Human-relevant near-organ neuromodulation of the immune system via the splenic nerve

Neuromodulation of immune function by stimulating the autonomic connections to the spleen has been demonstrated in rodent models. Consequently, neuroimmune modulation has been proposed as a new therapeutic strategy for the treatment of inflammatory conditions. However, demonstration of the translation of these immunomodulatory mechanisms in anatomically and physiologically relevant models is still lacking. Additionally, translational models are required to identify stimulation parameters that can be transferred to clinical applications of bioelectronic medicines. Here, we performed neuroanatomical and functional comparison of the mouse, rat, pig, and human splenic nerve using in vivo and ex vivo preparations. The pig was identified as a more suitable model of the human splenic innervation. Using functional electrophysiology, we developed a clinically relevant marker of splenic nerve engagement through stimulation-dependent reversible reduction in local blood flow. Translation of immunomodulatory mechanisms were then assessed using pig splenocytes and two models of acute inflammation in anesthetized pigs. The pig splenic nerve was shown to locally release noradrenaline upon stimulation, which was able to modulate cytokine production by pig splenocytes. Splenic nerve stimulation was found to promote cardiovascular protection as well as cytokine modulation in a high- and a low-dose lipopolysaccharide model, respectively. Importantly, splenic nerve–induced cytokine modulation was reproduced by stimulating the efferent trunk of the cervical vagus nerve. This work demonstrates that immune responses can be modulated by stimulation of spleen-targeted autonomic nerves in translational species and identifies splenic nerve stimulation parameters and biomarkers that are directly applicable to humans due to anatomical and electrophysiological similarities.

The inflammatory status of the body is monitored and regulated through the neuroimmune axis, connecting the brain to the immune system via both humoral and neural pathways (1–3). In particular, the inflammatory reflex (3) controls systemic immune responses; detection of inflammatory stimuli in the periphery is communicated to the brain that activates outflow of neural signals to promote peripheral immune responses proportional to the threat. Studies in rodent models have identified the cholinergic anti-inflammatory pathway (CAIP) as the brain’s efferent response to infection and inflammation through peripheral neurotransmitters released in lymphoid organs, mainly the spleen (4, 5). Within this pathway, the peripheral connection between the vagus nerve (VN), the splenic nerve (SpN), and its terminal release of noradrenaline (NA) into the spleen have been identified as crucial components of this neural circuit (6–8) (SI Appendix, Fig. S1A).Importantly, the CAIP can be harnessed to promote immune control. Activation of the cervical VN by electrical stimulation (vagus nerve stimulation—VNS; SI Appendix, Fig. S1A) has been shown to be effective in reducing lipopolysaccharide (LPS)-induced levels of tumor necrosis factor alpha (TNF-α) (4, 6, 7) and in preclinical rodent models of chronic inflammatory diseases (9, 10). Murine models have generally been used to demonstrate biological proof of concepts of novel neuromodulation therapies in this and other contexts. However, the development of clinical bioelectronic medicines requires the accurate estimation and validation of stimulation parameters in a histologically, surgically, and anatomically relevant model to define device and therapy requirements. The translation of stimulation parameters from rodent to human is hampered by anatomical (e.g., size of nerves), histological (e.g., number of axons, connective tissue thickness, proportion of adipose tissue), and physiological (e.g., immunological) differences. Therefore, it is suggested that the use of large animal models, human tissues, and in silico modeling are more appropriate for the optimization and scaling of human-relevant parameters (11, 12).Although early clinical feasibility studies have provided preliminary evidence of immunomodulatory effects of VNS in patients (13, 14), clear demonstration of the translation of the splenic anti-inflammatory pathway in clinically relevant species is currently lacking in the literature. The VN has a functionally and anatomically complex composition. In animals and humans, the VN contains both afferent and efferent axons of varying size (large, medium, and small) and degree of myelination (heavily myelinated, lightly myelinated, and unmyelinated axons) innervating multiple organs and muscles (15). As a consequence, currently used VNS results in activation of off-target circuits (SI Appendix, Fig. S1A) that can cause dysphonia, coughing, hoarseness, pain, and dyspnea (16–18); in some patients, these can be managed and can also improve over time (18). Further, it remains unclear which axons (efferent versus afferent, myelinated versus unmyelinated) within the VN relay immunomodulatory signals to peripheral organs (19, 20). As a result, it is difficult to optimize the stimulation parameters necessary to activate axons within the VN which carry signals to the spleen. Typically, clinical parameters are selected based on the individual patient’s tolerance of off-target effects (13, 21) without direct evidence of activation of the anti-inflammatory pathway because of a lack of an organ-specific biomarker. Since the SpN directly transmits neural signals to the spleen and is the fundamental nodal circuit in mediating the anti-inflammatory response (22), SpN stimulation (SpNS) may represent an alternative modality providing the opportunity for near-organ modulation of the immune system (SI Appendix, Fig. S1 B and C). Proof of concept experiments in rodents have shown that immune responses can indeed be modulated by stimulation of the SpN with comparable cytokine suppressive effects to VNS (7, 8, 23).Here, we anatomically, histologically, and functionally compared the mouse, rat, pig, and human SpN, demonstrating the superiority of the pig as a translational model of the human SpN. We then performed functional in vivo pig electrophysiological studies to identify organ-specific physiological biomarkers that can be used to assess nerve engagement and to refine stimulation parameters. Finally, we assessed the large animal translation of the spleen-dependent anti-inflammatory pathway in the pig using in vitro splenocyte preparations together with two in vivo models of acute inflammation.     

