Effect of Abdominal Lymphatic Pump Treatment on Disease Activity in a Rat Model of Inflammatory Bowel Disease.

CONTEXT: Inflammatory bowel disease (IBD), such as ulcerative colitis and Crohn disease, are chronic relapsing inflammatory diseases that affect 1.5 million people in the United States. Lymphatic pump treatment (LPT) techniques were designed to enhance the movement of lymph and can be used to relieve symptoms in patients with IBD and other gastrointestinal disorders. OBJECTIVE: To determine whether LPT would decrease gastrointestinal inflammation and reduce disease severity in rats with acute IBD. METHODS: On day 0, rats were randomized into control or experimental groups. Control rats received normal drinking water for days 0 to 9. On days 0 to 9, rats in the experimental groups received drinking water containing 3.5% dextran sodium sulfate (DSS). On day 3, experimental rats were randomized into 3 groups. On days 3 to 8, experimental rats received either (1) no treatment or anesthesia (DSS alone); (2) 4 minutes of LPT with anesthesia administration (DSS+LPT); or (3) 4 minutes of sham treatment (ie, light touch) and anesthesia (DSS+sham). On day 9, colons and gastrointestinal lymphoid tissue were collected. Colon weight, histologic changes, disease activity index (DAI) score, and the concentration of leukocytes were measured. RESULTS: At day 9, the mean (SD) DAI score in the DSS+LPT group (1.0 [0.1]) was significantly decreased (P<.01) compared with the DAI score of DSS-alone rats (1.5 [0.1]). While the DAI in DSS+LPT rats was reduced on days 8 to 9, this difference was not statistically different (P>.05) compared with DSS+sham (1.3 [0.1]). No significant differences were found in colon weight, histopathologic findings, or the concentration of gastrointestinal leukocytes between DSS alone, DSS+sham, or DSS+LPT (P>.05). CONCLUSION: While DSS+LPT reduced IBD compared with DSS+sham, the decrease was not statistically significant. Considering the growing use of adjunctive treatment for the management of IBD, it is important to identify the effect of osteopathic manipulative medicine on IBD progression.

Lymphatic Pump Treatment Mobilizes Bioactive Lymph That Suppresses Macrophage Activity In Vitro.

CONTEXT: By promoting the recirculation of tissue fluid, the lymphatic system preserves tissue health, aids in the absorption of gastrointestinal lipids, and supports immune surveillance. Failure of the lymphatic system has been implicated in the pathogenesis of several infectious and inflammatory diseases. Thus, interventions that enhance lymphatic circulation, such as osteopathic lymphatic pump treatment (LPT), should aid in the management of these diseases. OBJECTIVE: To determine whether thoracic duct lymph (TDL) mobilized during LPT would alter the function of macrophages in vitro. METHODS: The thoracic ducts of 6 mongrel dogs were cannulated, and TDL samples were collected before (baseline), during, and 10 minutes after LPT. Thoracic duct lymph flow was measured, and TDL samples were analyzed for protein concentration. To measure the effect of TDL on macrophage activity, RAW 264.7 macrophages were cultured for 1 hour to acclimate. After 1 hour, cell-free TDL collected at baseline, during LPT, and after TDL was added at 5% total volume per well and co-cultured with or without 500 ng per well of lipopolysaccharide (LPS) for 24 hours. As a control for the addition of 5% TDL, macrophages were cultured with phosphate-buffered saline (PBS) at 5% total volume per well and co-cultured with or without 500 ng per well of LPS for 24 hours. After culture, cell-free supernatants were assayed for nitrite (NO2-), tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10). Macrophage viability was measured using flow cytometry. RESULTS: Lymphatic pump treatment significantly increased TDL flow and the flux of protein in TDL (P<.001). After culture, macrophage viability was approximately 90%. During activation with LPS, baseline TDL, TDL during LPT, and TDL after LPT significantly decreased the production of NO2-, TNF-α, and IL-10 by macrophages (P<.05). However, no significant differences were found in viability or the production of NO2-, TNF-α, or IL-10 between macrophages cultured with LPS plus TDL taken before, during, and after LPT (P>.05). CONCLUSION: The redistribution of protective lymph during LPT may provide scientific rationale for the clinical use of LPT to reduce inflammation and manage edema.

Connecting (T)issues: How Research in Fascia Biology Can Impact Integrative Oncology.

