Decreased monocyte shedding of the migration inhibitor soluble CD18 in alcoholic hepatitis.

OBJECTIVES: During alcoholic hepatitis (AH) monocytes traverse the vascular boundaries and massively invade the liver. In principle, tissue extravasation can be limited through shedding of CD18 integrins from leukocytes, including monocytes. The soluble (s) product sCD18 conceals adhesion receptors on the endothelium, which reduces monocyte extravasation. In AH, monocytes are dysfunctional, but whether this involves their self-generated anti-migration is unknown. Our aim was, therefore, to investigate monocyte CD18 dynamics in AH. METHODS: We studied 50 AH patients and 20 healthy controls. We measured monocyte expression and conformational activation of CD18, plasma (P)-sCD18, stimulated in vitro CD18 shedding and P-sCD18 in a short-term chronic-binge mouse model. RESULTS: AH-derived monocytes had a 30-60% higher expression of active CD18 receptors (p < 0.01), but the sCD18 concentration per monocyte was reduced in vivo by 30% and in vitro by 120% (p < 0.01). Ethanol reduced the in vitro shedding of CD18 in the patients only. TNFα increased sCD18 concentration per monocyte, but less so in the patients (p < 0.04). P-sCD18 per monocyte was inversely related to disease severity. In early alcoholic liver disease, P-sCD18 was decreased in the mouse model. CONCLUSIONS: The monocyte CD18 integrins are highly activated in AH and the single monocyte shedding of CD18 was decreased favoring tissue extravasation. Alcohol in itself and altered monocyte responsiveness to TNFα may explain this lowered shedding. TRANSLATIONAL IMPACT: The contribution of this mechanism to the excessive monocyte liver infiltration in AH should be further explored as it may serve as a potential therapeutic target to limit liver inflammation.

[Modified tests basophil degranulation and leukocyte migration inhibition factor in drug allergy. Study 2009-2014]. / Pruebas modificadas de degranulación de basófilos y factor de inhibición de migración de leucocitos en alergia a fármacos. Estudio de 2009 a 2014.

BACKGROUND: Adverse reactions to drugs are increasing and there are few studies for the diagnosis. OBJECTIVE: To determine the utility of modified basophil degranulation (MBD) test and modified leukocyte migration inhibition factor (MLMIF) test to prove drug hypersensitivity. METHODS: 177 patients of both sexes were studied with the diagnosis of drug hypersensitivity, determining MBD, MLMIF, or both, between 2009 and 2014. They were matched with positive and negative controls and the non-allergic population. Applications are issued according to the type of hypersensitivity, considering type I MBD and type IV MLMIF. RESULTS: 170 patients (96.04%) were positive to at least one drug (RR = 4.71). 561 MBD (73.62%) and 201 MLMIF (26.37%) were performed. Female sex was more frequent (64.41%); the average age was 38.5. MBD was positive in 70.23% and MLMIF in 67.16%. The test sensitivity was increased complementarily and with two dilutions. The correlation of MBD and MLMIF was positive and highly significant. CONCLUSIONS: Women have more drug reactions. Modified MBD test is useful at any age. Since medications can activate one or other hypersensitivity mechanism, it is important to request the tests simultaneously.

Antecedentes: Existe incremento de reacciones adversas a medicamentos y pocos estudios para el diagnóstico. Objetivo: Determinar la utilidad de pruebas modificadas de degranulación de basófilos (DB) y del factor inhibidor de la migración de leucocitos (LIF, leukocyte migration inhibition factor) para comprobar la hipersensibilidad a medicamentos. Métodos: Se estudiaron 177 pacientes, de uno y otro sexo, con diagnóstico de hipersensibilidad a medicamentos, en quienes se determinó pruebas modificadas de DB, LIF, o ambas entre 2009 y 2014. Se parearon con controles positivos, negativos y población no alérgica. Las solicitudes se emitieron de acuerdo con el tipo de hipersensibilidad, considerando tipo I a DB y tipo IV a LIF Resultados: 170 pacientes (96.04%) fueron positivos al menos a un medicamento (RR, 4.71). Se realizaron 561 pruebas modificadas de DB (73.62%) y 201 de LIF (26.37%). El sexo femenino fue más frecuente (64.41%); la edad promedio fue de 38.5 años. La prueba modificada de DB resultó positiva en 70.23% y la de LIF en 67.16%. La sensibilidad de las pruebas se incrementó en forma complementaria y a dos diluciones. La correlación de las pruebas fue altamente significativa. Conclusiones: Las mujeres presentan más reacciones a fármacos. La prueba modificada de DB es útil en cualquier edad. Como los medicamentos pueden activar uno u otro mecanismo de hipersensibilidad es importante solicitar las pruebas simultáneamente.