Moderate-Vigorous Physical Activity in Older People in Northern Ireland: Levels, Demographic Patterns and Types of Moderate-Vigorous Physical Activity Undertaken

Work on the health benefits of physical activity currently recommends participation in 2.5 h of moderate-vigorous physical activity/week, and advocates consideration of the physical activity gained from activities of daily living in this total. Using the inclusion of activities of daily living, this analysis aimed to investigate the physical activity undertaken by a representative sample of older people living in Northern Ireland (NI). Using a telephone questionnaire, 426 individuals (representative of the NI older population) reported participating in a mean 4.1?±?6.3 h of moderate–vigorous physical activity/week, but 225 (53 %) of these individuals reported participation in less than 2.5 h of moderate-vigorous activity/week, and 126 of these individuals reported none. Regression analyses revealed greater participation by males, younger individuals and those living in less deprived areas (smallest B?=??0.11, p?=?0.03). Males were also found to participate in more Do-It-Yourself, cycling, heavy gardening and exercise/sport in summer and less heavy housework, than females (smallest B?=??0.03, p?=?0.05), but no differences were found dependent on age or deprivation of residential area. These findings suggest a need to increase participation in moderate-vigorous physical activity in this population, with specific emphasis on females, older individuals and those living in more deprived areas. Analysis of the types of moderate-vigorous physical activity undertaken suggests that females may benefit particularly from increased opportunity or promotion of exercise/sport as a leisure activity.     

Comparison of insulin aspart with buffered regular insulin and insulin lispro in continuous subcutaneous insulin infusion: a randomized study in type 1 diabetes

