Significant Leukocyte Migration Inhibition Research Articles

Platelets as inflammatory mediators in a murine model of periodontitis.

To investigate the role of platelets during the development of ligature-induced experimental periodontitis in mice. Experimental periodontitis was induced by placement of sterilized 5-0 cotton ligatures around the maxillary and mandibular second molars of C57BL/6 wild-type mice. Flow cytometry was used to analyse platelet activation and platelet-leucocyte aggregate formation, and histologic analysis was used to evaluate inflammation and localization of platelets and leucocytes in periodontal tissues during the development of experimental periodontitis and in experimental periodontitis with and without antiplatelet drug treatment. Experimental periodontitis induced platelet activation and platelet-leucocyte interaction. Platelets and leucocytes gradually infiltrated in inflammatory gingival tissues during the development of experimental periodontitis. The inhibition of platelet activation via drug therapy led to significant inhibition of leucocyte migration and marked reduction in periodontal inflammation. This study revealed that platelets are critical for inflammation and tissue injury in periodontitis and serve as mediators of inflammation in periodontal tissue.

Bioassay-guided evaluation of Dioscorea villosa - an acute and subchronic toxicity, antinociceptive and anti-inflammatory approach.

BackgroundDioscorea villosa (DV) has been used in Brazil as an alternative medicine to attenuate menopause symptoms, as well as for the treatment of joint pain and rheumatoid arthritis. In spite of the popular use of DV for the treatment of various disorders, there are limited scientific data regarding safety aspects of this herb. In this regard, we carried out to evaluated both antinociceptive and anti-inflammatory activities in experimental models and assess the toxic effects of the acute (single dose) and subchronic (30 days) oral administration of dry extract of Dioscorea villosa in rodents.MethodsThe LC analyses were performed to assess the presence of the diosgenin in samples of DV. The antinociceptive study of DV was performed using models of acetic acid-induced writhing and formalin-induced pain in mice. The anti-inflammatory study was accomplished by leukocyte migration to the peritoneal cavity. A dry extract of DV was tested at doses of 100, 200 and 400 mg/kg (per os or p.o.). The toxicological properties of the dry extract were evaluated by toxicity assays of acute (5 g/kg, single dose) and subchronic (1 g/kg/day, 30 days) treatment. Haematological, biochemical, and histopathological parameters were studied. The results are expressed as mean ± S.D., and statistical analysis of the data were performed with the Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s test. In all cases differences were considered significant if p < 0.05.ResultsHPLC-DAD analysis of the extract from DV revealed the presence of diosgenin as the major compound. Doses of 200 and 400 mg⁄kg significantly reduced the amount of acetic acid-induced writhing in relation to the vehicle (p < 0.0001). In the first phase, using the formalin-induced neurogenic pain test, only the 400 mg/kg dose of DV showed significant inhibition of neurogenic pain (p < 0.001). In the second phase, 200 and 400 mg/kg of DV showed significant inhibition of inflammatory pain (p < 0.0001). Significant inhibition of leukocyte migration was observed with doses of 100 (p < 0.001), 200 (p < 0.01) and 400 mg/kg (p < 0.01). Haematological, biochemical and histopathological data obtained in both acute and subchronic toxicological assays revealed only unremarkable changes, which are unlikely to indicate DV toxicity with oral administration.ConclusionWe found that DV possesses antinociceptive and anti-inflammatory properties in rodent models. In addition, no acute or subchronic toxicity was evident when the herbal extract was administered orally. These results supporting the folkloric usage of the plant to treat various inflammatory diseases.

In vivo and in vitro anti-inflammatory activity of red clover Trifolium pratense dry extract

Red clover Trifolium pratense L., Fabaceae, contains four isoflavones, mainly formononetin and biochanin A, and in smaller concentrations, daidzein and genistein. These compounds have gained a lot interest due to its human health benefits, such as estrogenic and progestogenic activities, antioxidant, anti-cancer and others. The objective of this study was to determine in vivo and in vitro anti-inflammatory activity of red clover dry extract. The in vitro anti-inflammatory activity was assayed by the technique using the Boyden chamber method, evaluating the leukocyte migration inhibition (chemotaxis). The in vivo anti-inflammatory activity was tested by a carrageenan-induced rat paw edema test. The results of anti-inflammatory in vitro test showed that there was a significant inhibition of leukocyte migration at the concentrations of 100, 50, 25, 10 and 5 µg/mL of red clover dry extract, these doses resulted in 94.73, 95.39, 94.73, 84.68 and 78.75% of inhibition for each dose, respectively. The anti-inflammatory in vivo test resulted in a significant activity in both tested doses (100 and 50 mg/kg of red clover dry extract) and at each tested time. The average percentage of edema inhibition was 63.37%. The findings of this study suggested that red clover extract might be suitable for the treatment of inflammatory diseases.

