Modulation of cell migration and cell tracking of the gilthead seabream (Sparus aurata) SAF-1 cells by probiotics.

Cell migration is an essential process in immunity and wound healing. The in vitro scratch assay was optimized for the SAF-1 cell line, obtained from gilthead seabream (Sparus aurata) fin. In addition, selected cells from the cell front were tracked for detailed individual cell movement and morphological analysis. Modulation of migration and cell tracking of the SAF-1 cell line by probiotics was evaluated. Cells were cultured and incubated for 24 h with three species of extremophilic yeasts [Yarrowia lipolytica (D1 and N6) and Debaryomyces hansenii (CBS004)] and the bacterium Shewanella putrefaciens (known as SpPdp11) and then scratch and cell tracking assays were performed. The results indicated that the forward velocity was significantly (p < 0.05) increased in SAF-1 cells incubated with CBS004 or SpPdp11. However, cell velocity, cumulative distance and Euclidean distance were only significantly increased in SAF-1 cells incubated with SpPdp11. Furthermore, to increase our understanding of the genes involved in cell movement, the expression profile of ten structural proteins (α-1ß tubulin, vinculin, focal adhesion kinase type, alpha-2 integrin, tetraspanin, integrin-linked kinase 1, tensin 3, tensin 4, paxillin, and light chain 2) was studied by real time-PCR. The expression of these genes was modulated as a function of the probiotic tested and the results indicate that CBS004 and SpPdp11 increase the movement of SAF-1 cells.

Adhesion of the ulcerative pathogen Mycobacterium ulcerans to DACC-coated dressings.

OBJECTIVE: Mycobacterium ulcerans is the causative agent of Buruli ulcer disease, the third most common mycobacteriosis after tuberculosis and leprosy and an emerging public health threat in sub-Saharan Africa. The bacteria produce a diffusible cytotoxin called mycolactone, which triggers the formation of necrotic lesions in cutaneous and subcutaneous tissues. The principal aim of this study was to characterise the cell surface hydrophobicity of Mycobacterium ulcerans and determine if bacteria bind to dialkyl carbamoyl chloride (DACC)-coated dressings through hydrophobic interactions in vitro. Since mycolactone displays hydrophobic groups, a secondary aim was to compare mycolactone binding to hydrophobic and standard dressings. METHODS: We used hydrophobic interaction chromatography to evaluate the cell surface hydrophobicity of Mycobacterium ulcerans, compared to that of other microorganisms colonising wounds. The binding of Mycobacterium ulcerans bacteria to DACC-coated and control dressings was then assessed quantitatively by measurement of microbial adenosine triphosphate (ATP), while that of mycolactone was evaluated by fluorescence spectroscopy. RESULTS: Compared to Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, Mycobacterium ulcerans displayed the highest cell surface hydrophobicity, irrespective of the bacterial production of mycolactone. Mycobacterium ulcerans bacteria bound to DACC-coated dressings [corrected] better than untreated controls. Mycolactone did not bind stably to hydrophobic, nor standard dressings, in the conditions tested. CONCLUSION: Retention of Mycobacterium ulcerans and other wound pathogens to DACC-coated dressings may help reduce the bacterial load in Buruli ulcers and thereby improve healing. Dressings efficiently capturing mycolactone may bring an additional clinical benefit, by accelerating the elimination of the toxin during the course of antibiotic treatment.

Reprogramming adult Schwann cells to stem cell-like cells by leprosy bacilli promotes dissemination of infection.

Differentiated cells possess a remarkable genomic plasticity that can be manipulated to reverse or change developmental commitments. Here, we show that the leprosy bacterium hijacks this property to reprogram adult Schwann cells, its preferred host niche, to a stage of progenitor/stem-like cells (pSLC) of mesenchymal trait by downregulating Schwann cell lineage/differentiation-associated genes and upregulating genes mostly of mesoderm development. Reprogramming accompanies epigenetic changes and renders infected cells highly plastic, migratory, and immunomodulatory. We provide evidence that acquisition of these properties by pSLC promotes bacterial spread by two distinct mechanisms: direct differentiation to mesenchymal tissues, including skeletal and smooth muscles, and formation of granuloma-like structures and subsequent release of bacteria-laden macrophages. These findings support a model of host cell reprogramming in which a bacterial pathogen uses the plasticity of its cellular niche for promoting dissemination of infection and provide an unexpected link between cellular reprogramming and host-pathogen interaction.

Oxidative stress and leukocyte migration inhibition response in cutaneous adverse drug reactions.

