High-Throughput CAMP Assay (HiTCA): A Novel Tool for Evaluating the Vitamin D-Dependent Antimicrobial Response.

Vitamin D is known to modulate human immune responses, and vitamin D deficiency is associated with increased susceptibility to infection. However, what constitutes sufficient levels or whether vitamin D is useful as an adjuvant therapeutic is debated, much in part because of inadequate elucidation of mechanisms underlying vitamin D's immune modulatory function. Cathelicidin antimicrobial peptide (CAMP) has potent broad-spectrum activity, and the CAMP gene is regulated in human innate immune cells by active 1,25(OH)2D3, a product of hydroxylation of inactive 25(OH)D3 by CYP27B1-hydroxylase. We developed a CRISPR/Cas9-edited human monocyte-macrophage cell line containing the mCherry fluorescent reporter gene at the 3' end of the endogenous CAMP gene. The High Throughput CAMP Assay (HiTCA) developed here is a novel tool for evaluating CAMP expression in a stable cell line that is scalable for a high-throughput workflow. Application of HiTCA to serum samples from a small number of human donors (n = 10) showed individual differences in CAMP induction that were not fully accounted for by the serum vitamin D metabolite status of the host. As such, HiTCA may be a useful tool that can advance our understanding of the human vitamin D-dependent antimicrobial response, which is being increasingly appreciated for its complexity.

Conventional Type 1 Dendritic Cells (cDC1) in Human Kidney Diseases: Clinico-Pathological Correlations.

Background: cDC1 is a subset of conventional DCs, whose most recognized function is cross-presentation to CD8+ T cells. We conducted this study to investigate the number and location of cDC1s in various human kidney diseases as well as their correlation with clinico-pathological features and CD8+ T cells. Methods: We analyzed 135 kidney biopsies samples. Kidney diseases included: acute tubular necrosis (ATN), acute interstitial nephritis (AIN), proliferative glomerulonephritis (GN) (IgA nephropathy, lupus nephritis, pauci-immune GN, anti-GBM disease), non-proliferative GN (minimal change disease, membranous nephropathy) and diabetic nephropathy. Indirect immunofluorescence staining was used to quantify cDC1s, CD1c+ DCs, and CD8+ T cells. Results: cDC1s were rarely present in normal kidneys. Their number increased significantly in ATN and proliferative GN, proportionally much more than CD1c+ DCs. cDC1s were mainly found in the interstitium, except in lupus nephritis, pauci-immune GN and anti-GBM disease, where they were prominent in glomeruli and peri-glomerular regions. The number of cDC1s correlated with disease severity in ATN, number of crescents in pauci-immune GN, interstitial fibrosis in IgA nephropathy and lupus nephritis, as well as prognosis in IgA nephropathy. The number of CD8+ T cells also increased significantly in these conditions and cDC1 number correlated with CD8+ T cell number in lupus nephritis and pauci-immune GN, with many of them closely co-localized. Conclusions: cDC1 number correlated with various clinic-pathological features and prognosis reflecting a possible role in these conditions. Their association with CD8+ T cells suggests a combined mechanism in keeping with the results in animal models.

Morphine produces better thermal analgesia in young Huntington mice and are associated with less neuroinflammation in spinal cord.

BACKGROUND: Huntington's disease (HD) is an inherited disease characterized by both mental and motor dysfunctions. Our previous studies showed that HD mice demonstrate a diminished pain response. However, few studies have focused on the relationship between HD and morphine analgesia. The purpose of this study is to investigate and compare the analgesic effects of morphine in HD and wild-type (WT) mice. METHODS: We used clinically similar transgenic HD mice (7-10 weeks of age with motor dysfunction at 8-9 mo of age) carrying a mutant Huntington CAG trinucleotide repeats to evaluate morphine analgesia. The morphine (10 mg/kg subcutaneously) analgesia was evaluated with a tail-flick in hot water (52°C). Mice spinal cords were harvested at the end of the analgesia studies. An immunofluorescence assay and western blotting were used to identify changes in the cells and cytokines. RESULTS: Our data demonstrate that preonset young HD mice exhibited a better analgesic response to morphine than the WT mice. Western blotting and an immunohistological examination of the lumbar spinal cord tissue indicated less activation of glial cells and astrocytes in the HD mice compared with the WT mice. The production levels of tumor necrosis factor α and interleukine-1ß were also lower in the young HD mice. CONCLUSION: Our data demonstrate better morphine analgesic and less pain-related cytokine responses at the spinal cord level for HD mice. Further studies are needed to determine the morphine analgesia mechanism in HD.

