A 20-Year Journey from Axonal Injury to Neurodegenerative Diseases and the Prospect of Immunotherapy for Combating Alzheimer's Disease.

The understanding of the dialogue between the brain and the immune system has undergone dramatic changes over the last two decades, with immense impact on the perception of neurodegenerative diseases, mental dysfunction, and many other brain pathologic conditions. Accumulated results have suggested that optimal function of the brain is dependent on support from the immune system, provided that this immune response is tightly controlled. Moreover, in contrast to the previous prevailing dogma, it is now widely accepted that circulating immune cells are needed for coping with brain pathologies and that their optimal effect is dependent on their type, location, and activity. In this perspective, we describe our own scientific journey, reviewing the milestones in attaining this understanding of the brain-immune axis integrated with numerous related studies by others. We then explain their significance in demonstrating the possibility of harnessing the immune system in a well-controlled manner for the treatment of neurodegenerative diseases.

Maternal Type-I interferon signaling adversely affects the microglia and the behavior of the offspring accompanied by increased sensitivity to stress.

Viral infection during pregnancy is often associated with neuropsychiatric conditions. In mice, exposure of pregnant dams to the viral mimetic poly(I:C), serves as a model that simulates such pathology in the offspring, through a process known as Maternal Immune Activation (MIA). To investigate the mechanism of such effect, we hypothesized that maternal upregulation of Type-I interferon (IFN-I), as part of the dam's antiviral response, might contribute to the damage imposed on the offspring. Using mRNA sequencing and flow cytometry analyses we found that poly(I:C) treatment during pregnancy caused reduced expression of genes related to proliferation and cell cycle in the offspring's microglia relative to controls. This was found to be associated with an IFN-I signature in the embryonic yolk sac, the origin of microglia in development. Neutralizing IFN-I signaling in dams attenuated the effect of MIA on the newborn's microglia, while systemic maternal administration of IFNß was sufficient to mimic the effect of poly(I:C), and led to increased vulnerability of offspring's microglia to subsequent stress. Furthermore, maternal elevation of IFNß resulted in behavioral manifestations reminiscent of neuropsychiatric disorders. In addition, by adopting a "two-hit" experimental paradigm, we show a higher sensitivity of the offspring to postnatal stress subsequent to the maternal IFNß elevation, demonstrated by behavioral irregularities. Our results suggest that maternal upregulation of IFN-I, in response to MIA, interferes with the offspring's programmed microglial developmental cascade, increases their susceptibility to postnatal stress, and leads to behavioral abnormalities.

Obesity impacts the regulation of miR-10b and its targets in primary breast tumors.

BACKGROUND: Obesity increases breast cancer (BC) risk in post-menopausal women by mostly unknown molecular mechanisms which may partly be regulated by microRNAs (miRNAs). METHODS: We isolated RNA from paired benign and malignant biopsies from 83 BC patients and determined miRNA profiles in samples from 12 women at the extremes of the BMI distribution by RNA-seq. Candidates were validated in all samples. Associations between miR-10b expression and validated target transcript levels, and effects of targeted manipulation of miR-10b levels in a primary BC cell line on proliferation and invasion potential, were explored. RESULTS: Of the 148 miRNAs robustly expressed in breast tissues, the levels of miR-21, miR-10b, miR-451a, miR-30c, and miR-378d were significantly associated with presence of cancer. Of these, miR-10b showed a stronger down-regulation in the tumors of the obese subjects, as opposed to the lean. In ductal but not lobular tumors, significant inverse correlations were observed between the tumor levels of miR-10b and miR-30c and the mRNA levels of cancer-relevant target genes SRSF1, PIEZO1, MAPRE1, CDKN2A, TP-53 and TRA2B, as well as tumor grade. Suppression of miR-10b levels in BT-549 primary BC-derived cells increased cell proliferation and invasive capacity, while exogenous miR-10b mimic decreased invasion. Manipulation of miR-10b levels also inversely affected the mRNA levels of miR-10b targets BCL2L11, PIEZO1 and NCOR2. CONCLUSIONS: Our findings suggest that miR-10b may be a mediator between obesity and cancer in post-menopausal women, regulating several known cancer-relevant genes. MiR-10b expression may have diagnostic and therapeutic implications for the incidence and prognosis of BC in obese women.

