), heterozygous HbA–HbS (AS) and homozygous HbS (SS) Townes SCD mice and detect lentiviral vector-encoded hβAS3-globin in transduced mouse erythroid cell cultures and transduced human CD34⁺ cells after erythroid differentiation. hβAS3-globin was also detected in peripheral blood 6 weeks post-transplant of transduced Townes SS bone marrow cells into syngeneic Townes SS mice and persisted for over 20 weeks post-transplant. As several genome-editing and gene therapy approaches for severe hemoglobin disorders are currently in clinical trials, this MS method will be useful for patient assessment before treatment and during follow-up.     

Alterations of Glial Cells in the Mouse Hippocampus During Postnatal Development

We investigated the postnatal alterations of neurons, astrocyte, oligodendrocyte, and microglia in the mouse hippocampal CA1 sector and dentate gyrus under the same conditions using immunohistochemistry. Neuronal nuclei (NeuN), Glial fibrillary acidic protein (GFAP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), and ionized calcium binding adaptor molecule 1 (Iba 1) immunoreactivity were measured in 1-, 2-, 4-, and 8-week-old mice. Total number of NeuN-positive neurons was unchanged in the mouse hippocampal CA1 sector and dentate gyrus from 1 to 8 weeks of birth. In contrast, a significant increase in the number of GFAP-positive astrocytes was observed only in the hippocampal CA1 sector of 1-week-old mice when compared with 8-week-old animals. Thereafter, total number of GFAP-positive astrocytes was unchanged in the hippocampal CA1 sector and dentate gyrus from 2 to 8 weeks of birth. For microglia, a significant increase in the number of Iba 1-positive microglia was observed in the hippocampal CA1 sector and dentate gyrus of 1-, 2-, and 4-week-old mice as compared with 8-week-old animals. On the other hand, a significant decrease in the area of expression of CNPase-positive fibers was observed in the hippocampal CA1 sector of 1- and 2-week-old mice as compared with 8-week-old animals. In dentate gyrus, a significant decrease in the area of expression of CNPase-positive fibers was found in 1-, 2-, and 4-week-old mice. Furthermore, our double-labeled immunostaining showed that brain-derived neurotrophic factor (BDNF) immunoreactivity was observed in GFAP-positive astrocytes and Iba 1-positive microglia in the hippocampal CA1 sector and dentate gyrus of 1- and 2-week-old mice. These results show that glial cells may play some role in the maintenance and neuronal functions of hippocampal CA1 pyramidal neurons and granule cells of dentate gyrus during postnatal development. Furthermore, our results demonstrate that glial BDNF may play an important role in the maturation of oligodendrocyte in the hippocampal CA1 sector and dentate gyrus during postnatal development. Thus, our findings provide valuable information on the developmental processes.     

CD40L protects against mouse hepatitis virus-induced neuroinflammatory demyelination

Neurotropic mouse hepatitis virus (MHV-A59/RSA59) infection in mice induces acute neuroinflammation due to direct neural cell dystrophy, which proceeds with demyelination with or without axonal loss, the pathological hallmarks of human neurological disease, Multiple sclerosis (MS). Recent studies in the RSA59-induced neuroinflammation model of MS showed a protective role of CNS-infiltrating CD4⁺ T cells compared to their pathogenic role in the autoimmune model. The current study further investigated the molecular nexus between CD4⁺ T cell-expressed CD40Ligand and microglia/macrophage-expressed CD40 using CD40L^-/- mice. Results demonstrate CD40L expression in the CNS is modulated upon RSA59 infection. We show evidence that CD40L^-/- mice are more susceptible to RSA59 induced disease due to reduced microglia/macrophage activation and significantly dampened effector CD4⁺ T recruitment to the CNS on day 10 p.i. Additionally, CD40L^-/- mice exhibited severe demyelination mediated by phagocytic microglia/macrophages, axonal loss, and persistent poliomyelitis during chronic infection, indicating CD40-CD40L as host-protective against RSA59-induced demyelination. This suggests a novel target in designing prophylaxis for virus-induced demyelination and axonal degeneration, in contrast to immunosuppression which holds only for autoimmune mechanisms of inflammatory demyelination.     

