Three-step isolation of human blood monocytes using discontinuous density gradients of Percoll

A three step method for the purification of normal human blood monocytes is described. The procedure consists of a combination of dextran sedimentation, Ficoll-Isopaque (F-I) centrifugation and isopycnic centrifugation on discontinuous gradients of Percoll. No selective loss of monocytes was observed after the first step, and after F-I centrifugation mononuclear cells (MNC) were obtained, of which 20 +/- 6% were monocytes. The MNC were further separated on hyper-osmotic and iso-osmotic discontinuous density gradients of Percoll. The best purification of monocytes occurred on hyper-osmotic density gradients and the density interface between 1.074 and 1.066 g/ml yielded 85 +/- 7% monocytes, 13 +/- 7% lymphocytes and 1 +/- 1% granulocytes. 77 +/- 16% of the monocytes obtained after F-I centrifugation, were recovered in this interface. The purified monocytes were viable and retained their capacity to mature into macrophages. The whole procedure takes about 5 h, is reproducible and can be applied to small and large volumes (500 ml) of blood.     

Stimulation of Intestinal Mucosal Afferent Nerves Increases Superior Mesenteric Artery and Decreases Mesenteric Adipose Tissue Blood Flow

We tested the hypothesis that stimulation of intestinal mucosal afferent nerves produces an increase in superior mesenteric artery (SMA) but a decrease in mesenteric adipose tissue (MAT) blood flow. In anesthetized rats, blood flow in the SMA (pulsed Doppler flowmetry) and MAT (hydrogen gas clearance) was measured simultaneously before and after administration of 0.9% saline, 640 M capsaicin, or 5% dextrose into the intestinal lumen. The changes in the SMA were 3.8 ± 3.0, 15.9 ± 4.0, and 18.8 ± 7.6%; and those in the MAT, 4.7 ± 4.0, –11.5 ± 3.4, and –0.07 ± 3.4% of baseline, respectively. The data indicate that exposure of the intestinal lumen to an afferent nerve stimulant or nutrient induced a dichotomous pattern of blood flow changes, an increase in the SMA and a reduction in MAT. The capsaicin-sensitive afferent nerves may be instrumental in mediating these energy responses.     

Treatment of Temporomandibular Dysfunction With Hypertonic Dextrose Injection (Prolotherapy): A Randomized Controlled Trial With Long-term Partial Crossover

ObjectiveTo assess the efficacy and longer-term effectiveness of dextrose prolotherapy injections in participants with temporomandibular dysfunction.Patients and MethodsA randomized controlled trial with masked allocation was conducted from January 14, 2013, through December 19, 2015. Forty-two participants (with 54 joints) meeting temporomandibular dysfunction criteria were randomized (1:1) to 3 monthly intra-articular injections (20% dextrose/0.2% lidocaine or 0.2% lidocaine) followed by as-needed dextrose/0.2% lidocaine injections through 1 year. Primary and secondary outcome measures included a 0 to 10 Numerical Rating Scale score for facial pain and jaw dysfunction; maximal interincisal opening (MIO) measured in millimeters, percentage of joints with 50% or more change (improvement) in pain and function, and satisfaction.ResultsRandomization produced a control group with more female participants (P=.03), longer pain duration (P=.01), and less MIO (P=.01). Upon 3-month analysis, including pertinent covariates, dextrose group participants reported decreased jaw pain (4.3±2.9 points vs 1.8±2.7 points; P=.02), jaw dysfunction (3.5±2.8 points vs 1.0±2.1 points; P=.008), and improved MIO (1.5±4.1 mm vs ?1.8±5.1 mm; P=.006). Control group participants received dextrose injections beginning at 3 months. No between-group differences were noted at 12 months; pooled data suggested that jaw pain, jaw function, and MIO improved by 5.2±2.7 points (68%), 4.1±2.8 points (64%), and 2.1±5.5 mm, respectively. Pain and dysfunction improved by at least 50% in 38 of 54 (70%) and 39 of 54 (72%) jaws, respectively.ConclusionIntra-articular dextrose injection (prolotherapy) resulted in substantial improvement in jaw pain, function, and MIO compared with masked control injection at 3 months; clinical improvements endured to 12 months. Satisfaction was high.Trial Registrationclinicaltrials.gov Identifier: NCT01706172     

