Reactivity Of Peripheral Blood Leukocytes Research Articles

Tumor size, leukocyte adherence inhibition and serum levels of tumor antigen in dogs with the canine transmissible venereal sarcoma.

Tumor antigen (TA) associated with the canine transmissible venereal sarcoma (CTVS) was detected in the sera of dogs bearing the tumor. Rabbit antisera specific for tumor antigen and 3 M KCl extracts of CTVS cells were used in both a competitive enzyme-linked immunosorbent assay (ELISA) and antigen-capture ELISA to quantify levels of circulating TA. In a study of 29 dogs bearing the transplanted CTVS, levels of circulating TA correlated positively with tumor volume. In a longitudinal study of four dogs receiving a transplant of 10(8) viable CTVS cells, circulating CTVS antigen was detected transiently 2 days after transplantation, while persistent levels of TA associated with increasing tumor volume were demonstrable 19-34 days after transplantation. In three of four tumor-bearing dogs, levels of serum TA correlated inversely with values obtained with peripheral blood leukocytes in the leukocyte adherence inhibition (LAI) assay; elevated levels of circulating TA found in dogs with large (greater than 7 cm3) tumors were associated with decreased LAI reactivity of peripheral blood leukocytes. TA could not be detected in sera 48-72 h after surgical removal of CTVS whereas LAI reactivity of peripheral blood leukocytes to CTVS antigen rebounded 1-3 weeks following tumor excision. Results of this study support the use of the competitive ELISA and LAI techniques in assessing levels of circulating tumor antigen, tumor burden and tumor-specific immunity.

Immune reactions in patients with superficial bladder cancer after intradermal and intravesical treatment with bacillus Calmette-Guérin.

The immune reactivity of patients with strongly recurrent superficial bladder cancer was followed after combined intravesical and intradermal bacillus Calmette-Guérin (BCG) immunotherapy. All patients in this study were previously treated without success with intravesical chemotherapy. The BCG treatment regimen consisted of weekly administrations with BCG (RIVM) for six consecutive weeks, both intravesically and intradermally. In this study, sera and peripheral blood leukocytes (PBL) of patients were tested serially. Besides BCG-antigen-specific reactions, e.g. skin reactivity to purified protein derivatives of Mycobacterium tuberculosis (PPD), antibody formation and antigen stimulation of PBL in vitro, non-antigen-specific immune reactivities were also measured, e.g. mitogen response and spontaneous cytotoxic activity of PBL. In addition the antibody response to bladder carcinoma antigens and the cytotoxic activity of PBL for the bladder carcinoma cell line T24 and the natural-killer-sensitive K562 cell line were investigated. The results obtained from the various assays were evaluated for their prognostic value in relation to the length of the tumor-free interval after the BCG treatment. Because sera and PBL were only obtained during the first 6 months after the BCG treatment, the immune reactivity was compared to the clinical results at that same time. At 6 months after therapy 12 out of 40 BCG-treated patients were tumor-free whereas 28 out of 40 showed a recurrence. Skin reactivity to tuberculin PPD was measured in 40 patients during a period of 3-6 months after therapy. Of patients who showed a recurrence of the tumor within 6 months, 48% of them showed a transient response or developed no response at all to PPD. In the group of patients with a longer tumor-free period (n = 10), only one patient lost the response to tuberculin PPD. Although PBL of a limited number of patients were tested, it was observed that the cytotoxicity to the bladder carcinoma cell line T24, and the natural-killer-sensitive K562 cell line increased in a number of the patients (7 out of 14, and 9 out of 14 respectively). Reactivity of PBL to mitogens and subset distribution (ratio T-helper: T-suppressor/cytotoxic) were not influenced by the BCG treatment. Antibody response to mycobacterial antigen was detected in 9 out of 23 patients investigated. Of these 9 patients, 8 belonged to the group with a recurrence of the tumor within 6 months (n = 17). There was no correlation between the skin reactivity and the antibody response to tuberculin PPD.(ABSTRACT TRUNCATED AT 400 WORDS)

Modification of luminol-dependent chemiluminescence reactivity of peripheral blood leukocytes from patients with lymphoreticular tumor, solid cancer, or healthy blood donors by interferon-alpha.