Newly Formed Endothelial Cells Regulate Myeloid Cell Activity Following Spinal Cord Injury via Expression of CD200 Ligand.

The central nervous system (CNS) is endowed with several immune-related mechanisms that contribute to its protection and maintenance in homeostasis and under pathology. Here, we discovered an additional mechanism that controls inflammatory responses within the CNS milieu under injurious conditions, involving CD200 ligand (CD200L) expressed by newly formed endothelial cells. We observed that CD200L is constitutively expressed in the mouse healthy CNS by endothelial cells of the blood-cerebrospinal fluid barrier and of the spinal cord meninges, but not by the endothelium of the blood-spinal cord barrier. Following spinal cord injury (SCI), newly formed endothelial cells, located only at the epicenter of the lesion site, expressed CD200L. Moreover, in the absence of CD200L expression by CNS-resident cells, functional recovery of mice following SCI was impaired. High throughput single-cell flow cytometry image analysis following SCI revealed CD200L-dependent direct interaction between endothelial and local CD200R+ myeloid cells, including activated microglia and infiltrating monocyte-derived macrophages (mo-MΦ). Absence of CD200L signaling, both in vitro and in vivo, resulted in a higher inflammatory response of the encountering macrophages, manifested by elevation in mRNA expression of Tnfα and Il1ß, increased intracellular TNFα immunoreactivity, and reduced expression levels of macrophage factors that are associated with resolution of inflammation, Dectin-1, CD206 (mannose receptor), and IL-4R. Collectively, our results highlight the importance of CD200-mediated immune dialogue between endothelial cells and the local resident microglia and infiltrating mo-MΦ within the lesion area, as a mechanism that contributes to regulation of inflammation following acute CNS injury. SIGNIFICANCE STATEMENT: This manuscript focuses on a novel mechanism of inflammation-regulation following spinal cord injury (SCI), orchestrated by CD200-ligand (CD200L) expressed by newly formed endothelial cells within the lesion site. Our study reveals that, in homeostasis, CD200L is expressed by endothelial cells of the mouse blood-cerebrospinal fluid barrier and of the blood-leptomeningeal barrier, but not by endothelial cells of the blood-spinal cord barrier. Following SCI, newly formed endothelial cells located within the epicenter of the lesion site were found to express CD200L at time points that were shown to be critical for repair. Our results reveal a direct interaction between CD200L+ endothelial cells and CD200R+ microglia and macrophages, resulting in attenuated inflammation, biasing macrophage phenotype toward inflammation-resolving cells, and promotion of functional recovery following SCI.

Elevated Levels of miR-122 in Serum May Contribute to Improved Endothelial Function and Lower Oncologic Risk Following Bariatric Surgery.

BACKGROUND: Weight loss surgery is the most effective treatment for obesity, and it reduces cardiovascular and cancer risk through poorly understood mechanisms. MicroRNAs (miRNAs) are short RNA molecules that regulate the stability and translation of many mRNAs. We hypothesized that levels of specific circulating miRNAs are altered following surgery and may contribute to lower cancer risk. OBJECTIVES: To investigate the change of miRNA following surgery. METHODS: All patients underwent gastric "sleeve operation". RNA was isolated from sera of 21 patients (14 men, 7 women) before and 3 months after surgery. Sera were combined into two pools, which served for cDNA library construction followed by miSeq sequencing. The levels of candidate miRNAs were validated in the individual samples by QRT-PCR. RESULTS: Serum miR-122 was significantly up-regulated 3 months post-bariatric surgery in sera of patients, whose endothelial function had greatly improved. In addition, serum miR-122 levels correlated positively with endothelial function as measured by FMD. The changes in miR-122 levels from pre-surgery to 3 months post-surgery also tended to correlate with the respective changes in FMD. CONCLUSIONS: The serum miR-122/miR-451 ratio may serve as a marker for endothelial function in obese patients. miR-122 is the dominant miRNA in the liver and a known tumor suppressor. Our findings suggest a role for circulating miR-122 in the maintenance of vascular endothelial cells (VECs) and in the prevention of cancer. Further studies are required to elucidate the mechanism of its secretion into circulation and its absorption by VECs, as well as its relevant cellular targets.