CD8+ T cells are increased in the subventricular zone with physiological and pathological aging

Age‐related cognitive decline and neurodegenerative diseases are associated with less functional neurogenic niches. It has been recently shown that aged subventricular zone (SVZ) suffers an infiltration of T cells, which affects neural stem cell activity in mice. Whether this occurs in human neurogenic niches or to which extent T‐cell infiltration is also taking place in neurodegenerative diseases remains unknown. In this work, we studied the presence of T cells in both human neurogenic niches in young and old individuals. There was a significant increase in the number of CD3⁺ and CD8⁺ T cells in the SVZ of elderly individuals, which was not detected in the dentate gyrus. Moreover, we also found CD3⁺ and CD8⁺ T cells in the SVZ of individuals with neurodegenerative diseases. However, T‐cell count was similar when compared non‐neuropathological elderly with disease diagnosed patients. Our study reveals the infiltration of T cells in old human brains, particularly in the SVZ under non‐pathological conditions and also in neurodegenerative contexts.     

Activation of α-7 Nicotinic Acetylcholine Receptor Reduces Ischemic Stroke Injury through Reduction of Pro-Inflammatory Macrophages and Oxidative Stress

Activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) has a neuro-protective effect on ischemic and hemorrhagic stroke. However, the underlying mechanism is not completely understood. We hypothesized that α-7 nAchR agonist protects brain injury after ischemic stroke through reduction of pro-inflammatory macrophages (M1) and oxidative stress. C57BL/6 mice were treated with PHA568487 (PHA, α-7 nAchR agonist), methyllycaconitine (MLA, nAchR antagonist), or saline immediately and 24 hours after permanent occlusion of the distal middle cerebral artery (pMCAO). Behavior test, lesion volume, CD68⁺, M1 (CD11b⁺/Iba1⁺) and M2 (CD206/Iba1⁺) microglia/macrophages, and phosphorylated p65 component of NF-kB in microglia/macrophages were quantified using histological stained sections. The expression of M1 and M2 marker genes, anti-oxidant genes and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were quantified using real-time RT-PCR. Compared to the saline-treated mice, PHA mice had fewer behavior deficits 3 and 7 days after pMCAO, and smaller lesion volume, fewer CD68⁺ and M1 macrophages, and more M2 macrophages 3 and 14 days after pMCAO, whereas MLA''s effects were mostly the opposite in several analyses. PHA increased anti-oxidant genes and NADPH oxidase expression associated with decreased phosphorylation of NF-kB p65 in microglia/macrophages. Thus, reduction of inflammatory response and oxidative stress play roles in α-7 nAchR neuro-protective effect.     

Ethanol-Induced TLR4/NLRP3 Neuroinflammatory Response in Microglial Cells Promotes Leukocyte Infiltration Across the BBB

We reported that the ethanol-induced innate immune response by activating TLR4 signaling triggers gliosis and neuroinflammation. Ethanol also activates other immune receptors, such as NOD-like-receptors, and specifically NLRP3-inflammasome in astroglial cells, to stimulate caspase-1 cleavage and IL-1β and IL-18 cytokines production. Yet, whether microglia NLRs are also sensitive to the ethanol effects that contribute to neuroinflammation is uncertain. Using cerebral cortexes of the chronic alcohol-fed WT and TLR4^?/? mice, we demonstrated that chronic ethanol treatment enhanced TLR4 mediated-NLRP3/Caspase-1 complex activation, and up-regulated pro-inflammatory cytokines and chemokines levels. Ethanol-induced NLRP3-inflammasome activation and mitochondria-ROS generation were also observed in cultured microglial cells. The up-regulation of CD45^high/CD11b⁺ cell populations and matrix metalloproteinase-9 levels was also noted in the cortexes of the ethanol-treated WT mice. Notably, elimination of the TLR4 function abolished most ethanol-induced neuroinflammatory effects. Thus, our results demonstrate that ethanol triggers TLR4-mediated NLRP3-inflammasome activation in glial cells, and suggest that microglia stimulation may compromise the permeability of blood–brain barrier events to contribute to ethanol-induced neuroinflammation and brain damage.     