The effect of interferon-alpha (rIFN-alpha 2) on modulating the luminol-dependent chemiluminescence (CL) response evoked by Zymosan or phorbol myristate acetate (PMA) has been studied in heparinized whole blood as well as isolated peripheral blood leukocytes derived from cancer patients and healthy blood donors. The cancer patients, diagnosed with chronic leukemia or lymphoma or solid cancer, were under rIFN-alpha 2 therapy. The CL emission to both Zymosan or PMA was enhanced in the whole blood of the patient's group in comparison to whole blood of healthy blood donors. CL reactivity induced in Dextran-sedimented leukocytes derived from neoplasia patients and healthy blood donors did not differ in the two groups. CL reactivity to Zymosan or PMA could be potentiated when whole blood samples or isolated leukocytes of patients or healthy blood donors were incubated in vitro with rIFN-alpha 2 in the range of 0.001-1000 U/ml prior to addition of CL-stimulating agents. rIFN-alpha 2 itself without adding further agents did not stimulate CL reactivity. Therefore, enhanced CL reactivity to PMA or Zymosan observed in the whole blood of the patients' population appears to be due to the presence of interferon in serum blood components as a consequence of IFN-infusion rather than to other symptoms or treatments of the neoplastic diseases, since the relative CL enhancement in patients is abrogated when isolated blood leukocytes are used for CL assay.

Blastogenic responses of peripheral blood leukocytes from owl monkeys experimentally infected with Leishmania braziliensis panamensis.

The relationship of the progression and regression of cutaneous lesions of 6 owl monkeys (Aotus trivirgatus) to the responses of their peripheral blood leukocytes (PBL) in vitro to mitogens and to leishmanial antigens, as well as their delayed skin test responses (DTH) in vivo to leishmanin antigen, were studied after primary and challenge infections with Leishmania braziliensis panamensis (WR 128 or WR 539). All 6 infected monkeys developed primary and satellite cutaneous leishmanial lesions which were measured for up to 30 weeks in 3 of the monkeys and up to 52 weeks in the other 3 monkeys. Two owl monkeys which had recovered from cutaneous leishmaniasis demonstrated acquired resistance when challenged with an intradermal inoculation of L. b. panamensis (WR 128). Reactivity of PBL from infected owl monkeys to PHA, Con A, and PWM was similar during primary and challenge infections to that observed prior to infection. Reactivity to leishmanial antigens was detected at 20 to 28 weeks post-infection (PI), became statistically significant after 28 weeks and remained elevated up to 52 weeks PI and after challenge infections. During primary infections DTH responses to leishmanin antigen were detected as early as 8 weeks PI, and continued up to 27 weeks PI. After challenge infections DTH reactivity was positive at 25 and 37 weeks, the only times the response was evaluated. The immunological responses of owl monkeys to L. b. panamensis were similar in many respects to those observed in humans with localized cutaneous leishmaniasis. This nonhuman primate model should be useful for future studies involving the immunology and chemotherapy of cutaneous leishmaniasis.

Leishmania donovani: Cellular and humoral immune responses after primary and challenge infections in squirrel monkeys, Saimiri sciureus

Cellular and humoral immune responses were studied in squirrel monkeys after primary and challenge infection with a Khartoum strain (WR 378) of Leishmania donovani. Each of 7 squirrel monkeys, Saimiri sciureus, was inoculated intravenously with 5 × 10 7 amastigotes/ kg body weight, and one other monkey (control) was inoculated with uninfected hamster spleen homogenate. Five infected monkeys recovered from visceral leishmaniasis and two infected monkeys died. Three of the five squirrel monkeys which recovered from the primary infection demonstrated acquired resistance when challenged with an intravenous inoculation of 1.0 × 10 8 amastigotes of L. donovani/kg of body weight. Each of these same three monkeys, the two remaining monkeys which recovered from the primary infection and an uninfected control monkey, were challenged subsequently with an intradermal injection of 2.2 × 10 7 promastigotes of L. braziliensis panamensis (WR539) and developed cutaneous lesions. The reactivity of peripheral blood leukocytes from infected squirrel monkeys to phytohemagglutinin was depressed 2 to 10 weeks after infection, and the reactivity to concanavalin A was not affected. Data on responses to pokeweed mitogen were inconclusive. Reactivity to leishmanial antigens was detected at 12 weeks after infection, which coincided with a marked decrease or disappearance of parasites in liver imprints. Two of five surviving squirrel monkeys developed weak delayed skin test responses to leishmanin antigens after 23 weeks; the three remaining monkeys were anergic during the primary infection but developed strong delayed skin test responses to leishmanin antigens at 7 weeks after a challenge with L. donovani. All squirrel monkeys inoculated with L. donovani developed a hyperproteinemia, hypergammaglobulinemia, hypoalbuminemia, and a reversal of the albumin/globulin ratio between 4 to 18 weeks after infection. Plasma IgM and IgG levels were increased between 2 to 18 weeks after infection; much of this increase was due to IgG. Class-specific antileishmanial antibodies, with generally low IgM and high IgG titers, reached a maximum after 14 and 16 weeks, respectively. A correlation was observed between concentrations of -γ-globulins and plasma IgM and IgG levels, but not between γ-globulin concentrations and maximum titers of class-specific antileishmanial antibodies. Squirrel monkeys challenged with L. donovani again developed hyperproteinemia, hypergammaglobulinemia, and increased concentrations of plasma IgM and IgG which correlated with high titers of IgG class-specific antileishmanial antibody 4 weeks after reinoculation. Squirrel monkeys subsequently challenged with L. braziliensis panamensis developed persistent cutaneous lesions indicating a lack of resistance to the heterologous challenge.

