Indirect leukocyte migration inhibition in breast cancer and benign breast disease patients by mouse mammary tumor virus grown in feline kidney cells.

Indirect migration inhibition assays were performed with normal and mammary tumor-bearing C3H/HeN mice and patients with breast disease to assess cellular immunity against three different mouse mammary tumor virus (MTV) preparations grown in feline kidney cell cultures and against a mouse-derived MTV preparation. MTV obtained after passage through feline kidney cells and the mouse-derived MTV were capable of eliciting macrophage migration inhibitory factor production by mouse spleen cells obtained from normal or mammary tumor-bearing C3H/HeN mice, thus demonstrating a similar degree of antigenicity of these preparations. In experiments with human breast cancer patients' leukocytes, leukocyte inhibitory factor (LIF) was produced by 32-50% of these patients in response to the mouse-derived MTV or to three different MTV preparations obtained after passage through feline kidney cells. A significant proportion (31-54%) of benign breast disease patients also reacted with both the mouse-derived and feline-derived MTV preparations. Patients with both malignant and benign breast disease, however, had a significantly different (P less than .05) pattern of reactivity to mouse- and feline-derived MTV preparations from that observed with normal donors. Finally, some LIF activity was also observed (but not statistically significant with the use of nonparametric analysis methods) when feline leukemia virus was used as antigen with these patients. The data suggest that both breast cancer and benign breast disease patients were reactive against antigens largely specific for MTV in the feline cells and, presumably, were not reactive against feline cellular components, although the second possibility cannot be completely ruled out.

Indirect leukocyte migration inhibition reactions to a 3-M KCl extract of lung adenocarcinoma by lung cancer patients.

Mononuclear (MN) cells from the peripheral blood of lung cancer patients were tested for their ability to respond to a 3-M KCl extract of adenocarcinoma of lung with the use of an indirect leukocyte migration inhibition (LMl) assay. Antigen-stimulated MN cell cultures were evaluated for leukocyte inhibitory factor production by their ability to inhibit the migration of indicator polymorphonuclear cells from agarose droplets. When supernatants were prepared in conventional round-bottomed tubes (5X10(6) cell/tube), 25 of 44 (57%) lung cancer patients had positive indirect LMl responses to the 7661 antigen as compared to only 2 of the 30 (7%) normal donors. When supernatants were prepared in conical microtubes, with 10 times fewer MN cells, similar results were obtained. Patients with all histologic types of lung cancer had a similar incidence of reactivity, and reactivity of untreated patients did not appear to be related to stage of disease or degree of tumor burden. Surgical removal of the tumor appeared to decrease the incidence of reactivity in the 1- to 12-month postoperative period. These results strongly suggest that the LMl reactivity against lung tumor extracts is lymphokine mediated, inducing cellular responses by the patients against such antigens.

[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.

Evidence for cell-mediated immunity and specific suppressor T lymphocyte dysfunction in Graves' disease and diabetes mellitus.

Migration inhibition of purified peripheral T lymphocytes in response to pancreatic islet cell antigen or thyroid antigen was used to study cell-mediated immune mechanisms in patients with diabetes mellitus (IDDM) and Graves' disease (GD). In response to islet cell antigen, T lymphocytes of subjects with IDDM for less than 3 yr exhibited migration inhibition, whereas those of normal subjects, noninsulin dependent diabetics, and subjects with IDDM for longer than 3 yr did not. Admixture of T lymphocytes from normal subjects with T lymphocytes from patients with IDDM for less than 3 yr substantially ameliorated the migration inhibition of the IDDM subjects to islet cell antigen. Migration of T lymphocytes from GD subjects was markedly inhibited by thyroid antigen and marginally inhibited by islet cell antigen. Admixture of GD T lymphocytes significantly ameliorated the migration inhibition of IDDM T lymphocytes to islet cell antigen, despite sensitization to thyroid antigen of the GD T lymphocytes. We conclude: 1) sensitization to islet cell antigen in IDDM of recent onset is confirmed; 2) the ability of normal and GD T lymphocytes to ameliorate the migration inhibition of IDDM T lymphocytes strongly suggests correction of deficient suppressor T lymphocyte function; 3) the ability of GD T lymphocytes to ameliorate migration inhibition of IDDM T lymphocytes to islet cell antigen is evidence for an antigen-specific rather than a generalized suppressor T lymphocyte defect in GD; and 4) similarly, the normalization of migration index of GD T lymphocytes in response to thyroid antigen by those IDDM T lymphocytes not sensitized to thyroid antigen is again evidence for an antigen-specific and not a generalized suppressor T lymphocyte defect in IDDM.