BACKGROUND: Cutaneous adverse drug reactions (CADRs) may either be immunological or non-immunological. The precise mechanisms, however, are largely obscure. Other concomitant mechanisms may amplify and/or contribute to the severity and duration of a reaction. One such mechanism could be oxidative stress, a state of imbalance between reactive oxygen species, and their subsequent detoxification by antioxidants. AIMS: (a) to assess the oxidative stress status in the blood of cutaneous drug reaction patients by assaying for reduced glutathione (GSH) and malondialdehyde (MDA) levels, (b) to determine the leukocyte migration inhibition (LMI) response in these patients in response to the suspected drug (s), and (c) to look for the association between oxidative stress parameters and LMI. METHODS: Ethical committee approval was obtained for this study. Fresh venous blood samples were obtained from the patients of CADRs (group A) during the acute phase of reaction and healthy control subjects (group B). MDA levels, a measure of oxidative lipid damage, and reduced GSH levels, a measure of anti-oxidant capacity, were assayed in the blood samples of both groups using spectrophotometry. LMI response was measured by challenging the patients' peripheral blood mononuclear cells with the suspected drug to confirm immunological perturbation. RESULTS: Totally 66 participants, 33 cases in group A and equal number of controls in group B, were studied. The mean MDA levels were found to be raised (P < 0.001), but GSH levels were significantly reduced in group A when compared with group B (P = <0.001). LMI response against drug(s) was performed in 33 cases (group A), out of which 25 cases showed a positive LMI response as follows: fixed drug eruption (10/25), SJS (5/25), urticaria (3/25), exfoliative dermatitis (2/25), morbilliform rash (2/25), erythroderma (1/25), vasculitis (1/25), and dapsone syndrome (1/25). The mean MDA levels were found to be significantly higher in the LMI positive CADRs (P < 0.001) when compared with LMI-negative ones, while no significant difference was seen for GSH (P = 0.100). Furthermore, there was a significant positive correlation between MDA levels and LMI response (r = 0.831, P < 0.001). On the other hand, a negative but statistically insignificant correlation was found between GSH and LMI response (r = -0.248, P = 0.271). CONCLUSION: CADR patients were found to be under oxidative stress based on MDA and GSH levels in the peripheral blood. There is a significant positive correlation of LMI response (against the causative drug) with MDA levels, which strongly associates oxidative stress with the immunopathogenesis in CADRs.

Reductions in laminin beta2 mRNA translation are responsible for impaired IGFBP-5-mediated mesangial cell migration in the presence of high glucose.

Insulin-like growth factor binding protein-5 (IGFBP-5) mediates mesangial cell migration through activation of cdc42, and laminin421 binding to alpha(6)beta(1)-integrin (Berfield AK, Hansen KM, Abrass CK. Am J Physiol Cell Physiol 291: C589-C599, 2006). Because glomerular expression of laminin beta(2) is reduced in diabetic rats (Abrass CK, Spicer D, Berfield AK, St. John PL, Abrahamson DR. Am J Pathol 151: 1131-1140, 1997), we directly examined the effect of hyperglycemia on mesangial cell migration and laminin beta2 expression. Migration mediated by IGFBP-5 is impaired in the presence of 25 mM glucose. This reduction in migration was found to result from a loss in mesangial cell synthesis of laminin421, and IGFBP-5-induced migration could be restored by replacing laminin421. Additional studies showed that there was selective reduction in mRNA translation of laminin beta2 in the presence of high glucose. Preserved synthesis of laminin beta1 indicates that not all proteins are reduced by high glucose and confirms prior data showing that laminin411 cannot substitute for laminin421 in IGFBP-5-mediated migration. Given the importance of mesangial migration in the reparative response to diabetes-associated mesangiolysis, these findings provide new insights into abnormalities associated with diabetic nephropathy and the potential importance of differential control of protein translation in determination of alterations of protein expression.

High matrix metalloproteinase production correlates with immune activation and leukocyte migration in leprosy reactional lesions.