Leukocyte adherence inhibition (LAI) in rats bearing transplantable syngeneic tumors of different immunogenicity

The tube leukocyte adherence inhibition (LAI) assay was used to follow the LAI response in inbred BN rats with subcutaneously (s.c.) transplanted syngeneic liposarcoma (LS 175) tumor and in inbred WAG rats with s.c. transplanted syngeneic colon (CC 531) and skin ( 1618) tumors. Initially, the tube LAI assay was used to follow the LAI reactivity of peripheral blood leukocytes (PBL) from BN rats bearing tumor LS 175. Sporadic LAI reactivity was observed in the PBL of these rats when incubated with a specific crude tumor extract for either 2 or 20 hr. No definite conclusion regarding the lack of significant tumor-specific LAI reactivity could be drawn since LS 175 is a non-immunogenic tumor. Therefore additional LAI studies were performed with the weakly immunogenic tumor CC 531 and the highly immunogenic tumor 1618 in WAG rats. Surprisingly, only sporadic LAI reactivity was observed in the PBL of rats bearing either the CC 531 or the 1618 tumors using both the 2 and 20 hr LAI assays. This absence of tumor-specific LAI reactivity was particularly remarkable in the highly immunogenic 1618 tumor-bearing rats, since it would suggest that the antigen(s) responsible for its high immunogenicity could not evoke any consistent LAI reactivity. Since oxidative metabolites of arachidonic acid have been shown to be the final mediators of chemoattractant-induced LAI, the adherence inhibition effect of leukotriene B 4 (LTB 4) on the PBL of BN and WAG rats was also investigated in order to exclude the possibility that the lack of consistent LAI reactivity was due to a lack of responsive cell population in the PBL. PBL of both BN and WAG rats showed a significant increase in the adherence inhibition in the presence of LTB4, suggesting that the sporadic LAI reactivity was not due to a lack of responsive cell population.

Sequential changes in peripheral blood leukocyte adherence inhibition (LAI) reactivity during progressive growth and spontaneous regression of canine transmissible venereal sarcoma.

Sequential changes in canine transmissible venereal sarcoma (CTVS) extract-induced leukocyte adherence inhibition (LAI) reactivity of peripheral blood leukocytes (PBL) were monitored in dogs from the time of tumor inoculation through progressive growth and spontaneous regression or surgical excision of CTVS. The time-course profiles of LAI reactivity of tumor dog PBL correlated with the clinical stage of tumor growth, substantiating our previous findings in a cross-sectional study. The kinetics of the time-course appearance of LAI reactivity observed and the demonstration of a rapid anamnestic response following tumor challenge indicate further that LAI is a reliable in vitro measure of in vivo tumor immunity. A significant increase in CTVS antigen extract-induced LAI reactivity observed in the PBL during tumor regression suggest that LAI reflects a function effector cell mechanism associated with spontaneous regression of CTVS. By contrast, absence of significant LAI reactivity of PBL during progressive tumor growth suggests the presence of serum blocking factors. In addition a substantial rebound in LAI reactivity observed in the PBL of dogs 3 to 7 days following surgical excision of progressively growing CTVS provides further evidence that tumor-cell components are associated with progressive tumor growth and diminished LAI reactivity.

Leukocyte adherence inhibition and immunoreactivity in prostatic cancer. II. Tissue- and disease-specificity of anti-tumour cell-mediated immunity

Tissue- and disease-specificity of the reactivity of peripheral blood leukocytes from 20 prostatic cancer patients to malignant prostatic tissue have been evaluated. Results of leukocyte adherence inhibition, employed previously, had suggested antitumour cell-mediated immunity. Absence of significant reactivity in prostatic cancer patients to tissues other than malignant prostate and in non-prostatic cancer patients (including patients with benign disease of the prostate) to malignant prostate confirms earlier observations. These had suggested the identification of prostatic tumour-associated antigens and of tumour-associated immunity in prostatic carcinoma.

