Novel mechanisms of action contributing to benralizumab's potent anti-eosinophilic activity.

BACKGROUND: Benralizumab is a humanised, anti-interleukin-5 receptor α monoclonal antibody with anti-eosinophilic activity. Lack of fucose (afucosylation) increases its affinity to CD16a and significantly enhances antibody-dependent cell-mediated cytotoxicity by natural killer (NK) cells. Although benralizumab proved clinically efficacious in clinical trials for patients with severe asthma and hypereosinophilic syndrome, in-depth characterisation of its anti-eosinophilic mechanisms of action remains elusive. METHODS: Here, we further investigated the mechanisms involved in benralizumab's anti-eosinophilic activities by employing relevant primary human autologous cell co-cultures and real-time-lapse imaging combined with flow cytometry. RESULTS: In the presence of NK cells, benralizumab induced potent eosinophil apoptosis as demonstrated by the upstream induction of Caspase-3/7 and upregulation of cytochrome c. In addition, we uncovered a previously unrecognised mechanism whereby benralizumab can induce eosinophil phagocytosis/efferocytosis by macrophages, a process called antibody-dependent cellular phagocytosis. Using live cell imaging, we unravelled the stepwise processes leading to eosinophil apoptosis and uptake by activated macrophages. Through careful observations of cellular co-culture assays, we identified a novel role for macrophage-derived tumour necrosis factor (TNF) to further enhance benralizumab-mediated eosinophil apoptosis through activation of TNF receptor 1 on eosinophils. TNF-induced eosinophil apoptosis was associated with cytochrome c upregulation, mitochondrial membrane depolarisation and increased Caspase-3/7 activity. Moreover, activated NK cells were found to amplify this axis through the secretion of interferon-γ, subsequently driving TNF expression by macrophages. CONCLUSIONS: Our data provide deeper insights into the timely appearance of events leading to benralizumab-induced eosinophil apoptosis and suggest that additional mechanisms may contribute to the potent anti-eosinophilic activity of benralizumab in vivo. Importantly, afucosylation of benralizumab strongly enhanced its potency for all mechanisms investigated.

Neurosensory Re-education following Gender-affirming Phalloplasty: A Novel Treatment Protocol.

Sensory nerve transfers are performed as part of phalloplasty surgery. Despite this, sensory re-education to rehabilitate these nerve transfers has not been employed. Additionally, little attention has been paid to the role of the central nervous system in experienced sensation following phalloplasty. The purpose of this article is to report on the development of a targeted rehabilitation protocol to integrate and optimize peripheral and central contributions to sensation following phalloplasty. Methods: This neurosensory re-education protocol was constructed over four phases by a multi-disciplinary team (phalloplasty/peripheral nerve surgeon, reconstructive urologist, pelvic floor physiotherapists, nerve therapist, sex therapist, sexual medicine physician) and individuals with lived phalloplasty experience. The final protocol was approved by all team members and is supported here by qualitative narratives from people with lived phalloplasty experience. Results: The protocol is built to follow each stage of phalloplasty surgery. In each stage, exercises are grouped into three core themes: visualization, tactile stimulation, and sexual/erogenous stimulation. Visualization exercises progress from static to dynamic. Tactile exercises start at simple touch and progress toward targeted sensory stimulation. Sexual stimulation focuses on developing erogenous sensation in the phallus that is separate from erogenous sensation in the natal clitoral tissue. By recommendation of individuals with phalloplasty, the protocol is now integrated into our center's phalloplasty care pathway for all individuals undergoing phalloplasty surgery. Conclusion: We introduce a novel protocol targeting peripheral and central contributions to sensation to provide a tool to help optimize experienced sensation for transmasculine individuals undergoing phalloplasty.

B-cell depletion in vitro and in vivo with an afucosylated anti-CD19 antibody.