The PPARδ agonist GW0742 inhibits neuroinflammation,but does not restore neurogenesis or prevent early delayed hippocampal-dependent cognitive impairment after whole-brain irradiation

Brain tumor patients often develop cognitive impairment months to years after partial or fractionated whole-brain irradiation (WBI). Studies suggest that neuroinflammation and decreased hippocampal neurogenesis contribute to the pathogenesis of radiation-induced brain injury. In this study, we determined if the peroxisomal proliferator-activated receptor (PPAR) δ agonist GW0742 can prevent radiation-induced brain injury in C57Bl/6 wild-type (WT) and PPARδ knockout (KO) mice. Dietary GW0742 prevented the acute increase in IL-1β mRNA and ERK phosphorylation measured at 3 h after a single 10-Gy dose of WBI; it also prevented the increase in the number of activated hippocampal microglia 1 week after WBI. In contrast, dietary GW074 failed to prevent the radiation-induced decrease in hippocampal neurogenesis determined 2 months after WBI in WT mice or to mitigate their hippocampal-dependent spatial memory impairment measured 3 months after WBI using the Barnes maze task. PPARδ KO mice exhibited defects including decreased numbers of astrocytes in the dentate gyrus/hilus of the hippocampus and a failure to exhibit a radiation-induced increase in activated hippocampal microglia. Interestingly, the number of astrocytes in the dentate gyrus/hilus was reduced in WT mice, but not in PPARδ KO mice 2 months after WBI. These results demonstrate that, although dietary GW0742 prevents the increase in inflammatory markers and hippocampal microglial activation in WT mice after WBI, it does not restore hippocampal neurogenesis or prevent early delayed hippocampal-dependent cognitive impairment after WBI. Thus, the exact relationship between radiation-induced neuroinflammation, neurogenesis, and cognitive impairment remains elusive.     

Cyclosporine A Reduces Dendritic Outgrowth of Neuroblasts in the Subgranular Zone of the Dentate Gyrus in C57BL/6 Mice

In the present study, we observed the effects of cyclosporine A (CsA), an efficient immunosuppressant, on cell proliferation and neuroblast differentiation in the subgranular zone of the dentate gyrus (SZDG) in normal C57BL/6 mice using Ki67 and doublecortin (DCX) immunohistochemical staining, respectively. At 8 weeks of age, vehicle (physiological saline) or CsA was daily administered (40 mg/kg, i.p.) for 1 week. Animals were sacrificed at 2 weeks after last administration. CsA treatment did not show any influences in neurons, astrocytes and microglia based on immunohistochemistry for its markers, respectively. However, in the CsA-treated group, Fluoro-Jade B, a marker for neurodegeneration, positive cells were found in the SZDG, not in the vehicle-treated group. In the vehicle-treated group, Ki67 immunoreactive (⁺) nuclei were clustered in the SZDG, whereas in the CsA-treated group Ki67⁺ nuclei were scattered in the SZDG, showing no difference in cell numbers. Numbers of DCX⁺ neuroblasts with well-developed processes (tertiary dendrites) were much lower in the CsA-treated group than those in the vehicle-treated group; however, numbers of DCX⁺ neuroblasts with secondary dendrites were similar in both the groups. These results suggest that CsA significantly reduces dendritic outgrowth and complexity from neuroblasts in the SZDG without any affecting in neurons, astrocytes and microglia in normal mice.     

