Clinical Biomarker of Sterile Inflammation, HMGB1, in Patients with Chronic Non-Specific Low Back Pain: A Pilot Cross-Sectional Study.

The present study explores whether the inflammatory biomarker of sterile inflammation, high mobility box 1 (HMGB1), contributes to the inflammatory/nociceptive pathophysiology that characterizes chronic non-specific low back pain (LBP). Patients with chronic LBP (N = 10, >3 pain score on a 11-point Visual Analogue Scale, VAS) and asymptomatic participants (N = 12) provided peripheral blood (PB) samples. The proportion of classical CD14++ monocytes within PB leukocytes was determined by flow cytometry. The plasma and extracellular HMGB1 levels in unstimulated adherent cell (AC) cultures were measured using specific immunoassays. HMGB1 localization in ACs was assessed by immunofluorescent staining. The relative gene expression levels of tumor necrosis factor α (TNFα), interleukin-1 beta (IL-1ß) and HMGB1 were determined by quantitative polymerase chain reaction (qRT-PCR) in relation to the pain intensity (11-point VAS scores) in patients with LBP. The extracellular release of HMGB1 in the LBP patient AC cultures was significantly elevated (p = 0.001) and accompanied by its relocation into the cytoplasm from the nuclei. The number of CD14++ monocytes in the patients' PB was significantly (p = 0.03) reduced, while the HMGB1 plasma levels remained comparable to those of the controls. The mRNA levels of TNFα, IL-1ß and HMGB1 were overexpressed relative to the controls and those of HMGB1 and IL-1ß were correlated with the VAS scores at a significant level (p = 0.01-0.03). The results suggest that HMGB1 may play an important role in the pathophysiology of chronic non-specific LBP.

Effects of spinal manipulative therapy on inflammatory mediators in patients with non-specific low back pain: a non-randomized controlled clinical trial.

BACKGROUND: The inflammatory profiles of patients with acute and chronic nonspecific low back pain (LBP) patients are distinct. Spinal manipulative therapy (SMT) has been shown to modulate the production of nociceptive chemokines differently in these patient cohorts. The present study further investigates the effect(s) of SMT on other inflammatory mediators in the same LBP patient cohorts. METHODS: Acute (n = 22) and chronic (n = 25) LBP patients with minimum pain scores of 3 on a 10-point numeric scale, and asymptomatic controls (n = 24) were recruited according to stringent exclusion criteria. Blood samples were obtained at baseline and after 2 weeks during which patients received 6 SMTs in the lumbar or lumbosacral region. The in vitro production of tumor necrosis factor (TNFα), interleukin-1 ß (IL-1ß), IL-6, IL-2, interferon É£ (IFNÉ£), IL-1 receptor antagonist (IL-1RA), TNF soluble receptor type 2 (sTNFR2) and IL-10 was determined by specific immunoassays. Parametric as well as non-parametric statistics (PAST 3.18 beta software) was used to determine significance of differences between and within study groups prior and post-SMT. Effect size (ES) estimates were obtained using Cohen's d. RESULTS: Compared with asymptomatic controls, SMT-related change scores were significant (P = 0.03-0.01) in reducing the production levels of TNFα in both patient cohorts and those of IL-6, IFNÉ£ and sTNFR2 (P = 0.001-0.02) in patients with chronic LBP. Above-moderate to large ES (d > 0.6-1.4) was observed for these mediators. Compared with respective baselines, a significant post-SMT reduction (P = 0.01) of IL-6 production was detected only in patients with chronic LBP while a significant increase of IL-2 production (P = 0.001 vs. control, and P = 0.004 vs. chronic LBP group) and a large ES (d = 0.87) were observed in patients with acute LBP. Pain and disability scores declined significantly (P < 0.001) in all LBP patients, and were positively correlated (P = 0.03) with IFNÉ£ and IL-2 levels in the acute LBP cohort. CONCLUSION: The short course of SMT treatments of non-specific LBP patients resulted in significant albeit limited and diverse alterations in the production of several of the mediators investigated in this study. This exploratory study highlights the potential of SMT to modulate the production of inflammatory components in acute and chronic non-specific LBP patients and suggests a need for further, randomized controlled clinical trials in this area. TRIAL REGISTRATION: This study was prospectively registered April 2012 with Clinical Trials.gov ( #NCT01766141 ). https://register.clinicaltrials.gov/prs/app/action/SelectProtocol?sid=S0003ZIL&selectaction=Edit&uid=U0001V74&ts=2&cx=-axvqtg.

