Cannabinoid Mixture Affects the Fate and Functions of B Cells through the Modulation of the Caspase and MAP Kinase Pathways.

Cannabis use is continuously increasing in Canada, raising concerns about its potential impact on immunity. The current study assessed the impact of a cannabinoid mixture (CM) on B cells and the mechanisms by which the CM exerts its potential anti-inflammatory properties. Peripheral blood mononuclear cells (PBMCs) were treated with different concentrations of the CM to evaluate cytotoxicity. In addition, flow cytometry was used to evaluate oxidative stress, antioxidant levels, mitochondrial membrane potential, apoptosis, caspase activation, and the activation of key signaling pathways (ERK1/2, NF-κB, STAT5, and p38). The number of IgM- and IgG-expressing cells was assessed using FluoroSpot, and the cytokine production profile of the B cells was explored using a cytokine array. Our results reveal that the CM induced B-cell cytotoxicity in a dose-dependent manner, which was mediated by apoptosis. The levels of ROS and those of the activated caspases, mitochondrial membrane potential, and DNA damage increased following exposure to the CM (3 µg/mL). In addition, the activation of MAP Kinase, STATs, and the NF-κB pathway and the number of IgM- and IgG-expressing cells were reduced following exposure to the CM. Furthermore, the exposure to the CM significantly altered the cytokine profile of the B cells. Our results suggest that cannabinoids have a detrimental effect on B cells, inducing caspase-mediated apoptosis.

Evaluation of pediatric-specific resources to support utilization of the Wheelchair Skills Training Program by the users of the resources: a descriptive qualitative study.

BACKGROUND: Children's ability to engage in meaningful activities is positively influenced by their ability to move independently. Preliminary evidence in children suggests that wheelchair skills training improves wheelchair skills, which are important for independent mobility. The Wheelchair Skills Training Program is a standardized program to teach wheelchair skills. However, it is underutilized in pediatric rehabilitation settings. To increase its utilization, 3 pediatric-specific Wheelchair Skills Training Program resources related to indoor skills were developed (i.e., a storybook, four instructional posters, and a training workbook). This study aimed to describe occupational therapists' (OTs) and pediatric manual wheelchair users' (PMWUs) perceived satisfaction with the storybook, instructional posters and training workbook, and to explore their perceptions regarding the usability, relevance, and feasibility of these resources in pediatric rehabilitation settings. METHODS: A descriptive qualitative design was used. Convenience samples of OTs and PMWUs were recruited in a rehabilitation center and affiliated schools. A focus group with OTs and semi-structured interviews with PMWUs were conducted by videoconference to obtain participants' feedback on the resource prototypes and suggestions for improvement. Data were deductively analyzed using the Framework method. RESULTS: Eight OTs and 5 PMWUs expressed general satisfaction with the resources, describing them as usable, relevant, and feasible to integrate into wheelchair skills training with novice wheelchair users and younger children. All OTs and 3 PMWUs expressed the desire to use the resources for wheelchair skills training. Two PMWUs perceived the resources were not relevant to them because they already mastered the skills. The participants suggested minor modifications for improving the resources (e.g., more action in the story, increased precision of illustrations related to the characters' position in the wheelchair). CONCLUSION: OTs and PMWUs were satisfied with the resources, perceiving them to be applicable for training wheelchair skills among young children and novice wheelchair users. The resources represent a concrete solution to facilitate the use of the Wheelchair Skills Training Program in pediatric rehabilitation settings. Additional resources are needed to better reach older and more experienced PMWUs (i.e., of intermediate and advanced skill levels).

Short-Term exposure of umbilical cord blood CD34+ cells to human platelet lysate and cytokines enhances engraftment.

BACKGROUND: Intra bone marrow (IBM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic progenitor stem cell (HSPC) transplantation and thus increases the number of HSPC that engraft. Despite physical delivery into the bone marrow cavity, many donor cells are rapidly redistributed by vascular perfusion. Thus, the objective of our study was to evaluate the ability of human platelet lysates (hPL) to improve HSPC retention into the bone marrow and consequently to improve engraftment. STUDY DESIGN AND METHODS: HSPC were isolated from human umbilical cord blood. HSPC were seeded in the wells of a 24-well microplate and exposed to increasing concentrations of hPL with or without cytokines for 24 hours. Following priming, HSPC cells chemotaxis to rhSDF-1 was determined in vitro and engraftment in NSG mice was evaluated. RESULTS: Priming of cord blood CD34+ cells to a combination of hPL and cytokines resulted in a significant increase (up to 3-fold) in the expression of the CD34 antigen on HSPC. This effect was closely correlated to a significantly increased (up to 7-fold) migration toward a rhSDF-1 concentration gradient. In addition, IBM injection of CD34+ cells previously primed with hPL+cytokines into NSG mice showed significantly increased engraftment as measured by human platelet numbers, human CD45 and human CD34+ cells for unprimed and primed cells, respectively. CONCLUSION: The use of hPL + cytokines as a short-term priming treatment for UCB could be an advantageous strategy to improve clinical outcomes following IBM injection.

