Galectin-3 absence alters lymphocytes populations dynamics behavior and promotes functional recovery after spinal cord injury in mice.

Spinal cord injury (SCI) results from various mechanisms that damage the nervous tissue and the blood-brain barrier, leading to sensory and motor function loss below the injury site. Unfortunately, current therapeutic approaches for SCI have limited efficacy in improving patients outcomes. Galectin-3, a protein whose expression increases after SCI, influences the neuroinflammatory response by favoring pro-inflammatory M1 macrophages and microglia, while inhibiting pro-regenerative M2 macrophages and microglia, which are crucial for inflammation resolution and tissue regeneration. Previous studies with Galectin-3 knock-out mice demonstrated enhanced motor recovery after SCI. The M1/M2 balance is strongly influenced by the predominant lymphocytic profiles (Th1, Th2, T Reg, Th17) and cytokines and chemokines released at the lesion site. The present study aimed to investigate how the absence of galectin-3 impacts the adaptive immune system cell population dynamics in various lymphoid spaces following a low thoracic spinal cord compression injury (T9-T10) using a 30 g vascular clip for one minute. It also aimed to assess its influence on the functional outcome in wild-type (WT)and Galectin-3 knock-out (GALNEG) mice. Histological analysis with hematoxylin-eosin and Luxol Fast Blue staining revealed that WT and GALNEG animals exhibit similar spinal cord morphology. The absence of galectin-3 does not affect the common neuroanatomy shared between the groups prompting us to analyze outcomes between both groups. Following our crush model, both groups lost motor and sensory functions below the lesion level. During a 42-day period, GALNEG mice demonstrated superior locomotor recovery in the Basso Mouse Scale (BMS) gait analysis and enhanced motor coordination performance in the ladder rung walk test (LRW) compared to WT mice. GALNEG mice also exhibited better sensory recovery, and their electrophysiological parameters suggested a higher number of functional axons with faster nerve conduction. Seven days after injury, flow cytometry of thymus, spleen, and blood revealed an increased number of T Reg and Th2 cells, accompanied by a decrease in Th1 and Th17 cells in GALNEG mice. Immunohistochemistry conducted on the same day exhibited an increased number of Th2 and T Reg cells around the GALNEG's spinal cord lesion site. At 42-day dpi immunohistochemistry analyses displayed reduced astrogliosis and greater axon preservation in GALNEG's spinal cord seem as a reduction of GFAP immunostaining and an increase in NFH immunostaining, respectively. In conclusion, GALNEG mice exhibited better functional recovery attributed to the milder pro-inflammatory influence, compensated by a higher quantity of T Reg and Th2 cells. These findings suggest that galectin-3 plays a crucial role in the immune response after spinal cord injury and could be a potential target for clinical therapeutic interventions.

Age-Associated Upregulation of Glutamate Transporters and Glutamine Synthetase in Senescent Astrocytes In Vitro and in the Mouse and Human Hippocampus.

Aging is marked by complex and progressive physiological changes, including in the glutamatergic system, that lead to a decline of brain function. Increased content of senescent cells in the brain, such as glial cells, has been reported to impact cognition both in animal models and human tissue during normal aging and in the context of neurodegenerative disease. Changes in the glutamatergic synaptic activity rely on the glutamate-glutamine cycle, in which astrocytes handle glutamate taken up from synapses and provide glutamine for neurons, thus maintaining excitatory neurotransmission. However, the mechanisms of glutamate homeostasis in brain aging are still poorly understood. Herein, we showed that mouse senescent astrocytes in vitro undergo upregulation of GLT-1, GLAST, and glutamine synthetase (GS), along with the increased enzymatic activity of GS and [3H]-D-aspartate uptake. Furthermore, we observed higher levels of GS and increased [3H]-D-aspartate uptake in the hippocampus of aged mice, although the activity of GS was similar between young and old mice. Analysis of a previously available RNAseq dataset of mice at different ages revealed upregulation of GLAST and GS mRNA levels in hippocampal astrocytes during aging. Corroborating these rodent data, we showed an increased number of GS + cells, and GS and GLT-1 levels/intensity in the hippocampus of elderly humans. Our data suggest that aged astrocytes undergo molecular and functional changes that control glutamate-glutamine homeostasis upon brain aging.

Adipose tissue of control and ex-obese patients exhibit differences in blood vessel content and resident mesenchymal stem cell population.