Reactivity of peripheral blood leukocytes against human foetal cells. II. Cytotoxic potential of preparations enriched or depleted of different leukocyte populations

Leukocytes separated initially by Ficoll-Triosil sedimentation from the peripheral blood of 62 healthy donors and 64 patients with a variety of cancers were tested for cytotoxicity against cells from a single human foetal lung, by means of a microplate technique based on visual enumeration of surviving target cells. Under these conditions cytotoxicity was primarily observed to be a function of the granulocyte content of the preparations, which was extremely variable (range 0-60%). When the influence of these cells was greatly diminished through depletion by adherence, residual cytotoxicity was still detectable in the majority of samples tested. Complete abrogation of this cytotoxicity by pretreatment with carrageenan implicated cells of the monocyte-macrophage series, which persisted as a small minority (about 2%) in the already depleted effector cell populations. However some cytotoxicity could also be demonstrated in populations, purified by passage through columns containing nylon fibre or Degalan-coated (human Ig-anti Ig) beads, which consisted almost exclusively (about 98%) of lymphocytes, mainly of thymus (T)-dependent type (greater than or equal to 80%). Under these conditions and those in which T-cells were concentrated by formation of spontaneous E rosettes, cytotoxicity appeared to decline with T cell enrichment, suggesting that the cytolytic effects were attributable to cells of the non T-compartment which were present to a variable extent in the different T-cell-enriched populations. Although the absolute identity of these cells was not established, the experiments illustrate potential sources of non-specific cytotoxicity among different effector cell populations against target cells expressing antigens to which the majority of leukocyte donors--in health or disease--were presumably non-sensitized. As such these data have several implications for the interpretation of in vitro cytotoxicity tests against human neoplasms.

Serum modulation ofin vitro cellular immune reactivity to Epstein-Barr virus-associated antigen

The reactivity of peripheral blood leukocytes to Epstein-Barr virus-associated antigen was assessed employing two in vitro tests, leukocyte adherence inhibition (LAI) and leukocyte migration inhibition (LMI). In both tests, autologous serum from some normal subjects was shown to confer strong reactivity on weak or non-responder cells. Reactivity to purified protein derivative of tuberculin (PPD) assessed in parallel was shown to be unaffected by autologous serum. Employing the LAI system, variations in the effects of autologous serum samples collected on consecutive days was shown to be dependent on the reactivity of the leukocytes themselves, rather than changes in the activity or concentration of factors present in the serum.

Antigen-reactive cells to tumor antigens in normal and tumor patients

The leukocyte migration technique (LMT) has been used to assess the cell-mediated response in patients with malignant disease. The response of patients with malignant melanoma and adenocarcinoma of the gastrointestinal tract and breast has been compared to that of normal controls. The reactivity of peripheral blood leukocytes to tumor antigen and to a bacterial antigen (BCG) was measured. No significant difference was detectable between the response of controls and of the cancer patients at any stage of their disease. The results were not altered by serum factors, by the removal of phagocytic cells from the peripheral leukocytes, or by trypsin pretreatment of the cells. The tumor antigen reactivity detectable in the patients did not correlate with the progression of the malignant disease. The response of patients with malignant melanoma and adenocarcinoma of the gastrointestinal tract and breast has been compared to that of normal controls. The reactivity of peripheral blood leukocytes to tumor antigen and to a bacterial antigen (BCG) was measured. No significant difference was detectable between the response of controls and of the cancer patients at any stage of their disease. The results were not altered by serum factors, by the removal of the phagocytic cells from the peripheral leukocytes, or by trypsin pretreatment of the cells. The tumor antigen reactivity detectable in the patients did not correlate with the progression of the malignant disease.

Dissociation of correlates of cellular immunity in man: functional heterogeneity within the antigen-reactive cell population?

Cellular immune responsiveness (CMI) to ubiquitous bacterial antigens was assessed in serial peripheral blood samples collected from normal subjects, employing three in vitro correlates of CMI in parallel. Delayed cutaneous hypersensitivity (DCH) was also assessed on a number of occasions in these subjects. These experiments showed that the reactivity of peripheral blood leukocytes from normal individuals as measured in each of the in vitro tests fluctuated from day to day, and that the reactivity of cells functioning in each of the tests fluctuated independently of one another. In contrast, DCH reactivity in these individuals was not subject to such fluctuations. The results are discussed in terms of the periodic appearance of functionally distinct subpopulations of T lymphocytes in the peripheral circulation.

Cell-mediated immunity to human tumors. Abrogation by serum factors and nonspecific effects of oral BCG therapy.

The reactivity of peripheral blood leukocytes from patients with carcinoma of the gastrointestinal tract and malignant melanoma was assessed by the leukocyte migration technique to membrane fractions of histologically similar and unrelated tumors. Inhibition of migration was observed when the tumor membranes were derived from histologically similar tumors but not with those from unrelated tumors. Normal volunteers' leukocytes failed to inhibit with either type of tumor membrane antigen. Inhibition of migration was noted when the leukocytes were cultured in serum-free medium. Autologous serum produced complete abrogation of the response, but decreased inhibition of migration was also noted when allogeneic serum was added to the culture medium. Seven patients with disseminated melanoma were treated with BCG therapy administered repeatedly by the oral route. This type of treatment effected a regression in three of seven patients and had prevented tumor progression in an additional two patients. No alteration in the reactivity of these patients' leukocytes to tumor membrane antigens was noted during the course of the therapy.