The pan B-cell surface antigen CD19 is an attractive target for therapeutic monoclonal antibody (mAb) approaches. We have generated a new afucosylated anti-human (hu)CD19 mAb, MEDI-551, with increased affinity to human FcγRIIIA and mouse FcγRIV and enhanced antibody-dependent cellular cytotoxicity (ADCC). During in vitro ADCC assays with B-cell lines, MEDI-551 is effective at much lower mAb concentrations than the fucosylated parental mAb anti-CD19-2. Furthermore, the afucosylated CD19 mAb MEDI-551 depleted B cells from normal donor peripheral blood mononuclear cell samples in an autologous ADCC assay, as well as blood and tissue B cells in human CD19/CD20 double transgenic (Tg) mice at lower concentrations than that of the positive control mAb rituximab. In huCD19/CD20 Tg mice, both macrophage-mediated phagocytosis and complement-dependent cytotoxicity contribute to depletion with rituximab; MEDI-551 did not require complement for maximal B-cell depletion. Furthermore, extended B-cell depletion from the blood and spleen was achieved with MEDI-551, which is probably explained by bone marrow B-cell depletion in huCD19/CD20 Tg mice relative to the control mAb rituximab. In summary, MEDI-551 has potent B-cell-depleting activity in vitro and in vivo and may be a promising new approach for the treatment of B-cell malignancies and autoimmune diseases.

Implementation of a Pelvic Floor Physical Therapy Program for Transgender Women Undergoing Gender-Affirming Vaginoplasty.

OBJECTIVE: To describe the incidence of pelvic floor dysfunction in transgender women undergoing gender-affirming vaginoplasty and outcomes in a program providing pelvic floor physical therapy (PT). METHODS: We conducted a retrospective, single-institution study on vaginoplasty patients between May 1, 2016, and February 28, 2018; all were referred for pelvic floor PT. We reviewed medical records for baseline demographics, medical comorbidities, prior surgeries, insurance data, attendance at pelvic floor PT, and dilation success at 3 and 12 months. RESULTS: Seventy-two of 77 patients (94%) attended pelvic floor PT at least once. Preoperative pelvic floor PT identified a high incidence of potential problems: 42% had pelvic floor dysfunction, 37% had bowel dysfunction. Of those patients found to have dysfunction preoperatively, the rate of resolution by the first postoperative visit of pelvic floor and bowel dysfunction were 69% and 73%, respectively. There were significantly lower rates of pelvic floor dysfunction postoperatively for those patients who attended pelvic floor PT both preoperatively and postoperatively compared with only postoperatively (28% vs 86%, P=.006). Patients reporting a history of abuse had a significantly higher rate of preoperative pelvic floor muscle dysfunction (91% vs 31%, P<.001). Successful dilation at 3 months in all patients was 89%. CONCLUSION: Pelvic floor physical therapists identify and help patients resolve pelvic floor-related problems before and after surgery. We find strong support for pelvic floor PT for patients undergoing gender-affirming vaginoplasty.

Hyperexcitability of distal dendrites in hippocampal pyramidal cells after chronic partial deafferentation.

Traumatic injury to the CNS results in chronic partial deafferentation of subsets of surviving neurons. Such injuries are often followed by a delayed but long-lasting period of aberrant hyperexcitability. The cellular mechanisms underlying this delayed hyperexcitability are poorly understood. We developed an in vitro model of deafferentation and reactive hyperexcitability using organotypic hippocampal slice cultures to study the underlying cellular mechanisms. One week after transection of the Schaffer collateral and temporoammonic afferents to CA1 neurons, brief tetanic stimulation of the residual excitatory synapses produced abnormally prolonged depolarizations, compared with responses in normally innervated neurons. Responses to weak stimulation, in contrast, were unaffected after deafferentation. Direct stimulation of distal apical dendrites using focal photolysis of caged glutamate triggered abnormally prolonged plateau potentials in the deafferented neurons when strong stimulation was given, but responses to weak stimulation were not different from controls. An identical phenotype was produced by chronic "chemical deafferentation" with glutamate receptor antagonists. Responses to strong synaptic and photolytic stimulation were selectively prolonged by small-conductance (SK-type) calcium-activated potassium channel blockers in normally innervated cells but not after deafferentation. No significant changes in SK2 mRNA or protein levels, GABAergic inhibition, glutamate receptor function, input resistance, or action potential parameters were observed after chronic deafferentation. We suggest that a posttranslational downregulation of SK channel function in thin distal dendrites is a significant contributor to deafferentation-induced reactive hyperexcitability.