Association between apolipoprotein ε4 allele,factor V Leiden,and plasma lipid and lipoprotein levels with sickle cell disease in southern Iran

To investigate whether there is any association between various APOE alleles and factor V Leiden (FVL) with lipid profiles and sickle cell disease (SCD) in Southern Iran. 65 SCD patients consisting of 35 sickle cell anemia homozygous (SS), 15 sickle cell heterozygous (AS) and 15 sickle cell/βThalassemia (S/βthal) patients and 68 healthy individuals with normal hematological indices were studied. APOE and FVL polymorphisms were detected by PCR–RFLP and serum lipid level was measured enzymatically. The frequencies of FVL and APOE-ε4 allele were significantly higher in SCD patients than in control (15.4 vs. 4.4 and 13.7% vs. 3.3%, respectively). The distributions of APOE ε3ε3, ε2ε3 and ε2ε4 + ε3ε4 alleles in SCD patients were significantly different from those in the control group. The SCD subjects particularly SS/S βthal (SS + S/βthal) and SS patients have significantly lower frequency of APOE ε3ε3 allele (P < 0.05) whereas SCD, SS patients and AS individuals have a significantly higher frequency of APOE ε4 allele (ε2ε4 + ε3ε4; P = 0.003, P = 0.011 and P = 0.035, respectively) compared to the control group. The LDL-C (P = 0.006) and total cholesterol (P < 0.001) levels in SCD subjects were found to be significantly lower than those in the control group. In addition, the presence of non-APOE ε4 allele (ε2ε3 + ε3ε3) resulted in a significant decrease in the level of LDL-C and total cholesterol in SCD subjects in general and in SS and SS/S βthal patients in particular compared to controls. Furthermore, the presence of APOE ε4 allele (ε2ε4 + ε3ε4) was found to be associated with the risk of sickle cell anemia [OR = 4.1, P = 0.04]. The presence of either FVL mutation (OR = 4.6; CI: 0.91–24, P = 0.07) or APOE-ε4 allele (OR = 4.07; CI: 1.01–16.4, P = 0.048) is associated with the risk of sickle cell disease in Southern Iran. This data suggest that the activation of coagulation system enhances thrombus generation and decreases antioxidant activity in SCD patients from Southern Iran.     

Absence of CD14 Delays Progression of Prion Diseases Accompanied by Increased Microglial Activation

Prion diseases are fatal neurodegenerative disorders characterized by accumulation of PrP^Sc, vacuolation of neurons and neuropil, astrocytosis, and microglial activation. Upregulation of gene expressions of innate immunity-related factors, including complement factors and CD14, is observed in the brains of mice infected with prions even in the early stage of infections. When CD14 knockout (CD14^−/−) mice were infected intracerebrally with the Chandler and Obihiro prion strains, the mice survived longer than wild-type (WT) mice, suggesting that CD14 influences the progression of the prion disease. Immunofluorescence staining that can distinguish normal prion protein from the disease-specific form of prion protein (PrP^Sc) revealed that deposition of PrP^Sc was delayed in CD14^−/− mice compared with WT mice by the middle stage of the infection. Immunohistochemical staining with Iba1, a marker for activated microglia, showed an increased microglial activation in prion-infected CD14^−/− mice compared to WT mice. Interestingly, accompanied by the increased microglial activation, anti-inflammatory cytokines interleukin-10 (IL-10) and transforming growth factor β (TGF-β) appeared to be expressed earlier in prion-infected CD14^−/− mice. In contrast, IL-1β expression appeared to be reduced in the CD14^−/− mice in the early stage of infection. Double immunofluorescence staining demonstrated that CD11b- and Iba1-positive microglia mainly produced the anti-inflammatory cytokines, suggesting anti-inflammatory status of microglia in the CD14^−/− mice in the early stage of infection. These results imply that CD14 plays a role in the disease progression by suppressing anti-inflammatory responses in the brain in the early stage of infection.     