Leptin and insulin up-regulate miR-4443 to suppress NCOA1 and TRAF4, and decrease the invasiveness of human colon cancer cells.

BACKGROUND: Obesity is a risk factor for colorectal cancer (CRC). Normal and tumor cells respond to metabolic hormones, such as leptin and insulin. Thus, obesity-associated resistance to these hormones likely leads to changes in gene expression and behavior of tumor cells. However, the mechanisms affected by leptin and insulin signaling in CRC cells remain mostly unknown. METHODS: We hypothesized that microRNAs (miRNAs) are involved in the regulation of tumorigenesis-related gene expression in CRC cells by leptin and insulin. To test this hypothesis, miRNA levels in the CRC-derived cell lines HCT-116, HT-29 and DLD-1 were profiled, following leptin and insulin treatment. Candidate miRNAs were validated by RT-qPCR. Predicted miRNA targets with known roles in cancer, were validated by immunoblots and reporter assays in HCT-116 cells. Transfection of HCT-116 cells with candidate miRNA mimic was used to test in vitro effects on proliferation and invasion. RESULTS: Of ~800 miRNAs profiled, miR-4443 was consistently up-regulated by leptin and insulin in HCT-116 and HT-29, but not in DLD-1, which lacked normal leptin receptor expression. Dose response experiments showed that leptin at 100 ng/ml consistently up-regulated miR-4443 in HCT-116 cells, concomitantly with a significant decrease in cell invasion ability. Transfection with miR-4443 mimic decreased invasion and proliferation of HCT-116 cells. Moreover, leptin and miR-4443 transfection significantly down-regulated endogenous NCOA1 and TRAF4, both predicted targets of miR-4443 with known roles in cancer metastasis. miR-4443 was found to directly regulate TRAF4 and NCOA1, as validated by a reporter assay. The up-regulation of miR-4443 by leptin or insulin was attenuated by the inhibition of MEK1/2. CONCLUSIONS: Our findings suggest that miR-4443 acts in a tumor-suppressive manner by down-regulating TRAF4 and NCOA1 downstream of MEK-C/EBP-mediated leptin and insulin signaling, and that insulin and/or leptin resistance (e.g. in obesity) may suppress this pathway and increase the risk of metastatic CRC.

Inversely Regulated Inflammation-Related Processes Mediate Anxiety-Obesity Links in Zebrafish Larvae and Adults.

Anxiety and metabolic impairments are often inter-related, but the underlying mechanisms are unknown. To seek RNAs involved in the anxiety disorder-metabolic disorder link, we subjected zebrafish larvae to caffeine-induced anxiety or high-fat diet (HFD)-induced obesity followed by RNA sequencing and analyses. Notably, differentially expressed (DE) transcripts in these larval models and an adult zebrafish caffeine-induced anxiety model, as well as the transcript profiles of inherently anxious versus less anxious zebrafish strains and high-fat diet-fed versus standard diet-fed adult zebrafish, revealed inversely regulated DE transcripts. In both larval anxiety and obesity models, these included long noncoding RNAs and transfer RNA fragments, with the overrepresented immune system and inflammation pathways, e.g., the "interleukin signaling pathway" and "inflammation mediated by chemokine and cytokine signaling pathway". In adulthood, overrepresented immune system processes included "T cell activation", "leukocyte cell-cell adhesion", and "antigen processing and presentation". Furthermore, unlike adult zebrafish, obesity in larvae was not accompanied by anxiety-like behavior. Together, these results may reflect an antagonistic pleiotropic phenomenon involving a re-adjusted modulation of the anxiety-metabolic links with an occurrence of the acquired immune system. Furthermore, the HFD potential to normalize anxiety-upregulated immune-related genes may reflect the high-fat diet protection of anxiety and neurodegeneration reported by others.