Alterations in the function of circulating mononuclear cells derived from patients with Crohn's disease treated with mastic.

AIM: To assess the effects of mastic administration on cytokine production of circulating mononuclear cells of patients with active Crohn's disease (CD). METHODS: The study was conducted in patients with established mildly to moderately active CD, attending the outpatient clinics of the hospital, and in healthy controls. Recruited to a 4 wk treatment with mastic caps (6 caps/d, 0.37 g/cap) were 10 patients and 8 controls, all of who successfully completed the protocol. Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), monocyte chemotactic protein-1 (MCP-1), macrophage migration inhibitory factor (MIF) and intracellular antioxidant glutathione (GSH) were evaluated in peripheral blood mononuclear cells (PBMC) before and after treatment. RESULTS: Treating CD patients with mastic resulted in the reduction of TNF-alpha secretion (2.1 +/- 0.9 ng/mL vs 0.5 +/- 0.4 ng/mL, P = 0.028). MIF release was significantly increased (1.2 +/- 0.4 ng/mL vs 2.5 +/- 0.7 ng/mL, P = 0.026) meaning that random migration and chemotaxis of monocytes/macrophages was inhibited. No significant changes were observed in IL-6, MCP-1 and GSH concentrations. CONCLUSION: This study shows that mastic acts as an immunomodulator on PBMC, acting as a TNF-alpha inhibitor and a MIF stimulator. Although further double-blind, placebo-controlled studies in a large number of patients is required to clarify the role of this natural product, this finding provides strong evidence that mastic might be an important regulator of immunity in CD.

Overexpression of SOCS3 inhibits astrogliogenesis and promotes maintenance of neural stem cells.

To investigate the effects of suppressors of cytokine signaling 3 (SOCS3) on neural stem cell fate, stem cells were infected with an adenoviral vector expressing SOCS3. Three days later, western blot analysis and immunocytochemical analysis revealed that the protein level of MAP2 and the number of MAP2-positive cells were significantly increased in SOCS3-transfected cells, whereas the protein level of GFAP and the number of GFAP-positive cells were significantly decreased. Furthermore, promoter assay revealed a significant reduction in the transcriptional level of signal transducer and activator of transcription 3 (Stat3) in the transfected cells. In addition, the mRNA levels of Notch family member (notch1) and inhibitory basic helix-loop-helix (bHLH) factors (hes5 and id3) were significantly up-regulated 1 day after overexpression of SOCS3. Three days after transfection, the mRNA level of hes5 was significantly decreased, whereas that of notch1 was still up-regulated. Moreover, all of SOCS3-positive cells expressed Nestin protein but did not express MAP2 or GFAP proteins. These data indicate that overexpression of SOCS3 induced neurogenesis and inhibited astrogliogenesis in neural stem cells. Our data also show that SOCS3 promoted maintenance of neural stem cells.

Modulación del tráfico leucocitario: papel de las quimiocinas y de los opioides / Modulation of leukocyte trafficking: role of chemokines and opioids

El correcto movimiento y posicionamiento celular son elementosclaves en el desarrollo, determinantes tanto en situacionesfisiológicas como patológicas. Probablemente sea en el sistemainmunológico donde más extensamente se han estudiado losprocesos de migración celular, ya que muchos aspectos de la respuestainmune están directamente relacionados con la regulacióndel tráfico leucocitario. La migración de leucocitos es un procesoaltamente coordinado en el que participan muchas moléculas ysus correspondientes receptores. En esta revisión, nos centramosprincipalmente en las quimiocinas, moléculas con capacidad quimioatrayente,y en los opioides, ya que, aunque normalmente suactividad se ha considerado restringida al sistema nervioso, tambiénafectan a las células inmunes y modulan su movilidad. Larespuesta inmune es un proceso muy dinámico que depende dela concentración de una gran variedad de moléculas que aparentementepueden no tener ninguna relación así como de la presenciade los receptores para esas moléculas en la membrana celular