In vitro effect of interferon alpha-2b on T lymphocyte transformation and leukocyte migration inhibition in patients with chronic brucellosis.

The in vitro effect of IFN-a on lymphocyte transformation and specific immune response against Brucella antigens was studied in 33 patients with chronic brucellosis and 10 normal controls. The following immunologic in vitro tests were applied: PHA activated lymphocyte transformation test using Bromodeoxyuridine and a monoclonal antibody in the presence and absence of 50 and 100 IU IFN Alpha-2b and leukocyte migration inhibition test against Brucella antigens in the presence and absence of 100 and 500 IU of IFN Alpha-2b. Patients were further divided to 2 subgroups according to a positive or negative migration inhibition test. Our results showed that T lymphocyte transformation was similar in patients and controls and that the addition of 50 IU IFN resulted in a significant increase of transforming cells whereas in the concentration of 100 IU IFN only anergic patients and controls responded positively. IFN also resulted in a significant leukocyte migration inhibition only in anergic patients and controls. These findings suggest that the chronic infection is not due to a generalized cellular immunodeficiency state and that IFN Alpha-2b might be a promising therapeutic approach in anergic patients.

Laboratory Evidence for Impaired Cellular Immunity in Different Stages of Syphilis

Abundant evidence suggests that Treponema pallidum (T.p.) escapes humoral immune defence despite the host produces antibodies early in the infection. Since the serologic responses in syphilis have been studied in detail this paper focuses on the cellular immune mechanisms. For this purpose the leukocyte migration inhibition was investigated in 17 patients in different stages of syphilis. Leukocyte migration inhibition assay was performed before, and 7 days, 3 weeks, 2 mo. and 1 yr after start of treatment. Ultrasonicated T.p. were used as antigen corresponding to 5 X 10(6) to 2 X 10(7) Treponema pallida per ml. Controls without antigen, with addition of Concanavalin. A instead of T.p. and using cells of normal volunteers were run. There was no leukocyte migration inhibition before treatment, suggesting nonexistent or depressed cellular immunity in the untreated syphilitic patient. Significant leukocyte migration inhibition, however, was observed as early as 2 days after start of treatment, being most pronounced after 1 week. Hypothetical circulating blocking factors for cellular immune reactions might be present in the untreated syphilitic patient, which become abolished after therapy. Since stimulation with Con A of syphilitic leukocytes gave normal results even before treatment in the syphilitic patient, there might be a specific block of leukocyte migration inhibition against T.p.

Immunological Response of Chickens to Mycoplasma gallisepticum Infection

The cellular, humoral, and local immune responses of chickens to Mycoplasma gallisepticum infection were studied at weekly intervals for 10 weeks. A cellular response was indicated by significant leukocyte migration inhibition (LMI) was demonstrated as early as one week postinfection (PI), but the maximum LMI of 36.4% was observed at seven weeks PI. Induction of cellular response was further confirmed by positive-delayed type of hypersensitivity reaction, observed in all infected birds tested. The antibody response, as determined by the hemagglutination-inhibition (HI) test, was found to be provoked very early in infection, and the mean HI titer peaked seven weeks PI. A highly significant correlation (p less than 0.05) was found between LMI percentages and mean HI titers. In the infected chickens a significant elevation of mean serum immunoglobulin (Ig) G level was noted from the first week PI onwards; this correlated well with the rise of HI antibodies. There was no change in the serum IgA and IgM levels. A low level of M. gallisepticum-specific HI activity was detected in tracheal washings (TW) of infected birds from the third week PI onwards; the levels peaked six weeks PI. The IgA/IgG ratio in TW of infected chickens did not differ significantly from those of controls. The recovery of chickens as assessed by regression of lesions and disappearance of organisms from trachea coincided with peak LMI, maximum HI titers in serum and TW, and elevated levels of serum IgG.