Nonspecific Low Back Pain: Inflammatory Profiles of Patients With Acute and Chronic Pain.

BACKGROUND: The pathogenesis of low back pain (LBP) remains unclear. However, recent studies suggest that the inflammatory response may be inherent in spinal pain. The purpose of this study was to discern inflammatory profiles in patients with nonspecific acute and chronic LBP in relation to those in asymptomatic individuals. MATERIALS AND METHODS: Peripheral blood samples were obtained from asymptomatic controls and patients with nonspecific acute and chronic LBP reporting a minimum pain score of 3 on a 10-point Visual Analogue Scale (VAS). The levels of in vitro production of proinflammatory (tumor necrosis factor α [TNFα], interleukin [IL] 1ß, IL-6, IL-2, interferon γ) and anti-inflammatory (IL-1 receptor antagonist, soluble receptors of TNF2, and IL-10) mediators were determined by specific immunoassays. RESULTS: The mean VAS scores were comparable between the acute and chronic LBP patient groups. Compared with asymptomatic group, the production of TNFα, IL-1ß, IL-6 and their ratios to IL-10 levels were significantly elevated in both patient groups (P=0.0001 to 0.003). In acute LBP group, the ratio of IL-2:IL-10 was also significantly increased (P=0.02). In contrast, the production of interferon γ was significantly reduced compared with the other study groups (P=0.005 to 0.01), nevertheless, it was positively correlated (P=0.006) with pain scores. In chronic LBP patients, the production of TNFα, IL-1 receptor antagonist, and soluble receptors of TNF2 was significantly increased (P=0.001 to 0.03) in comparison with the control and acute LBP groups, and TNFα and IL-1ß levels were positively correlated (P<0.001) with VAS scores. CONCLUSIONS: The inflammatory profiles of patients with acute and chronic LBP are distinct. Nonetheless, in both patient groups, an imbalance between proinflammatory and anti-inflammatory mediator levels favors the production of proinflammatory components.

Elevated Production of Nociceptive CC Chemokines and sE-Selectin in Patients With Low Back Pain and the Effects of Spinal Manipulation: A Nonrandomized Clinical Trial.

BACKGROUND: The involvement of inflammatory components in the pathophysiology of low back pain (LBP) is poorly understood. It has been suggested that spinal manipulative therapy (SMT) may exert anti-inflammatory effects. PURPOSE: The purpose of this study was to determine the involvement of inflammation-associated chemokines (CC series) in the pathogenesis of nonspecific LBP and to evaluate the effect of SMT on that process. METHODS: Patients presenting with nonradicular, nonspecific LBP (minimum pain score 3 on 10-point visual analog scale) were recruited according to stringent inclusion criteria. They were evaluated for appropriateness to treat using a high velocity low amplitude manipulative thrust in the lumbar-lumbosacral region. Blood samples were obtained at baseline and following the administration of a series of 6 high velocity low amplitude manipulative thrusts on alternate days over the period of 2 weeks. The in vitro levels of CC chemokine ligands (CCL2, CCL3, and CCL4) production and plasma levels of an inflammatory biomarker, soluble E-selectin (sE-selectin), were determined at baseline and at the termination of treatments 2 weeks later. RESULTS: Compared with asymptomatic controls baseline production of all chemokines was significantly elevated in acute (P=0.004 to <0.0001), and that of CCL2 and CCL4 in chronic LBP patients (P<0.0001). Furthermore, CCL4 production was significantly higher (P<0.0001) in the acute versus chronic LBP group. sE-selectin levels were significantly higher (P=0.003) in chronic but not in acute LBP patients. Following SMT, patient-reported outcomes showed significant (P<0.0001) improvements in visual analog scale and Oswestry Disability Index scores. This was accompanied by a significant decline in CCL3 production (P<0.0001) in both groups of patients. Change scores for CCL4 production differed significantly (P<0.0001) only for the acute LBP cohort, and no effect on the production of CCL2 or plasma sE-selectin levels was noted in either group. CONCLUSIONS: The production of chemotactic cytokines is significantly and protractedly elevated in LBP patients. Changes in chemokine production levels, which might be related to SMT, differ in the acute and chronic LBP patient cohorts.

Effect of Interactive Neurostimulation Therapy on Inflammatory Response in Patients With Chronic and Recurrent Mechanical Neck Pain.