Effects of glycyrrhetinic acid and liquorice extract on cell proliferation and prostate-specific antigen secretion in LNCaP prostate cancer cells.

Glycyrrhetinic acid (GA) is the active metabolite of glycyrrhizic acid, one of the components of liquorice extract. It has been shown to possess anti-inflammatory activity and to inhibit hepatic tumour growth. In this preliminary study, we have shown that GA could significantly reduce the rate of proliferation of LNCaP androgen dependent prostate cancer cells, whereas it had no effect on proliferation of PC3 and DU145 androgen-independent prostate cancer cells. Additionally, GA could significantly reduce the production of prostate-specific antigen by LNCaP cells maintained in-vitro. This study provides a sound platform for further investigation.

A comparison of smoking cessation treatments for persons with schizophrenia and other serious mental illnesses.

Adults with any DSM-IV diagnosed mental illness smoke nearly half of the cigarettes consumed in the U.S. (Lasser et al. 2000). This study compared two smoking cessation interventions for persons with schizophrenia or other serious mental illness because national data suggests that: (1) they smoke at two to three times the rate of the general population; (2) cessation interventions for this population are understudied; (3) most cessation studies exclude persons with serious mental illness; and (4) cessation results in public health care savings and disposable income savings for clients. This study included a large number of persons with serious mental illness (N=181) who were randomly assigned to one of three groups: contingent reinforcement (CR), CR plus nicotine patch (21 mg, CR+NRT) for 16 weeks, and a minimal intervention, self-quit control group. These participants were followed for 36 weeks. CR was accomplished with escalating financial compensation for achieving and maintaining abstinence as verified by expired carbon monoxide (CO). Quit rates, as measured by expired CO, were higher and discordant with saliva cotinine quit rates. Cotinine showed lower quit rates and small differences between intervention and control participants at weeks 20 and 36. There was, however, evidence of reduced smoking and importantly, no evidence of psychiatric exacerbation.

Moderate magnesium deprivation results in calcium retention and altered potassium and phosphorus excretion by postmenopausal women.

Enzyme and or hormone actions have not been shown to be consistently changed by consuming a low-magnesium diet similar to one that may occur in the general population. Thus, a human metabolic study was performed to determine whether deficient intakes of magnesium similar to those that occur naturally have pathophysiological effects through altering calcium retention and the metabolism of other minerals (sodium, potassium, phosphorus) involved in cellular ionic balance. Fifteen postmenopausal Caucasian women were recruited by advertisement throughout the United States. Eleven women (ages 49 to 71 years) completed the study as designed. The women resided in a metabolic research unit and consumed a basal Western-type diet that resulted in a mean intake of 4.40 mmol (107 mg) magnesium/d. The women were fed the basal diet supplemented with 9.05 mmol (220 mg) magnesium/d for 18 d (equilibration) before being assigned to one of two groups in an experiment with a double blind, crossover design. One group was fed the basal diet and supplemented with a lactose placebo while the other group continued consuming the basal diet supplemented with 9.05 mmol magnesium/d for 72 d, then each group switched to the other's diet, which they consumed for 72 d. Magnesium was supplemented as magnesium gluconate. Magnesium deprivation resulted in a non-positive magnesium balance (-0.21 mmol or -5 mg/d) that was highly positive during magnesium supplementation (+2.22 mmol or +54 mg/d). Magnesium deprivation decreased red blood cell membrane magnesium (2.5 versus 2.7 nmol or 0.061 versus 0.065 microg/mg protein; p < or = 0.05). Magnesium deprivation increased calcium balance (+0.82 mmol or +35 mg/d versus -0.02 or -1 mg/d; p < or = 0.009); decreased the fecal excretion of phosphorus (28.9% versus 32.3% of intake; p < or =0.0001); increased the urinary excretion of phosphorus (73.4% versus 71.0%; p < 0.003); and decreased the urinary excretion of potassium (40.4 mmol or 1.58 g/d versus 41.9 mmol or 1.64 g/d; p < 0.04). Non-positive magnesium balance and decreased red blood cell membrane magnesium concentration apparently are indicators of magnesium deprivation. Moderate magnesium deprivation achieved through diet alone results in increased calcium retention. Magnesium deprivation also alters phosphorus and potassium excretion. The changes indicate that an intake of 4.40 mmol (107 mg) magnesium/d is inadequate for postmenopausal women because of changes in cellular ionic balance that may lead to pathophysiological conditions.

