Nociceptive scores and endorphin-containing cells reduced by low-level laser therapy (LLLT) in inflamed paws of Wistar rat

Objective: This study aimed to investigate how local pain relief is mediated by laser therapy and how dose affects the relationship. Methods: Inflammation was induced in the hind-paws of Wistar rats. Two groups of rats received 780-nm laser therapy (Spectra-Medics Pty Ltd.) at one of two doses (2.5 and 1 J/cm(2)). One group acted as a control. Scores of nociceptive threshold were recorded using paw pressure and paw thermal threshold measures. Results: A dose of 1 J/cm(2) had no statistically significant effect on antinociceptive responses. A dose of 2.5 J/cm(2) demonstrated a statistically significant effect on paw pressure threshold (p < 0.029) compared to controls. There was no difference in paw thermal threshold responses and paw volumes at either dose. Immunohistochemistry in control animals demonstrated normal beta-endorphin containing lymphocytes in control inflamed paws but no beta-endorphin containing lymphocytes in rats that received laser at 2.5 J/cm(2). Conclusion: The results confirm previous findings that the effect of laser therapy is dose-related. The mechanism of effect may occur via a differentiated pressure-sensitive neural pathway rather than a thermal-sensitive neural pathway. The significance of the immunohistochemistry findings remains unknown.

Mast cells and oral inflammation

Mast cells are mobile granule-containing secretory cells that are distributed preferentially about the microvascular endothelium in oral mucosa and dental pulp. The enzyme profile of mast cells in oral tissues resembles that of skin, with most mast cells expressing the serine proteases tryptase and chymase. Mast cells in oral tissues contain the pro-inflammatory cytokine tumour necrosis factor-alpha in their granules, and release of this promotes leukocyte infiltration during evolving inflammation in several conditions, including lichen planus, gingivitis, pulpitis, and periapical inflammation, through induction of endothelial-leukocyte adhesion molecules. Mast cell synthesis and release of other mediators exerts potent immunoregulatory effects on other cell types, while several T-lymphocyte-derived cytokines influence mast cell migration and mediator release. Mast cell proteases may contribute to alterations in basement membranes in inflammation in the oral cavity, such as the disruptions that allow cytotoxic lymphocytes to enter the epithelium in oral lichen planus. A close relationship exists among mast cells, neural elements, and laminin, and this explains the preferential distribution of mast cells in tissues. Mast cells are responsive to neuropeptides and, through their interaction with neural elements, form a neural immune network with Langerhans cells in mucosal tissues. This facilitates mast cell degranulation in response to a range of immunological and non-immunological stimuli. Because mast cells play a pivotal role in inflammation, therapies that target mast cell functions could have value in the treatment of chronic inflammatory disorders in the oral cavity.

Neointimal formation by circulating bone marrow cells

The origin of smooth muscle cells involved in vascular healing was examined. Eighteen C57BL/6 (Ly 5.2) female mice underwent whole body irradiation followed by transfusion with 10(6) bone nucleated marrow cells from congenic (Ly 5.1) male donors. Successful repopulation by donor marrow was demonstrated after 4 weeks by flow cytometry with FITC-conjugated A20.1/Ly 5.1 monoclonal antibody. The iliac artery of six of the chimeric mice was scratch-injured by five passes of a probe, causing severe medial damage. After 4 weeks the arterial lumen was obliterated by a cell-rich neointima, with alpha-smooth muscle actin-containing cells present around the residual lumen. Approximately half of these cells were of male donor origin, as evidenced by in situ hybridization with a Y chromosome-specific probe. An organized arterial thrombus was formed in the remaining 12 chimeric mice by inserting an 8.0 silk suture into the left common carotid artery. Donor cells staining with alpha-smooth muscle actin were found in those arteries sustaining serious damage but not in arteries with minimal damage. Our results suggest that bone marrow-derived cells are recruited in vascular healing as a complementary source of smooth muscle-like cells when the media is severely damaged and few resident smooth muscle cells are available to effect repair.