Complementary and integrative treatments, such as massage, acupuncture, and yoga, are used by increasing numbers of cancer patients to manage symptoms and improve their quality of life. In addition, such treatments may have other important and currently overlooked benefits by reducing tissue stiffness and improving mobility. Recent advances in cancer biology are underscoring the importance of connective tissue in the local tumor environment. Inflammation and fibrosis are well-recognized contributors to cancer, and connective tissue stiffness is emerging as a driving factor in tumor growth. Physical-based therapies have been shown to reduce connective tissue inflammation and fibrosis and thus may have direct beneficial effects on cancer spreading and metastasis. Meanwhile, there is currently little knowledge on potential risks of applying mechanical forces in the vicinity of tumors. Thus, both basic and clinical research are needed to understand the full impact of integrative oncology on cancer biology as well as whole person health. Cancer Res; 76(21); 6159-62. ©2016 AACR.

Lymphatic pump treatment as an adjunct to antibiotics for pneumonia in a rat model.

BACKGROUND: Lymphatic pump treatment (LPT) is a technique used by osteopathic physicians as an adjunct to antibiotics for patients with respiratory tract infections, and previous studies have demonstrated that LPT reduces bacterial load in the lungs of rats with pneumonia. Currently, it is unknown whether LPT affects drug effcacy. OBJECTIVE: To determine whether the combination of antibiotics and LPT would reduce bacterial load in the lungs of rats with acute pneumonia. METHODS: Rats were infected intranasally with 5×107 colony-forming units (CFU) of Streptococcus pneumoniae. At 24, 48, and 72 hours after infection, the rats received no therapy (control), 4 minutes of sham therapy, or 4 minutes of LPT, followed by subcutaneous injection of 40 mg/kg of levofoxacin or sterile phosphate-buffered saline. At 48, 72, and 96 hours after infection, the spleens and lungs were collected, and S pneumoniae CFU were enumerated. Blood was analyzed for a complete blood cell count and leukocyte differential count. RESULTS: At 48 and 72 hours after infection, no statistically significant differences in pulmonary CFU were found between control, sham therapy, or LPT when phosphate-buffered saline was administered; however, the reduction in CFU was statistically significant in all rats given levofoxacin. The combination of sham therapy and levofoxacin decreased bacterial load at 72 and 96 hours after infection, and LPT and levofoxacin significantly reduced CFU compared with sham therapy and levofoxacin at both time points (P<.05). Colony-forming units were not detected in the spleens at any time. No statistically significant differences in hematologic findings between any treatment groups were found at any time point measured. CONCLUSION: The results suggest that 3 applications of LPT induces an additional protective mechanism when combined with levofoxacin and support its use as an adjunctive therapy for the management of pneumonia; however, the mechanism responsible for this protection is unclear.

Antigen-pulsed bone marrow-derived and pulmonary dendritic cells promote Th2 cell responses and immunopathology in lungs during the pathogenesis of murine Mycoplasma pneumonia.

Mycoplasmas are a common cause of pneumonia in humans and animals, and attempts to create vaccines have not only failed to generate protective host responses, but they have exacerbated the disease. Mycoplasma pulmonis causes a chronic inflammatory lung disease resulting from a persistent infection, similar to other mycoplasma respiratory diseases. Using this model, Th1 subsets promote resistance to mycoplasma disease and infection, whereas Th2 responses contribute to immunopathology. The purpose of the present study was to evaluate the capacity of cytokine-differentiated dendritic cell (DC) populations to influence the generation of protective and/or pathologic immune responses during M. pulmonis respiratory disease in BALB/c mice. We hypothesized that intratracheal inoculation of mycoplasma Ag-pulsed bone marrow-derived DCs could result in the generation of protective T cell responses during mycoplasma infection. However, intratracheal inoculation (priming) of mice with Ag-pulsed DCs resulted in enhanced pathology in the recipient mice when challenged with mycoplasma. Inoculation of immunodeficient SCID mice with Ag-pulsed DCs demonstrated that this effect was dependent on lymphocyte responses. Similar results were observed when mice were primed with Ag-pulsed pulmonary, but not splenic, DCs. Lymphocytes generated in uninfected mice after the transfer of either Ag-pulsed bone marrow-derived DCs or pulmonary DCs were shown to be IL-13(+) Th2 cells, known to be associated with immunopathology. Thus, resident pulmonary DCs most likely promote the development of immunopathology in mycoplasma disease through the generation of mycoplasma-specific Th2 responses. Vaccination strategies that disrupt or bypass this process could potentially result in a more effective vaccination.