Isothiocyanates inhibit psoriasis-related proinflammatory factors in human skin.

OBJECTIVE: 4-Methylthiobutylisothiocyanate (MTBI), the main rocket (Eruca sativa) seed isothiocyanate (ITC), and its oxidized form, sulforaphane (SFN), were assessed for their potential effects on psoriasis-related factors. METHODS: MTBI and SFN were evaluated for their effect on mRNA expression and cytokine secretion in vitro in human monocytes and macrophage-like cells and ex vivo in topically treated inflamed human skin. In addition, they were assayed in vivo for morphological changes in topically treated psoriasiform human skin in severe-combined immunodeficient (SCID) mice. RESULTS: MTBI and SFN contributed to the prevention of inflammation development and reduced ongoing inflammation by downregulating lipopolysaccharide (LPS)-induced mRNA expression of the psoriasis-related cytokines, interleukin (IL)-12/23p40 (25-58 %), tumor necrosis factor (TNF)-α (15-37 %) and IL-6 (25-71 %), in human macrophage-like cells. In monocytes, they tended to act additively on cytokine mRNA and reduced IL-12/23p40 (51 %) secretion. In an ex-vivo inflamed human skin organ culture, MTBI (1 µg/ml) reduced the secretion of IL-1 (39 %) and IL-6 (32 %). Moreover, 2/8 and 3/8 of the MTBI- and SFN-treated psoriasiform SCID mice, respectively, recovered partially or entirely from the psoriasiform process. CONCLUSIONS: Results from these models indicate the potential of rocket seed ITCs as biological agents in the therapy of psoriasis and inflammation-related skin diseases.

Key role of the CCR2-CCL2 axis in disease modification in a mouse model of tauopathy.

BACKGROUND: For decades, dementia has been characterized by accumulation of waste in the brain and low-grade inflammation. Over the years, emerging studies highlighted the involvement of the immune system in neurodegenerative disease emergence and severity. Numerous studies in animal models of amyloidosis demonstrated the beneficial role of monocyte-derived macrophages in mitigating the disease, though less is known regarding tauopathy. Boosting the immune system in animal models of both amyloidosis and tauopathy, resulted in improved cognitive performance and in a reduction of pathological manifestations. However, a full understanding of the chain of events that is involved, starting from the activation of the immune system, and leading to disease mitigation, remained elusive. Here, we hypothesized that the brain-immune communication pathway that is needed to be activated to combat tauopathy involves monocyte mobilization via the C-C chemokine receptor 2 (CCR2)/CCL2 axis, and additional immune cells, such as CD4+ T cells, including FOXP3+ regulatory CD4+ T cells. METHODS: We used DM-hTAU transgenic mice, a mouse model of tauopathy, and applied an approach that boosts the immune system, via blocking the inhibitory Programmed cell death protein-1 (PD-1)/PD-L1 pathway, a manipulation previously shown to alleviate disease symptoms and pathology. An anti-CCR2 monoclonal antibody (αCCR2), was used to block the CCR2 axis in a protocol that partially eliminates monocytes from the circulation at the time of anti-PD-L1 antibody (αPD-L1) injection, and for the critical period of their recruitment into the brain following treatment. RESULTS: Performance of DM-hTAU mice in short-term and working memory tasks, revealed that the beneficial effect of αPD-L1, assessed 1 month after a single injection, was abrogated following blockade of CCR2. This was accompanied by the loss of the beneficial effect on disease pathology, assessed by measurement of cortical aggregated human tau load using Homogeneous Time Resolved Fluorescence-based immunoassay, and by evaluation of hippocampal neuronal survival. Using both multiparametric flow cytometry, and Cytometry by Time Of Flight, we further demonstrated the accumulation of FOXP3+ regulatory CD4+ T cells in the brain, 12 days following the treatment, which was absent subsequent to CCR2 blockade. In addition, measurement of hippocampal levels of the T-cell chemoattractant, C-X-C motif chemokine ligand 12 (Cxcl12), and of inflammatory cytokines, revealed that αPD-L1 treatment reduced their expression, while blocking CCR2 reversed this effect. CONCLUSIONS: The CCR2/CCL2 axis is required to modify pathology using PD-L1 blockade in a mouse model of tauopathy. This modification involves, in addition to monocytes, the accumulation of FOXP3+ regulatory CD4+ T cells in the brain, and the T-cell chemoattractant, Cxcl12.