The leucocyte migration inhibition test and subpopulations of peripheral lymphocytes in insulin-dependent diabetics.

The leucocyte migration inhibition test (LMT) was performed by the agarose plate method with thyroid and pancreatic antigens in patients with insulin-dependent or independent diabetes mellitus. The mean migration indices with thyroglobulin, thyroid mitochondria and beef insulin were not significantly different in insulin-dependent diabetics from those in insulin-independent diabetics or normal controls. However, significant inhibition of leucocyte migration was observed in insulin-dependent diabetics when thyroid microsome or pancreatic extract was used as antigen. Although no significant difference was found in the percentages of T and B lymphocytes between insulin-dependent diabetics and insulin-independent diabetics or normal controls, the results of LMT strongly suggest the presence of cellular immunity against the thyroid and pancreas in insulin-dependent juvenile-onset diabetes.

Kinetics of Cellular and Humoral Response of Rabbits Immunized withMycoplasma Pneumoniae

The cellular and humoral responses of rabbits immunized with Mycoplasms pneumoniae antigen in saline or incorporated in Freund's complete adjuvant were examined. Peripheral blood leukocytes were used for the leukocyte migration inhibition (LMI) test. Both groups of animals showed significant LMI activity in the presence of M. pneumoniae as well as cross-reacting M. salivarium antigens but response to M. pneumoniae antigen was more pronounced. In the humoral response no such cross-reactivity was observed. Although some of the animals (3/8) demonstrated antibodies to M. salivarium prior to immunization the titers were not influenced by the immunization with M. pneumoniae antigen. Both groups of animals produced antibodies to M. pneumoniae antigen only, but significantly higher titers were observed in the adjuvant group. Cold hemagglutinins in both groups appeared earlier than the specific antibodies to mycoplasma. The adjuvant had no effect on the production of the cold agglutinins.

Detection of human tumor-associated antigens by the leukocyte migration in agarose assay.

Twenty-three of 36 (64%) lung cancer patients, 19 of 36 (54%) melanoma patients and 18 of 27 (66%) sarcoma patients tested in the leukocyte migration in agarose assay against soluble extracts of histologically similar tumors showed significant inhibition of leukocyte migration. Reactivity to extracts of dissimilar tumors was low. Sera of only 1/13 (7%) lung cancer patients, 2/19 (10%) melanoma patients and 7/21 (33%) sarcoma patients were inhibited by extracts of histologically dissimilar tumors. Only 7-9% of cancer patients reacted to paired extracts of normal tissue from the tumor donors. An average of 13% of sera from normal controls reacted to tumor extracts. Stage of disease and mode of therapy appeared to have little effect on overall reactivity in this assay, although the number of patients within the various categories was small for purposes of statistical analysis. The leukocyte migration in agarose assay shows a sensitivity and specificity to tumor-associated antigens comparable to that of the older capillary tube method in general use and may facilitate performance of migration inhibition. This assay may not be useful as a prognostic test due to the lack ofcorrelation with stage of disease and treatment modality. However, its high specificity and economical use of tumor antigen suggest applications in tumor antigen purification. The use of soluble tumor antigen preparations may make it possible to purify these antigens further to increase specificity and reactivity.

Lymphocyte sensitization to Aspergillus fumigatus antigens in pulmonary diseases in man.

In vitro studies of T-lymphocyte responses using five different batches of Aspergillus fumigatus antigens, were undertaken in twelve patients with A. fumigatus related lung disease and in three normal controls. Using a leucocyte migration method, five of the twelve patients showed significant inhibition of leucocyte migration with a migration index of 0-80 or less to A. fumigatus, but in only three was this demonstrated with more than one batch of antigen (one patient with aspergilloma and two with bronchopulmonary aspergillosis). The same antigens were used in lymphocyte transformation tests. Only two patients, one with aspergilloma and one with bronchopulmonary aspergillosis, showed clearly significant transformation although there were several borderline results. Only three patients had evidence of delayed skin responses to A. fumigatus antigens in vivo, one with aspergilloma, one with bronchopulmonary aspergillosis and one with atypical bronchopulmonary aspergillosis. Two of these three patients also had one or more positive in vitro test results. Thus T-lymphocyte sensitization to A. fumigatus as demonstrated by these in vitro methods, although present in occasional patients, was not clearly related to any one particular clinical syndrome in this small group of patients with aspergillus related pulmonary disease. There was, however, one of the three aspergilloma patients with positive lymphocyte transformation to all five batches of antigens and having higher transformation indices than in any other patient. This suggest that lymphocyte studies should be extended in this group. In contrast to the frequent negative results using A. fumigatus antigens, evidence of T-lymphocyte sensitization to either Candida albicans or Mycobacterium tuberculosis or both, was shown by positive delayed hypersensitivity skin responses and in vitro inhibition of leucocyte migration in the majority of the patients, despite which lymphocyte transformation was often negative. The possibility of impaired capacity to transform is supported by the finding of an impaired response to phytohaemagglutinin (PHA) in four of the twelve cases. This information, together with other data discussed, helps to complete the contrasting immunological profiles seen in different Aspergillus fumigatus related lung diseases.