CD47 limits antibody dependent phagocytosis against non-malignant B cells.

Recent studies have demonstrated the importance of CD47 in protecting malignant B cells from antibody dependent cellular phagocytosis (ADCP). Combined treatment of anti-CD47 and -CD20 antibodies synergistically augment elimination of tumor B cells in xenograft mouse models. This has led to the development of novel reagents that can potentially enhance killing of malignant B cells in patients. B cell depleting therapy is also a promising treatment for autoimmune patients. In the current study, we aimed to investigate whether or not CD47 protects non-malignant B cells from ADCP. We show that CD47 is expressed on all B cells in mice, with the highest level on plasma cells in bone marrow and spleen. Although its expression is dispensable for B cell development in mice, CD47 on B cells limits antibody mediated phagocytosis. B cell depletion following in vivo anti-CD19 treatment is more efficient in CD47-/- mice than in wild type mice. In vitro, both naïve and activated B cells from CD47-/- mice are more sensitive to ADCP than wild type B cells. Lastly, we show in an ADCP assay that blocking CD47 can enhance anti-CD19 antibody mediated phagocytosis of wild type B cells. These results suggest that in addition to its already demonstrated benefit in cancer, targeting CD47 may be used as an adjunct in combination with B cell depletion antibodies for treatment of autoimmune diseases.

Comparing the CAAPE, a new assessment tool for co-occurring disorders, with the SCID.

Co-occurring mental health and substance use disorders (COD) are common and frequently under-detected, which may lead to less than optimal treatment for persons in psychosocial rehabilitation settings. A new, relatively brief instrument, the Comprehensive Addictions and Psychological Evaluation (CAAPE) was compared with the Structured Clinical Interview for DSM-IV (SCID). The CAAPE required less time to administer than the SCID, efficiently explored DSM substance use disorder criteria and served as a screen for psychiatric disorders. The CAAPE promises to be a useful screening and diagnostic instrument for persons with co-occurring disorders, especially suited for use in psychosocial rehabilitation.

Inebilizumab, a B Cell-Depleting Anti-CD19 Antibody for the Treatment of Autoimmune Neurological Diseases: Insights from Preclinical Studies.

Exaggerated or inappropriate responses by B cells are an important feature in many types of autoimmune neurological diseases. The recent success of B-cell depletion in the treatment of multiple sclerosis (MS) has stimulated the development of novel B-cell-targeting therapies with the potential for improved efficacy. CD19 has emerged as a promising target for the depletion of B cells as well as CD19-positive plasmablasts and plasma cells. Inebilizumab (MEDI-551), an anti-CD19 antibody with enhanced antibody-dependent cell-mediated cytotoxicity against B cells, is currently being evaluated in MS and neuromyelitis optica. This review discusses the role of B cells in autoimmune neurological disorders, summarizes the development of inebilizumab, and analyzes the recent results for inebilizumab treatment in an autoimmune encephalitis mouse model. The novel insights obtained from these preclinical studies can potentially guide future investigation of inebilizumab in patients.