Potential skin antiinflammatory effects of 4-methylthiobutylisothiocyanate (MTBI) isolated from rocket (Eruca sativa) seeds.

Isothiocyanates (ITCs), which are organosulfur compounds present in cruciferous vegetables, have anticarcinogenic, antiinflammatory, and antiproliferative activities. These biological activities, and the knowledge that rocket seed (Eruca sativa) extract is used in skin disorders in traditional Middle Eastern medicine, led to the isolation and assessment of 4-methylthiobutylisothiocyanate (MTBI), the major ITC in rocket seeds, for its potential in the prevention of inflammatory skin diseases, such as psoriasis. MTBI was found to depress the growth of activated keratinocytes and to arrest the activated THP-1 monocytes in the G2 stage. Both MTBI and its oxidized derivative sulforaphane (SFN), which was found in the rocket seed at a low concentration, downregulated the expression of the proinflammatory genes, tumor necrosis factor (TNF)-alpha and interleukin (IL)-12/23 p40, as well as that of intercellular adhesion molecule-1, in activated THP-1 cells. These results demonstrate that MTBI may deter the inflammation process, as has been reported for SFN. Furthermore, pretreatment with MTBI hindered the induction of the inflammatory state in the THP-1 cells, as shown by the inhibition of cytokine mRNA expression of IL-1beta, IL-12/23 p40, and TNF-alpha. Overall, our results imply that MTBI may represent a new family of natural compounds possessing significant skin inflammation-preventive activities.

Potential immunotherapy for Alzheimer disease and age-related dementia.

Emerging results support the concept that Alzheimer disease (AD) and age-related dementia are affected by the ability of the immune system to contain the brain's pathology. Accordingly, well-controlled boosting, rather than suppression of systemic immunity, has been suggested as a new approach to modify disease pathology without directly targeting any of the brain's disease hallmarks. Here, we provide a short review of the mechanisms orchestrating the cross-talk between the brain and the immune system. We then discuss how immune checkpoint blockade directed against the PD-1/PD-L1 pathways could be developed as an immunotherapeutic approach to combat this disease using a regimen that will address the needs to combat AD.

PD-1/PD-L1 checkpoint blockade harnesses monocyte-derived macrophages to combat cognitive impairment in a tauopathy mouse model.

Alzheimer's disease (AD) is a heterogeneous disorder with multiple etiologies. Harnessing the immune system by blocking the programmed cell death receptor (PD)-1 pathway in an amyloid beta mouse model was shown to evoke a sequence of immune responses that lead to disease modification. Here, blocking PD-L1, a PD-1 ligand, was found to have similar efficacy to that of PD-1 blocking in disease modification, in both animal models of AD and of tauopathy. Targeting PD-L1 in a tau-driven disease model resulted in increased immunomodulatory monocyte-derived macrophages within the brain parenchyma. Single cell RNA-seq revealed that the homing macrophages expressed unique scavenger molecules including macrophage scavenger receptor 1 (MSR1), which was shown here to be required for the effect of PD-L1 blockade in disease modification. Overall, our results demonstrate that immune checkpoint blockade targeting the PD-1/PD-L1 pathway leads to modification of common factors that go awry in AD and dementia, and thus can potentially provide an immunotherapy to help combat these diseases.