Gelatinases A and B (matrix metalloproteinase 2 [MMP-2] and MMP-9, respectively) can induce basal membrane breakdown and leukocyte migration, but their role in leprosy skin inflammation remains unclear. In this study, we analyzed clinical specimens from leprosy patients taken from stable, untreated skin lesions and during reactional episodes (reversal reaction [RR] and erythema nodosum leprosum [ENL]). The participation of MMPs in disease was suggested by (i) increased MMP mRNA expression levels in skin biopsy specimens correlating with the expression of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), (ii) the detection of the MMP protein and enzymatic activity within the inflammatory infiltrate, (iii) increased MMP levels in patient sera, and (iv) the in vitro induction of MMP-9 by Mycobacterium leprae and/or TNF-alpha. It was observed that IFN-gamma, TNF-alpha, MMP-2, and MMP-9 mRNA levels were higher in tuberculoid than lepromatous lesions. In contrast, interleukin-10 and tissue inhibitor of MMP (TIMP-1) message were not differentially modulated. These data correlated with the detection of the MMP protein evidenced by immunohistochemistry and confocal microscopy. When RR and ENL lesions were analyzed, an increase in TNF-alpha, MMP-2, and MMP-9, but not TIMP-1, mRNA levels was observed together with stronger MMP activity (zymography/in situ zymography). Moreover, following in vitro stimulation of peripheral blood cells, M. leprae induced the expression of MMP-9 (mRNA and protein) in cultured cells. Overall, the present data demonstrate an enhanced MMP/TIMP-1 ratio in the inflammatory states of leprosy and point to potential mechanisms for tissue damage. These results pave the way toward the application of new therapeutic interventions for leprosy reactions.

Involvement of NO in the inhibitory effect of Calotropis procera latex protein fractions on leukocyte rolling, adhesion and infiltration in rat peritonitis model.

AIM OF THE STUDY: The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. It comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). MATERIAL AND METHODS: The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LP(PI), LP(PII) and LP(PIII)) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. RESULTS: LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LP(PI), the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LP(PI) on neutrophil influx. CONCLUSIONS: Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LP(PI) was most potent in inhibiting neutrophil functions and its effects are mediated through NO production.

M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells.

M. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.

Anetoderma occurring after hepatitis B vaccination.

Anetoderma is an elastolytic disorder of unknown origin. To our knowledge, anetoderma secondary to hepatitis B immunization has been described only once in the literature, in two siblings vaccinated at the same time. We describe, what we believe to be an additional case of such a rare disorder in a 21-year-old man. He presented with white spots and papules on his neck, upper limbs and trunk, that had developed gradually within the last 6 years without any symptoms. The initial lesions were red macules, which gradually enlarged in size and number, becoming pale. Biopsy of a sack-like lesion revealed normal epidermis and a discrete mononuclear infiltrate throughout the dermis. Association of anetoderma with hepatitis B vaccination is speculated here, as suggested by history of vaccination two weeks prior to the onset of skin eruption and ruling out other possible causes of anetoderma.

Effect of Mycobacterium leprae lipids on BCG- and carrageenan-induced cellular recruitment in mouse pleurisy.

Pathogenic mycobacteria survive inside macrophages and deactivate these cells, using a mechanism that is still poorly understood. Mycobacterial cell wall lipids constitute the first contact with the host cell. Although Mycobaterium leprae and M. bovis BCG share common antigens, they induce opposite inflammatory responses. Apolar M. leprae lipids have been shown to be anti-inflammatory by down-regulating macrophage activation and T-cell functions. We wonder if these lipids would influence cellular migration to BCG or to other inflammatory agent. We investigated the effect of M. leprae, its lipids or delipidated bacteria on acute and chronic BCG- or carrageenan-induced pleurisy. Previous injection of intact or delipidated M. leprae did not alter either the BCG- or carrageenan-induced pleural inflammatory reaction. However, M. leprae lipids enhanced carrageenan-induced acute cellular migration without impairing BCG inflow; moreover, they reduced BCG chronic response. Together these data suggest distinct mechanisms for intracellular deactivation and pleural cell recruitment exerted by mycobacterial structures.

Comparison between C57Bl/6 and C57Bl/10 mycobacterial mouse pleurisy with respect to cellular migration and nitric oxide production.

Mycobacterium bovis-BCG (BCG) and Mycobacterium leprae (ML) have opposite inflammatory properties. Mycobacteria-induced pleurisy in C57Bl/6 and C57Bl/10 mice was evaluated to establish if their innate responses could be comparable, verifying cellular migration and nitrite production. Kinetic responses after ML or BCG intrathoracic injection were compared in those mice, sharing the H-2(b) MHC haplotype. BCG led to acute eosinophilia and late neutrophilia in both mice. In C57Bl/6 late pleurisy, monocytes and neutrophil recruitment was dose- and iNOS-dependent, inhibited by methotrexate but not by indomethacin. Pleural macrophages released nitrites ex vivo after 7 days of BCG stimulus, without "priming" and blocked by the nitrite inhibitor L-N5-(1-iminoethyl)-ornithine (L-NIO). ML did not induce cellular migration or nitrite production, independent of the mouse strain, timing, or number of bacilli. Although these mycobacteria have high homology, there was no effect of ML on BCG-evoked secondary cellular recruitment. Both C57Black mice trigger similar onset of inflammatory responses to these mycobacteria, so far can alternatively be used in experimental studies.

