Leukocyte Migration Inhibition Test Research Articles

Immunomodulatory activity of Punica granatum in rabbits—a preliminary study

Punica granatum L. (Punicaceae) fruit rind powder (PGFRP) at the dose of 100 mg/kg orally as aqueous suspension was found to stimulate the cell-mediated and humoral components of the immune system in rabbits. PGFRP elicited an increase in antibody titer to typhoid-H antigen. It also enhanced the inhibition of leucocyte migration in Leucocyte Migration Inhibition test and induration of skin in delayed hypersensitivity test with Purified Protein Derivative (PPD) confirming its stimulatory effect on cell-mediated immune response.

小柴胡湯のヒトリンパ球に対する免疫薬理作用 薬剤添加リンパ球刺激試験と白血球遊走阻止試験における小柴胡湯の抗原調製の検討

We performed drug-induced lymphocyte stimulation tests (DLST) and leukocyte migration inhibition tests (LMIT) in an analysis of a traditional Chinese medicine, Xiao-chai-hu-tang (Japanese name : Shosaiko-to) and seven kinds of the constituent herbs in healthy volunteers to study their immunopharmacological effects on the peripheral blood lymphocytes.The DLST findings were positive at concentrations of more than 0.01mg/mL for the heated suspension of Shosaiko-to and more than 0.1mg/mL for the centrifuged supernatant and the 0.22μm membrane-filtrate. On the other hand, the LMIT findings were positive at concentrations of more than 0.1mg/mL for three kinds of Shosaiko-to preparations. The activities of Shosaiko-to in DLST and LMIT decreased after removing insoluble materials using centrifugation and filtration and were higher in enriched T-cells than in unfractionated peripheral blood mononuclear cells. Scutellariae Radix, Glycyrrhizae Radix and Bupleuri Radix showed higher DLST and LMIT activities among the constituent herbs of Shosaiko-to.Our findings indicate that DLST should be performed at concentrations of less than 0.001mg/mL using the heated suspension of Shosaiko-to or less than 0.01mg/mL using the centrifuged supernatant and LMIT at less than 0.01mg/mL using either the heated suspension or the centrifuged supernatant to determine whether patients have hypersensitivity to Shosaiko-to, because high concentrations of Shosaiko-to cause in vitro polyclonal stimulation of the peripheral blood mononuclear cells isolated from healthy donors. Furthermore, our results also suggest that the above activities of Shosaiko-to may effect T-cells due to high molecular weight insoluble particles most likely contained in the Scutellariae Radix, Glycyrrhizae Radix and Bupleuri Radix.

Antisperm response in rams experimentally infected with Brucella ovis

Brucella ovis infection causes chronic epididymitis in rams and may result in the formation of spermatic granulomas and a major reduction in fertility. To study the relationship between bacterial genital infection and antisperm response, 14 adult rams were experimentally inoculated via conjunctival ( n = 7; G1) or preputial route ( n = 7; G2) with a B. ovis strain. Serological response to B. ovis was evaluated by means of complement fixation (CF) and gel diffusion (GD). Autoimmunity was estimated by determination of antisperm antibodies (ASab) and the leukocyte migration inhibition test (MIT) using three different antigens: autologous sperm, ovine testicle, and B. ovis antigen. Immune responses were analyzed in relation to clinical signs, histopathological features, and bacterial isolations from semen and genital organs. Seven non-infected rams served as controls. Specific antibody titers were detected by CF during the second week post-inoculation (PI) in 100% of the inoculated rams. The CF values reached a peak during the 8th and 6th week PI for groups G1 and G2, respectively. The percentage of seropositive animals decreased progressively in both groups until weeks 45 and 30 PI. Specific antisperm reaction was verified by detection of immobilizing ASab and by MIT. ASab were detected in the serum of 85.7% and 71.4% of rams in G1 and G2, respectively, from week 3 PI onwards. Genital alterations were clinically detected in 71.4% of the inoculated rams. Gross epididymal lesions consisted of multiple caseous masses and cysts with purulent content. In those rams that presented clinical lesions, numerous spermatic granulomas were observed. 80% of these granulomas were located in the epididymis tail. Epididymitis, seminal vesiculitis, and chronic ampullitis were histologically detected in 71.4% of the animals of G1 and G2. B. ovis was isolated from 57% of semen cultures and was recovered from genital tissues in 75% of rams in both G1 and G2. At week 50 PI, four rams from each inoculated group (G1 and G2) received antibiotic treatment with long-acting oxytetracycline. In those animals treated with antibiotics, all semen and tissue cultures became negative to B. ovis at the time of the necropsy. However, cellular immunity was positive for autologous, testicular, and bacterial antigen and all rams demonstrated to be positive for ASab until week 80 PI. These data showed that rams with genital lesions caused by B. ovis developed a long-standing antisperm immune reaction. This autoimmune process could be significant in the pathogenesis of reduced fertility observed in B. ovis infected rams.