The use of human lymphoid cell line lymphokine preparations (LCL-LK) as working standards in the bioassay of human leucocyte migration inhibition factor (LIF).

The leucocyte migration inhibition test in agarose as described by Clausen (1971) was modified into a statistically designed assay of LIF activity using human polymorphonuclear leucocytes from single blood donors. Individual assays included a laboratory standard of lymphokine with LIF activity prepared from the culture supernatants of the RPMI 1788 human lymphoblastoid cell line (LCL-LK). Analysis of 157 LIF assays revealed simple criteria by which the acceptability of an individual assay could be judged before subjecting it to statistical analysis. The failure of LIF assays to meet these criteria of acceptability was particularly associated with low areas of control polymorph migration in the absence of lymphokine ('spontaneous migration'). We demonstrate that the statistically designed assay permits the measurement, with precision, of LIF activity in units/ml by reference to a working standard of LCL-LK. We illustrate the use of this assay in the measurement of LIF activity generated by tuberculin-stimulated human peripheral blood lymphocytes.

In vitro cell-mediated immunologic assay for cow's milk allergy.

The production of a lymphokine, the leukocyte-migration-inhibition factor (LIF), by peripheral blood lymphocytes in response to an in vitro challenge with bovine beta-lactoglobulin was assayed in infants and children suspected of having allergy to cow's milk protein. Of the patients studied, 24 had cow's milk allergy, 24 were normal control subjects, 18 had recovered from milk allergy, 10 were newborns, and 10 were babies suffering from acute gastroenteritis. All patients with milk allergy demonstrated significant LIF production in response to beta-lactoglobulin (23.5% +/- 6.4%). In the normal control subjects, LIF was 3.1% +/- 4.3% (P < .0005). Only two of the 24 control subjects and two of the ten newborns had high-normal values bordering on the positive. None of the ten babies with acute gastroenteritis gave a positive response. Most of the children who had recovered from milk allergy and were ingesting cow's milk had negative assays. This cell-mediated immune assay is shown to be a reliable test for the diagnosis of sensitivity to milk protein in infants and children, and for determining dietary treatment and when this treatment can be safely terminated. In most cases, its use should eliminate the need for the potentially dangerous and ethically questionable provocation test, as well as the need for repeated intestinal biopsies.

Modulation of immune response in vitro and in vivo by human granulocyte factors.

The adherence of granulocytes induces secretion of specific granule contents. The secreted proteins were termed granulocyte factors (GF). The experiments in vivo provide evidence that GF play an essential role in the stimulation of PFC in BALB/c mice immunized with SRBC when applied before challenge three times (5 micrograms per mouse), but 50 micrograms per mouse given in the same way diminishes the response. To elucidate this discrepancy, the effect of GF on the generation of suppressor cells (SC) and helper cells (HC) in vitro has been investigated. Antigen specific nonadherent SC or HC were induced in vitro using CBA mice spleen cells incubated with 100 micrograms/ml or 0.1 mg/ml of TNP-KLH, respectively, for 4 days. GF in concentrations of 0.1 to 1 microgram/ml abolish antigen specific SC generation. SC and HC activity was tested in cooperative cultures. Antigen specific SC in delayed hypersensitivity (DTH) to BCG were induced in an in vitro system as above using normal BALB/c spleen cells and 100 micrograms/ml PPD. Nonadherent suppressor cells were transferred intravenously into cyclophosphamide (CY)-treated syngeneic recipients. The recipients were immunized to BCG immediately after the cell transfer. DTH was measured by foot-pad reaction. This reaction was positive to PPD in CY treated mice immunized to BCG, while it was suppressed by the transfer of in vitro induced SC. When the SC were induced in the presence of 1 microgram/ml GF, the suppression was abrogated. The higher GF concentrations stimulated SC activities when they were measured in response to a nonrelated antigen and in specific anti-PPD response, but the HC inhibition could not be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunologic studies on myasthenia gravis. II: A new chemotactic factor for lymphocytes found in patients with myasthenia gravis.