OBJECTIVE: To compare the safety and efficacy of insulin aspart (IAsp), buffered regular insulin (BR), and insulin lispro administered by continuous subcutaneous insulin infusion (CSII) in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: After completing a 4-week run-in period with BR, 146 adult patients with type 1 diabetes (with pretrial CSII experience) were randomly assigned (2:2:1) to CSII treatment with IAsp, BR, or lispro for 16 weeks in a multicenter, open-label, randomized, parallel-group study. Bolus insulin doses were administered 30 min before meals (BR) or immediately before meals (IAsp or lispro). RESULTS: Treatment groups had similar baseline HbA(1c) (7.3% +/- 0.7 for IAsp, 7.5% +/- 0.8 for BR, and 7.3% +/- 0.7 for lispro). After 16 weeks of treatment, HbA1c values were relatively unchanged from baseline, and the mean changes in baseline HbA1c values were not significantly different between the three groups (0.00 +/- 0.51, 0.15 +/- 0.63, and 0.18 +/- 0.84 for the IAsp, BR, and lispro groups, respectively). The rates of hypoglycemic episodes (blood glucose <50 mg/dl) per patient per month were similar (3.7, 4.8, and 4.4 for the IAsp, BR, and lispro groups, respectively). Clogs/blockages in pumps or infusion sets were infrequent; most subjects (76, 83, and 75% in the IAsp, BR, and lispro groups, respectively) had < or = 1 clog or blockage per 4 weeks during the trial. CONCLUSIONS: Insulin aspart in CSII was as efficacious and well tolerated as BR and lispro and is a suitable insulin for continuous subcutaneous insulin infusion using external pumps.     

Body weight changes with beta-blocker use: results from GEMINI

Purpose

Patients with type 2 diabetes are commonly overweight, which can contribute to poor cardiovascular outcomes. β-blockers may promote weight gain, or hamper weight loss, and are a concern in high-risk patients. The current analysis of the Glycemic Effect in Diabetes Mellitus: Carvedilol-Metoprolol Comparison in Hypertensives (GEMINI) trial evaluates the effects of carvedilol and metoprolol tartrate on weight gain in patients with type 2 diabetes and hypertension.

Methods

This prespecified secondary analysis of the GEMINI study (n=1106) evaluated change in body weight after 5 months.

Results

Mean (±SE) baseline weights were 97.5 (±20.1) kg for carvedilol and 96.6 (±20.1) kg for metoprolol tartrate. Treatment difference (c vs m) in mean (±SE) weight change from baseline was −1.02 (±0.21) kg (95% confidence interval [CI], −1.43 to −0.60; P <.001). Patients taking metoprolol had a significant mean (±SE) weight gain of 1.19 (±0.16) kg (P <.001); patients taking carvedilol did not (0.17 [±0.19] kg; P =.36). Metoprolol tartrate-treated patients with body mass index (BMI) >30 kg/m² had a statistically significant greater weight gain than comparable carvedilol-treated patients. Treatment differences (c vs m) in the obese (BMI >30 kg/m²) and morbidly obese groups (BMI >40 kg/m²) were −0.90 kg (95% CI, −1.5 to −0.3; P =.002) and −1.84 kg (95% CI, −2.9 to −0.8; P =.001), respectively. Pairwise correlation analyses revealed no significant associations between weight change and change in HbA_1c, HOMA-IR, or blood pressure.

Conclusions

Metoprolol tartrate was associated with increased weight gain compared to carvedilol; weight gain was most pronounced in subjects with hypertension and diabetes who were not taking insulin therapy.     