Effectiveness of nalbuphine, a κ-opioid receptor agonist and µ-opioid receptor antagonist, in the inhibition of INa , IK(M) , and IK(erg) unlinked to interaction with opioid receptors.

Nalbuphine (NAL) is recognized as a mixer with the κ-opioid receptor agonist and the µ-opioid receptor antagonist. However, whether this drug causes any modifications in neuronal ionic currents is unclear. The effects of NAL on ionic currents in mHippoE-14 hippocampal neurons were investigated. In the whole-cell current recordings, NAL suppressed the peak amplitude of voltage-gated Na+ current (INa ) with an IC50 value of 1.9 µM. It shifted the steady-state inactivation curve of peak INa to the hyperpolarized potential, suggesting that there is the voltage dependence of NAL-mediated inhibition of peak INa . In continued presence of NAL, subsequent application of either dynorphin A1-13 (1 µM) or naloxone (30 µM) failed to modify its suppression of peak INa . Tefluthrin (Tef; 10 µM), a pyrethroid known to activate INa , increased peak INa with slowed current inactivation; however, further application of NAL suppressed Tef-mediated suppression of peak INa followed by an additional slowing of current inactivation. In addition, NAL suppressed the amplitude of M-type K+ current [IK(M) ] with an IC50 value of 5.7 µM, while it slightly suppressed erg-mediated and delayed-rectifier K+ currents. In the inside-out current recordings, NAL failed to modify the activity of large-conductance Ca2+ -activated K+ channels. In differentiated NG108-15 neuronal cells, NAL also suppressed the peak INa , and subsequent addition of Tef reversed NAL-induced suppression of INa . Our study highlights the evidence that in addition to modulate opioid receptors, NAL has the propensity to interfere with ionic currents including INa and IK(M) , thereby influencing the functional activities of central neurons.

Magnetic field distribution modulation of intrathecal delivered ketorolac iron-oxide nanoparticle conjugates produce excellent analgesia for chronic inflammatory pain.

BACKGROUND: Nanoparticles have become one of the most promising among the potential materials used for biomedical applications. However, few researchers have focused on their effects on analgesia. Despite the fact that various nanoparticles have been evaluated for drug delivery and MRI imaging contrast enhancement in clinical settings, no reports have investigated the in vivo synergy of ketorolac iron-oxide nanoparticle conjugates to improve the analgesic effect. METHODS: Ketorolac conjugated magnetic iron oxide nanoparticles (Keto-SPIO) were synthesized via two-stage additions of protective agents and chemical co-precipitation. ICR mice were used to develop inflammatory pain models induced by Complete Freund's adjuvant (CFA) injection in the hind paw. Different magnet field strengths and polarities were applied to the spinal cord after injecting Keto-SPIO into the theca space. Analgesia behavior was evaluated with the up-down method via von Frey microfilament measurement. Spinal cord tissues were harvested at the end analgesia time point upon induction of the inflammatory pain. The presence of the two cyclooxygenases (COX) in the spinal cord was examined via Western blotting to quantify the changes after intra-thecal Keto-SPIO administration. RESULTS: Intrathecal Keto-SPIO administration demonstrated a magnetic field-dependent analgesia effect in CFA pain model with a significant reduction in COX expression. CONCLUSIONS: Our results indicated that intrathecal administration of the Keto-SPIO combined magnet field modulated delivery significantly promoted an analgesia effect with suppression of COX in the mice inflammatory pain model.

Magnetic Resonance Classification System of Cervical Intervertebral Disk Degeneration: Its Validity and Meaning.