Comparison of perineural platelet‐rich plasma and dextrose injections for moderate carpal tunnel syndrome: A prospective randomized,single‐blind,head‐to‐head comparative trial

Recent studies demonstrated the utility of perineural injection with platelet‐rich plasma (PRP) and 5% dextrose (D5W) as novel strategies for treatment of carpal tunnel syndrome (CTS). The present study comprised a prospective, randomized, single‐blind, head‐to head comparative trial to compare the 6‐month outcome of perineural injection with PRP or D5W in patients with moderate CTS. Fifty‐two patients with unilateral moderate CTS were enrolled and randomized into two groups: The PRP group received a single 3‐cc perineural injection of PRP under ultrasound guidance, and dextrose group received a single 3‐cc perineural injection of D5W under ultrasound guidance. The Boston Carpal Tunnel Syndrome Questionnaire score was used as the primary outcome. Secondary outcomes included cross‐sectional area (CSA) of the median nerve and electrophysiological assessments. Evaluations were performed at baseline and at 1, 3, and 6 months postinjection. All patients (26 patients per group) completed the study. Compared with the dextrose group, the PRP group demonstrated significant reductions in Boston Carpal Tunnel Syndrome Questionnaire function at 3 months (p = .044), distal motor latency at 6 months (p = .028), and CSA at 3 and 6 months (p = .010 and.018, respectively). A single perineural injection of PRP reduced the CSA of the median nerve more effectively than injection of D5W at 3 and 6 months postinjection for patients with moderate CTS.     

Early inflammatory response of knee ligaments to prolotherapy in a rat model

Prolotherapy is an alternative injection‐based therapy for chronic musculoskeletal pain. Three different proliferants, D ‐glucose (dextrose), phenol‐glucose‐glycerine (P2G), and sodium morrhuate, used in prolotherapy are hypothesized to strengthen and reorganize chronically injured soft tissue and decrease pain through modulation of the inflammatory process. Our hypothesis is that commonly used prolotherapy solutions will induce inflammation (leukocyte and macrophage infiltration) in medial collateral ligaments (MCLs) compared to needlestick, saline injection, and no‐injection controls. MCLs of 84 Sprague‐ Dawley rats were injected one time at both the tibial and femoral insertions. Immunohistochemistry (IHC) was used to determine the inflammatory response at three locations (tibial and femoral insertions and midsubstance) 6, 24, and 72 h after dextrose injection compared to saline‐ and no‐injection controls and collagenase (positive control) (n = 4). qPCR was used to analyze gene expression 24 h postinjection (n = 4). Sodium morrhuate, P2G, and needlestick control were also investigated after 24 h (n = 4). In general, inflammation (CD43+, ED1+, and ED2+ cells) increased after prolotherapy injection compared to no‐injection control but did not increase consistently compared to saline and needlestick control injections. This response varied by both location and proliferant. Inflammation was observed at 6 and 24 h postinjection but was resolved by 72 h compared to no‐injection controls (p < 0.05). CD43+ leukocytes and ED2+ macrophages increased compared to needlestick and saline‐injection control, respectively, 24 h postinjection (p < 0.05). Prolotherapy injections created an inflammatory response, but this response was variable and overall, not uniformly different from that caused by saline injections or needlestick procedures. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:816–823, 2008     

Honey ameliorates influence of hemorrhage and food restriction on renal and hepatic functions,and hematological and biochemical variables