Prognostic efficacy of cardiac biomarkers for mortality in dialysis patients

Background: The high prevalence of cardiovascular mortality in the end‐stage renal disease population is well established. The aim of this current study was to document the relative prognostic significance of established cardiac biomarkers troponin T (TnT), troponin I (TnI), B‐type natriuretic peptide (BNP) and N‐terminal pro‐BNP (NT‐pro‐BNP) in this population. Methods: A prospective cohort study of dialysis patients undertaken in a single tertiary centre in Australia. Relevant clinical and biochemical information was collected at entry and all patients followed up prospectively without any loss to follow up. End‐point of interest was all‐cause mortality. Statistical analysis using Cox proportional hazards was used to study relationship between competing covariates and outcome. A total of 143 patients with a mean age of 59.67 ± 15.49 years was followed up for a median duration of 30 months. Of these patients, 89.3% were white Australians of European ancestry. Twenty‐seven per cent had an established diagnosis of diabetes mellitus. The mean concentrations (±SD) of TnT, TnI, BNP and N‐terminal peptide pro‐BNP (NT‐pro‐BNP) were 0.08 ± 0.04 µg/L, 0.09 ± 0.2 µg/L, 270 ± 117 ng/L and 1434 ± 591 ng/L respectively. Results: Twenty‐eight subjects died during the period of follow up. By univariate analysis, all cardiac markers (TnT, TnI, BNP, NT‐pro‐BNP and C‐reactive protein) were significantly associated with an increase in mortality. On Cox proportionate hazards analysis, only albumin and NT‐pro‐BNP showed a significant association with mortality, with hazard ratios of 0.834, 95% confidence interval (CI) 0.779–0.893, P < 0.001, and 1.585, 95%CI 1.160–20165, P = 0.004 respectively. Conclusion: In patients with end‐stage renal failure on dialysis NT‐pro‐BNP provides greater prognostic information compared with TnT and TnI.     

Anemia and the role of erythropoietin in diabetes

Anemia is more common in patients with diabetes than without diabetes, and the problem is magnified in patients with renal impairment. Diabetic patients with anemia may be at increased risk of adverse outcomes from diabetic retinopathy, nephropathy, neuropathy, and cardiovascular disease. The etiology of anemia in diabetes is multifactorial and includes inflammation, nutritional deficiencies, concomitant autoimmune diseases, drugs, and hormonal changes in addition to kidney disease. Anemia that is associated with erythropoietin deficiency may have prognostic significance for persons with nephropathy or heart failure. In early diabetic nephropathy, damage to the peritubular fibroblasts can occur and lead to erythropoietin deficiency and anemia prior to the loss of filtration. Correction of the anemia not only leads to less fatigue, greater exercise tolerance, and an improved quality of life but also to a reduction in mortality and hospital admissions for congestive heart failure (CHF). Data are accumulating that suggest that treatment of anemia will slow the progression of microvascular and macrovascular complications, including postural hypotension from autonomic neuropathy, retinopathy, and loss of renal function from diabetic nephropathy. Promptly diagnosing and treating anemia in patients with diabetes may result in an improved quality of life and decreased morbidity and mortality.     

Extent of nucleus pulposus migration in the annulus of porcine intervertebral discs exposed to cyclic flexion only versus cyclic flexion and extension

Background

Repeated flexion of an intervertebral disc has been identified as a mechanism to produce posterior herniations. Repeated extension under certain conditions has also been shown to cause the nucleus of partially herniated discs to reverse and migrate anteriorly. While research shows that the nucleus pulposus migrates anteriorly in extension and infiltrates the annulus posteriorly in flexion, it is not known if a cycle of flexion followed by a cycle of extension produces more or less annular damage compared to pure flexion alone.

Methods

Two groups of porcine spinal motion segments were exposed to either repeated flexion with extension or just repeated flexion. Digitized photographs of dissected specimens enhanced with a radio-opaque blue dye enabled the quantification of the area of annulus infiltrated with nucleus pulposus.

Findings

Specimens exposed to both flexion and extension showed significantly more annular damage and axial creep compared to those exposed to flexion alone.

Interpretation

It would appear that while flexion alone can still cause nucleus pulposus to track through the annulus of an intervertebral disc, the effects are compounded when it is followed by a subsequent cycle of extension. Thus, movements which require both repetitive flexion and extension, have the potential to produce more annular damage than those which require merely flexion.     