Peripheral adaptive immunity of the triple transgenic mouse model of Alzheimer's disease.

BACKGROUND: Immunologic abnormalities have been described in peripheral blood and central nervous system of patients suffering from Alzheimer's disease (AD), yet their role in the pathogenesis still remains poorly defined. AIM AND METHODS: We used the triple transgenic mouse model (3xTg-AD) to reproduce Aß (amyloid plaques) and tau (neurofibrillary tangles) neuropathologies. We analyzed important features of the adaptive immune system in serum, primary (bone marrow) as well as secondary (spleen) lymphoid organs of 12-month-old 3xTg-AD mice using flow cytometry and ELISPOT. We further investigated serum cytokines of 9- and 13-month-old 3xTg-AD mice using multiplex ELISA. Results were compared to age-matched non-transgenic controls (NTg). RESULTS: In the bone marrow of 12-month-old 3xTg-AD mice, we detected decreased proportions of short-term reconstituting hematopoietic stem cells (0.58-fold, P = 0.0116), while lymphocyte, granulocyte, and monocyte populations remained unchanged. Our results also point to increased activation of both B and T lymphocytes. Indeed, we report elevated levels of plasma cells in bone marrow (1.3-fold, P = 0.0405) along with a 5.4-fold rise in serum IgG concentration (P < 0.0001) in 3xTg-AD animals. Furthermore, higher levels of interleukin (IL)-2 were detected in serum of 9- and 13-month-old 3xTg-AD mice (P = 0.0018). Along with increased concentrations of IL-17 (P = 0.0115) and granulocyte-macrophage colony-stimulating factor (P = 0.0085), these data support helper T lymphocyte activation with Th17 polarization. CONCLUSION: Collectively, these results suggest that the 3xTg-AD model mimics modifications of the adaptive immunity changes previously observed in human AD patients and underscore the activation of both valuable and harmful pathways of immunity in AD.

Transient warming events occurring after freezing impairs umbilical cord-derived mesenchymal stromal cells functionality.

BACKGROUND: Mesenchymal stromal cells (MSCs) have shown promising results for the treatment of refractory acute graft-versus-host disease. While safety of MSC infusion has been demonstrated, the use of cryopreserved MSCs in clinical trials has raised concerns regarding the retention of their functional activity. This has led to the recommendation by experts in the field to use freshly harvested MSCs, even though this approach is much less practical from a logistic point of view. In the present study, we revisited the impact of cryopreservation on MSC functionality and addressed the possibility that warming events on frozen cells rather than cryopreservation per se could impact MSC functionality. METHODS: Following controlled-rate freezing to -130°C, umbilical cord-derived MSCs were left at room temperature (RT) for 2-10 min or on dry ice for 10 min, before being transferred into liquid nitrogen (LqN2). MSCs of each group were subsequently tested (viability, functionality and cellular damage) and compared with their freshly harvested counterparts. RESULTS: We demonstrated that freshly harvested MSCs as well as cryopreserved MSCs that were left on dry ice following step-down freezing have comparable viability, functionality and integrity. In contrast, cryopreserved MSCs that were left at RT before being transferred into LqN2 were functionally impaired and showed cellular damage upon thawing even though they exhibited high viability. DISCUSSION: Warming events after freezing and not cryopreservation per se significantly impair MSC functionality, indicating that cryopreserved MSCs can be an advantageous alternative to freshly harvested cells for therapeutic purposes.

MiR-146a potentially promotes IVIg-mediated inhibition of TLR4 signaling in LPS-activated human monocytes.