OBJECTIVE: The purpose of this study is to evaluate the effect of treatment with a novel noninvasive interactive neurostimulation device (InterX5000) on the production of inflammatory biomarkers in chronic and recurrent mechanical neck pain (NP) syndrome. METHODS: This study represents pilot biological data from a randomized controlled clinical trial. Twenty-five NP patients and 14 asymptomatic subjects included for baseline comparison only completed the study. The patients received 6 InterX5000 or placebo treatments within 2 weeks, and pretreatment and post-treatment blood samples were collected for in vitro determination of biomarker production. Whole blood cell cultures were activated by lipopolysaccharide or by the combination of lipopolysaccharide and phytohemagglutinin for 24 to 48 hours. The levels of tumor necrosis factor α (TNFα) and its soluble type II receptor (sTNFR II), interleukin (IL) 1, IL-1 receptor antagonist (IL-1RA), IL-6, IL-10, and monocyte chemotactic protein (CCL2/MCP-1) were determined by specific immunoassays. RESULTS: Compared with asymptomatic subjects, baseline production levels of all proinflammatory mediators (TNFα, IL-1ß, IL-6, and CCL2/MCP-1) were significantly augmented or trended higher (P = .000-.008) in patients with NP. Of the anti-inflammatory markers, only IL-1RA was significantly elevated (P = .004). The increase in IL-10 and tumor necrosis factor receptor II levels did not reach statistical significance. Neither InterX5000 nor placebo therapy had any significant effect on the production of the inflammatory mediators over the study period. CONCLUSION: This investigation determined that inflammatory cytokine pathways are activated in NP patients but found no evidence that a short course of InterX5000 treatment normalized the production of inflammatory biomarkers.

Elevated production of inflammatory mediators including nociceptive chemokines in patients with neck pain: a cross-sectional evaluation.

OBJECTIVE: This study investigated whether the production of inflammatory mediators and chemotactic cytokines (chemokines) is altered in patients with chronic and recurrent neck pain (NP). METHODS: Cross-sectional data evaluating blood and serum samples were obtained from 27 NP patients and 13 asymptomatic (control) subjects recruited from a chiropractic outpatient clinic. Cell cultures were activated by lipopolysaccharide (LPS) and phytoheamagglutinin for 24 to 48 hours. The levels of tumor necrosis factor α (TNF-α), monocyte chemotactic protein 1, also known as CCL2 (CCL2/MCP-1), and macrophage inflammatory protein 1α or CCL3 (CCL3/MIP-1α) were determined by specific immunoassays. Serum levels of nitric oxide metabolites were evaluated simultaneously, in vanadium III-reduced samples, by Griess reaction. RESULTS: Low levels of constitutive (spontaneous) TNF-α production were present in 7 of the 27 cultures from patients with NP. Both LPS-induced TNF-α production and inducer (LPS/phytoheamagglutin)-stimulated production of CCL2 were significantly elevated (P = .00) in patients compared with controls. In patients, the constitutive synthesis of CCL3 occurred significantly more frequently (P = .00) and ranged from 30 to more than 2000 pg/mL. Finally, serum levels of nitric oxide were significantly elevated (P = .00) in NP patients. CONCLUSIONS: Production of inflammatory mediators was consistently elevated in NP patients in this study, both in vitro and in vivo, and activation of inflammatory pathways was accompanied by up-regulation of CC chemokine synthesis. This suggests that, in NP patients, CC chemokines may be involved in regulation of local inflammatory response through recruitment of immune cells to the inflamed tissue and exert pronociceptive effects.

Interleukin 2-regulated in vitro antibody production following a single spinal manipulative treatment in normal subjects.