Correct cell movement and positioning are central elementsin development, and influence both normal physiology and diseasestates. Cell movement has probably been studied most extensivelyin the immune system, where many aspects of the immuneresponse are closely related to coordination of leukocyte trafficking.Leukocyte migration is thus a highly regulated processthat implicates many molecules and receptors. In this reviewwe focus our attention on chemokines, classical chemoattractantmolecules and on opioids, molecules that usually act on thenervous system but that also affect immune cells and modulatetheir movement. The final immune response is a very dynamicprocess that depends on the amount of a variety of different apparentlyunrelated molecules and on the presence at the cell membraneof their corresponding receptors

Humira®: terapia biológica humana / Humira®: human biological therapy

Humira®: (AU)

An unexplained sequestration of latrunculin A is required in neutrophils for inhibition of actin polymerization.

Latrunculin A (LatA) is a toxic natural product that causes disruption of the actin cytoskeleton in many eukaryotic cells at submicromolar concentrations. LatA has been found to bind G-actin with a dissociation constant of 0.2 microM, and more recently to bind profilin-G-actin and, weakly, thymosin beta4-G-actin. A number of investigators have used LatA as a G-actin sequestering agent. Thus, we studied neutrophil chemotaxis and its requisite conversion of G-actin to F-actin, supported by an extensive pool of G-actin, mainly bound to thymosin beta4. Calculations suggest that the affinity of LatA is insufficient to cause significant sequestration of this pool, and the pool's buffering action should protect neutrophils from depletion of productive G-actin species by submicromolar LatA. Nonetheless, we found that both chemoattractant stimulated migration and F-actin polymerization in neutrophils were inhibited by LatA at these concentrations. The latter effect was accompanied by sequestration of LatA and showed a cell density dependence that was consistent with G-actin sequestration. The apparent contradiction between the calculations and the experimental observations could be reconciled by assuming the presence of an accessory species, of unknown normal function, which forms a high affinity ternary complex with LatA and G-actin, thus causing the cells to concentrate LatA. Other models that could not be ruled out also invoke new actions of LatA, suggesting caution in the interpretation of its effects on cells.

The evaluation of non-specific cellular immunological parameters amongst children with Schistosoma haematobium infection in Nigeria.

This study used the leucocyte migration index to assess cellular immune function in children with urinary schistosomiasis. Migration inhibitory factor was produced (with other lymphokines) by sensitizing mitogens. The production of antigen-induced migration inhibitory factor in vitro correlated with the in vivo state of cellular hypersensitivity of the lymphocyte donor. The percentage positive leucocyte migration rate using three mitogens was least with inactivated measles haemagglutinin virus (IMV) and highest with Bacillus Calmette-Guerin (BCG) in the control group, while highest with tuberculin purified protein derivative (PPD) and least with IMV in the test group. The measurement of the migration index of leucocytes comparing the control with lightly- and heavily-infected children on activation using three mitogens was significantly reduced, except in the case of the control versus lightly-infected children using IMV. Using IMV, the leucocyte migration index for control versus lightly-infected children and heavily-infected children was significant (p > 0.002 and p < 0.001, respectively). Using BCG the difference between controls and lightly- and heavily-infected children were significant (p < 0.02). PPD showed no significant difference in leucocyte migration between control and the lightly- or heavily-infected children. In all leucocyte migration index decreased with intensity of infection except in the case of PPD (p < 0.002 for BCG; p < 0.001 for the IMV). There was a significant correlation between egg count and leucocyte migration index; for BCG (r = -0.20, p < 0.005); for IMV (r = -0.3, p < 0.001); for PPD (r = -0.38,p < 0.001). Patients with schistosomiasis infection can express normal and effective cellular immune responses to non-schistosomal antigens and also have equal immunological ability to combat pathogens as S. haematobium-free controls.