Leucocyte migration inhibition in myasthenia gravis

Cellular hypersensitivity in myasthenia gravis (MG) to the thymus, muscles and peripheral nerves was examined by the method of the leucocyte migration inhibition test. The group of MG patients without thymoma had inhibition of leucocyte migration by thymus antigens. After thymectomy, they had a normal value of leucocyte migration. However, in the group of MG patients with thymoma, the inhibition of leucocyte migration by thymus antigens was observed after thymectomy. No significant inhibition of leucocyte migration was observed using muscle and peripheral nerve antigens. Cellular immunity in myasthenia gravis and the pathogenesis of the disease was discussed.

Cell mediated immunity to herpes simplex in man. IV. A correlation of lymphocyte stimulation and inhibition of leukocyte migration

The cell mediated immune response to herpes simplex virus was assessed by a modification of the leukocyte migration inhibition test, developed to increase its sensitivity and correlated with the in vitro stimulation of lymphocytes. The introduction of a preincubation step allowed us to demonstrate significant inhibition of leukocyte migration in 15 out of 24 subjects suceptible to recurrent herpes labialis. This was not present in any of the 10 subjects without such infections. However lymphocytes from all 24 subjects demonstrated stimulation in response to herpes virus antigen. The degrees of lymphocyte stimulation and migration inhibition in the susceptible subjects were well correlated and do not support the suggestion that there is a focal defect in this aspect of the cell mediated immune response to herpes simplex virus in those patients.

Cell-Mediated Immunity to Melanoma-Associated Antigens in Patients With Ocular Malignant Melanoma

We tested 11 patients with choroidal melanomas and 32 control subjects for cell-mediated immunity to melanoma-associated antigens by an in vitro leukocyte migration inhibition assay. Five of seven patients with choroidal melanomas, who received two melanoma extracts used in this series of experiments, had significant leukocyte migration inhibition as compared with none of 17 normal subjects. Four of five melanoma patients who received a third melanoma extract had significant leukocyte migration inhibition as did three of four normal controls, indicating that the extract showed nonspecific inhibition. These data agreed with the concept that patients with choroidal melanomas have cell-mediated immunity to common melanoma-associated antigens.

The modifying effect of autologous serum on leukocyte migration inhibition by liver antigens in systemic lupus erythematosus.

The leukocyte migration test was used to examine patients with systemic lupus erythematosus (SLE) for evidence of cell-mediated immunity to human liver microsomes and mitochondria. Testing of both these antigens with leukocytes from patients was carried out simultaneously in culture medium enriched by either 20% fetal bovine serum (the standard test) or substituted 20% autologous serum. Normal controls showed little or no reactivity in the standard test whereas the SLE group revealed a high incidence of significant leukocyte migration inhibition to both antigens which, however, was considerably reduced by the substitution of autologous serum. The results suggest that the sera of patients with SLE contain factor(s) capable of blocking the in vitro expression of cell-mediated immunity to human tissue antigens. These factor(s), if active in vivo, might have a role in controlling some of the auto-immune manfestations of the disease.

Migration of peripheral leukocytes in the presence of carcinoembryonic antigen. Studies in patients with chronic inflammatory diseases of the intestine and carcinoma of the colon and pancreas (32568).

The leukocyte migration technique was employed to study in vitro cell-mediated immune responses to purified CEA in patients with Crohn's disease and active ulcerative colitis and in those with colonic and pancreatic carcinoma. No significant inhibition of leukocyte migration was demonstrated by CEA, with the exception of one patient with pancreatic carcinoma. Thus, with the leukocyte migration technique, no consistent in vitro cell-mediated immunity to CEA was demonstrated supporting the hypothesis that CEA is not the antigen toward which cell-mediated host response phenomena are directed.