Early behavioral changes and quantitative analysis of neuropathological features in murine prion disease: Stereological analysis in the albino Swiss mice model

Behavioral and neuropathological changes have been widely investigated in murine prion disease but stereological based unbiased estimates of key neuropathological features have not been carried out. After injections of ME7 infected (ME7) or normal brain homogenates (NBH) into dorsal CA1 of albino Swiss mice and C57BL6, we assessed behavioral changes on hippocampal-dependent tasks. We also estimated by optical fractionator at 15 and 18 weeks post-injections (w.p.i.) the total number of neurons, reactive astrocytes, activated microglia and perineuronal nets (PN) in the polymorphic layer of dentate gyrus (PolDG), CA1 and septum in albino Swiss mice. On average, early behavioral changes in albino Swiss mice start four weeks later than in C57BL6. Cluster and discriminant analysis of behavioral data in albino Swiss mice revealed that four of nine subjects start to change their behavior at 12 w.p.i. and reach terminal stage at 22 w.p.i and the remaining subjects start at 22 w.p.i. and reach terminal stage at 26 w.p.i. Biotinylated dextran-amine BDA-tracer experiments in mossy fiber pathway confirmed axonal degeneration and stereological data showed that early astrocytosis, microgliosis and reduction in the perineuronal nets are independent of a change in the number of neuronal cell bodies. Statistical analysis revealed that the septal region had greater levels of neuroinflammation and extracellular matrix damage than CA1. This stereological and multivariate analysis at early stages of disease in an outbred model of prion disease provided new insights connecting behavioral changes and neuroinflammation and seems to be important to understand the mechanisms of prion disease progression.Key words: prion disease, optical fractionator, neuropathology, behavioral changes, albino Swiss mice     

Weakened Intracellular Zn<Superscript>2+</Superscript>-Buffering in the Aged Dentate Gyrus and Its Involvement in Erasure of Maintained LTP

Memory is lost by the increased influx of extracellular Zn²⁺ into neurons. It is possible that intracellular Zn²⁺ dynamics is modified even at non-zincergic medial perforant pathway-dentate granule cell synapses along with aging and that vulnerability to the modification is linked to age-related cognitive decline. To examine these possibilities, vulnerability of long-term potentiation (LTP) maintenance, which underlies memory retention, to modification of synaptic Zn²⁺ dynamics was compared between young and aged rats. The influx of extracellular Zn²⁺ into dentate granule cells was increased in aged rats after injection of high K⁺ into the dentate gyrus, but not in young rats. This increase impaired maintained LTP in aged rats. However, the impairment was rescued by co-injection of CaEDTA, an extracellular Zn²⁺ chelator, or CNQX, an AMPA receptor antagonist, which suppressed the Zn²⁺ influx. Maintained LTP was also impaired in aged rats after injection of ZnAF-2DA into the dentate gyrus that chelates intracellular Zn²⁺, but not in young rats. Interestingly, the capacity of chelating intracellular Zn²⁺ with intracellular ZnAF-2 was almost lost in the aged dentate gyrus 2 h after injection of ZnAF-2DA into the dentate gyrus, suggesting that intracellular Zn²⁺-buffering is weakened in the aged dentate gyrus, compared to the young dentate gyrus. In the dentate gyrus of aged rats, maintained LTP is more vulnerable to modification of intracellular Zn²⁺ dynamics than in young rats, probably due to weakened intracellular Zn²⁺-buffering.     