BACKGROUND: The normal function of white adipose tissue is disturbed in obesity. After weight loss that follows bariatric surgery, ex-obese patients undergo plastic surgery to remove residual tissues and it is not known whether their adipose tissue returns to its original state. The aim of this study was to compare the white adipose tissue composition of ex-obese with control patients with regard to blood vessels and resident mesenchymal stem cells (MSC). METHODS: Quantification of blood vessels was performed on histological sections of adipose tissue stained with hematoxylin and eosin and for von Willebrand antigen. MSC were induced to the adipogenic and osteogenic lineages by specific inductive culture media. Expression of PPARgamma2 was analyzed by reverse transcription polymerase chain reaction. RESULTS: Ex-obese adipose tissue showed a higher number (p = 0.0286) of small (107.3 +/- 22.0) and large (22.5 +/- 6.4) blood vessels, when compared to control patients (42.0 +/- 24.4 and 7.2 +/- 2.2, respectively) and they also occupied a larger area (control versus ex-obese, p = 0.0286). Adipose tissue MSC from both groups of patients expressed PPARgamma2 and were equally able to differentiate to the osteogenic lineage, but ex-obese MSC showed a higher adipogenic potential when induced in vitro (p < 0.05). CONCLUSIONS: The higher number of adipose tissue blood vessels in ex-obese patients explains the excessive bleeding observed during their plastic surgery. The presence of more committed cells to the adipogenic lineage may favor the easy weight regain that occurs in ex-obese patients. These results show that, after extensive weight loss, adipose tissue cell composition was not totally restored.

A Randomized Controlled Trial Investigating the Effects of Undulatory, Staggered, and Linear Load Manipulations in Aerobic Training on Oxygen Supply, Muscle Injury, and Metabolism in Male Recreational Runners.

BACKGROUND: Taper is considered as a strategy to avoid overtraining and increase peak performance in athletes. Because aerobic exercise increases the level and duration of independence during the lifespan, the participation of recreational athletes has increased in running events around the world. However, the effects of using load reduction in their training and aerobic performance are still not well known. OBJECTIVES: Our goal was to compare load manipulations, using tapering-like periods in the end of recreational athletes training evaluating alterations in oxygen supply, muscle injury, and metabolism markers. METHODS: Healthy males (n = 88, 20-35 years old) were randomly distributed in groups using a combination of two mesocycles of 4 weeks, undulatory and staggered, with two daily microcycles, undulatory and linear. Undulatory-undulatory (Und-Und) and undulatory-linear (Und-Lin) groups had load reduction in the final week of training while the staggered-undulatory (Sta-Und) and staggered-linear (Sta-Lin) groups did not. Cardiorespiratory capacity (V̇O2max), body mass index (BMI), and body fat % were evaluated. Blood samples were also collected to analyze hematocrit (Ht), mean corpuscular hemoglobin (MCHC), circulating levels of hemoglobin (Hb), creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea (U), cortisol (C), free testosterone (FT), and free T/C ratio. RESULTS: After 8 weeks of training, Und-Und had the highest trend to increase V̇O2max. Both Und-Und and Sta-Lin reduced body fat %. Und-Und showed a significant increase in MCHC, T and Free T/C ratio, a positive trend to increase Ht% and Hb levels, and a trend to decrease CK, LDH, and C circulating levels. Sta-Lin presented a trend to decrease Ht% and Hb levels, a significant increase in CK, LDH, AST, ALT circulating levels, and a decrease in Free T/C ratio. CONCLUSION: The use of daily undulatory training load provides better gains for aerobic fitness while the use of staggered load, mainly associated with linear load, promotes inadequate recovery in recreational runners.

Age-associated alterations in the levels of cytotoxic lipid molecular species and oxidative stress in the murine thymus are reduced by growth hormone treatment.

During age-associated thymic involution, thymocytes decrease and lipid-laden cells accumulate. However, if and how aging affects the thymic lipid profile is not well understood, nor is it known if the hormonal milieu modifies this process. Here we demonstrate a correlation between reduced thymocyte numbers and markers of inflammation and oxidative stress with age. Evaluating the lipidomics profile of the whole thymus, between the ages of 4 (young) and 18 months (old), we found increased amounts of triacylglycerides, free cholesterol, cholesterol ester and 4-hydroxynonenal (4-HNE) with age. Moreover, levels of C24:0 and C24:1 sphingomyelins and ceramide C16:0 were elevated in 12-14 month-old (middle-aged) mice while the levels of sulfatide ceramide and ganglioside GD1a increased in the old thymus. Evaluating isolated thymocytes, we found increased levels of cholesterol ester and 4-HNE adducts, as compared to young mice. Next, we treated middle-aged mice with growth hormone (GH), which has been considered a potent immunomodulator. GH reduced thymic levels of TNF-α and 4-HNE and increased the number of thymocytes as well as the thymic levels of dihydroceramide, a ceramide precursor and autophagic stimuli for cell survival. In conclusion, GH treatment attenuated inflammation and age-related increases in oxidative stress and lipotoxicity in the thymus.