Experimental Inactivated Concentrated Adjuvant Rabies Vaccine. Evaluation of its Efficacy in Cattle

Experimental Inactivated Concentrated Adjuvant Rabies Vaccine. Evaluation of its Efficacy in Cattle

Assessment of the immune response to duck plague vaccinations

An experiment was conducted to assess the immune responses of ducks to duck plague ( DP) vaccinations employing one commercial and one laboratory-adapted (LA) DP vaccines. Virus neutralisation and leucocyte migration-inhibition tests were conducted at regular intervals before and after vaccinations. Similarly, ducks in vaccinated and control groups were subjected to challenge infection with virulent DP virus. The commercial vaccine yielded a poor immune response and partial protection on challenge whereas satisfactory responses were obtained in ducks receiving two doses of LA vaccine. The humoral as well as cellular factors were stimulated indicating possible involvement of both the immune responses in the protection from duck plague.

Protection, humoral and cell-mediated immune responses in calves immunized with multiple emulsion haemorrhagic septicaemia vaccine

A multiple emulsion (ME), vaccine against Pasteurella multocida (P52) infection in cattle was prepared and the efficiency in terms of immunity to direct challenge, duration of this immunity for up to 1 year and the role of humoral and cell-mediated immune mechanisms were studied. ME vaccine was sterile, safe and was potent when tested in rabbits and calves. Nineteen calves were immunized with a single 4 ml dose of ME vaccine intramuscularly. Group of these calves were challenge infected with virulent P. multocida (P52) (10 −1 18 h old broth culture) given by the subcutaneous route at 21 days, 3 months, 6 months, 9 months and 1 year. All the immunized calves withstood challenge infection and showed 100% protection. Humoral immune response was measured by indirect haemagglutination test (IHA) and enzyme-linked immunosorbant assay (ELISA). Statistically ELISA values were found to be superior to IHA values because of small coefficient of variance. A fall in mean antibody titres during 24 h, 48 h post-challenge infection was recorded whereas a steady increase in the titre after 72 h up to 10 days was noticed. The prechallenge mean titre in animals correlated with survival of animals. Humoral antibodies were detected as early as 7 day post-immunization and persisted to 1 year after immunization. Leucocyte migration inhibition test showed > 20% migration inhibition during all pre- and post-challenge periods in animals suggesting an involvement of cell-mediated immune mechanism in protection. Our findings suggested that both humoral and cell-mediated immune responses contribute to protection in vaccinated animals. The results of these studies of ME vaccine showed that it can be successfully used for the effective control of haemorrhagic septicaemia.

Activity of a crude extract formulation in experimental hepatic amoebiasis and in immunomodulation studies

The activity of a crude extract formulation was evaluated in experimental amoebic liver abscess in golden hamsters and in immunomodulation studies. The formulation comprises the following five plants — Boerhavia diffusa, Tinospora cordifolia, Berberis aristata, Terminalia chebula and Zingiber officinale. The formulation had a maximum cure rate of 73% at a dose of 800 mg/kg/day in hepatic amoebiasis reducing the average degree of infection (ADI) to 1.3 as compared to 4.2 for sham-treated controls. In immunomodulation studies humoral immunity was enhanced as evidenced by the haemagglutination titre. The T-cell counts remained unaffected in the animals treated with the formulation but cell-mediated immune response was stimulated as observed in the leukocyte migration inhibition (LMI) tests.