Dendritic branching patterns of pyramidal cells in the visual cortex of the new world marmoset monkey, with comparative notes on the old world macaque monkey

The basal dendritic arbors of 442 supragranular pyramidal cells in visual cortex of the marmoset monkey were compared by fractal analyses. As detailed in a previous study,(1) individual cells were injected with Lucifer Yellow and processed for a DAB reaction product. The basal dendritic arbors were drawn, in the tangential plane, and the fractal dimension (D) determined by the dilation method. The fractal dimensions were compared between cells in ten cortical areas containing cells involved in visual processing, including the primary visual area (Vi), the second visual area (V2), the dorsoanterior area (DA), the dorsomedial area (DM), the dorsolateral. area (DL), the middle temporal area (MT), the posterior parietal area (PP), the fundus of the superior temporal area (FST) and the caudal and rostral subdivisions of inferotemporal cortex (ITc and ITr, respectively). Of 45 pairwise interareal comparisons of the fractal dimension of neurones, 20 were significantly different. Moreover, comparison of data according to previously published visual processing pathways revealed a trend for cells with greater fractal dimensions in higher cortical areas. Comparison of the present results with those in homologous cortical areas in the macaque monkey(2) revealed some similarities between the two species. The similarity in the trends of D values of cells in both species may reflect developmental features which, result in different functional attributes.

Immune-derived opioids and peripheral antinociception

1. Recent findings have suggested a significant involvement of the immune system in the control of pain. Immune cells contain opioid peptides that are released within inflamed tissue and act at opioid receptors on peripheral sensory nerve endings. It is also apparent that different types of lymphocytes contain P-endorphin, memory T cells containing more beta -endorphin than naive cells. 2. These findings highlight an integral link between immune cell migration and inflammatory pain, The present review highlights immune system involvement in the site-directed control of inflammatory pain. 3. Full-length mRNA transcripts for opioid precursor proteins are expressed in immune cells. Increased expression of pro-opiomelanocortin mRNA and beta -endorphin has been demonstrated in stimulated lymphocytes and lymphocytes from animals with inflammation. 4. Cytokines and corticotropin-releasing factor (CRF) release opioids from immune cells, Potent peripheral analgesia due to direct injection of CRF can be blocked by antagonists to CRF, antibodies to opioid peptides, antisense to CRF and opioid receptor-specific antagonists. The release of opioid peptides from lymphocytes is calcium dependent and opioid receptor specific. Furthermore, endogenous sources of opioid peptides produce potent analgesia when implanted into the spinal cord. 5. Activated immune cells migrate directly to inflamed tissue using cell adhesion molecules to adhere to the epithelial surface of the vasculature in inflamed tissue. Lymphocytes that have been activated can express opioid peptides, Memory type T cells that contain opioid peptides are present within inflamed tissue; naive cells are not present in inflamed tissue and do not contain opioid peptides, Inhibiting the migration of memory type T cells into inflamed tissue by blocking selectins results in reduced numbers of beta -endorphin containing cells, a reduced quantity of beta -endorphin in inflamed paws and reduced stress- and CRF-induced peripheral analgesia. 6. Immunosuppression is associated with increased pain in patients. Moreover, immunosuppression results in decreased lymphocyte numbers as well as decreased analgesia in animal models.

The role of insect cell lines in an artificial diet for the parasitold wasp, Trichogramma pretiosum