Pharmacological profile of MEDI-551, a novel anti-CD19 antibody, in human CD19 transgenic mice.

B cell depletion therapy is beneficial for patients with B cell malignancies and autoimmune diseases. CD19, a transmembrane protein, is expressed on a vast majority of normal and neoplastic B cells, making it a suitable target for monoclonal antibody (MAb) mediated immunotherapy. We have developed MEDI-551, an affinity optimized and afucosylated IgG1 MAb targeting human CD19 for B cell depletion. MEDI-551 is currently under investigation in multiple clinical trials. Because MEDI-551 does not cross react with rodent and non-human primate CD19, the pharmacological characteristics of the MAb were evaluated in human CD19 transgenic mice (hCD19 Tg). Here we show that MEDI-551 potently depletes tissue and circulating B cells in hCD19 Tg mice and is more efficacious than the anti-CD19 MAb with intact fucose. The length of B cell depletion depends on MEDI-551 dose; and, B cell recovery in the circulation follows stepwise phenotypic maturation. Furthermore, intravenous (IV) and subcutaneous (SC) administration of MEDI-551 results in comparable efficacy. Lastly, the combination of MEDI-551 with the anti-CD20 MAb, rituximab, further prolongs the duration of B cell depletion. In summary, the pharmacological profile of MEDI-551 presented in hCD19 Tg mice supports further testing of MEDI-551 in clinical trials involving B cell malignancies and autoimmune diseases.

MEDI-551 Treatment Effectively Depletes B Cells and Reduces Serum Titers of Autoantibodies in Mice Transgenic for Sle1 and Human CD19.

OBJECTIVE: To evaluate treatment with MEDI-551, a humanized anti-human CD19 monoclonal antibody, in a model of autoimmunity involving mice transgenic (Tg) for Sle1 and human CD19 (hCD19). METHODS: Sle1.hCD19-Tg mice were given either a single intravenous dose of MEDI-551 or repeated doses of MEDI-551 biweekly for up to 12 weeks. The numbers of B cells in the blood, spleen, and bone marrow were determined by flow cytometry assay. In the spleen and bone marrow, the number of IgM- and IgG-specific antibody-secreting cells (ASCs) and the number of ASCs specific for anti-double-stranded DNA (anti-dsDNA) were determined by enzyme-linked immunospot assay. Serum autoantibody and total immunoglobulin levels were determined by enzyme-linked immunosorbent assay, and levels of inflammatory proteins were tested using a multianalyte profiling platform. RESULTS: MEDI-551 treatment of Sle1.hCD19-Tg mice resulted in effective and sustained B cell depletion throughout the duration of the experiment. The frequency of IgM and IgG ASCs in the spleen was reduced by ≥90%, whereas in the bone marrow, the total ASC frequency was not changed. Levels of autoantibodies specific for dsDNA as well as antihistone and antinuclear antibodies were each reduced by 40-80%, but total serum immunoglobulin levels were largely unchanged at the end of 12 weeks of treatment. CONCLUSION: These findings highlight the ability of MEDI-551 to deplete B cells and ASCs in autoimmune Sle1.hCD19-Tg mice. MEDI-551 treatment resulted in a robust reduction of autoantibodies but had minimal effect on total serum immunoglobulins. Thus, the novel ability of MEDI-551 to remove a broad range of B cells as well as to lower most disease-driving autoantibodies in an autoimmune disease mouse model warrants continued research. Several clinical studies to explore the safety and activity of MEDI-551 in autoantibody-associated autoimmune diseases are ongoing.

Methods to track leukocyte and erythrocyte transit through the bronchial vasculature in sheep.