The objectives were to assess the effects of various diets, including total food restriction with 50% honey feeding, total food restriction with 50% dextrose feeding or adlibitum (control group) commercial regular diet, on the hematology and biochemical variables, and to assess the effects of the various diets on the influence of acute blood loss on the same parameters. Thirty Sprague–Dawley albino rats were divided into three groups, 10 rats each: group A, fed a commercial regular diet; group B, total food restriction with 50% dextrose feeding; and group C, total food restriction with 50% honey feeding. After 8 days of feeding, rats were subjected to acute blood loss (6 ml/kg) and blood investigations were performed. After acute blood loss, the same feedings were continued for a further 8 days and the blood tests were repeated at day 8 post-bleeding. Total food restriction with 50% dextrose feeding compared with commercial regular diet reduces hematological and biochemical variables. Total food restriction with 50% honey feeding compared with total food restriction with 50% dextrose feeding causes a greater reduction in fasting blood glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and triacylglycerol. Acute blood loss causes elevation of white blood cells, lymphocyte percentage, fasting blood sugar, blood urea nitrogen, alkaline phosphatase and triacylglycerol, and a reduction in serum albumen, protein, cholesterol, AST, serum creatinine and hemoglobin; the results are significant (P<0.05) concerning fasting blood glucose, AST, alkaline phosphatase, serum albumin and protein. A significant reduction in fasting blood glucose, white blood cells, BUN, AST, ALT, alkaline phosphatase and triacylglycerol, and a significant elevation of hemoglobin and serum albumin are obtained after acute blood loss in rats on total food restriction with 50% honey feeding as compared with the other two groups. Total food restriction with 50% honey feeding increases serum albumin, serum protein, fasting blood glucose, and causes lower reduction in hemoglobin as compared with the other groups. Conclusively, honey feeding during total food restriction significantly modifies and ameliorates biochemical and hematological changes observed after acute blood loss. This will pave the way to use honey as part of bleeding management and during a food restriction regimen.     

Variations of the phosphorylation of 1-β-d-arabinofuranosylcytosine (ara-C) in human myeloid leukemic cells related to the cell cycle

Bone marrow cells of five patients with acute myeloid leukemia were fractionated by means of counterflow centrifugation (elutriation). The different fractions were enriched with cells belonging to subsequent stages of the cell cycle. Cytokinetic evaluation of these cell fractions was performed by [³H]thymidine autoradiography, [³H]thymidine incorporation and DNA/RNA-flow cytometry.Phosphorylation of cytosine arabinoside (ara-C, 1-β-d-arabinofuranosylcytosine) in the different fractions was measured by incubation of the cells for 30 min with 1.07 μM [³H]ara-C. Phosphorylation of ara-C in the whole bone marrow samples ranged from 5.9 to 33.2 pmol/10⁶ cells. In the fractions containing only G₁-phase cells, phosphorylation ranged from 1.2 to 19.5 pmol/10⁶ cells. The phosphorylation seems to increase before DNA synthesis starts. Maximal activities were found in the fractions enriched with cells in late G₁- or S-phase of the cell cycle. In these fractions the ara-C phosphorylating activity was 1.5–8 times higher compared to the fractions with the lowest activity.One may therefore assume that not only S-phase cells are killed by ara-C, but that G₁-phase cells which can phosphorylate ara-C, may also be doomed when they enter S-phase, since the elimination of the intracellular cytosine arabinoside tri-phosphate (ara-CTP) is a relatively slow process. The fraction of G₁-phase cells phosphorylating ara-C, may be an important determinant in the extent of the cell-killing effect of ara-C treatment in the different leukemias.     

Transport Refusal by Hypoglycemic Patients after On-scene Intravenous Dextrose

OBJECTIVES: Administration of intravenous (IV) dextrose to hypoglycemic patients is delegated to advanced care paramedics in Ontario. Following a quality assurance review, which revealed that 47% of patients refused transport after receiving IV dextrose, the authors studied whether such patients seek additional medical care in the three days following the initial refusal. METHODS: Sequential ambulance call reports for on-scene treatments of hypoglycemia were examined, and a standardized telephone survey of the patients was conducted. Patient satisfaction was assessed using a five-point Likert scale. Data were collected from April 1999 to March 2000. RESULTS: One hundred patients were studied, with ages ranging from 20 to 92 years (mean 53.2 years). The average Glasgow Coma Scale (GCS) score on presentation was 8.7 +/- 3.5. The average blood glucose level before administration of IV dextrose was 1.91 +/- 0.63 mmol/L. Sixty-eight percent of the patients refused transport. Significant differences between the transported group and the refusal group were age (transported 64.7 years, refused 47.8 years, p = 0.002) and initial blood glucose (transported 1.8, refused 2.1, p = 0.001). No difference was found in terms of repeat access to health care for related complaints. Patient satisfaction was high in both groups, with no difference in the overall satisfaction with paramedics' care (4.76 +/- 0.58 vs 4.75 +/- 0.45). CONCLUSIONS: The practice of treating patients for symptomatic hypoglycemia and leaving them at the scene appears to be safe. Further study is required to confirm this.