Trichogramma species are mass-produced for biological control using host eggs. Artificial diets have been developed to reduce production costs, however, most include insect haemolymph as a major component, which still results in a significant expense. Medium conditioned with insect cell lines has produced some success as a haemolymph replacement in artificial diets for several parasitoid wasp species. Trichogramma australicum Girault (Hymenoptera: Trichogrammatidae) was the first species to develop successfully to the adult stage on diets containing concentrated HeliothiS zea (Boddie) (Lepidoptera: Noctuidae) cells. Tricho-gramma pretiosum Riley (Hymenoptera: Trichogrammatidae) was subsequently grown to the adult stage on a similar cell line diet. This success encouraged a systematic investigation into the use of insect cell lines in Trichogramma artificial diets. We compared the effect of diets containing insect cells with diets containing conditioned cell line media. Diets containing insect cells produced significantly more pupae than diets containing conditioned medium and, although not significant, produced a higher number of adults. Second, we compared the effect of diets containing cell lines established from ovary-associated tissue of H. zea and embryo tissue of Aedes albopictus (Skuse) (Diptera: Culicidae) on T pretiosum development. Trichogramma pretiosum development was not significantly different on diets containing cells from the two origins and tissue types. Third, the effect of cell storage on T pretiosum development was observed. HeliothiS zea cells in medium were stored at 4 degrees C and room temperature (22 degrees C for one, two, four and seven days before addition to artificial diets. Cell viability was calculated for these storage treatments. HeliothiS zea cells could be stored at 4 degrees C for up to seven days with no detrimental effect on T pretiosum development. Tricho-gramma pretiosum development did not depend on cell viability. The use of insect cell lines as a haemolymph replacement has the potential to significantly reduce production costs and simplify Trichogramma artificial diets with the eventual aim of replacing host production in mass rearing facilities. (c) 2005 Elsevier Inc. All rights reserved.

Effects of alcohol exposure during early life on neuron numbers in the rat hippocampus. I. Hilus neurons and granule cells

We previously showed that 16-day-old rats exposed to a relatively high dose of ethanol at 10-15 postnatal days of age have fewer neurons in the hilus region of the hippocampus compared with controls. Dentate gyrus granule cell numbers, however, showed no statistically significant changes attributable to the ethanol treatment. It is possible that some of the changes in brain morphology, brought about as a result of the exposure to ethanol during early life, may not be manifested until later in life. This question has been further addressed in an extension to our previous study. Wistar rats were exposed to a relatively high daily dose of ethanol on postnatal days 10-15 by placement in a chamber containing ethanol vapour, for 3 h/day. The blood ethanol concentration was found to be similar to430 mg/dl at the end of the period of exposure. Groups of ethanol-treated (ET), separation control (SC), and mother-reared control (MRC) rats were anaesthetised and killed either at 16 or 30 days of age by perfusion with phosphate-buffered 2.5% glutaraldehyde. The Cavalieri principle and the physical disector methods were used to estimate, respectively, the regional volumes and neuron cell numerical densities in the hilus and granule cell regions of the dentate gyrus. The total numbers of neurons in the hilus region and granule cell layer were computed from these estimates. It was found that 16-day-old animals had 398,000-441,000 granule cells, irrespective of group. The numbers of granule cells increased such that by 30 days of age, rats had 487,000-525,500 granule cells. However, there were no significant differences between ethanol-treated rats and their age-matched controls in granule cell numbers. In contrast, ethanol-treated rats had slightly but significantly fewer neurons in the hilus region than did control animals at 16 days of age, but not at 30 days of age. Therefore, it appears that a short period of ethanol exposure during early life can have effects on neuron numbers of some hippocampal neurons, but not others. The effects on hilar neuron numbers, observed as a result of such short periods of ethanol treatment, appeared to be transitory. (C) 2003 Wiley-Liss, Inc.

Activation of the peroxisome proliferator-activated receptor-alpha enhances cell death in cultured cerebellar granule cells