Effect of microbial heat shock proteins on airway inflammation and hyperresponsiveness.

Microbial heat shock proteins (hsp) have been associated with the generation and induction of Th1-type immune responses. We tested the effects of treatment with five different microbial hsp (Mycobacterium leprae, Streptococcus pneumoniae, Helicobacter pylori, bacillus Calmette-Guérin, and Mycobacterium tuberculosis) in a murine model of allergic airway inflammation and airway hyperresponsiveness (AHR). Mice were sensitized to OVA by i.p. injection and then challenged by OVA inhalation. Hsp were administered to each group by i.p. injection before sensitization and challenge. Sensitized and challenged mice developed increased serum levels of OVA-specific IgE with significant airway eosinophilia and heightened responsiveness to methacholine when compared with nonsensitized animals. Administration of M. leprae hsp prevented both development of AHR as well as bronchoalveolar lavage fluid eosinophilia in a dose-dependent manner. Treatment with M. leprae hsp also resulted in suppression of IL-4 and IL-5 production in bronchoalveolar lavage fluid, while IL-10 and IFN-gamma production were increased. Furthermore, M. leprae hsp treatment significantly suppressed OVA-specific IgE production and goblet cell hyperplasia/mucin hyperproduction. In contrast, treatment with the other hsp failed to prevent changes in airway responsiveness, lung eosinophilia, or cytokine production. Depletion of gamma/delta T lymphocytes before sensitization and challenge abolished the effect of M. leprae hsp treatment on AHR. These results indicate selective and distinctive properties among the hsp, and that M. leprae hsp may have a potential therapeutic role in the treatment of allergic airway inflammation and altered airway function.

Post-traumatic migration and emergence of a novel cell line upon the ependymal surface of the third cerebral ventricle in the adult mammalian brain.

This investigation describes the migration and emergence of significant numbers of what appear to be neuron-like cells upon the surface of the median eminence of the adult rodent neurohypophyseal system of the endocrine hypothalamus following the trauma of hypophysectomy. These cells appear to migrate through the neuropil of the underlying median eminence and emerge in large numbers upon the surface of the third cerebral ventricle within 7 days following hypophysectomy (axotomy) of supraoptic (SON) and paraventricular neurites (PVN) of the adult neurohypophyseal system. Previous investigations have demonstrated regeneration of the neural stem and neural lobe in a variety of mammalian species (Adams et al., J Comp Neurol, 1969;135:121-144; Beck et al., Neuroendocrinology, 1969;5:161-182; Scott et al., Exp Neurol, 1995;131-1:23-39; Scott and Hansen, Vir Med 1997;124:249-261). It also has been demonstrated that the process of regeneration is invariably accompanied by the up-regulation of nitric oxide synthase (NOS), the enzyme that catalyzes arginine to nitric oxide (NO) and that both neurohypophyseal regeneration, as well as migration and emergence of neuron-like cells upon the surface of the adjacent third cerebral ventricle, is associated with the up-regulation of NOS and increased expression of NO. It also has been amply demonstrated that this entire process of neurohypophyseal regeneration and cell migration is completely inhibited by the introduction of the antagonist of nitric oxide, namely, nitroarginine (Scott et al., Exp Neurol, 1995;131-1:23-39; Scott and Hansen, Vir Med, 1997;124:249-261). The emergence and migratory dynamics of this novel cell line upon the floor of the rodent third cerebral ventricle are discussed with respect to the role of the ubiquitous free radical NO and the implications and potential clinical applications of neuronal migration following trauma in the human central nervous system (CNS).

Opposite cellular accumulation and nitric oxide production in vivo after pleural immunization with M. leprae or M. bovis BCG.