High Prevalence and Diversity of Kidney Dysfunction in Patients With Type 2 Diabetes Mellitus and Coronary Artery Disease: The BARI 2D Baseline Data

BackgroundWe describe baseline renal function and albumin excretion rate in patients enrolled in Bypass Angioplasty Revascularization Investigation 2 Diabetes, a randomized clinical trial comparing the impact of revascularization and medical therapy with medical therapy alone and deferred or no revascularization and the impact of glycemic control with either insulin-providing or insulin-sensitizing drugs, on 5-year mortality.MethodsStudy participants had type 2 diabetes mellitus, documented coronary artery disease, and creatinine <2 mg/dL. Albuminuria status (albumin/creatinine ratio [ACR]) and estimated glomerular filtration rate (eGFR), using the abbreviated Modified Diet in Renal Disease equation, were determined at baseline. Univariate and multivariate relationships between baseline clinical characteristics and the presence of albuminuria and reduced eGFR rate were estimated.ResultsTwo thousand one hundred forty-six subjects were included in the analysis. Forty-three percent of the cohort had evidence of kidney dysfunction at baseline: 23% had an eGFR ≥60 mL/min/1.73 m² with either microalbuminuria (>30 ACR; 17%) or macroalbuminuria (>300 ACR; 6%). Twenty-one percent had a reduced eGFR <60 mL/min/1.73 m²; 52% with reduced eGFR had no albuminuria; 28% had microalbuminuria, and 20% had macroalbuminuria. Race, smoking status, duration of diabetes, hypertension, hemoglobin A1c, triglycerides, vascular disease, abnormal ejection fraction, and reduced eGFR were associated with greater albuminuria. Age, sex, duration of diabetes, ACR, hemoglobin A1c, high density lipoprotein, and number of hypertensive medications were associated with reduced eGFR.ConclusionKidney dysfunction is common in older patients with type 2 diabetes mellitus and coronary artery disease; Albuminuria was present in 33%. Reduced eGFR was present in 21%, and half the patients with reduced eGFR had no evidence of albuminuria.     

Which diabetic patients should receive podiatry care? An objective analysis

INTRODUCTION: Diabetes is the leading cause of lower limb amputation in Australia. However, due to limited resources, it is not feasible for everyone with diabetes to access podiatry care, and some objective guidelines of who should receive podiatry is required. METHODS: A total of 250 patients with neuropathy (Biothesiometer; Biomedical Instruments, Newbury, Ohio, USA) ( > 30, age < 65)) but no active foot lesion, and 222 without neuropathy matched for age, type of diabetes, gender and duration, was followed prospectively for 2 years. Sensation was also tested using a 10 g Semmes Weinstein monofilament (Royal Prince Alfred Hospital Diabetes Centre). After the baseline examination, patients were contacted at 6 months and thereafter yearly to determine ulcer status. Incidence of foot ulceration across different risk categories was calculated using Kaplan-Meier survival curve. Log-rank test and Cox's proportional model were used to compare groups. The Number Needed to Treat (NNT) to prevent one ulcer per year was calculated using the standard formulae. RESULTS: During the follow-up period, 34 new ulcers occurred in the neuropathy group and three ulcers in the control group (chi2 (1df) = 21.3; P < 0.0001), equating to an annual incidence of 6.3% and 0.5%, respectively. Fifty-four per cent of the ulcers were due to trauma from footwear. Further stratification of the neuropathy group showed annual incidence of ulceration to be 4% for those with abnormal biothesiometer reading, but who could still feel the monofilament, 10% for those who cannot feel the monofilament and 26% for those with previous ulceration or amputation. Predictors of ulceration were past history of ulceration/amputation (chi2 = 27.8; P < 0.0001) and the presence of neuropathy (chi2 = 4.7; P = 0.03). Assuming a 55% relative risk reduction in ulceration from podiatry care (mean of estimates from 10 reports), the NNT to prevent one foot ulcer per year was: no neuropathy (vibration perception threshold (VPT) < 30)), NNT = 367; neuropathy (VPT > 30) alone, NNT = 45; +cannot feel monofilament, NNT = 18; +previous ulcer/amputation, NNT = 7. CONCLUSION: Provision of podiatry care to diabetic patients should not be only economically based, but should also be directed to those with reduced sensation, especially where there is a previous history of ulceration or amputation.