Peroxisome proliferator-activated receptor-alpha (PPAR alpha) is a member of the steroid hormone receptor superfamily. In rodents, PPAR alpha. alters genes involved in cell cycle regulation in hepatocytes. Some of these genes are implicated in neuronal cell death. Therefore, in this study, we examined the toxicological consequence of PPAR alpha activation in rat primary cultures of cerebellar granule neurons. Our studies demonstrated the presence of PPAR alpha mRNA in cultures by reverse transcriptase-polymerase chain reaction. After 10 days in vitro, cerebellar granule neuron cultures were incubated with the selective PPAR alpha activator 4-chloro-6-(2,3-xylidino)2-pyrimidinylthioacetic acid (Wy-14,643). The inherent toxicity of Wy-14,643 and the effect of PPAR alpha activation following toxic stimuli were assessed. In these studies, neurotoxicity was induced through reduction of extracellular [KCl] from 25 mM to 5.36 mM. We observed no inherent toxicity of Wy-1 4,643 (24 hr) in cultured cerebellar granule cells. However, after reduction of [KCl], cerebellar granule cell cultures incubated with Wy-14,643 showed significantly greater toxicity than controls. These results suggest a posssible role for PPAR(x in augmentation of cerebellar granule neuronal death after toxic stimuli. (C) 2001 Wiley-Liss, Inc.

Reduction of β-Endorphin-Containing Immune Cells in Inflamed Paw Tissue Corresponds with a Reduction in Immune-Derived Antinociception: Reversible by Donor Activated Lymphocytes

The functional integrity of the immune system is essential for peripheral antinociception. Previous studies have demonstrated that immune cells elicit potent antinociception in inflamed tissues and that corticotropin-releasing factor-induced antinociception is significantly inhibited in animals that have undergone cyclosporin A (CsA)-induced immunosuppression. In this study, we examined the effect of a single bolus of CsA on inflammatory nociception. CsA-treated rats had substantially increased nociception compared with nonimmunosuppressed rats, consistent with a reduction in circulating and infiltrating lymphocytes. Furthermore, CsA-treated rats had inhibition of corticotropin-releasing factor-induced immune-derived antinociception, which was dose-dependently reversed by IV injection of concanavalin A-activated donor lymphocytes (1.0-7.0 X 10(6) cells/0.1 mL). In conclusion, our findings provided further evidence that opioid-containing immune cells are essential for peripheral analgesia. It is evident from these findings that control of inflammatory pain relies heavily on a functioning immune system.

Liquid distribution as a means to describing the granule growth mechanism

Copper concentrate (chalcopyrite) was granulated in a rotating drum with a diameter of 0.3 m and a length of 0.2 m. Water was used as the binder and it was sprayed onto the powder bed with a nozzle. This material exhibited induction type behaviour, which was defined by Iveson and Litster [AIChE J. 44 (1998) 1510]. Induction type behaviour is characterized by the occurrence of an induction stage, during which the granules are gradually being compacted and little or no growth occurs. At the end of this induction stage, binder liquid is squeezed from the interior of the granules onto the granule surface and the granules are then surface-wet. This results in a rapid growth rate of the granules. Different types of experiments were conducted. The influence of the nozzle pressure and the distance from the nozzle to the powder bed on the growth behaviour of the granules as well as on the binder distribution was examined. The results of these experiments led to the postulation of a modified mechanism for induction type behaviour: it was found that after the binder was delivered, there were large granules containing a high amount of binder and small granules containing less binder. During the induction stage, the granules are compacted and binder liquid continuously appears at the surface of the large granules. These wet spots that are continuously being formed pick up the dry and small granules. When all the small granules have been picked up, further expulsion of binder liquid onto the granules' surface results in granules that remain surface-wet. This phenomenon marks the end of the induction stage and it coincides with the disappearance of the small granules. The hypothesis was tested by selectively removing the smaller granules during an experiment. As expected, this resulted in a shorter induction time.