Growth hormone modulates thymocyte development in vivo through a combined action of laminin and CXC chemokine ligand 12.

Previous evidence indicates that GH modulates thymic cell migration. In this study we approached this issue in vivo, studying thymocyte migration in GH transgenic animals and in normal mice treated intrathymically with GH. Extracellular matrix and chemokines are involved in thymocyte migration. In this respect, thymocyte adhesion to laminin was higher in GH-treated animals than controls, and the numbers of migrating cells in laminin-coated Transwells was higher in GH-transgenic and GH-injected mice. Additionally, CXC chemokine ligand 12 (CXCL12)-driven migration was higher in GH-Tg and GH-treated animals compared with controls. Interestingly, although CXCR4 expression on thymocytes did not change in GH-Tg mice, the CXCL12 intrathymic contents were higher. We found that CXCL12, in conjunction with laminin, would additionally enhance the migration of thymocytes previously exposed to high concentrations of GH in vivo. Lastly, there was an augmentation of recent thymic emigrants in lymph nodes from GH-Tg and GH-injected animals. In conclusion, enhanced thymocyte migration in GH transgenic mice as well as GH-injected mice results at least partially from a combined action of laminin and CXCL12. Considering that GH is presently being used as an adjuvant therapeutic agent in immunodeficiencies, including AIDS, the concepts defined herein provide important background knowledge for future GH-based immune interventions.

Lipid-Laden Multilocular Cells in the Aging Thymus Are Phenotypically Heterogeneous.

Intrathymic lipid-laden multilocular cells (LLMC) are known to express pro-inflammatory factors that might regulate functional activity of the thymus. However, the phenotype of age-associated intrathymic LLMC is still controversial. In this study, we evaluated LLMC density in the aging thymus and better characterized their distribution, ultrastructure and phenotype. Our results show an increased density of LLMC in the thymus from 03 to 24 months of age. Morphologically, intrathymic LLMC exhibit fibroblastoid fusiform, globular or stellate shapes and can be found in the subcapsular region as well as deeper in the parenchyma, including the perivascular area. Some parenchymal LLMC were like telocytes accumulating lipids. We identified lipid droplets with different electrondensities, lipofuscin granules and autolipophagosome-like structures, indicating heterogeneous lipid content in these cells. Autophagosome formation in intrathymic LLMC was confirmed by positive staining for beclin-1 and perilipin (PLIN), marker for lipid droplet-associated proteins. We also found LLMC in close apposition to thymic stromal cells, endothelial cells, mast cells and lymphocytes. Phenotypically, we identified intrathymic LLMC as preadipocytes (PLIN+PPARγ2+), brown adipocytes (PLIN+UCP1+), macrophages (PLIN+Iba-1+) or pericytes (PLIN+NG2+) but not epithelial cells (PLIN- panCK+). These data indicate that intrathymic LLMC are already present in the young thymus and their density significantly increases with age. We also suggest that LLMC, which are morphologically distinct, establish direct contact with lymphocytes and interact with stromal cells. Finally, we evidence that intrathymic LLMC correspond to not only one but to distinct cell types accumulating lipids.

Interesterified fat or palm oil as substitutes for partially hydrogenated fat in maternal diet can predispose obesity in adult male offspring.