STUDY DESIGN: Retrospective analysis of kinetic magnetic resonance images (kMRIs). OBJECTIVE: (1) To analyze the changes seen on MRI related to disk degeneration and to develop a new grading system for cervical disk degeneration. (2) To evaluate the reliability and validity of the grading system. SUMMARY OF BACKGROUND DATA: Few have studied the relationship between changes seen on MRI with cervical disk degeneration and the chronological order of disk degeneration. A few grading systems for cervical disk degeneration have been reported; however, there have been problems related to subjectivity and lack of a clear, reliable algorithm. METHODS: A total of 300 cervical intervertebral disks were graded for nucleus color, structure, disk bulge, and disk height. On the basis of the analysis, a new grading system consisting of 4 grades (grade 0-III) and algorithm were developed. Intraobserver and interobserver reliabilities were assessed. A total of 2802 intervertebral disks were then evaluated using the grading system to correlate disk degeneration grades with patient age and function and to evaluate the validity of the new system. RESULTS: On the basis of cross-table analysis, disk degeneration presents in the following order: (1) decrease and/or change of nucleus intensity; (2) loss of distinction between nucleus and annulus; (3) positive disk bulge; and (4) disk height decrease. The κ-coefficients for intraobserver and interobserver agreements were 0.96 and 0.90, respectively. Severe disk degeneration is most common at C5/C6 followed by C6/C7 and C4/C5, and total disk degeneration grade is correlated with age (R=0.467). There was a decrease of angular motion in grades I-III and an increase in translational motion and decrease of space available for the cord in grades II-III. CONCLUSIONS: We developed a new classification system of cervical disk degeneration based on analysis of the changes seen on MRI. Reliability tests indicated high reproducibility of this system, and further analysis confirmed its validity and clinical significance.

Prevention of Chronic Post-Thoracotomy Pain in Rats By Intrathecal Resolvin D1 and D2: Effectiveness of Perioperative and Delayed Drug Delivery.

Thoracotomy results in a high frequency of chronic postoperative pain. Resolvins are endogenous molecules, synthesized and released by activated immune cells, effective against inflammatory and neuropathic pain. Different resolvins have differential actions on selective neuronal and glial receptors and enzymes. This article examines the ability of intrathecal resolvin D1 and resolvin D2 to reduce chronic post-thoracotomy pain in rats. Thoracotomy, involving intercostal incision and rib retraction, resulted in a decrease in the mechanical force threshold to induce nocifensive behavior, an enlargement of the pain-sensitive area, and an increase in the fraction of rats showing nocifensive behavior, all for at least 5 weeks. The qualitative nature of the behavioral responses to tactile stimulation changed dramatically after thoracotomy, including the appearance of vigorous behaviors, such as turning, shuddering, and squealing, all absent in naive rats. Intrathecal delivery of resolvin D1 (30 ng/30 µL), at surgery or 4 days later, halved the spread of the mechanosensitive area, lowered by 60% the percent of rats with tactile hypersensitivity, and reduced the drop in threshold for a nocifensive response, along with a reduction in the occurrence of vigorous nocifensive responses. Resolvin D2's actions on threshold changes were statistically the same. These findings suggest that intrathecal resolvins, delivered preoperatively or several days later, can prevent chronic postoperative hyperalgesia. PERSPECTIVE: In studies of rats, the injection of the proresolving compounds of the resolvin-D series into spinal fluid, before or just after thoracotomy surgery, prevents the occurrence of acute and chronic pain. If these chemicals, which have shown no side-effects, were used in humans it might greatly reduce chronic postoperative pain.

Relationship between visuospatial neglect and kinesthetic deficits after stroke.

BACKGROUND: After stroke, visuospatial and kinesthetic (sense of limb motion) deficits are common, occurring in approximately 30% and 60% of individuals, respectively. Although both types of deficits affect aspects of spatial processing necessary for daily function, few studies have investigated the relationship between these 2 deficits after stroke. OBJECTIVE: We aimed to characterize the relationship between visuospatial and kinesthetic deficits after stroke using the Behavioral Inattention Test (BIT) and a robotic measure of kinesthetic function. METHODS: Visuospatial attention (using the BIT) and kinesthesia (using robotics) were measured in 158 individuals an average of 18 days after stroke. In the kinesthetic matching task, the robot moved the participant's stroke-affected arm at a preset direction, speed, and magnitude. Participants mirror-matched the robotic movement with the less/unaffected arm as soon as they felt movement in their stroke affected arm. RESULTS: We found that participants with visuospatial inattention (neglect) had impaired kinesthesia 100% of the time, whereas only 59% of participants without neglect were impaired. For those without neglect, we observed that a higher percentage of participants with lower but passing BIT scores displayed impaired kinesthetic behavior (78%) compared with those participants who scored perfect or nearly perfect on the BIT (49%). CONCLUSIONS: The presence of visuospatial neglect after stroke is highly predictive of the presence of kinesthetic deficits. However, the presence of kinesthetic deficits does not necessarily always indicate the presence of visuospatial neglect. Our findings highlight the importance of assessment and treatment of kinesthetic deficits after stroke, especially in patients with visuospatial neglect.