Effects of the selective adenosine A2A receptor antagonist, SCH 412348, on the parkinsonian phenotype of MitoPark mice.

Adenosine A2A receptors are predominantly localized on striatopallidal gamma-aminobutyric acid (GABA) neurons, where they are colocalized with dopamine D2 receptors and are involved in the regulation of movement. Adenosine A2A receptor antagonists have been evaluated as a novel treatment for Parkinson's disease and have demonstrated efficacy in a broad spectrum of pharmacological and toxicological rodent and primate models. Fewer studies have been performed to evaluate the efficacy of adenosine A2A receptor antagonists in genetic models of hypodopaminergic states. SCH 412348 is a potent and selective adenosine A2A receptor antagonist that shows efficacy in rodent and primate models of movement disorders. Here we evaluated the effects of SCH 412348 in the MitoPark mouse, a genetic model that displays a progressive loss of dopamine neurons. The dopamine cell loss is associated with a profound akinetic phenotype that is sensitive to levodopa (l-dopa). SCH 412348 (0.3-10mg/kg administered orally) dose dependently increased locomotor activity in the mice. Moreover, SCH 412348 retained its efficacy in the mice as motor impairment progressed (12-22 weeks of age), demonstrating that the compound was efficacious in mild to severe Parkinson's disease-like impairment in the mice. Additionally, SCH 412348 fully restored lost functionality in a measure of hind limb bradykinesia and partially restored functionality in a rotarod test. These findings provide further evidence of the anti-Parkinsonian effects of selective adenosine A2A receptor antagonists and predict that they will retain their efficacy in both mild and severe forms of motor impairment.

Lymphatic pump treatment repeatedly enhances the lymphatic and immune systems.

BACKGROUND: Osteopathic practitioners utilize manual therapies called lymphatic pump techniques (LPT) to treat edema and infectious diseases. While previous studies examined the effect of a single LPT treatment on the lymphatic system, the effect of repeated applications of LPT on lymphatic output and immunity has not been investigated. Therefore, the purpose of this study was to measure the effects of repeated LPT on lymphatic flow, lymph leukocyte numbers, and inflammatory mediator concentrations in thoracic duct lymph (TDL). METHODS AND RESULTS: The thoracic ducts of five mongrel dogs were cannulated, and lymph samples were collected during pre-LPT, 4 min of LPT, and 2 hours post-LPT. A second LPT (LPT-2) was applied after a 2 hour rest period. TDL flow was measured, and TDL were analyzed for the concentration of leukocytes and inflammatory mediators. Both LPT treatments significantly increased TDL flow, leukocyte count, total leukocyte flux, and the flux of interleukin-8 (IL-8), keratinocyte-derived chemoattractant (KC), nitrite (NO2(-)), and superoxide dismutase (SOD). The concentration of IL-6 increased in lymph over time in all experimental groups; therefore, it was not LPT dependent. CONCLUSION: Clinically, it can be inferred that LPT at a rate of 1 pump per sec for a total of 4 min can be applied every 2 h, thus providing scientific rationale for the use of LPT to repeatedly enhance the lymphatic and immune system.

Thoracic and abdominal lymphatic pump techniques inhibit the growth of S. pneumoniae bacteria in the lungs of rats.

BACKGROUND: Osteopathic physicians utilize manual medicine techniques called lymphatic pump techniques (LPT) to improve lymphatic flow and enhance immunity. Clinical studies report that LPT enhances antibody responses to bacterial vaccines, shortens duration of cough in patients with respiratory disease, and shortens the duration of intravenous antibiotic therapy and hospital stay in patients with pneumonia. The purpose of this study was to identify if thoracic LPT (Th-LPT) or abdominal LPT (Ab-LPT) would reduce Streptococcus pneumoniae colony-forming units (CFU) in the lungs of rats with acute pneumonia. METHODS AND RESULTS: Rats were nasally infected with S. pneumoniae and received either control, sham, Ab-LPT, or Th-LPT once daily for 3 consecutive days. On day 4 post-infection, lungs were removed and bacteria were enumerated. Three daily applications of either Ab-LPT or Th-LPT were able to significantly (p<0.05) reduce the numbers of pulmonary bacteria compared to control and sham. There were no significant differences in the percentage or concentration of leukocytes in blood between groups, suggesting neither Ab-LPT nor Th-LPT release leukocytes into blood circulation. CONCLUSIONS: Our data demonstrate that LPT may protect against pneumonia by inhibiting bacterial growth in the lung; however, the mechanism of protection is unclear. Once these mechanisms are understood, LPT can be optimally applied to patients with pneumonia, which may substantially reduce morbidity, mortality, and frequency of hospitalization.