BACKGROUND & AIMS: Palm oil (PO) and interesterified fat (IF) have been used to replace partially hydrogenated fat (PHF), which is rich in trans isomers, in processed foods. The purpose of this study was to investigate whether normolipidic diets containing PHF, IF, or PO consumed during pregnancy and lactation affect total body adiposity and adipose tissue morphology of adult offspring mice. METHODS: Four groups of female C57BL/6 mice were fed, during pregnancy and lactation, a control diet (control group, CG), a PHF diet (trans group, TG), a PO diet (PG group), or an IF diet (IG group). After weaning (at 21 days), male pups received the control diet for 70 days. Food intake and body weight were monitored in all groups throughout the experimental period. At 3 months of age, mice were sacrificed and the inguinal (IWAT), epididymal (EWAT), retroperitoneal (RPWAT), and mesenteric (MWAT) adipose fat pads were removed and weighed. Adiposity was quantified by micro computed tomography (micro-CT), and adipocyte areas and cell number were analyzed by histology. RESULTS: PG and IG offspring gained more weight than CG and TG groups (p < 0.01) during the first 10 weeks after weaning, resulting in higher final body weights (p < 0.05). IG mice and PG mice had respectively heavier EWAT and IWAT than TG and CG mice. Micro-CT scanning revealed that the total volumes of internal, external, and total fat depots were greater in IG animals, as compared to the other groups. Larger adipocyte areas were observed in EWAT and IWAT in IG and TG, respectively, in comparison to CG and PG mice. PG mice showed increased adipocyte numbers in IWAT. CONCLUSIONS: Maternal intake of IF and/or PO during pregnancy and lactation predisposes the offspring to the development of obesity in adult life in mice.

Quantitative differences in lipid raft components between murine CD4+ and CD8+ T cells.

BACKGROUND: Lipid rafts have been shown to play a role in T cell maturation, activation as well as in the formation of immunological synapses in CD4+ helper and CD8+ cytotoxic T cells. However, the differential expression of lipid raft components between CD4+ and CD8+ T cells is still poorly defined. To examine this question, we analyzed the expression of GM1 in T cells from young and aged mice as well as the expression of the glycosylphosphatidylinositol (GPI)-linked protein Thy-1 and cholesterol in murine CD4+ and CD8+ T cell subpopulations. RESULTS: We found that CD4+CD8- and CD8+CD4- thymocytes at different stages of maturation display distinct GM1 surface expression. This phenomenon did not change with progressive aging, as these findings were consistent over the lifespan of the mouse. In the periphery, CD8+ T cells express significantly higher levels of GM1 than CD4+ T cells. In addition, we observed that GM1 levels increase over aging on CD8+ T cells but not in CD4+ T cells. We also verified that naïve (CD44lo) and memory (CD44hi) CD8+ T cells as well as naïve and memory CD4+ T cells express similar levels of GM1 on their surface. Furthermore, we found that CD8+ T cells express higher levels of the GPI-anchored cell surface protein Thy-1 associated with lipid raft domains as compared to CD4+ T cells. Finally, we observed higher levels of total cellular cholesterol in CD8+ T cells than CD4+ T cells. CONCLUSION: These results demonstrate heterogeneity of lipid raft components between CD4+ and CD8+ T cells in young and aged mice. Such differences in lipid raft composition may contribute to the differential CD4 and CD8 molecule signaling pathways as well as possibly to the effector responses mediated by these T cell subsets following TCR activation.

Age-associated alterations in CXCL1 chemokine expression by murine B cells.

BACKGROUND: The CXCL1 chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), have been shown to play a role in a number of pathophysiological disease states including endotoxin-induced inflammation and bacterial meningitis. While the expression of these chemokines has been identified in a variety of cell types in the mouse, little is known about their expression with murine B-lymphocytes. RESULTS: Here, we demonstrate that highly purified murine splenic B cells are capable of expressing both MIP-2 and KC protein and mRNA upon activation with lipopolysaccharide (LPS) but not in response to anti-micro and anti-CD40 in combination with interleukin-4 (IL-4) stimulation. Moreover, these chemokines are expressed at higher levels in B cells derived from young (4 m) compared to old (24-29 m) mice. Upon fractionation into distinct B-cell subsets, we found that the expression of MIP-2 and KC by aged follicular (FO) B cells is significantly decreased when compared to the same cells from younger mice, while only MIP-2 production was found to be diminished in aged marginal zone (MZ) B cells. Interestingly, MIP-2 and KC production by newly formed (NF) B cells did not significantly differ with age. Moreover, the potential relevance of these findings is supported by the poor ability of LPS-activated aged B cells to specifically mediate CXCL1-dependent leukocyte recruitment when compared to younger B cells. CONCLUSION: Overall, the decreased expression of CXCL1 chemokines by aged B cells in response to LPS may have potential implications on the secondary recruitment of leukocytes to sites of microbial infections and inflammation possibly contributing to the increased susceptibility of older subjects to pathogen challenge.

Enhanced Aß(1-40) production in endothelial cells stimulated with fibrillar Aß(1-42).