Study of the underlying mechanisms of leukocyte recruitment in the airway circulation is a crucial aspect of understanding the pathology of inflammatory airways disease. However, few in vivo studies have focused on leukocyte kinetics through the systemic airway vasculature. Because the bronchial vasculature of the sheep shows anatomical similarity to that of the human as well as being easily accessible for perfusion, we developed methods to study leukocyte transit through the sheep bronchial circulation. Leukocytes were isolated from whole blood after hypotonic lysis of red cells and labeled with 5-(and 6) carboxyfluorescein succinimidylester (CFSE; 0.1 microM), a green fluorescent dye. Red blood cells were labeled using PKH26-GL Red Fluorescent Cell Linker and served as a marker for blood flow and volume. Labeled leukocytes were tested for activation during the labeling process by monitoring surface levels of leukocyte adhesion molecules CD11b and L-selectin. When activated with phorbol myristate acetate (10 ng/ml), sheep leukocytes showed a marked increase in CD11b expression and a decrease in L-selectin. However, sheep leukocytes labeled with CFSE showed no significant increase of CD11b or shedding of L-selectin, suggesting that the labeling process did not significantly activate the adhesion properties of the cells. Aliquots containing both labeled erythrocytes and leukocytes were infused into the bronchial vasculature of the sheep through the cannulated bronchial artery at normal bronchial flow (0.6 ml/kg). Serial blood samples were withdrawn from the outflow of the bronchial vasculature (the left atrium) and analyzed using conventional flow cytometry. Retention of leukocytes and transit relative to red cell transit could then be evaluated with this technique. In addition, since cells are stably labeled, airway tissue samples can be removed and analyzed histologically to determine sites of extravasation. These methods offer an approach for examining leukocyte kinetics in situ in the airways of a relevant animal model.

Human mast cells induce caspase-independent DNA fragmentation in leukemic T cells.

Mast cells are known to play an important role in inflammation and host defense. Recent evidence suggests that mast cells may also participate in immune surveillance against cancer cells. In this study, we demonstrate that human mast cells are able to trigger apoptosis in Jurkat T leukemia cells. Fragmentation of Jurkat cell DNA was detectable by JAM assay within 4 h of exposure to HMC-1 mast cells or cultured human cord blood-derived mast cells. HMC-1 mast cells that were fixed with paraformaldehyde retained the ability to induce apoptosis in Jurkat cells while cell-free conditioned supernatants from HMC-1 cell cultures failed to elicit DNA fragmentation, suggesting that mast cells mediate anti-tumor activity via a cell-surface molecule. Surprisingly, the apoptosis-inducing activity of HMC-1 mast cells was not mediated by known tumor necrosis factor (TNF) superfamily members (TNF-alpha, Fas ligand, TRAIL, TWEAK), nor did the fragmentation of Jurkat cell DNA by mast cells require the activity of caspase-3, -8, or -9. Collectively, these data indicate that mast cells trigger caspase-independent apoptosis in leukemic cells via a novel cell-surface molecule, and are consistent with a role for mast cells as anti-tumor effector cells.

Germinal center B cell depletion diminishes CD4+ follicular T helper cells in autoimmune mice.

BACKGROUND: Continuous support from follicular CD4(+) T helper (Tfh) cells drives germinal center (GC) responses, which last for several weeks to produce high affinity memory B cells and plasma cells. In autoimmune Sle1 and NZB/W F1 mice, elevated numbers of Tfh cells persist, promoting the expansion of self-reactive B cells. Expansion of circulating Tfh like cells have also been described in several autoimmune diseases. Although, the signals required for Tfh differentiation have now been well described, the mechanisms that sustain the maintenance of fully differentiated Tfh are less understood. Recent data demonstrate a role for GC B cells for Tfh maintenance after protein immunization. METHODS AND FINDING: Given the pathogenic role Tfh play in autoimmune disease, we explored whether B cells are required for maintenance of autoreactive Tfh. Our data suggest that the number of mature autoreactive Tfh cells is controlled by GC B cells. Depletion of B cells in Sle1 autoimmune mice leads to a dramatic reduction in Tfh cells. In NZB/W F1 autoimmune mice, similar to the SRBC immunization model, GC B cells support the maintenance of mature Tfh, which is dependent mainly on ICOS. The CD28-associated pathway is dispensable for Tfh maintenance in SRBC immunized mice, but is required in the spontaneous NZB/W F1 model. CONCLUSION: These data suggest that mature Tfh cells require signals from GC B cells to sustain their optimal numbers and function in both autoimmune and immunization models. Thus, immunotherapies targeting B cells in autoimmune disease may affect pathogenic Tfh cells.