BACKGROUND: Our recent investigations have demonstrated that cell cultures from subjects, who received a single spinal manipulative treatment in the upper thoracic spine, show increased capacity for the production of the key immunoregulatory cytokine, interleukin-2. However, it has not been determined if such changes influence the response of the immune effector cells. Thus, the purpose of the present study was to determine whether, in the same subjects, spinal manipulation-related augmentation of the in vitro interleukin-2 synthesis is associated with the modulation of interleukin 2-dependent and/or interleukin-2-induced humoral immune response (antibody synthesis). METHODS: A total of seventy-four age and sex-matched healthy asymptomatic subjects were studied. The subjects were assigned randomly to: venipuncture control (n = 22), spinal manipulative treatment without cavitation (n = 25) or spinal manipulative treatment associated with cavitation (n = 27) groups. Heparinized blood samples were obtained from the subjects before (baseline) and then at 20 minutes and 2 hours post-treatment. Immunoglobulin (antibody) synthesis was induced in cultures of peripheral blood mononuclear cells by stimulation with conventional pokeweed mitogen or by application of human recombinant interleukin-2. Determinations of the levels of immunoglobulin G and immunoglobulin M production in culture supernatants were performed by specific immunoassays. RESULTS: The baseline levels of immunoglobulin synthesis induced by pokeweed mitogen or human recombinant interleukin-2 stimulation were comparable in all groups. No significant changes in the production of pokeweed mitogen-induced immunoglobulins were observed during the post-treatment period in any of the study groups. In contrast, the production of interleukin-2 -induced immunoglobulin G and immunoglobulin M was significantly increased in cultures from subjects treated with spinal manipulation. At 20 min post-manipulation, immunoglobulin G synthesis was significantly elevated in subjects who received manipulation with cavitation, relative to that in cultures from subjects who received manipulation without cavitation and venipuncture alone. At 2 hr post-treatment, immunoglobulin M synthesis was significantly elevated in subjects who received manipulation with cavitation relative to the venipuncture group. There were no quantitative alterations within the population of peripheral blood B or T lymphocytes in the studied cultures. CONCLUSION: Spinal manipulative treatment does not increase interleukin-2 -dependent polyclonal immunoglobulin synthesis by mitogen-activated B cells. However, antibody synthesis induced by interleukin-2 alone can be, at least temporarily, augmented following spinal manipulation. Thus, under certain physiological conditions spinal manipulative treatment might influence interleukin-2 -regulated biological responses.

Enhancement of in vitro interleukin-2 production in normal subjects following a single spinal manipulative treatment.

BACKGROUND: Increasing evidence supports somato-visceral effects of manual therapies. We have previously demonstrated that a single spinal manipulative treatment (SMT) accompanied by audible release has an inhibitory effect on the production of proinflammatory cytokines in asymptomatic subjects. The purpose of this study is to report on SMT-related changes in the production of the immunoregulatory cytokine interleukin 2 (IL-2) and to investigate whether such changes might differ with respect to the treatment approach related to the presence or absence of an audible release (joint cavitation). METHODS: Of 76 asymptomatic subjects, 29 received SMT with cavitation (SMT-C), 23 were treated with SMT without cavitation (SMT-NC) and 24 comprised the venipuncture control (VC) group. The SMT-C and SMT-NC subjects received a single, similar force high velocity low amplitude manipulation, in the upper thoracic spine. However, in SMT-NC subjects, positioning and line of drive were not conducive to cavitation. Blood and serum samples were obtained before and then at 20 and 120 min post-intervention. The production of IL-2 in peripheral blood mononuclear cell cultures was induced by activation for 48 hr with Staphylococcal protein A (SPA) and, in parallel preparations, with the combination of phorbol ester (TPA) and calcium ionophore. The levels of IL-2 in culture supernatants and serum were assessed by specific immunoassays. RESULTS: Compared with VC and their respective baselines, SPA-induced secretion of IL-2 increased significantly in cultures established from both SMT-C and SMT-NC subjects at 20 min post-intervention. At 2 hr post-treatment, significant elevation of IL-2 synthesis was still apparent in preparations from SMT-treated groups though it became somewhat attenuated in SMT-NC subjects. Conversely, IL-2 synthesis induced by TPA and calcium ionophore was unaltered by either type of SMT and was comparable to that in VC group at all time points. No significant alterations in serum-associated IL-2 levels were observed in any of the study groups. CONCLUSION: The present study demonstrates that, the in vitro T lymphocyte response to a conventional mitogen (SPA), as measured by IL-2 synthesis, can become enhanced following SMT. Furthermore, within a period of time following the manipulative intervention, this effect may be independent of joint cavitation. Thus the results of this study suggest that, under certain physiological conditions, SMT might influence IL-2-regulated biological responses.

Spinal manipulative therapy reduces inflammatory cytokines but not substance P production in normal subjects.

OBJECTIVE: To examine the effect of a single spinal manipulation therapy (SMT) on the in vitro production of inflammatory cytokines, tumor necrosis factor alpha, and interleukin (IL) 1beta, in relation to the systemic (in vivo) levels of neurotransmitter substance P (SP). METHODS: Sixty-four asymptomatic subjects were assigned to SMT, sham manipulation, or venipuncture control group. SMT subjects received a single adjustment in the thoracic spine. Blood and serum samples were obtained from subjects before and then at 20 minutes and 2 hours after intervention. Whole-blood cultures were activated with lipopolysaccharide (LPS) for 24 hours. Cytokine production in culture supernatants and serum SP levels were assessed by specific immunoassays. RESULTS: Over the study period, a significant proportion (P