Osteopathic manual treatment in patients with diabetes mellitus and comorbid chronic low back pain: subgroup results from the OSTEOPATHIC Trial.

CONTEXT: Chronic pain is often present in patients with diabetes mellitus. OBJECTIVE: To assess the effects of osteopathic manual treatment (OMT) in patients with diabetes mellitus and comorbid chronic low back pain (LBP). DESIGN: Randomized, double-blind, sham-controlled, 2×2 factorial trial, including OMT and ultrasound therapy (UST) interventions. SETTING: University-based study in Dallas-Fort Worth, Texas. PATIENTS: A subgroup of 34 patients (7%) with diabetes mellitus within 455 adult patients with nonspecific chronic LBP enrolled in the OSTEOPAThic Health outcomes In Chronic low back pain (OSTEOPATHIC) Trial. MAIN STUDY MEASURES: The Outpatient Osteopathic SOAP Note Form was used to measure somatic dysfunction at baseline. A 100-mm visual analog scale was used to measure LBP severity over 12 weeks from randomization to study exit. Paired serum concentrations of tumor-necrosis factor (TNF)-α obtained at baseline and study exit were available for 6 subgroup patients. RESULTS: Key osteopathic lesions were observed in 27 patients (79%) with diabetes mellitus vs 243 patients (58%) without diabetes mellitus (P=.01). The reduction in LBP severity over 12 weeks was significantly greater in 19 patients with diabetes mellitus who received OMT than in 15 patients with diabetes mellitus who received sham OMT (mean between-group difference in changes in the visual analog scale pain score, -17 mm; 95% confidence interval [CI], -32 mm to -1 mm; P=.04). This difference was clinically relevant (Cohen d=0.7). A corresponding significantly greater reduction in TNF-α serum concentration was noted in patients with diabetes mellitus who received OMT, compared with those who received sham OMT (mean between-group difference, -6.6 pg/mL; 95% CI, -12.4 to -0.8 pg/mL; P=.03). This reduction was also clinically relevant (Cohen d=2.7). No significant changes in LBP severity or TNF-α serum concentration were associated with UST during the 12-week period. CONCLUSION: Severe somatic dysfunction was present significantly more often in patients with diabetes mellitus than in patients without diabetes mellitus. Patients with diabetes mellitus who received OMT had significant reductions in LBP severity during the 12-week period. Decreased circulating levels of TNF-α may represent a possible mechanism for OMT effects in patients with diabetes mellitus. A larger clinical trial of patients with diabetes mellitus and comorbid chronic LBP is warranted to more definitively assess the efficacy and mechanisms of action of OMT in this population.

Osteopathic lymphatic pump techniques to enhance immunity and treat pneumonia.

Pneumonia is a common cause of morbidity and mortality worldwide. While antibiotics are generally effective for the treatment of infection, the emergence of resistant strains of bacteria threatens their success. The osteopathic medical profession has designed a set of manipulative techniques called lymphatic pump techniques (LPT), to enhance the flow of lymph through the lymphatic system. Clinically, LPT is used to treat infection and oedemaand might be an effective adjuvant therapy in patients with pneumonia.The immune system uses the lymphatic and blood systems to survey to rid the body of pathogens; however, only recently have the effects of LPT on the lymphatic and immune systems been investigated. This short review highlightsclinical and basic science research studies that support the use of LPT to enhance the lymphatic and immune systems and treat pneumonia, and discusses the potential mechanisms by which LPT benefits patients with pneumonia.

Associations of cytokine concentrations with key osteopathic lesions and clinical outcomes in patients with nonspecific chronic low back pain: results from the OSTEOPATHIC Trial.