Mycobacteria as intracellular pathogens have evolved mechanisms to survive within macrophages. Our previous data showed that M. leprae (ML), unlike M. bovis BCG, did not induce an inflammatory response in the mice subcutaneous tissue. Further, ML inhibited BCG-induced foot pad oedema and seemed to transform macrophages in epithelioid cells. Since these mycobacteria share common antigens, here we seeked to compare the acute and chronic cellular response evoked by ML and BCG in pleurisy of a mycobacteria-susceptible mice (BALB/c). The total leukocytes, the cell type that migrated to the pleural cavity and macrophage activation assayed by nitric oxide release were determined. Live or dead BCG Moreau recruited the same extent of cells, essentially monocytes and neutrophils, dose-dependently, in both acute and chronic pleurisy. BCG-induced eosinophilia was observed only in the acute response (after 24 h of injection). A significant nitric oxide release by pleural macrophages was triggered by BCG Moreau without previous activation. Nevertheless, ML failed to recruit leukocytes to the pleural space or to lead to nitric oxide production despite the number of bacilli used and the time studied (1, 7 or 14 days after injection). Although these mycobacteria have common antigens that cross-react, these data show a distinct ability of ML or BCG to recruit cells to the pleural space and to activate pleural macrophage for nitric oxide production in vivo.

Inflammation induced by a glycolipid fraction from Mycobacterium leprae.

1. The inflammatory properties of a glycolipid fraction isolated from human recovered Mycobacterium leprae were investigated. The inflammatory reaction induced in mouse lung by the inoculation of the glycolipid fraction adsorbed to charcoal particles was characterized by a large influx of macrophages at various stages of maturation and of epithelioid cells around the particles. 2. When injected as an aqueous emulsion into the footpad of mice, the same fraction evoked a dose-dependent massive influx of mononuclear (MN) cells. The inflammatory reaction reached a peak at 6 days. The minimal effective dose of glycolipid was 0.1 micrograms. 3. The kinetics of inflammatory cell migration was studied by total and differential counts of leucocytes that migrated to the peritoneal cavity of mice inoculated intraperitoneally with the glycolipid fraction. This fraction initially induced intense polymorphonuclear (PMN) migration, which was later reduced, with a simultaneous increase in MN cells. 4. Adherent peritoneal cells (APC) incubated with glycolipid released one or more soluble factor(s) which induce active PMN and MN cell chemotaxis in vivo as well as in vitro. Thus, the MN cells may be attracted to the site of glycolipid inoculation by factor(s) released through the interaction of macrophages with the glycolipid fraction. 5. The present results demonstrate that a glycolipid containing trehalose and mycolic acid isolated from M. leprae reproduces some aspects of the fundamental lesion of leprosy.

Diversity in migration of CD4 and CD8 lymphocytes in different microanatomical compartments of the skin in the tuberculin reaction in man.

The lymphocytes in the perivascular foci of tuberculin skin tests have a similar CD4:CD8 ratio to those in the peripheral blood, suggesting that these subsets do not show bias in their initial emigration. By contrast, the diffusely infiltrating lymphocytes show a relative preponderance of CD4 cells which is progressively greater in successive 250 micron layers into the dermis. A generally similar pattern is seen in healthy controls and in patients with untreated pulmonary tuberculosis, treated leprosy, haemophilia A and chronic obstructive lung disease (COLD) patients treated with prednisolone, but the gradient of increasing CD4:CD8 ratio with depth into the dermis is significantly less steep in patients with tuberculosis, haemophilia and prednisolone-treated COLD than in the healthy controls. Selective migration results in a relative preponderance of CD4 cells in the diffuse infiltrate and it is suggested that this is a mechanism likely to potentiate defensive reaction to Mycobacterium tuberculosis: any deficiency in selective migration may make immunological defences less effective and so contribute to the chronicity of the lesions of tuberculosis.

Epidermal keratinocyte Ia expression, Langerhans cell hyperplasia and lymphocytic infiltration in skin lesions of leprosy.

Epidermal changes, Ia expression on keratinocytes, Langerhans cell hyperplasia and lymphocyte infiltration were sought in skin lesions of leprosy: 15 borderline tuberculoid (BT), six borderline lepromatous (BL), 17 lepromatous (LL), 13 erythema nodosum leprosum (ENL), six Lucio reactions and nine reversal reactions. All three changes were well developed in BT and reversal reactions. ENL showed well developed keratinocyte Ia and Langerhans cell hyperplasia, but little lymphocytic infiltration. LL and Lucio tissues had some Langerhans cell hyperplasia but little or no keratinocyte Ia or lymphocytic infiltration. BL tissues were so diverse as to suggest two distinct subgroups. These findings are consistent with the hypothesis that keratinocyte Ia expression is an immunohistological sign of a cell-mediated immune (CMI) response. However, the Ia keratinocyte expression found in BL and ENL tissues appears contrary to the undifferentiated macrophages and numerous bacilli found in the lesions. Thus, if a sign of CMI, keratinocyte Ia expression is not a measure of the effectiveness of the response.