Immune Response to Synthetic Materials

Metallic orthopaedic devices are composed of elements that are known to be skin sensitizers in the general population. There is concern about the possibility of sensitivity reactions in patients bearing these implants. Blood samples were drawn from 22 patients having primary total joint replacement and who had no known prior metal allergies or exposure. Repeat blood samples were drawn 3 months to 1 year later. All preoperative blood samples showed no immune reactions against titanium, cobalt, chromium, or nickel ion solutions in a leukocyte migration inhibition test. Thirty two percent (7 of 22) of the patients developed sensitivity to at least 1 of the antigens, but only 5 percent (1 of 22) developed a severe reaction. Review of the literature and these studies has indicated that such reactions can occur. However, the incidence seems to be very low.

Transfer factor--current status and future prospects.

We have detected new clues to the composition and function of "Transfer Factor" using the direct Leucocyte Migration Inhibition (LMI) test as an in vitro assay of Dialysates of Leucocyte Extracts (DLE). This approach has revealed two opposing antigen-specific activities to be present in the same > 3500 < 12,000 DA dialysis fraction - one activity is possessed of Inducer/Helper function (Inducer Factor). The opposing activity is possessed of Suppressor function (Suppressor Factor). When non-immune leucocyte populations are cultured with Inducer Factor they acquire the capacity to respond to specific antigen and inhibition of migration occurs. This conversion to reactivity is antigen-specific and dose-dependent. When immune leucocyte populations are cultured with Suppressor Factor their response to specific antigen is blocked and Inhibition of Migration is prevented.

Evaluation of Paracoccidioides brasiliensis exoantigen in the detection of delayed dermal hypersensitivity in experimental and human paracoccidioidomycosis.

The exoantigen of Paracoccidioides brasiliensis standardized by Camargo et al. (AgR) was used to evaluate the in vivo and in vitro cell immune response of experimental animals and of patients with paracoccidioidomycosis (PBM). Fava Netto antigen (AgF) was tested in parallel as a control antigen. The study was conducted with mice and guinea pigs infected with P. brasiliensis or immunized with its fungal antigens, on patients with PBM and on their respective control groups. The cell immune response was analysed by skin tests, and by the macrophage and leucocyte migration inhibition tests (MMIT and LMIT) in the animals and in the patients, respectively. The skin test with AgR as paracoccidioidin was positive in infected or immunized mice and guinea pigs and negative in control animals. The skin tests with AgR (24 h) showed 96.7% positivity in patients with PBM and were negative in control individuals. Histopathological study of the in vivo tests in the different experimental models was consistent with a delayed hypersensitivity response (DHR). Immunohistochemical study of the skin tests of PBM patients demonstrated a predominance of T lymphocytes, confirming the nature of a DHR to the fungal antigens. The in vitro cell immune response showed variable results for the various experimental models, i.e. significant rates of MMIT in immunized mice, a tendency to positivity in infected guinea pigs, and the absence of migration inhibition in PBM patients. Taken together, the data indicate that the AgR is efficient as paracoccidioidin in the evaluation of DHR in PBM, with an optimum time of reading the test of 24 h.

Azathioprine-induced pulmonary haemorrhage in a child after renal transplantation

We report a case of azathioprine-induced haemorrhagic alveolitis, in a 14-year-old boy, after renal transplantation. On day 25 the patient developed haemoptysis, fever and hypoxaemia. Chest X-ray showed diffuse reticulo-nodular shadows in both lung fields. Bronchoalveolar lavage samples were haemorrhagic and demonstrated a relative neutrophilia and a mild lymphocytosis, with a normal CD4/CD8 ratio. Azathioprine was discontinued on day 26. The patient required mechanical ventilation for 4 days. A positive leucocyte migration inhibition test and the recurrence of the symptoms after a second short course of azathioprine therapy suggested a cell-mediated mechanism. This patient is, to our knowledge, the first child to suffer from azathioprine-induced pulmonary haemorrhage.