CONTEXT: Little is known about the role that cytokines play in osteopathic manual treatment (OMT) of patients with chronic low back pain (LBP). OBJECTIVE: To measure the baseline concentrations of interleukin (IL)-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α in patients with chronic LBP; the correlations of these cytokine concentrations with clinical measures, including the number of key osteopathic lesions; the changes in cytokine concentrations with OMT; and the association of such changes with clinical outcomes. DESIGN: Substudy nested within a randomized controlled trial of OMT for nonspecific chronic LBP. SETTING: University-based study in Dallas-Fort Worth, Texas. PATIENTS: Seventy adult research patients with nonspecific chronic LBP. MAIN OUTCOME MEASURES: A 10-cm visual analog scale, the Roland-Morris Disability Questionnaire, and the Medical Outcomes Study Short Form-36 Health Survey were used to measure LBP severity, back-specific functioning, and general health, respectively. RESULTS: At baseline, IL-1ß (ρ = 0.33; P = .005) and IL-6 (ρ = 0.32; P = .006) were each correlated with the number of key osteopathic lesions; however, only IL-6 was correlated with LBP severity (ρ = 0.28; P = .02). There was a significantly greater reduction of TNF-α concentration after 12 weeks in patients who received OMT compared with patients who received sham OMT (Mann-Whitney U = 251.5; P = .03). Significant associations were found between OMT and a reduced TNF-α concentration response at week 12 among patients who achieved moderate (response ratio, 2.13; 95% confidence interval [CI], 1.11-4.06; P = .006) and substantial (response ratio, 2.13; 95% CI, 1.07-4.25; P = .01) LBP improvements, and improvement in back-specific functioning (response ratio, 1.68; 95% CI, 1.04-2.71; P = .03). CONCLUSIONS: This study found associations between IL-1ß and IL-6 concentrations and the number of key osteopathic lesions and between IL-6 and LBP severity at baseline. However, only TNF-α concentration changed significantly after 12 weeks in response to OMT. These discordant findings indicate that additional research is needed to elucidate the underlying mechanisms of action of OMT in patients with nonspecific chronic LBP.

A potent and selective metabotropic glutamate receptor 4 positive allosteric modulator improves movement in rodent models of Parkinson's disease.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGluR4) have been proposed as a novel therapeutic approach for the treatment of Parkinson's disease. However, evaluation of this proposal has been limited by the availability of appropriate pharmacological tools to interrogate the target. In this study, we describe the properties of a novel mGluR4 PAM. 5-Methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine (ADX88178) enhances glutamate-mediated activation of human and rat mGluR4 with EC(50) values of 4 and 9 nM, respectively. The compound is highly selective for mGluR4 with minimal activities at other mGluRs. Oral administration of ADX88178 in rats is associated with high bioavailability and results in cerebrospinal fluid exposure of >50-fold the in vitro EC(50) value. ADX88178 reverses haloperidol-induced catalepsy in rats at 3 and 10 mg/kg. It is noteworthy that this compound alone has no impact on forelimb akinesia resulting from a bilateral 6-hydroxydopamine lesion in rats. However, coadministration of a low dose of L-DOPA (6 mg/kg) enabled a robust, dose-dependent reversal of the forelimb akinesia deficit. ADX88178 also increased the effects of quinpirole in lesioned rats and enhanced the effects of L-DOPA in MitoPark mice. It is noteworthy that the enhancement of the actions of L-DOPA was not associated with an exacerbation of L-DOPA-induced dyskinesias in rats. ADX88178 is a novel, potent, and selective mGluR4 PAM that is a valuable tool for exploring the therapeutic potential of mGluR4 modulation. The use of this novel tool molecule supports the proposal that activation of mGluR4 may be therapeutically useful in Parkinson's disease.

Lymphatic pump manipulation mobilizes inflammatory mediators into lymphatic circulation.