Hippocampal neurogenesis and neuroinflammation after cranial irradiation with (56)Fe particles

Exposure to heavy-ion radiation is considered a potential health risk in long-term space travel. In the central nervous system (CNS), loss of critical cellular components may lead to performance decrements that could ultimately compromise mission goals and long-term quality of life. Hippocampal-dependent cognitive impairments occur after exposure to ionizing radiation, and while the pathogenesis of this effect is not yet clear, it may involve the production of newly born neurons (neurogenesis) in the hippocampal dentate gyrus. We irradiated mice with 0.5-4 Gy of (56)Fe ions and 2 months later quantified neurogenesis and numbers of activated microglia as a measure of neuroinflammation in the dentate gyrus. Results showed that there were few changes after 0.5 Gy, but that there was a dose-related decrease in hippocampal neurogenesis and a dose-related increase in numbers of newly born activated microglia from 0.5-4.0 Gy. While those findings were similar to what was reported after X irradiation, there were also some differences, particularly in the response of newly born glia. Overall, this study showed that hippocampal neurogenesis was sensitive to relatively low doses of (56)Fe particles, and that those effects were associated with neuroinflammation. Whether these changes will result in functional impairments or if/how they can be managed are topics for further investigation.     

An Oriental Medicine,Hyungbangpaedok-San Attenuates Motor Paralysis in an Experimental Model of Multiple Sclerosis by Regulating the T Cell Response

The preventive and therapeutic mechanisms in multiple sclerosis are not clearly understood. We investigated whether Hyungbangpaedok-san (HBPDS), a traditional herbal medicine, has a beneficial effect in experimental autoimmune encephalomyelitis (EAE) mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG_35-55). Onset-treatment with 4 types of HBPDS (extracted using distilled water and 30%/70%/100% ethanol as the solvent) alleviated neurological signs, and HBPDS extracted within 30% ethanol (henceforth called HBPDS) was more effective. Onset-treatment with HBPDS reduced demyelination and the recruitment/infiltration and activation of microglia/macrophages in the spinal cord of EAE mice, which corresponded to the reduced mRNA expression of pro-inflammatory cytokines (TNF-α, IL–6, and IL–1β), iNOS, and chemokines (MCP–1, MIP–1α, and RANTES) in the spinal cord. Onset-treatment with HBPDS inhibited changes in the components of the blood-brain barrier such as astrocytes, adhesion molecules (ICAM–1 and VCAM–1), and junctional molecules (claudin–3, claudin–5, and zona occludens–1) in the spinal cord of EAE mice. Onset-treatment with HBPDS reduced the elevated population of CD4⁺, CD4⁺/IFN-γ⁺, and CD4⁺/IL–17⁺ T cells in the spinal cord of EAE mice but it further increased the elevated population of CD4⁺/CD25⁺/Foxp3⁺ and CD4⁺/Foxp3⁺/Helios⁺ T cells. Pre-, onset-, post-, but not peak-treatment, with HBPDS had a beneficial effect on behavioral impairment in EAE mice. Taken together, HBPDS could alleviate the development/progression of EAE by regulating the recruitment/infiltration and activation of microglia and peripheral immune cells (macrophages, Th1, Th17, and Treg cells) in the spinal cord. These findings could help to develop protective strategies using HBPDS in the treatment of autoimmune disorders including multiple sclerosis.     

Association of coagulation activation with clinical complications in sickle cell disease

Background

The contribution of hypercoagulability to the pathophysiology of sickle cell disease (SCD) remains poorly defined. We sought to evaluate the association of markers of coagulation and platelet activation with specific clinical complications and laboratory variables in patients with SCD.

Design and Methods

Plasma markers of coagulation activation (D-dimer and TAT), platelet activation (soluble CD40 ligand), microparticle-associated tissue factor (MPTF) procoagulant activity and other laboratory variables were obtained in a cohort of patients with SCD. Tricuspid regurgitant jet velocity was determined by Doppler echocardiography and the presence/history of clinical complications was ascertained at the time of evaluation, combined with a detailed review of the medical records.