Microglia development follows a stepwise program to regulate brain homeostasis.

Microglia, the resident myeloid cells of the central nervous system, play important roles in life-long brain maintenance and in pathology. Despite their importance, their regulatory dynamics during brain development have not been fully elucidated. Using genome-wide chromatin and expression profiling coupled with single-cell transcriptomic analysis throughout development, we found that microglia undergo three temporal stages of development in synchrony with the brain--early, pre-, and adult microglia--which are under distinct regulatory circuits. Knockout of the gene encoding the adult microglia transcription factor MAFB and environmental perturbations, such as those affecting the microbiome or prenatal immune activation, led to disruption of developmental genes and immune response pathways. Together, our work identifies a stepwise microglia developmental program integrating immune response pathways that may be associated with several neurodevelopmental disorders.

Thioredoxin-mimetic peptide CB3 lowers MAPKinase activity in the Zucker rat brain.

Diabetes is a high risk factor for dementia. High glucose may be a risk factor for dementia even among persons without diabetes, and in transgenic animals it has been shown to cause a potentiation of indices that are pre-symptomatic of Alzheimer's disease. To further elucidate the underlying mechanisms linking inflammatory events elicited in the brain during oxidative stress and diabetes, we monitored the activation of mitogen-activated kinsase (MAPKs), c-jun NH2-terminal kinase (JNK), p38 MAP kinases (p38(MAPK)), and extracellular activating kinsae1/2 (ERK1/2) and the anti-inflammatory effects of the thioredoxin mimetic (TxM) peptides, Ac-Cys-Pro-Cys-amide (CB3) and Ac-Cys-Gly-Pro-Cys-amide (CB4) in the brain of male leptin-receptor-deficient Zucker diabetic fatty (ZDF) rats and human neuroblastoma SH-SY5Y cells. Daily i.p. injection of CB3 to ZDF rats inhibited the phosphorylation of JNK and p38(MAPK), and prevented the expression of thioredoxin-interacting-protein (TXNIP/TBP-2) in ZDF rat brain. Although plasma glucose/insulin remained high, CB3 also increased the phosphorylation of AMP-ribose activating kinase (AMPK) and inhibited p70(S6K) kinase in the brain. Both CB3 and CB4 reversed apoptosis induced by inhibiting thioredoxin reductase as monitored by decreasing caspase 3 cleavage and PARP dissociation in SH-SY5Y cells. The decrease in JNK and p38(MAPK) activity in the absence of a change in plasma glucose implies a decrease in oxidative or neuroinflammatory stress in the ZDF rat brain. CB3 not only attenuated MAPK phosphorylation and activated AMPK in the brain, but it also diminished apoptotic markers, most likely acting via the MAPK-AMPK-mTOR pathway. These results were correlated with CB3 and CB4 inhibiting inflammation progression and protection from oxidative stress induced apoptosis in human neuronal cells. We suggest that by attenuating neuro-inflammatory processes in the brain Trx1 mimetic peptides could become beneficial for preventing neurological disorders associated with diabetes.

Endothelial dysfunction and procoagulant activity in acute ischemic stroke.