A specific chemotactic factor for lymphocytes was found in thymic tissue extract from myasthenic patients who had undergone thymectomy. Biologic activities and the biochemical nature of this factor were examined. The major findings were as follows: (1) The factor from myasthenic thymus was specifically chemotactic for T lymphocytes. Marker analysis of target cells revealed that only OKT4 positive cells (helper/inducer T cells) responded to the factor. Chemotactic activity was also found in the normal thymus, but its activity was different from that found in the myasthenic thymus. Interestingly, OKT8 positive cells (suppressor/cytotoxic T cells), as well as OKT4 positive cells, migrated toward the normal thymic extract (2) Chromatography of the extract by gel filtration gave three peaks with chemotactic activity; molecular size were 160,000 to 100,000, 25,000 to 15,000, and smaller than 1350 daltons (3) The factor was inactivated by heating for 30 minutes at 56 degree C and was also destroyed under conditions of pH 4 and pH 10. The factor was also sensitive to treatment by trypsin, sodium metaperiodata, 8 mol/L urea, reduction, or alkylation. (4) The chemotactic activity in the myasthenic thymus was distinct from other known chemotactic factors, including C5a, interleukin-1, and interleukin-2. (5) Chemotactic activity for lymphocytes was found in both the aqueous extract and the culture supernatant of thymic stromal cells, but not in thymocyte extract or in the serum. These findings indicate that the chemotactic factor specific for OKT4 positive cells may be secreted by stromal cells of the myasthenic thymus but not of the normal thymus.

Blood transfusions generate/increase previously absent/weak blocking antibody in women with habitual abortion.

Women who suffered recurrent spontaneous abortions of unknown cause were studied for cellular reactivity and blocking antibody in a one-way mixed lymphocyte culture. A defined group of 20 women whose serum displayed no blocking capacity was given three transfusions of leukocyte-rich erythrocyte concentrates. Serum from all women displayed significant blocking capacity 2 months after the third transfusion. Because blocking antibody seems to be one of the necessary prerequisites for successful pregnancy, leukocyte transfusions from third-party donors ought to be an effective cure for habitual abortion in selected cases.

Cellular and humoral responses in coeliac disease. 2. Protein extracts from different cereals.

The humoral and cellular immune responses to grain protein extracts from coeliac-toxic and non-toxic cereals were compared by use of a number of ELISA and immunoblotting methods and the indirect leucocyte migration inhibition factor (LMIF) assay. Both adult and child coeliacs had elevated levels of serum antibody to proteins from the coeliac-toxic cereals, namely bread wheat, durum wheat, rye and barley and low levels of proteins from other cereals. Using protein blotting techniques, antibody binding was greatest to gliadins/low mol mass glutenin subunits and homologous prolamins from rye and barley, consistent with the ELISA findings. Competition ELISA and preabsorption tests indicated that antibody reaction to maize storage proteins did not simply result from cross-reaction of antigliadin antibodies. In LMIF assays, only the wheat extracts had activity in coeliac patients. This is most likely partly due to loss of some of T-cell epitopes from the extraction technique required for these proteins, as well as the relatively small effects seen for even very active fractions in the LMIF assay.