Prolonged amelioration of experimental postoperative pain by bupivacaine released from microsphere-coated hernia mesh.

BACKGROUND AND OBJECTIVES: Postoperative pain alters physiological functions and delays discharge. Perioperative local anesthetics are effective analgesics in the immediate 1- to 2-day postoperative period, but acute pain often lasts longer. The goal of this work was to develop a local anesthetic formulation adhering to an intraoperative implanted device that reduces pain for at least 3 days after surgery. METHODS: Six groups, each with 8 rats, were studied. In a control group (group I), one 1.2-cm-long incision of the skin was followed by blunt dissection to separate the skin away from the underlying tissues and closing with 2 sutures. In 3 of the treatment groups, the same surgical procedure was used, with the subcutaneous space formed by the blunt dissection lined with a 1-cm square patch of hernia mesh coated with poly lactide co-glycolic acid microspheres containing approximately 17 mg of bupivacaine (group II), no drug (placebo; group III), or bupivacaine free-base powder (group IV). Uncoated mesh implants (group V) served as a secondary control. A standard bupivacaine solution (0.4 mL, 0.5%; 2-mg dose) was infiltrated subcutaneously 30 minutes before the surgery and served as a standard control (group VI). Mechanosensitivity of the skin was tested by the local subcutaneous muscle responses to cutaneous tactile stimulation by von Frey hairs with forces of 4 g (for allodynia) and 15 g (for hyperalgesia) preoperatively and for 7 postoperative days. RESULTS: Control rats (group I) showed mechanohypersensitivity, indicative of postoperative allodynia and hyperalgesia, for all 7 postoperative days. Mechanohyperalgesia in rats that received mesh coated with bupivacaine-releasing microspheres (group II) was reduced during this period to 13% of control postoperative values (P < 0.001); mesh coated with bupivacaine base (group IV) reduced it by 50% (P = 0.034). The placebo mesh (group III) and uncoated mesh (group V) caused no significant reduction of mechanohypersensitivity, and bupivacaine solution infiltrated before the incision (group VI) reduced hypersensitivity for only approximately 2 hours, an overall insignificant effect. CONCLUSIONS: Bupivacaine slowly released for 72 hours from microspheres adsorbed to the hernia mesh significantly suppresses evoked postoperative hypersensitivity for at least 1 week and is more effective than a suspension of these microspheres or preoperative single-shot infiltration of bupivacaine.

Is posterior fusion necessary with laminectomy in the cervical spine?

BACKGROUND: Cervical decompressive laminectomy is a common procedure for addressing multilevel cervical spine pathology. The most common reasons for performing simultaneous posterior cervical fusion include the prevention of progressive postlaminectomy kyphotic deformity or other types of instability which can contribute to late neurological deterioration. METHODS: The medical literature (Pub Med with MeSH) concerning cervical laminectomy, posterior cervical fusion, and complications of laminectomy/fusion was reviewed. Additionally, references from the articles were queried to find additional literature. RESULTS: Multiple studies concluded that cervical laminectomy versus laminectomy and fusion produced similar short-term postoperative outcomes. Careful patient selection was warranted to minimize the complications associated with cervical laminectomy alone; these included postoperative kyphosis (6-46%) and late deterioration (10-37%). The addition of a posterior cervical fusion was associated with relatively low complication rates, and avoided the evolution of late deformity or delayed neurological deterioration. CONCLUSION: Although the short-term results of cervical laminectomy versus laminectomy and fusion are similar, there appear to be more complications associated with performing laminectomy alone over the long term. Here, we reviewed the pros and cons of posterior cervical decompression alone versus decompression with fusion/instrumentation to treat cervical pathology, highlighting the complications associated with each surgical alternative.