Endothelium-dependent vasodilator function may be regarded as an index of inflammation. Endothelial dysfunction has been observed in stroke patients and has been related to stroke physiopathology, stroke subtypes, clinical severity, and outcome. Our aim was to measure systemic vascular function directly (using forearm flow mediated dilatation) in patients with acute ischemic stroke and to clarify whether recent acute ischemic stroke is associated with impaired vascular function. Patients who were not eligible for thrombolytic therapy because of delayed arrival were randomly recruited to the study after signing a consent form. All 43 patients were conscious and had an acute ischemic stroke. Brain CT was performed on admission, and clinical evaluation was carried out by a neurologist on admission and four days later. Vascular responsiveness was evaluated by ABI and by endothelial function measurements on admission. Levels of P-selectin were measured during the first 24 hrs and on day 4. Forty-three patients (28 men and 15 women) and 23 healthy men (control) were enrolled in the study. Patients were older (62.4±12.5 y vs 44.2±11.6 y, p=0.001), had worse endothelial dysfunction (-4.4±7.4% vs 16.6±7.6%, p=0.001), and had a higher BMI (28±6 vs 24±5, p=0.001). No gender effect was found in endothelial function (-5.1±7.8% vs -2.5±6.6%, p=0.25) and ABI (1.0±0.26 vs 1.0±0.5, p=0.29). However, men had lower BMIs compared to women (26.8±5.8 vs 31.4±5.5, p=0.01). The neurological scale decreased from 4.9±3.4 to 3.2±3.0 on day 4 (p=0.001). In men, it was 4.8±3.8 on admission, and decreased to 3.2±3.4 on day 4 (p=0.001). In women, it was 5.0±2.7, and decreased to 3.3±2.3 on day 4 (p=0.001). P-selectin levels were high on admission (68.0±55.5 pg/ml) and increased 4 days later (102.3±72.0 pg/ml) (p=0.01). Men had higher levels on admission (79.1± 66.7 pg/ml vs 48.9± 15.4 pg/ml, p=0.02) and rose on day 4 to 113.6±82.6 pg/ml (p=0.05); in women P-selectin increased from 48.9± 15.4 pg/ml to 83.5±46.4 pg/ml (p=0.01), without gender effect on day 4 (113.6±82.6 pg/ml [men] vs 83.5±46.4 pg/ml [women] (p=0.08)). None of the univariate models seemed statistically significant---gender (p=0.448), age (p=0.100), BMI (p=0.607), ABI (p=0.103), FMD% (p=0.456), and P-selectin (p=0.195). Patients with acute stroke had severe endothelial dysfunction during the first 24 hrs with high P-selectin levels that further increased over the first week. Vascular instability and procoagulant activity are still in progress in the first days following acute stroke and patients are at risk to develop more vascular events at that time.

Human atherosclerotic plaque lipid extract promotes expression of proinflammatory factors in human monocytes and macrophage-like cells.

OBJECTIVE: The potential of the atherogenic human carotid plaque to stimulate the inflammatory process was examined in human monocytes and macrophages, in vitro. METHODS AND RESULTS: Exposure of monocytes to human carotid plaque lipid extract (LE) elevated the transcription level of the proinflammatory cytokines, interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, by 2.9 and 100.2 fold, respectively (as determined by real time PCR), and induced TNF-α secretion (as measured by enzyme-linked immunosorbent assay). Furthermore, LE caused an increase of 1.3-3.1 fold in the mRNA expression of the proinflammatory factors, IL-1ß, IL-6, TNF-α, cyclooxygenase-2 and intercellular adhesion molecule-1, in macrophage-like cells. In order to investigate the proinflammatory components in the extract, two fractions, obtained by silica gel separation of LE, were characterized. The cholesterol-oxysterol rich fraction was found to have the most significant proinflammatory effect. It caused an increase in TNF-α expression in monocytes, and upregulated IL-6, TNF-α, intercellular adhesion molecule-1 and cyclooxygenase 2 by 1.5-2.5 fold in macrophages. The triglyceride fraction had almost no effect on the cells. CONCLUSIONS: The human carotid plaque lipid extract was demonstrated to promote inflammation, in vitro. These data support the atherogenic character of the plaque and imply that its lipid composition may have ramifications on the progress of atherosclerosis.

The effect of under- and overexpressed CoEXG1-encoded exoglucanase secreted by Candida oleophila on the biocontrol of Penicillium digitatum.