Directional migration of leukocytes: their pathological roles in inflammation and strategies for development of anti-inflammatory therapies.

Directional migration of leukocytes is indispensable to innate immunity for host defense. However, recruitment of leukocytes to a site of tissue injury also constitutes a leading cause for inflammatory responses. Mechanistically, it involves a cascade of cellular events precisely regulated by temporal and spatial presentation of a repertoire of molecules in the migrating leukocytes and their surroundings (microenvironments). Here I will summarize the emerging evidence that has shed lights on the underlying molecular mechanism for directional migration of leukocytes, which has guided the therapeutical development for innovative anti-inflammatory medicines.

Migration-inhibitory factor gene-deficient mice are susceptible to cutaneous Leishmania major infection.

To determine the role of endogenous migration-inhibitory factor (MIF) in the development of protective immunity against cutaneous leishmaniasis, we analyzed the course of cutaneous Leishmania major infection in MIF gene-deficient mice (MIF(-/-)) and wild-type (MIF(+/+)) mice. Following cutaneous L. major infection, MIF(-/-) mice were susceptible to disease and developed significantly larger lesions and greater parasite burdens than MIF(+/+) mice. Interestingly, antigen-stimulated lymph node cells from MIF(-/-) mice produced more interleukin-4 (IL-4) and gamma interferon (IFN-gamma) than those from MIF(+/+) mice, although the differences were statistically not significant. IFN-gamma-activated resting peritoneal macrophages from MIF(-/-) mice showed impaired macrophage leishmanicidal activity and produced significantly lower levels of nitric oxide and superoxide in vitro. The macrophages from MIF(-/-) mice, however, produced much more IL-6 than macrophages from wild-type mice. These findings demonstrate that endogenous MIF plays an important role in the development of protective immunity against L. major in vivo. Furthermore, they indicate that the susceptibility of MIF(-/-) mice to L. major infection is due to impaired macrophage leishmanicidal activity rather than dysregulation of Th1 and Th2 responses.

Conditioned immunosuppressive effect of cyclophosphamide on delayed-type hypersensitivity response and a preliminary analysis of its mechanism.

In the present study, camphor odor and intraperitoneal (i.p.) injection of cyclophosphamide (CY) were used as conditioned stimulus (CS) and unconditioned stimulus (US), respectively. In the unconditioned group, mice were exposed to camphor odor for 1 h followed by an i.p. injection of CY (75 mg/kg). On the next day, the above CS/US association trial session was repeated followed by smearing dinitrochlorobenzene (DNCB) on mouse abdominal skin for sensitizing the animal for delayed-type hypersensitivity (DTH) response. Five days after DNCB sensitization, mice were exposed to camphor odor (1 h), followed by an i.p. injection of CY, and then DNCB was smeared on the left ear of mice for the challenge of DTH response. Both the left/right ear weight ratio and the activity of leukocyte migration inhibitory factor (LMIF) were used as the index of DTH response, which was done 24 h after DNCB challenge. In the conditioned group, the treatment was the same as that in the unconditioned group, except that normal saline was injected on day 5 instead of CY. Furthermore, in order to analyze the mechanism of the conditioned response (CR), the mouse serum from the conditioned group (CR serum) was injected into normal mice 6 h prior to DNCB challenge. Results showed that in the conditioned group, left/right ear weight ratio and LMIF activity were statistically lower than that in the DTH group, and there was no difference between conditioned and unconditioned groups. Thus, an animal model of conditioned immunosuppressive response had been established. The results also showed that after CR serum was injected into normal mice, DTH response was also significantly suppressed. However, if CR serum was treated with dialysis (10,000 molecular weight cut-off), the suppressive effect of CR serum on DTH response disappeared. Taken together, the data suggested that a chemical compound(s) in serum, with a molecular weight less than 10,000, was important in mediating the conditioned immunosuppressive response. This may be a very important molecule(s) that could be very critical to our understanding of the interaction between the central nervous system and immune function.

Identification of macrophage migration inhibitory factor as a potent endothelial cell growth-promoting agent released by ectopic human endometrial cells.