Lymph stasis can result in edema and the accumulation of particulate matter, exudates, toxins and bacteria in tissue interstitial fluid, leading to inflammation, impaired immune cell trafficking, tissue hypoxia, tissue fibrosis and a variety of diseases. Previously, we demonstrated that osteopathic lymphatic pump techniques (LPTs) significantly increased thoracic and intestinal duct lymph flow. The purpose of this study was to determine if LPT would mobilize inflammatory mediators into the lymphatic circulation. Under anesthesia, thoracic or intestinal lymph of dogs was collected at resting (pre-LPT), during four minutes of LPT, and for 10 min following LPT (post-LPT), and the lymphatic concentrations of interleukin-2 (IL-2), IL-4, IL-6, IL-10, interferon-γ, tissue necrosis factor α,  monocyte chemotactic protein-1 (MCP-1), keratinocyte chemoattractant, superoxide dismutase (SOD) and nitrotyrosine (NT) were measured. LPT significantly increased MCP-1 concentrations in thoracic duct lymph. Further, LPT increased both thoracic and intestinal duct lymph flux of cytokines and chemokines as compared with their respective pre-LPT flux. In addition, LPT increased lymphatic flux of SOD and NT. Ten minutes following cessation of LPT, thoracic and intestinal lymph flux of cytokines, chemokines, NT and SOD were similar to pre-LPT, demonstrating that their flux was transient and a response to LPT. This re-distribution of inflammatory mediators during LPT may provide scientific rationale for the clinical use of LPT to enhance immunity and treat infection.

Lymphatic pump treatment enhances the lymphatic and immune systems.

The osteopathic medical profession has long advocated the use of osteopathic lymphatic pump treatments (LPT) to improve lymphatic circulation, reduce edema and combat infectious disease. However, until recently, there was no scientific evidence that LPT enhances function of the lymphatic and immune systems. This review discusses the physiological functions of the lymphatic system, the ability of LPT to increase lymph flow under normal and experimental conditions, the clinical benefits of LPT, current research models for the study of LPT and the potential mechanisms by which LPT enhances lymphatic and immune function.

Lymphatic pump treatment augments lymphatic flux of lymphocytes in rats.

BACKGROUND: Lymphatic pump techniques (LPT) are used by osteopathic practitioners for the treatment of edema and infection; however, the mechanisms by which LPT enhances the lymphatic and immune systems are poorly understood. METHODS AND RESULTS: To measure the effect of LPT on the rat, the cisterna chyli (CC) of 10 rats were cannulated and lymph was collected during 4 min of 1) pre-LPT baseline, 2) 4 min LPT, and 3) 10 min post-LPT recovery. LPT increased significantly (p < 0.05) lymph flow from a baseline of 24 ± 5 µl/min to 89 ± 30 µl/min. The baseline CC lymphocyte flux was 0.65 ± 0.21 × 106 lymphocytes/min, and LPT increased CC lymphocyte flux to 6.10 ± 0.99 × 106 lymphocytes/min (p < 0.01). LPT had no preferential effect on any lymphocyte population, since total lymphocytes, CD4+ T cells, CD8+ T cells, and B cell numbers were similarly increased. To determine if LPT mobilized gut-associated lymphocytes into the CC lymph, gut-associated lymphocytes in the CC lymph were identified by staining CC lymphocytes for the gut homing receptor integrin α4ß7. LPT significantly increased (p < 0.01) the flux of α4ß7 positive CC lymphocytes from a baseline of 0.70 ± 0.03 × 105 lymphocytes/min to 6.50 ± 0.10 × 105 lymphocytes/min during LPT. Finally, lymphocyte flux during recovery was similar to baseline, indicating the effects of LPT are transient. CONCLUSIONS: Collectively, these results suggest that LPT may enhance immune surveillance by increasing the numbers of lymphocytes released in to lymphatic circulation, especially from the gut associated lymphoid tissue. The rat provides a useful model to further investigate the effect of LPT on the lymphatic and immune systems.

Pyruvate-enriched cardioplegia suppresses cardiopulmonary bypass-induced myocardial inflammation.