The yeast, Candida oleophila, is acknowledged for its biocontrol activity against postharvest moulds. However, the mechanism of this activity is not fully understood. One of the conjectured modes of action is associated with extracellular lytic enzymes, such as beta-exoglucanase. The relationship of beta-exoglucanase in the biocontrol activity of C. oleophila was investigated by generating C. oleophila CoEXG1-knockouts and double-CoEXG1 transformants. The knockout transformants secreted 0-13% of the exoglucanase activity detected in the medium of the untransformed yeast (depending on the medium), indicating that CoEXG1 is the main gene responsible for the production of the secreted exoglucanase. Correspondingly, the double-CoEXG1 transformants secreted approximately twice as much 1,3-beta-exoglucanase as the untransformed C. oleophila. The biocontrol activity of the CoEXG1-knockout and the double-CoEXG1 transformants against Penicillium digitatum did not differ from that of the untransformed C. oleophila on kumquats. These results imply that the 1,3-beta-exoglucanase encoded by the gene CoEXG1 is not involved in the biocontrol activity of C. oleophila against P. digitatum under these experimental terms. However, these findings do not rule out the possibilities, that the participation of CoEXG1 in biocontrol is dependent on the activity of other gene products, or that its effect may be manifested under altered environmental conditions.

Cloning and analysis of CoEXG1, a secreted 1,3-beta-glucanase of the yeast biocontrol agent Candida oleophila.

Lytic enzymes may have a role in the biological control of fungi. The yeast biocontrol agent, Candida oleophila, is an excellent subject to research this matter. In the present study, CoEXG1, which encodes for a secreted 1,3-beta-glucanase, is the first gene to be cloned from C. oleophila. It was isolated from a partial genomic library and analysed. Its open reading frame and putative promoter were expressed in baker's yeast, Saccharomyces cerevisiae. The reading frame, expressed under the inducible GAL1 promoter, caused an increased secretion of beta-glucanase, and the putative promoter region activated the lacZ reporter gene, to which it was fused. Sequencing analysis revealed that CoEXG1 carries the signature pattern of the 5 glycohydrolases family and has a putative secretion leader, as well as a high degree of identity to yeast 1,3-beta-glucanases. The GenBank Accession No. of CoEXG1 is AF393806.

Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila.

The yeast Candida oleophila, the base of the commercial product Aspire, is recommended for the control of postharvest decay of citrus and pome fruit. Competition for nutrients and space is believed to be the major mode of action. Involvement of fungal cell wall-degrading enzymes is also suggested to play a role in the mechanism of action of yeast antagonists. The present study showed that the yeast C. oleophila is capable of producing and secreting various cell wall-degrading enzymes, including exo-beta-1,3-glucanase, chitinase and protease. Exo-beta-1,3-glucanase and chitinase were produced and maximized in the early stages of growth, whereas protease reached a maximum level only after 6-8 days. Production of exo-beta-1,3-glucanase, chitinase and protease was stimulated by the presence of cell wall fragments of Penicillium digitatum in the growth medium, in addition to glucose. This study also provided evidence that C. oleophila is capable of secreting exo-beta-1,3-glucanase into the wounded surface of grapefruit. The role of exo-beta-1,3-glucanase ( CoEXG1) in the biocontrol activity of C. oleophila was tested using CoEXG1-knockouts and double- CoEXG1 over-producing transformants. In vitro bioassays showed that wild-type C. oleophila and exo-beta-1,3-glucanase over-expressing transformants had similar inhibitory effects on spore germination and germ-tube elongation; and both were more inhibitory to the fungus than the knockout transformant. In experiments conducted on fruit to test the biocontrol activity against infection by P. digitatum, no significant difference in inhibition was observed between transformants and untransformed C. oleophila cells at the high concentrations of cells used, whereas at a lower concentration of yeast cells the knockout transformants appeared to be less effective.