IVIg is used as an immunomodulatory agent in inflammatory disorders such as sepsis. IVIg also affects monocyte differentiation and functions, two processes in which microRNAs play a crucial role. Monocytes detect microorganisms through pathogen recognition receptors (PRRs) such as TLR4. MiR-146a has been shown to supress NF-κB and IRF3 activity, two key components of TLR4 signaling. To evaluate whether miR-146a is involved in the anti-inflammatory effects of IVIg, monocytes were treated with LPS or IVIg alone or, alternately, first activated with LPS followed by washing and addition of IVIg. MiR-146a, IRF3, TNF-α, IL-1ß, IL-6, IL-10, IFN-ß, TGF-ß1 and IL-1Ra expression was analyzed by qPCR, while IRAK1, TRAF6 and IκBα expression was measured by Western blotting. We found that addition of IVIg to LPS-activated monocytes significantly upregulated the expression of miR-146a, which was associated with a significant reduction in the expression of its targets IRF3 and its regulated gene IFN-ß. Furthermore, expression of IRAK1, TRAF6, and consequently NF-κB activation, was also reduced in LPS-activated monocytes following addition of IVIg, whereas TGF-ß1, IL-10 and IL-1Ra were increased. Our results thus suggest that miR-146a is a mediator of IVIg effects in inflammatory disorders, point to an important role for miR-146a in the control of inflammation during sepsis and highlight a new mechanism by which IVIg exerts its anti-inflammatory effects in sepsis.

Perceptions of the first family physicians to adopt advanced access in the province of Quebec, Canada.

In Quebec, several primary care physicians have made the transition to the advanced access model to address the crisis of limited access to primary care. The objectives are to describe the implementation of the advanced access model, as perceived by the first family physicians; to analyze the factors influencing the implementation of its principles; and to document the physicians' perceptions of its effects on their practice, colleagues and patients. Qualitative methods were used to explore, through semi-structured interviews, the experiences of 21 family physicians who had made the transition to advanced access. Of the 21 physicians, 16 succeeded in adopting all five advanced access principles to varying degrees. Core implementation issues revolved around the dynamics of collaboration between physicians, nurses and other colleagues. Secretaries' functions, in particular, had to be expanded. Facilitating factors were mainly related to the physicians' leadership and the professional resources available in the organizations. Impediments related to resource availability and team functioning were also encountered. This is the first exploratory study to examine the factors influencing the adoption of the advanced access model conducted with early-adopter family physicians. The lessons drawn will inform discussions on scaling up to other settings experiencing the same problems. Copyright © 2016 John Wiley & Sons, Ltd.

IVIg protects the 3xTg-AD mouse model of Alzheimer's disease from memory deficit and Aß pathology.

BACKGROUND: Intravenous immunoglobulin (IVIg) is currently in clinical study for Alzheimer's disease (AD). However, preclinical investigations are required to better understand AD-relevant outcomes of IVIg treatment and develop replacement therapies in case of unsustainable supply. METHODS: We investigated the effects of IVIg in the 3xTg-AD mouse model, which reproduces both Aß and tau pathologies. Mice were injected twice weekly with 1.5 g/kg IVIg for 1 or 3 months. RESULTS: IVIg induced a modest but significant improvement in memory in the novel object recognition test and attenuated anxiety-like behavior in 3xTg-AD mice. We observed a correction of immunologic defects present in 3xTg-AD mice (-22% CD4/CD8 blood ratio; -17% IL-5/IL-10 ratio in the cortex) and a modulation of CX3CR1+ cell population (-13% in the bone marrow). IVIg treatment led to limited effects on tau pathology but resulted in a 22% reduction of the soluble Aß42/Aß40 ratio and a 60% decrease in concentrations of 56 kDa Aß oligomers (Aß*56). CONCLUSION: The memory-enhancing effect of IVIg reported here suggests that Aß oligomers, effector T cells and the fractalkine pathway are potential pharmacological targets of IVIg in AD.

Brain bioavailability of human intravenous immunoglobulin and its transport through the murine blood-brain barrier.

Intravenous immunoglobulin (IVIg) is currently evaluated in clinical trials for the treatment of various disorders of the central nervous system. To assess its capacity to reach central therapeutic targets, the brain bioavailability of IVIg must be determined. We thus quantified the passage of IVIg through the blood-brain barrier (BBB) of C57Bl/6 mice using complementary quantitative and qualitative methodologies. As determined by enzyme-linked immunosorbent assay, a small proportion of systemically injected IVIg was detected in the brain of mice (0.009±0.001% of injected dose in the cortex) whereas immunostaining revealed localization mainly within microvessels and less frequently in neurons. Pharmacokinetic analyses evidenced a low elimination rate constant (0.0053 per hour) in the cortex, consistent with accumulation within cerebral tissue. In situ cerebral perfusion experiments revealed that a fraction of IVIg crossed the BBB without causing leakage. A dose-dependent decrease of brain uptake was consistent with a saturable blood-to-brain transport mechanism. Finally, brain uptake of IVIg after a subchronic treatment was similar in the 3xTg-AD mouse model of Alzheimer disease compared with nontransgenic controls. In summary, our results provide evidence of BBB passage and bioavailability of IVIg into the brain in the absence of BBB leakage and in sufficient concentration to interact with the therapeutic targets.