Evaluation of host resistance and non‐specific cell mediated immune responses in mice exposed to sublethal subchronic doses of Aldrin

The effect of sublethal subchronic dose of Aldrin, an organochlorine pesticide, has been studied on non‐specific cell mediated immune responses and host resistance to an infection. Aldrin treatment in mice could result in significant suppression of stimulation of leukocytes to PHA as measured by the leukocyte migration inhibition and leukocyte adherence inhibition test. Further, it leads to a significant increase in parasitaemia, increased mortality rate and decreased survival time in mice infected with Plasmodium berghei. Our results reveal that a sublethal subchronic dose of Aldrin may affect non‐specific stimulation of cells involved in immune reactions and increase the rate of parasitaemia.

Factors influencing the manifestation of skin allergic reaction to microorganism antigens in healthy subjects

The patterns of variation in skin allergic reactions to antigens ofStaphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Escherichia coli, and tuberculin were studied in healthy subjects. The extent of skin reaction was found to vary within a wide range in different populations, with no relationship to genotypic factors. The results of the skin test were related to the type of antigen and did not correlate with the sex, age, or blood group of examinees or with the results of the leukocyte migration inhibition test. Effects of “familial” or climatic and geographic factors were also negligible.

Immunization against Porcine Cysticercosis in an Endemic Area in Mexico: a Field and Laboratory Study

An antigenic extract from Taenia solium metacestodes was evaluated for immunogenicity in pig populations from a large area of endemic porcine cysticercosis in the State of Guerrero, Mexico. A total of 3,295 pigs from 18 villages were immunized with a single dose of 250 micrograms of protein administered intramuscularly. Systematic immunization was also performed on pigs (1,076 immunizations) from two of the villages with the highest percentages of cysticercosis. A year after immunization, porcine cysticercosis decreased from 4.8% and 5.4% to 0%. Immunity against the T. solium metacestode was estimated in vitro by measurements of 3H-thymidine uptake and inhibition of leukocyte migration. Peripheral blood lymphocytes from immunized cysticercotic (pigs that had cysticercosis prior to immunization), cysticercotic immunized (pigs that acquired cysticercosis after immunization), and normal control pigs incorporated 3H-thymidine better than lymphocytes from cysticercotic pigs when stimulated with concanavalin A. A significant inhibition in the leukocyte migration inhibition test was also found in leukocytes from immunized cysticercotic pigs (P < 0.01). Histopathologic studies revealed granuloma formation surrounding the metacestodes of the immunized cysticercotic and cysticercotic immunized pigs. These metacestodes exhibited several stages of destruction. Large numbers of eosinophils were frequently observed in a close association with the degeneration and destruction of parasites. Metacestodes in control cysticercotic pigs were intact and surrounded by a minor inflammatory reaction. Finally, the rate of in vitro evagination of scolices was high in metacestodes obtained from cysticercotic pigs and low or absent in those from immunized pigs (P < 0.01).

Cellular immune responsiveness in rabbits with Setaria Digitata filarial antigen and TDM adjuvant

The purified surface antigens of the bovine filarial parasite Setaria digitata were used as an antigen to immunize rabbits. The aqueous suspensions of trehalose 6-6′ dimycolate (TDM) has been successfully used as an effective immunomodulator in experimental studies on filariasis. The effectiveness of such an antigen — TDM combination was demonstrated by enhanced humoral and cellular immunity. Administration of antigen alone shows only humoral immunity. The detectable cellular immune responses further confirm the effect of filarial antigen — TDM combination. The cell-mediated immunity was expressed in vivo by delayed skin reaction and in vitro by leukocyte and macrophage migration inhibition tests.

Immunokinetics of equine herpesvirus 1 in donkey mares: suppression of secondary cell-mediated response.