The growth of endometrial cells in ectopic locations (endometriosis) is dependent on the establishment of an adequate blood supply. Neovascularization (angiogenesis) is therefore a vital step toward the progression of this disease. We first revealed the presence of a potent mitogenic activity for human endothelial cells in the culture medium of immortalized human endometriotic cells. The activity, measured by the level of [(3)H]-thymidine incorporation into the DNA of human coronary artery endothelial cells, was then purified by anion exchange high-performance liquid chromatography. Electrophoretic analysis of one of the bioactive fractions that markedly enhanced endothelial cell proliferation showed three distinct bands with apparent molecular masses of 15.8, 12.6, and 6.5 kDa. N-terminal microsequencing of an internal peptide from the 12. 6-kDa protein showed 100% homology with human macrophage migration inhibitory factor (MIF). The protein was positively identified as MIF by Western blot analysis using a specific anti-MIF antibody. Anti-MIF antibody inhibited the bioactivity found in the evaluated fraction and the conditioned medium of primary endometriotic cell cultures, and commercial recombinant human MIF displayed a high mitogenic activity for endothelial cells. Our findings reveal that MIF is released by endometriotic cells and acts as a potent mitogenic factor for human endothelial cells in vitro. This may have a considerable interest, in view of the crucial role of angiogenesis in ectopic endometrial cell growth and activity and in numerous tissues undergoing dynamic physiological changes, such as human endometrium.

[Human chemokine receptors: structure and function]. / Ludzkie receptory chemokin: budowa i funkcja.

Chemokines are relatively small peptides with potent chemoattractant and activation activities for leukocytes. Several chemokine receptors, belonging to the G proteincoupled receptor superfamily, have been cloned and characterized and their functions have been partially elucidated.

Leucocyte migration inhibitory factor assay in Nigerians with urinary schistosomiasis.

Schistosome antigens (soluble egg antigen and adult worm antigen) and non-schistosome antigens (Bacille Calmette Guerin vaccine antigen and measles virus vaccine antigen) were used to assay for leucocyte migration inhibitory factors (LMIF) in Nigerian children with and without Schistosoma haematobium infection. The severity of S. haematobium infection was graded into light infection (1-49 eggs/ 10 ml urine) or heavy infection (more than or equal to 50 eggs/10 ml urine). The mean percentage migration indices were significantly reduced in heavily infected urinary schistosomiasis (USS) subjects compared with the controls or lightly infected subjects when non-antigens were used to stimulate LMIF production. When Schistosome antigens were used to stimulate LMIF production, there were no significant decreases in the % migration indices in heavily infected USS subjects compared with the light USS subjects. The mean percentage migration index was significantly increased when schistosome antigen was used to stimulate LMIF production in treated USS subjects compared with untreated USS subjects but the increase was not significant when non-schistosome antigen was used. The conclusion that could be drawn from this study is that LMIF assay using schistosome antigen(s) has epidemiological value in schistosomiasis.

The green tea extract epigallocatechin gallate is able to reduce neutrophil transmigration through monolayers of endothelial cells.

Green tea is widely used in Asia and has also become popular in Western countries. The influence of green tea extracts on leukocytes is not well understood. Leukocytes play a crucial role in the process of inflammation. They migrate from the intravascular space into the tissue to attack micro-organisms. The aim of the current study was to investigate the influence of epigallocatechin gallate on leukocyte transmigration through endothelial cell monolayers and thereby evaluate its potential role in the inflammatory process. Human umbilical vein endothelial cells were cultured on microporous membranes to achieve a monolayer. Freshly isolated neutrophils from healthy subjects were measured with a migration assay. The amount of untreated neutrophils migrating through untreated endothelial cell monolayers was used as control and set as 100%. Neutrophils and/or endothelial cell monolayers were pre-treated with epigallocatechin gallate using relevant, as well as higher and lower concentrations. The relevant plasma concentration of epigallocatechin gallate was able to significantly inhibit neutrophil migration through endothelial cell monolayers (69 +/- 6.4% SD; p < 0.05 compared to control), when both cell types (leukocytes and endothelial cell monolayer) were treated. This is similar to the situation after resorption in-vivo. Treating either neutrophils or endothelial cell monolayers alone led to significant reductions in migratory response (only neutrophils treated: 86 +/- 8.1% SD, p < 0.05; only endothelial cell monolayers: 77 +/- 6.1%, p < 0.05). In conclusion, epigallocatechin gallate was identified as a potent inhibitor of leukocyte migration through endothelial cell monolayers. The treatment of both cell types showed an additive effect. Endothelial cells seem to be more affected than neutrophils. Further clinical investigations are necessary to understand the potential clinical consequences.