BACKGROUND: Cardiopulmonary bypass-induced oxidative stress initiates inflammation that can damage the myocardium. This study tested whether cardioplegia enriched with the intermediary metabolite and antioxidant pyruvate dampens postbypass myocardial inflammation. METHODS: Pigs were maintained on cardiopulmonary bypass while their hearts were arrested for 60 minutes with 4:1 blood:crystalloid cardioplegia, in which the crystalloid contained 188 mM glucose ± 24 mM pyruvate. Pigs were weaned from bypass after 30 minutes of whole blood reperfusion and recovered for 4 hours. Glutathione (GSH) and glutathione disulfide (GSSG) were measured in coronary sinus plasma to indirectly monitor myocardial GSH redox state (GSH/GSSG). Left ventricular myocardium was sampled 4 hours after cardiopulmonary bypass for analyses of C-reactive protein, matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinase-2 (TIMP-2), and to assess neutrophil infiltration by histology and myeloperoxidase assay. RESULTS: Coronary sinus GSH/GSSG fell 70% after cardiopulmonary bypass with control cardioplegia, but pyruvate cardioplegia produced a robust increase in coronary sinus GSH/GSSG that persisted for 4 hours after bypass. Myocardial C-reactive protein content increased 5.6-fold after control bypass, and neutrophil infiltration and myeloperoxidase activity also increased, but pyruvate-fortified cardioplegia prevented these inflammatory effects. Control cardioplegia lowered myocardial TIMP-2 content by 59% and increased matrix metalloproteinase-9 activity by 35% versus nonbypass sham values, but pyruvate cardioplegia increased TIMP-2 content ninefold versus control cardioplegia and prevented the increase in matrix metalloproteinase-9. Matrix metalloproteinase-2 was not affected by bypass ± pyruvate. CONCLUSIONS: Pyruvate-enriched cardioplegia dampens cardiopulmonary bypass-induced myocardial inflammation. Increased GSH/GSSG and TIMP-2 may mediate pyruvate's effects.

Lymphatic pump treatment mobilizes leukocytes from the gut associated lymphoid tissue into lymph.

BACKGROUND: Lymphatic pump techniques (LPT) are used clinically by osteopathic practitioners for the treatment of edema and infection; however, the mechanisms by which LPT enhances lymphatic circulation and provides protection during infection are not understood. Rhythmic compressions on the abdomen during LPT compress the abdominal area, including the gut-associated lymphoid tissues (GALT), which may facilitate the release of leukocytes from these tissues into the lymphatic circulation. This study is the first to document LPT-induced mobilization of leukocytes from the GALT into the lymphatic circulation. METHODS AND RESULTS: Catheters were inserted into either the thoracic or mesenteric lymph ducts of dogs. To determine if LPT enhanced the release of leukocytes from the mesenteric lymph nodes (MLN) into lymph, the MLN were fluorescently labeled in situ. Lymph was collected during 4 min pre-LPT, 4 min LPT, and 10 min following cessation of LPT. LPT significantly increased lymph flow and leukocytes in both mesenteric and thoracic duct lymph. LPT had no preferential effect on any specific leukocyte population, since neutrophil, monocyte, CD4+ T cell, CD8+ T cell, IgG+B cell, and IgA+B cell numbers were similarly increased. In addition, LPT significantly increased the mobilization of leukocytes from the MLN into lymph. Lymph flow and leukocyte counts fell following LPT treatment, indicating that the effects of LPT are transient. CONCLUSIONS: LPT mobilizes leukocytes from GALT, and these leukocytes are transported by the lymphatic circulation. This enhanced release of leukocytes from GALT may provide scientific rationale for the clinical use of LPT to improve immune function.

PISA, a novel pharmacodynamic assay for assessing poly(ADP-ribose) polymerase (PARP) activity in situ.

INTRODUCTION: Poly ADP-ribose polymerase (PARP) maintains genomic integrity by repairing DNA strand breaks, however over-activation of PARP following neural tissue injury is hypothesized to cause neuronal death. Therefore, PARP inhibitors have potential for limiting neural injury under certain conditions. A reliable method for assessing PARP activity in brain is critical for development of novel inhibitors with CNS activity. We developed the PARP In Situ Activity (PISA) assay to provide a direct, quantitative assessment of CNS PARP activity in vitro or in vivo. METHODS: The assay utilized brain sections from rats with striatal kainic acid (KA) lesions and 3H- or biotinylated NAD+ as the substrate to assess PARP activity. Following optimization of the assay, it was used to assess in vitro and in vivo efficacy of known and novel PARP inhibitors. The assay also was used to assess PARP activity in male and female gonad-intact and ovariectomized rats. RESULTS: Using 3H-NAD+ as the substrate, PARP activity was greater (p<0.01) in tissue from KA-lesioned vs. non-lesioned rats. Using biotinylated NAD+ it was revealed that PARP activity was present ipsilateral to the KA injection site, and labeling was blocked by incubation with excess unlabeled NAD+ or PARP inhibitors. The PARP inhibitor, 3-aminobenzamide and several novel inhibitors reduced (p<0.01) polymerase activity in vitro. Furthermore, the inhibitor MRLSD303 reduced (p<0.001) PARP activity in vivo in both male and female rats. Finally, administration of the novel PARP inhibitor MRLIT115 dose-dependently reduced (p<0.001) polymerase activity in vivo. DISCUSSION: The PISA assay provides a direct, quantitative method for assessing PARP activity in vitro and provides critical information on factors underlying in vivo efficacy of chemical inhibitors including brain penetration and target engagement. These findings support use of the PISA assay as a screening tool for testing efficacy of PARP inhibitors in brain.