Results

No significant differences in the levels of D-dimer, TAT, soluble CD40 ligand, and MPTF procoagulant activity were observed between patients in the SS/SD/Sβ⁰ thalassemia and SC/Sβ⁺ thalassemia groups. Both TAT and D-dimer were significantly correlated with measures of hemolysis (lactate dehydrogenase, indirect bilirubin and hemoglobin) and soluble vascular cell adhesion molecule-1. In patients in the SS/SD/Sβ⁰ thalassemia group, D-dimer was associated with a history of stroke (p = 0.049), TAT was associated with a history of retinopathy (p = 0.0176), and CD40 ligand was associated with the frequency of pain episodes (p = 0.039). In multivariate analyses, D-dimer was associated with reticulocyte count, lactate dehydrogenase, NT-proBNP and history of stroke; soluble CD40 ligand was associated with WBC count and platelet count; and MPTF procoagulant activity was associated with hemoglobin and history of acute chest syndrome.

Conclusions

This study supports the association of coagulation activation with hemolysis in SCD. The association of D-dimer with a history of stroke suggests that coagulation activation may contribute to the pathophysiology of stroke in clinically severe forms of SCD. More research is needed to evaluate the contribution of coagulation and platelet activation to clinical complications in SCD.     

Tregs Modulate Lymphocyte Proliferation,Activation, and Resident-Memory T-Cell Accumulation within the Brain during MCMV Infection

Accumulation and retention of regulatory T-cells (Tregs) has been reported within post viral-encephalitic brains, however, the full extent to which these cells modulate neuroinflammation is yet to be elucidated. Here, we used Foxp3-DTR (diphtheria toxin receptor) knock-in transgenic mice, which upon administration of low dose diphtheria toxin (DTx) results in specific deletion of Tregs. We investigated the proliferation status of various immune cell subtypes within inflamed central nervous system (CNS) tissue. Depletion of Tregs resulted in increased proliferation of both CD8⁺ and CD4⁺ T-cell subsets within the brain at 14 d post infection (dpi) when compared to Treg-sufficient animals. At 30 dpi, while proliferation of CD8⁺ T-cells was controlled within brains of both Treg-depleted and undepleted mice, proliferation of CD4⁺ T-cells remained significantly enhanced with DTx-treatment. Previous studies have demonstrated that Treg numbers within the brain rebound following DTx treatment to even higher numbers than in untreated animals. Despite this rebound, CD8⁺ and CD4⁺ T-cells proliferated at a higher rate when compared to that of Treg-sufficient mice, thus maintaining sustained neuroinflammation. Furthermore, at 30 dpi we found the majority of CD8⁺ T-cells were CD127^hi KLRG1^- indicating that the cells were long lived memory precursor cells. These cells showed marked elevation of CD103 expression, a marker of tissue resident-memory T-cells (T_RM) in the CNS, in untreated animals when compared to DTx-treated animals suggesting that generation of T_RM is impaired upon Treg depletion. Moreover, the effector function of T_RM as indicated by granzyme B production in response to peptide re-stimulation was found to be more potent in Treg-sufficient animals. Taken together, our findings demonstrate that Tregs limit neuroinflammatory responses to viral infection by controlling cell proliferation and may direct a larger proportion of lymphocytes within the brain to be maintained as T_RM cells.     

Flow Cytometric Characterization of T Cell Subsets and Microglia After Repetitive Mild Traumatic Brain Injury in Rats

Although, there is growing awareness in the progressive neurodegeneration of chronic traumatic encephalopathy, changes of immune reactions remain equivocal at best. Thus, in a clinically relevant rat repetitive mild traumatic brain injury (rmTBI) model, some immunologic cells (T cell subsets, microglia) in the injured brain and peripheral blood were analyzed by flow cytometry and immunofluorescence. In the injured brain, CD3⁺ T cells showed a bimodal increase during 42 days post-injury (dpi). CD3⁺CD4⁺ T cells firstly increased and then decreased, while CD3⁺CD8⁺ T cells had reversed tendency. CD86⁺/CD11b⁺ M1-like microglia increased at 42 dpi and CD206⁺/CD11b⁺ M2-like microglia peaked at 7 dpi. In addition, peripheral immune suppression was implicated in the chronic phase after rmTBI. Taken together, the study provided useful information on long-term dynamic changes of some immune cells after rmTBI in rats.