Human B cells secrete migration inhibition factor (MIF) and present a naturally processed MIF peptide on HLA-DRB1*0405 by a FXXL motif.

A better knowledge of peptide structures interacting with major histocompatibility complex (MHC) molecules is of great interest for better understanding of the molecular basis of immune recognition. We have isolated naturally processed peptides from a continuously growing antigen-presenting Epstein-Barr virus-transformed human B-cell line. HLA-DR complexes were purified by specific affinity chromatography and complexed peptides were released by acid treatment. The isolated peptides were separated by reversed phase chromatography and fractions were analysed by Edman degradation at picomolar ranges. From 30 fractions that were examined seven peptides bound to the HLA-DRB1*0405 and two peptides from the human leucocyte antigen (HLA) class II associated invariant chain bound to HLA-DRB1*1302. In addition, a N-terminal beta-chain peptide of the 0405 allele was identified. Evaluation of amino acid sequences revealed a refined FXXL motif for the 0405 allele, in which F (phenylalanine) stands for any aromatic amino acid and L (leucine) can be exchanged by either I (isoleucine) or V (valine). In total, three fractions contained a peptide derived from the human migration inhibition factor (MIF), a pro-inflammatory cytokine that is normally produced by activated T lymphocytes and monocytes/macrophages. Indeed, cytokine analysis revealed high amounts of MIF secreted by the B-cell line, confirming that MHC class II expressing cells can present any intrinsic peptide that contains the distinct motif for HLA-binding. For MIF, the amino acid sequence Y36IAV39 represents the required binding motif for HLA-DRB1*0405. Nevertheless, it is the first time that cytokine fragments were found to bind to HLA molecules on human B cells.

Leucocyte migration inhibition factor (L-MIF) in malnourished Nigerian children.

Leucocyte Migration Inhibition Factor (L-MIF) was measured in 41 children with marasmus, 19 with kwashiorkor, 5 with marasmic-kwashiorkor and 35 well-fed healthy children serving as controls. For L-MIF assay, two different antigens (live attenuated measles virus vaccine and diptheria pertussis tetanus (DPT) vaccine were used. Percentage migration indices obtained with the two antigens were significantly higher in the malnourished than in the well-fed healthy sex and age-matched controls (P < 0.01). The total serum protein and albumin concentrations were significantly reduced in the malnourished children compared with the controls (P < 0.01). Mean total leucocyte numbers were not significantly different in marasmic and marasmic-kwashiorkor children compared with the controls (P > 0.21).

[Leukocyte migration during erythrocyte sedimentation]. / Leukocyták mozgása vörösvértest-süllyedés folyamán.

Sedimentation properties of leukocytes was measured with a new, simple and reproducible method. The increment of leukocyte concentration was determined in the upper 100 mm section of the sedimentation blood column after one hour gravity sedimentation of the whole blood. The result (leukocyte antisedimentation rate, LAR) was expressed in percentage of the original, presedimentation leukocyte concentration. Blood samples taken from 35 healthy adults were investigated and 12.5% and 17.4% increments were found in total leukocyte count and in granulocyte concentration respectively in the upper half of the sedimentation blood column. The mean coefficient of variation of LAR measurements was 3.2% LAR was found significantly higher in a mixed group of patients than in healthy controls. The sedimentation properties of leukocytes were in significant correlation with leukocyte adherence (p < 0.01), with whole blood viscosity, hematocrit, and erythrocyte sedimentation rate (each p < 0.05) when blood samples of healthy individuals and postoperative intensive care patients were analysed in combination. In vitro pre-treatment of patients' blood samples with prednisolone and lidocaine resulted in a significant diminishment of LAR in a concentration dependent manner.