Abdominal lymphatic pump treatment increases leukocyte count and flux in thoracic duct lymph.

BACKGROUND: Previous studies suggest that rhythmic compression of the abdomen (abdominal lymphatic pump techniques, LPT) enhances immunity and resistance to infectious disease, but direct evidence of this has not been documented. In this study, the thoracic duct of eight anesthetized mongrel dogs was catheterized, so the immediate effects of LPT on lymph flow and leukocyte output could be measured. METHODS AND RESULTS: Lymph flow was measured by timed collection or ultrasonic flowmeter, and lymph was collected over ice under 1) resting (baseline) conditions, and 2) during application of LPT. The baseline leukocyte count was 4.8 +/- 1.7 x 10(6) cells/ml of lymph, and LPT significantly increased leukocytes to 11.8 +/- 3.6 x 10(6) cells/ml. Flow cytometry and differential cell staining revealed that numbers of macrophages, neutrophils, total lymphocytes, T cells and B cells were similarly increased during LPT. Furthermore, LPT significantly enhanced lymph flow from 1.13 +/- 0.44 ml/min to 4.14 +/- 1.29 ml/min. Leukocyte flux, computed from the product of lymph flow and cell count, was increased by LPT from 8.2 +/- 4.1 x 10(6) to 60 +/- 25 x 10(6) total cells/min. Similar trends were observed in macrophages, neutrophils, total lymphocytes, T cells and B cells during LPT. CONCLUSIONS: LPT significantly increased both thoracic duct lymph flow and leukocyte count, so lymph leukocyte flux was markedly enhanced. Increased mobilization of immune cells is likely and important mechanism responsible for the enhanced immunity and recovery from infection of patients treated with LPT.

Cytokine and chemokine transcription profile during Mycoplasma pulmonis infection in susceptible and resistant strains of mice: macrophage inflammatory protein 1beta (CCL4) and monocyte chemoattractant protein 2 (CCL8) and accumulation of CCR5+ Th cells.

The progression of murine mycoplasma pneumonia is dependent on T cells and other immune cells. The role of cytokines in immunity are complex, and identifying the network of cytokines produced after infection of mice is essential in dissecting the key cytokine cascades involved mycoplasma disease pathogenesis. In the present study, mRNA expression of 143 different cytokines, chemokines, or receptors were evaluated in lung tissues from both susceptible (BALB/c and C3H/HeN) and resistant (C57BL/6) mice after Mycoplasma pulmonis infection. To accomplish this, membrane-based cDNA microarrays were used to monitor changes mRNA expression in lungs. There was a clear association with disease susceptibility and development of cytokine mRNA expression. In addition to proinflammatory cytokines, mRNA expression of an anti-inflammatory cytokine, interleukin-10, increased with disease severity, suggesting an attempt to moderate the severity of the inflammatory response. Furthermore, it is clear that an array of chemokines produced in susceptible mice could contribute to the recruitment and maintenance of inflammatory cells at the site of disease. In support of this, there was an increase in macrophage inflammatory protein 1beta (MIP-1beta; CCL4) and monocyte chemoattractant protein 2 (MCP-2; CCL8) mRNA levels from mycoplasma-infected mice and a corresponding accumulation of CD4+ Th cells expressing the MIP-1beta/MCP-2 receptor, CCR5, in the lungs of mice. Furthermore, MIP-1beta- and MCP-2-producing cells and CD4+ T cells were found to be in close association in pulmonary lesions. Thus, there was a significant cytokine response associated with disease pathogenesis, and these studies provide important leads and insights into ongoing cytokine- and chemokine-mediated processes in this persistent inflammatory disease.