Amyloid accumulation in the brain of Alzheimer's patients results from altered processing of the 39- to 43-amino acid amyloid ß protein (Aß). The mechanisms for the elevated amyloid (Aß(1-42)) are considered to be over-expression of the amyloid precursor protein (APP), enhanced cleavage of APP to Aß, and decreased clearance of Aß from the central nervous system (CNS). We report herein studies of Aß stimulated effects on endothelial cells. We observe an interesting and as yet unprecedented feedback effect involving Aß(1-42) fibril-induced synthesis of APP by Western blot analysis in the endothelial cell line Hep-1. We further observe an increase in the expression of Aß(1-40) by flow cytometry and fluorescence microscopy. This phenomenon is reproducible for cultures grown both in the presence and absence of serum. In the former case, flow cytometry reveals that Aß(1-40) accumulation is less pronounced than under serum-free conditions. Immunofluorescence staining further corroborates these observations. Cellular responses to fibrillar Aß(1-42) treatment involving eNOS upregulation and increased autophagy are also reported.

Growth hormone modulates migration of developing T cells.

In the context of the cross-talk between the neuroendocrine and immune systems, it is well known that growth hormone (GH) exerts physiological effects in central as well as peripheral compartments of the immune system. GH modulates a variety of thymic functions. For example, GH upregulates proliferation of thymocytes and thymic epithelial cells. Accordingly, GH-transgenic mice, as well as animals and humans treated with exogenous GH, exhibit an enhanced cellularity in the thymus organ. GH also stimulates the secretion of thymic hormones, cytokines, and chemokines by the thymic microenvironment as well as the production of extracellular matrix proteins. These effects lead to an increase in thymocyte migratory responses and intrathymic traffic of developing T cells, including the export of thymocytes from the thymus organ, as ascertained by experimental studies with intrathymic injection of GH in normal mice and with GH-transgenic animals. Because GH promotes a replenishment of the thymus and an increase of thymocyte export, it has been applied as a potential adjuvant therapeutic agent in the treatment of immunodeficiencies associated with thymic atrophy.

Gene expression profiling: from microarrays to medicine.

With the mapping of the human genome comes the ability to identify genes of interest in specific diseases and the pathways involved therein. Laboratory technology has evolved in parallel, providing us with the ability to assay thousands of these genes at once, a technique known as microarray analysis. The main question that this type of technology raises is how we can apply this powerful technology to clinical medicine. Recently, advances in data analysis, as well as standardization of the technology, have allowed us to examine this question, and indeed a few clinical trials currently being performed include microarrays as part of their protocol. In this review we outline the microarray technique and describe these types of studies in further detail.

Growth hormone stimulates the selective trafficking of thymic CD4+CD8- emigrants to peripheral lymphoid organs.

Growth hormone (GH) has been shown to stimulate T cell development. However, its mechanisms of action on the peripheral T cell pool remain unknown. To address this question, intrathymic injection of GH in combination with fluorescein isothiocyanate (FITC) was used to assess the effects of GH on T cell trafficking from the thymus to the periphery. GH promoted a significant increase in the percentage and differential distribution of thymic CD4+CD8-FITC+ cells in secondary lymphoid organs. A significantly higher percentage of CD4+CD8-FITC+ cells was observed in the lymph nodes, while a relative decrease of these cells was found in the spleen. Moreover, we verified that GH treatment resulted in increased numbers of CD62L+CD4+CD8-FITC+ T cells in the lymph nodes, while the same treatment resulted in a decline in the percentage of VLA-6+CD4+CD8-FITC+ T cells in the spleen. Together, these findings suggest that GH is a potent immunoregulatory molecule which selectively stimulates the preferential homing of CD4+CD8- thymic emigrants to the subcutaneous lymph nodes possibly via the differential expression of CD62L and VLA-6.

Functional insulin-like growth factor-1/insulin-like growth factor-1 receptor-mediated circuit in human and murine thymic epithelial cells.

Interactions between thymocytes and thymic epithelial cell (TEC) can be modulated by growth hormone via insulin-like growth factor-1 (IGF-1). In this study, we showed IGF-1 and IGF-1 receptor mRNA expression by human and murine TEC and thymocytes. Functionally, IGF-1 stimulates extracellular matrix production by human TEC. Moreover, pretreatment of murine TEC with IGF-1 increases their adhesion to thymocytes. Interestingly, we observed an increase in the frequency of CD4-CD8-CD90+ T cells which adhered to pretreated TEC, supporting the concept that IGF-1 may also act indirectly on intrathymic T cell differentiation and migration through the thymic epithelium.