Immunoglobulin G dimers and immune complexes are dispensable for the therapeutic efficacy of intravenous immune globulin in murine immune thrombocytopenia.

BACKGROUND: Several mechanisms have been proposed to explain the therapeutic effects of intravenous immunoglobulin (IVIG) in immune thrombocytopenia (ITP). Noteworthy, a major role has been attributed to immunoglobulin (Ig)G dimers present in IVIG. It has also been suggested that immune complexes formed between IVIG and the patient's proteins after infusion could contribute to the therapeutic effect of IVIG in several autoimmune disorders. We recently observed that in-house preparations of polyclonal human IgG derived from small pools of plasma and devoid of IgG dimers were as efficient as IVIG in a mouse model of thrombocytopenia. In this work, we revisited the role of IgG dimers in the therapeutic effects of IVIG in ITP. STUDY DESIGN AND METHODS: We used the passive mouse model of ITP to determine the therapeutic efficacy of human IgG preparations devoid of IgG dimers and of dimer-enriched and -depleted commercial IVIG. Immune complex formation between IVIG and mouse plasma proteins was evaluated using a combination of chromatography and immunoprecipitation procedures. RESULTS: All preparations tested showed the same efficacy to alleviate ITP, regardless of their dimer contents. Significant amounts of immune complexes formed between IVIG and mouse plasma proteins were detected. However, the amount of immune complexes detected using the in-house preparation of human polyclonal IgG and mouse plasma was significantly lower, although the in-house preparation exhibited the same therapeutic efficacy as commercial IVIG. CONCLUSION: IgG dimers and immune complexes are dispensable to prevent thrombocytopenia in a mouse model of the disease.

Impact of intravenous immunoglobulin on the dopaminergic system and immune response in the acute MPTP mouse model of Parkinson's disease.

Intravenous immunoglobulin (IVIg) is a blood-derived product, used for the treatment of immunodeficiency and autoimmune diseases. Since a range of immunotherapies have recently been proposed as a therapeutic strategy for Parkinson's disease (PD), we investigated the effects of an IVIg treatment in a neurotoxin-induced animal model of PD. Mice received four injections of MPTP (15 mg/kg) at 2-hour intervals followed by a 14-day IVIg treatment, which induced key immune-related changes such as increased regulatory T-cell population and decreased CD4(+)/CD8(+) ratio. The MPTP treatment induced significant 80% and 84% decreases of striatal dopamine concentrations (P < 0.01), as well as 33% and 40% reductions in the number of nigral dopaminergic neurons (P < 0.001) in controls and IVIg-treated mice, respectively. Two-way analyses of variance further revealed lower striatal tyrosine hydroxylase protein levels, striatal homovanillic acid concentrations and nigral dopaminergic neurons (P < 0.05) in IVIg-treated animals. Collectively, our results fail to support a neurorestorative effect of IVIg on the nigrostriatal system in the MPTP-treated mice and even suggest a trend toward a detrimental effect of IVIg on the dopaminergic system. These preclinical data underscore the need to proceed with caution before initiating clinical trials of IVIg in PD patients.

Interaction between intravenous immunoglobulin (IVIg) and the low-density lipoprotein receptor-related protein 1: a role for transcytosis across the blood brain barrier?

Intravenous immunoglobulin (IVIg) is a therapeutic preparation of plasma-derived human IgG and is increasingly used for the treatment of several neurological inflammatory disorders. However, it is not clear whether the IgG molecules contained in IVIg can actually cross the BBB in treated patients. We recently showed that LRP1, an endocytic receptor involved in transcytosis of several proteins across the BBB was able to interact with IVIg. In the present study, we show that LRP1 is involved in IVIg internalization inside living cells. Our data also suggest that following internalization, IVIg is recycled to the cell surface, raising the possibility that LRP1 can mediate IVIg transcytosis across the BBB. Finally, we show that IVIg-LRP1 interaction leads to LRP1 tyrosine phosphorylation.

Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2.

In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.