Cellular and humoral responses in coeliac disease. 1. Wheat protein fractions.

The humoral and cellular immune response of coeliac individuals to various wheat protein fractions was studied using serum antibody ELISA assays and the indirect leucocyte migration inhibition factor (LMIF) assays. Greater migration inhibition factor activity was seen in coeliacs on a gluten-free-diet having low serum antibody titres, and using purified T-cells instead of total peripheral blood mononucleocytes. Gliadin was the most active fraction in both assays. Raised antibodies to low-molecular weight and high-molecular weight glutenin polypeptides was observed, though these proteins had little migration inhibition factor activity. No cellular or humoral response was seen to albumins or globulins. Proteins associated with the granules of well-washed wheat starch are distinct from gluten proteins and had little T-cell activity, correlating with clinical observations that properly prepared wheat starch is devoid of coeliac toxicity. The greater specificity of the humoral response for individual wheat protein fractions in this study, compared with the earlier reports, likely results from cross-contamination in the earlier work of each fraction with gliadin.

Metal allergy and the surgical patient.

Allergy to metal implants is under study at the Dartmouth-Hitchcock Medical Center using in vitro examination of white blood cell migration. Retrospective data from 121 patients confirm that allergic responses do occur. Prospective data are being gathered to investigate the incidence, prevalence and relation to morbidity.

Specific cellular immunity in acoustic neuroma patients.

An indirect leukocyte migration agarose technique to detect cell-mediated immunity was modified to obtain a specific assay for release of human leukocyte migration inhibitory factor. Acoustic neuroma patients exhibited a significant cellular immune response against acoustic neuroma extract (P less than .01) as well as perilymph from acoustic neuroma patients (P less than .01) when compared to healthy control persons. All 19 patients tested reacted to acoustic neuroma extract. Seven of 21 perilymph samples did not elicit migration inhibition. Crossover determination of antigenicity of two negative and four positive perilymph samples against three patients revealed highly reproducible results, uncorrelated to perilymph concentration of potassium and protein. Flow cytofluorometry did not reveal malignant DNA patterns in 10 acoustic neuromas examined. Immunofluorescence studies did not reveal autoantibodies against acoustic neuromas in sera from 11 patients. The responsible antigen(s), the mechanism of immunization, and the diagnostic implications remain to be determined.

Cell-mediated immune injury to the heart.

A micro in vitro procedure was utilized to assess cell-mediated immunity of patients following myocardial infarction or cardiac surgery. For this purpose, peripheral blood leukocytes from patients were tested in a microdroplet assay with antigen preparations derived from human cardiac tissue. Whereas whole saline extract and human myoglobin preparations had little effect on the migration of peripheral blood leukocytes in vitro, mitochondrial preparations were markedly effective in inhibiting migration of the leukocytes in the presence of mitochondrial extracts appeared to reflect development of cell mediated immunity to heart antigens after the myocardial infarct or surgery. These results extend the observations that humoral antibody may appear in patient following cardiac injury. The role of either antibody or sensitized lymphoid cells in mediation of post-myocardial infarction or post-cardiotomy syndromes is not clear, but it appears clear that injury to the heart induced auto-reactive responses which may play a role in subsequent pathologic events following the initial cardiac injury.

Patient-specific cell-mediated immunity against human acoustic neuroma extract.

Lymphocytes from 8 patients with acoustic neuromas and 8 healthy control persons were exposed to acoustic neuroma extract. Indirect leukocyte migration agarose technique was applied for detection of leukocyte migration inhibition (LMI). The standard technique was additionally modified using a specific assay for release of human leukocyte migration inhibitory factor (LIF). Both the standard technique and the LIF-specific assay revealed a significant LMI exerted by lymphocytes from patients, as compared with control persons. The LIF-specific assay demonstrates that the LMI is an expression of cell-mediated immunity due to release of LIF. The immune reaction is raised against an unknown antigen in acoustic neuroma extract. Further studies must determine whether the immune reaction is specific for acoustic neuroma patients vis-a-vis patients with other tumours, especially other neuromas.