Leukocyte migration inhibition in nickel dermatitis.

Leukocyte migration inhibitory factor assay was employed as an in vitro diagnostic aid in nickel dermatitis, the second most common contact dermatitis in North America. 15 patch test-positive and 5 patch test-negative patients, all giving a past history suggestive of nickel dermatitis, were investigated. Significant inhibition of leukocyte migration in both groups of patients was obtained only with nickel sulfate-albumin conjugate and not with unconjugated nickel sulfate. Specificity of this system was tested by utilizing an unrelated metallic albumin complex, and no inhibition was found. When patch testing is equivocal or contraindicated, this in vitro technique may be a practical alternative.

Leukocyte migration inhibition in chorioretinitis.

Leukocyte migration inhibition (LMI) by choroid, retina, and uveal tract antigens was studied in patients with active and quiescent chorioretinal disease. LMI by retina was more frequent in both groups of patients, occurring in 9 of 16 patients with active disease and in 8 to 15 patients with disease in remission. Choroid was tested in 15 patients with active inflammation and led to LMI in 7 of them when they were off steroid therapy; it produced LMI in 3 of 13 patients whose disease appeared healed. Significant LMI was detected in three of six patients with active chorioretinitis and in one of five patients with evidence of past chorioretinopathy when their cells were tested with antigen made from pooled uveal tract tissue. The LMI test, regarded as an indicator of cell-mediated immunity, is briefly discussed. LMI by retina in over one-half of patients with chorioretinopathies appears to be a secondary event, and cellular immunity to retina may be of pathophysiological importance in the chronicity of the disorder.

Studies in myasthenia gravis: cellular hypersensitivity to skeletal muscle.

The direct leukocyte migration inhibition technic was used to investigate cell-mediated immunity to skeletal muscle components in patients with myasthenia gravis. Significant migration inhibition was observed in 14 of the 21 patients studied using crude skeletal muscle as antigen. Myosin-containing muscle fractions demonstrated similar or increased inhibitory activities. A strong positive correlation was found between presence and titer of circulating antiskeletal muscle antibodies (demonstrable by indirect immunofluorescence) and significant leukocyte migration inhibition. These findings indicate that patients with myasthenia gravis may demonstrate both humoral and cellular sensitization to skeletal muscle.

Postoperative Depression of Tumour-directed Cell-mediated Immunity in Patients with Malignant Disease

Leucocytes from 46 melanoma patients, 45 breast carcinoma patients, and 95 control donors were tested by the leucocyte migration test against the supernatants of homogenates of malignant melanomas, breast carcinomas, simple breast tumours, and breasts showing simple cystic disease. By comparison with controls inhibition of migration occurred significantly more frequently when tumour patients' leucocytes were exposed to extracts of histogenetically similar tumours.Cell-mediated immunity to tumour-associated antigens was measured in 12 patients with breast carcinoma and 12 with malignant melanoma immediately before surgical operation and in the postoperative period. All patients tested before operation showed significant inhibition of migration on contact with extracts of histogenetically similar tumours. Postoperatively the degree of leucocyte migration inhibition was reduced in all patients with melanoma and breast carcinoma. Significant inhibition of leucocyte migration returned in most patients 6-22 days after operation.

Quantitative estimation of mouse transplantation antigens by two in vitro methods.

SUMMARY The inhibition tests of leukocyte migration and of target cell destruction have been compared for their sensitivity for the detection of transplantation antigens. The migration inhibition test was performed with spleen cells of C57BL/6J animals which had been immunized with Sal cells of the A strain. Inhibition of target cell destruction was carried out with rabbit antisera, using guinea pig complement and 51Cr-labeled target lymphocytes; the inhibitory antigen was the crude membrane-lipoprotein preparation isolated from the spleens and lymph nodes of normal A/J animals. Significant inhibition of leukocyte migration was obtained with the antigen preparation at a concentration of 100 µg/ml or higher. The inhibition of target cell lysis depended on the cytotoxic titer of the antiserum; at 71 and 56% cytotoxic titers, complete inhibitions were obtained with 100 and 25 /µg of antigen, while 50% inhibitions were obtained with 70 and 14 /µg of antigen, respectively.