Dendritic cells in rheumatoid arthritis - A model autoimmune inflammatory site

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease in which unknown arthrogenic autoantigen is presented to CD4+ T cells. The strong association of the disease with an epitope within the HLA-DR chain shared between various alleles of HLA-DR4 and DR1 emphasizes the importance of antigen presentation. This immune response predominantly occurs in the synovial tissue and fluid of the joints and autoreactive T cells are readily demonstrable in both the synovial compartment and blood. Circulating dendritic cells (DC) are phenotypically and functionally identical with normal peripheral blood (PB) DC. In the synovial tissue, fully differentiated perivascular DC are found in close association with T cells and with B cell follicles, sometimes containing follicular DC. These perivascular DC migrate across the activated endothelium from blood and receive differentiative signals within the joint from monocyte-derived cytokines and CD40-ligand+ T cells. In the SF, DC manifest an intermediate phenotype, similar to that of monocyte-derived DC in vitro. Like a delayed-type hypersensitivity response, the rheumatoid synovium represents an effector site. DC at many effector sites have a characteristic pattern of infiltration and differentiation. It is important to note that the effector response is not self-limiting in RA autoimmune inflammation. In this article, we argue that the presentation of self-antigen by DC and by autoantibody-producing B cells is critical for the perpetuation of the autoimmune response. Permanently arresting this ongoing immune response with either pharmaceutical agents or immunotherapy is a major challenge for immunology.

Gene transfer and expression of a non-viral polycation-based vector in CD4(+) cells

CD4-selective targeting of an antibody-polycation-DNA complex was investigated The complex was synthesized with the anti-CD4 monoclonal antibody B-F5, polylysine(268) (pLL) and either the pGL3 control vector containing the luciferase reporter gene or the pGeneGrip vector containing the green fluorescent protein (GFP) gene. B-F5-pLL-DNA complexes inhibited the binding of I-125-B-F5 to CD4(+) Jurkat cells, while complexes synthesised either without B-F5 or using a non-specific mouse IgG1 antibody had little or no effect Expression of the luciferase reporter gene was achieved in Jurkat cells using the B-F5-pLL-pGL3 complex and was enhanced in the presence of PMA. Negligible luciferase activity was defected with the non-specific antibody complex in Jurkat cells or with the B-F5-pLL-pGL3 complex in the CD4(-) K-562 cells. Using complexes synthesised with the pGeneGrip vector, the transfection efficiency in Jurkat and K-562 cells was examined using confocal microscopy. More than 95% of Jurkat cells expressed GFP and the level of this expression was markedly enhanced by PMA. Negligible GFP expression was seen in K-562 cells or when B-F5 was replaced by a nonspecific antibody. Using flow cytometry, fluorescein-labelled complex showed specific targeting to CD4(+) cells in a mixed cell population from human peripheral blood. These studies demonstrate the selective transfection of CD4(+) T-lymphoid cells using a polycation-based gene delivery system. The complex may provide a means of delivering anti-HIV gene therapies to CD4(+) cells in vivo.

Differentiated dendritic cells expressing nuclear RelB are predominantly located in rheumatoid synovial tissue perivascular mononuclear cell aggregates

Objective. Differentiated dendritic cells (DC) and other antigen-presenting cells are characterized by the nuclear location of RelB, a member of the nuclear factor kappa B/Rel family. To characterize and enumerate differentiated DC in rheumatoid arthritis (RA) peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST), the expression and location of RelB were examined. Methods. RelB protein expression and cellular location were determined in RA PB, SF, and ST by flow cytometry and immunohistochemical analysis of purified cells or formalin-fixed tissue. DNA-binding activity of RelB was determined by electrophoretic: mobility shift-Western immunoblotting assays. Results. Circulating RA PBDC resembled normal immature PBDC in that they did not express intracellular RelB protein. In RA ST serial sections, cells containing nuclear RelB (nRelB) were enriched in perivascular regions. A mean +/- SD of 84 +/- 10% of these cells were DC. The remaining nRelB+,HLA-DR+ cells comprised B cells and macrophages. Only 3% of sorted SFDC contained nRelB, However, RelB present in the nucleus of these SFDC was capable of binding DNA, and therefore capable of transcriptional activity. Conclusion. Circulating DC precursors differentiate and express RelB after entry into rheumatoid ST. Differentiated DC can thus be identified by immunohistochemistry in formalin-fixed ST. Signals for DC maturation may differ between RA ST and SF, resulting in nuclear location of RelB predominantly in ST. This is likely to have functional consequences for the DC in these sites.