Local lymphokine production in ocular inflammatory diseases.

We took samples of the aqueous humour and subretinal fluid from 12 patients suffering from various types of ocular inflammation. The presence of leucocyte migration inhibition factor in these fluids was determined using the leucocyte migration inhibition test. Our results indicated that this test differentiated ocular inflammatory reactions due to cell mediated immunity from those due to other mechanisms.

Development of a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, using an antigen of Brucella abortus.

A study was conducted to develop a migration inhibitory factor assay under agarose of bovine mononuclear leukocytes, with an antigen of Brucella abortus. Different concentrations of mononuclear leukocytes were prepared by the Ficoll-Hypaque technique from the blood of nonvaccinated calves and from calves previously vaccinated with strain 19. Concentrations of 0.5, 1, 2, 3, 4, and 5 x 10(6) leukocytes were suspended in RPMI-1640 medium and various dilutions (20, 10, 1, and 0.1 microgram) of B abortus-soluble antigen, dispensed in triplicate wells cut in 1% agarose containing minimal essential medium and 10% bovine fetal serum. These agarose plates were incubated for 4-, 8-, 12-, 16-, 20-, and 24-hour periods and then were fixed; leukocytes were stained with Wright's stain. Migration distances were measured, and statistical analyses of the data revealed a concentration of 2 x 10(6) cells/well and an antigen concentration of 10 microgram/well. An incubation period of 20 hours was optimal for the assay.

[Evaluation of leukocyte migration inhibition (LIF) in patients with carcinoma of the lung before and after the use of levamisole in vitro]. / Valutazione dell'inibizione della migrazione leucocitaria (LIF) su pazienti portatori di carcinoma del polmone prima e dopo l'uso di levamisolo in vitro.

Aspecific immunosuppression in neoplasia has long been known, even though all its biological aspects are not yet fully understood. Inhibition of leukocyte migration (LIF) was studied before and after the use of levamisole in vitro to determine whether changes occurred in cell reactivity. The results of the investigation are discussed in the light of modern immunopathological theories.

[Autoimmune aspects of a case of chronic interstitial cystitis]. / Aspetti autoimmuni in un caso di cistite cronica interstiziale.

Chronic interstitial cystitis is a disease characterized by clinical recurring cystitis with histopathological aspects of a chronic aspecific inflammation of the bladder. Etiopathogenesis is still unknown, even if autoimmunity has been hypothesized in some cases. In this paper we report an immunopathologic and immunohistochemical study on a case of chronic interstitial cystitis. The inappropriate immunologic behaviour of the lymphocyte populations, the inhibition of leukocytes migration with bladder extracts and the immunofluorescent findings of IgG and C3 deposits in the mucosa support the hypothesis of an autoimmune pathogenesis of the disease.

[Myasthenia and pernicious anemia or Biermer's (author's transl)]. / Myasthénie et anémie pernicieuse dite de Biermer.

The association of myasthenia and Biermer's anemia is very rarely reported. In a series of 138 cases of myasthenia, this association was found in only one patient, in whom the anemia developed 19 years after the discovery of a calcified thymoma and 13 years after the appearance of the first signs of myasthenia. This led the authors to conduct a prospective study for the presence of intrinsic antifactor antibodies. A total of 81 patients (20 men and 61 women) with myasthenia were studied. The myasthenia had appeared after 35 years of age in 40 patients and 19 had a thymoma. The results of the study for the antibodies was positive in 3 women, as was the test of inhibition of leucocyte migration, but none of them had anemia, vitamin B12 malabsorption, achlorhydria, or gastric atrophy. The discovery of these immunological disorders raises the problem of their significance ; two hypotheses can be discussed : pre-Biermer state or immunological disturbance without pathogenetic significance. The problem can probably only be resolved by studying these antibody levels in a very much larger number of patients with myasthenia.