Antibody-dependent cell-mediated cytotoxicity effector-enhanced EphA2 agonist monoclonal antibody demonstrates potent activity against human tumors.

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated approximately 80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells.

Dietary magnesium deficiency induces heart rhythm changes, impairs glucose tolerance, and decreases serum cholesterol in post menopausal women.

OBJECTIVE: To determine whether or not dietary magnesium restriction to about 33% of the Recommended Dietary Allowance (RDA) causes changes in glucose, cholesterol and electrolyte metabolism that could lead to pathologic consequences. DESIGN: The length of the experiment was 136 days. Subjects were fed a basal Western-type diet that provided 4.16 mmol (101 mg) magnesium per 8.4 MJ (2000 kcal) for 78 days then replenished with magnesium by supplementing the diet with 200 mg magnesium as the gluconate per day for 58 days. If a subject exhibited adverse heart rhythm changes before 78 days of depletion were completed, she entered the repletion period early. SETTING: The metabolic research unit of the Grand Forks Human Nutrition Research Center. SUBJECTS: A total of 14 post menopausal women were recruited by advertisement throughout the United States. Thirteen women (ages 47 to 75 years) completed the study. RESULTS: During magnesium depletion, heart rhythm changes appeared in 5 women and resulted in 4 prematurely entering the magnesium repletion period (42 to 64 days of depletion instead of 78). Three women exhibited atrial fibrillation and flutter that responded quickly to magnesium supplementation. Magnesium deprivation resulted in a non-positive magnesium balance that became highly positive with magnesium repletion. Magnesium deprivation decreased red blood cell membrane magnesium, serum total cholesterol and erythrocyte superoxide dismutase concentrations, increased the urinary excretion of sodium and potassium, and increased serum glucose concentration. CONCLUSIONS: Magnesium balance may be a suitable indicator of magnesium depletion under experimental conditions. Magnesium deficiency resulting from feeding a diet that would not be considered having an atypical menu induces heart arrhythmias, impairs glucose homeostasis, and alters cholesterol and oxidative metabolism in post menopausal women. A dietary intake of about 4.12 mmol (100 mg) Mg/8.4 MJ is inadequate for healthy adults and may result in compromised cardiovascular health and glycemic control in post menopausal women.

Injury-induced axonal sprouting in the hippocampus is initiated by activation of trkB receptors.

Penetrating head injuries are often accompanied by the delayed development of post-traumatic epilepsy. Schaffer collateral transection leads to axonal sprouting and hyperexcitability in area CA3 of hippocampal slice cultures. We used this model to test the hypothesis that the injury-induced axonal sprouting results from increased neurotrophin signaling via trkB receptors near the lesion. Using rats and mice, we established that sprouting CA3 pyramidal cell axons are labeled with an antibody to the growth-associated protein GAP-43. We observed two- to threefold increases in the level of brain-derived neurotrophic factor and trkB protein in area CA3 by 24-48 h after Schaffer collateral transection, preceding the onset of axonal sprouting. Finally, we demonstrated that injury-induced axonal sprouting of GAP-43-immunoreactive axons is impaired in hippocampal slice cultures from mice expressing low levels of trkB receptors. We conclude that injury-induced axonal sprouting is initiated by brain-derived neurotrophic factor-trkB signaling and suggest that this process may be critical for the genesis of post-traumatic epilepsy.