To study the immunokinetics of equine herpesvirus 1 (EHV1), donkey mares were immunised with a laboratory strain of EHV1, or with recommended doses of Pneumabort-K vaccine (EHV1 Army 183 strain, formalin-inactivated, with an oil adjuvant) and a booster was given after three months. Humoral immune responses were studied by employing a virus neutralisation (VN) test. A leucocyte migration inhibition test (LMIT) was employed for the assay of cellular immune responses. The VN antibody titre reached 1:64 or 1:128 after primary immunisation and showed a marginal increase (1:256) after secondary immunisation with either of the immunogens. After the primary dose of immunogen, there was a gradual increase in host cellular response which persisted for up to three months. However, on secondary immunisation, cell-mediated immune response was short-lived and weak compared to the primary response with both immunogens. This could be one possible explanation for breakdown of anti-EHV1 immunity leading to abortion in immunised mares.

Adjuvant immunotherapy in melanoma: a new approach.

Patients with metastatic cutaneous melanoma to two or more regional lymph nodes have an extremely poor prognosis despite radical lymphadenectomy. In an attempt to improve the survival and to determine the safety of a new method of tumor specific adjuvant immunotherapy in such a high risk group of patients, nine patients were studied. Three to four weeks after regional lymphadenectomy, each of them received a single intradermal injection of Bacillus Calmette-Guérin. Three weeks later, they were immunized by allogenic melanoma cells obtained from live donors with distant metastases. Each patient received three vaccinations, each from a different donor (except in one), to avoid development of HLA response, but maintaining exposure to melanoma antigens. No cultured melanoma cells were used. Each vaccine consisted of mitomycin-C treated tumor cells mixed with purified protein derivative (PPD) of tuberculin given intradermally once per month for 3 months. The patients were then observed with no further treatment. Utilizing the leukocyte migration inhibition test, there was some in vitro evidence of tumor specific cell mediated response which seemed to disappear 1-2 months postimmunization. At 5 years, five of the nine patients (55%) were alive free of disease. No autoimmune diseases were detected in any of the immunized patients. A major hindering factor for such an approach was the limited availability of the allogenic melanoma cells.

Sensitization against pancreatic antigen in Sjögren's syndrome and chronic pancreatitis. Preliminary communication.

A fractionated pancreatic antigen was prepared using a monoclonal antibody, SP3-1, which reacts with the duct cells of various exocrine organs. The cell-mediated immune response to this antigen was studied by the leukocyte migration inhibition test (LMT) in patients with chronic pancreatitis (CP), Sjögren's syndrome (SjS), and chronic sialoadenitis (CSA). The migration index (MI) for the LMT was 0.97 +/- 0.07 in normal controls (mean +/- SD, n = 11, range: 0.88-1.08). A positive LMT result (MI less than 0.82 = mean -2 SD in controls) was obtained in 7/8 (88%) SjS patients, 4/22 (18%) CP patients, 2/3 CP patients with SjS, and 0/3 CSA patients. These results indicated sensitization against the pancreatic antigen in most SjS patients and some CP patients, and may suggest that an immune response to a common antigenic determinant of the duct cells of exocrine glands plays a role in the pathophysiology of both diseases.

Erratum to : Leukocyte Migration Inhibition Test in Drug-Induced Pneumonitis

Erratum to : Leukocyte Migration Inhibition Test in Drug-Induced Pneumonitis

Evaluation of the Cell-Mediated Immune Response of Chickens Vaccinated with Newcastle Disease Virus as Determined by the Under-Agarose Leukocyte-Migration-Inhibition Technique

The under-agarose leukocyte-migration-inhibition (LMI) assay for the evaluation of cell-mediated immunity (CMI) was developed for use in chickens. A significant CMI response was demonstrated in chickens vaccinated with Newcastle disease vaccine by using this assay. Results also indicated that the under-agarose LMI test was a reproducible and relatively easy assay to perform. Serologic antibody titers were determined to study the correlation between hemagglutination-inhibition (HI) titers and LMI levels using the HI microassay. The presence of the CMI response always correlated to the presence of the serologic immune response and vice versa. However, the quantitative level of the CMI response did not always correspond to the quantitative level of the serologic immune response. Going by results of this study, we were unable to predict humoral antibody level on the basis of a CMI response and vice versa.