Cellular aspects of tolerance. II. Unresponsiveness of B cells

The responsiveness of bone marrow cells from tolerant donors was examined by reconstitution of lethally irradiated tolerogen-free recipients. In these animals, stem cells from tolerant donors gave rise to immunologically competent antigen sensitive B cells. The antibody produced by these cells could be detected by a sensitive plaque assay in liquid and by antigen elimination. The antibody was not demonstrable by an assay which only detected plaque forming antibody which was highly avid or was formed in large quantity per cell. In lethally irradiated animals, partially purified B cells from a tolerant animal could not cooperate with T cells from normal donors to reconstitute immunological responsiveness to immunogenic doses of the tolerance inducing antigen. We concluded that antigen sensitive B cells in the bone marrow become unresponsive following administration of tolerogenic forms of antigen. Responsiveness of the reconstituted recipient animals was due to the differentiation of donor stem cells and subsequent antibody production by their descendants. Earlier contradictory findings could be unified in terms of these observations and conclusions.     

Thomsen's antigen in the leukocytes of leukemia patients

Leukocytes of 19 leukemic patients and 10 healthy donors were tested for the presence of Thomsen's antigen, using a method of specific adsorption of isogenic sera. Before the adsorption the cells were sonicated. Thomsen's antigen was found in leukocytes of leukemic patients but not in cells of healthy donors.     

The active E-rosette test: a sensitive in vitro correlate for human delayed-type hypersensitivity.

The "active" rosette test was adapted as an in vitro assay and correlated with human delayed cutaneous hypersensitivity (DCH) to two microbial antigens. Peripheral lymphocytes were purified from donors known to be responders or nonresponders to PPD-tuberculin or tularemia on the basis of prior DCH reactions. Skin test antigen, incubated with lymphocytes from antigen-sensitive donors, produced a significant increase (+2 S.D.) in the ability of the lymphocytes to form active rosette-forming cells (A-RFC) when compared to lymphocytes cultured without antigen. Skin test antigen incubated with lymphocytes from nonsensitive donors produced no increase in their A-RFC. The optimal dose of each antigen was approximately 100 ng/ml. The percentage of A-RFC rose to maximum levels between 3 and 4 hr after the addition of antigen to the lymphocytes incubated at 37 degrees C. The assay appears to be specific for the antigen to which the individual demonstrates DCH. This assay may provide a new in vitro method for investigating mechanisms of cell-mediated immunity and a rapid diagnostic test for sensitization to microbial antigens.     

Detection of B-cell antigen on the blast cells in chronic myeloid leukemia]

The presence of the common antigen on B lymphocytes of healthy donors and myeloblasts of patients with chronic myeloid leukemia in blastic crisis was observed with antimyeloblastic serum in the indirect surface immunofluorescence test. The cytotoxic test showed this antigen in the blastic cells in 27 out of 57 patients with CML BC, in 3 of 11 patients with acute lymphoid leukemia, in 1 of 8 patients with chronic lymphoid leukemia and in 2 of 2 patients with undifferentiated leukemia. The antigen was not found in the peripheral blood cells of healthy donors.     

Leukocyte procoagulant activity in man: an in vitro correlate of delayed-type hypersensitivity

Human mononuclear leukocytes generate cell-bound procoagulant activity (LPCA) after incubation with an antigen (mumps or tuberculin) to which the donor was previously sensitized. An inhibitor of coagulation appears to be liberated into the extracellular culture fluid during incubation of leukocytes with the sensitizing antigen. Removal of this activity before measuring LPCA resulted in a reliable test that correlated directly with delayed skin reactivity. The assay was particularly sensitive in that cells from weakly sensitized donors who reacted only to high doses of tuberculin (100 TU) in the delayed skin tests produced detectable LPCA in vitro. By contrast cells from weakly sensitized donors did not react to PPD in the lymphocyte blast transformation test or the direct macrophage migration inhibition factor test. The LPCA assay correlated closely with the blast transformation and MIF tests in which cells were used from more strongly sensitized donors who reacted in skin tests with lower doses of tuberculin (1 or 10 TU). The assays were antigen-specific in that cells from donors sensitive to mumps antigen but not to tuberculin reacted only to mumps antigen in vitro. The assay was extremely reproducible; cells from individual donors reacted to the same extent over a period of 8 mo). We propose that the assay system reported here represents an improved method for the measurement of cell-mediated immunity in vitro because it requires fewer donor cells, is technically simpler, and is more sensitive than previously described methods.     

Antibodies against the common polysaccharide and protein protective antigens of Pseudomonas aeruginosa in normal blood donors.

Screening of normal plasma obtained from 172 blood donors from the Helsinki area and from 46 blood donors from the Moscow area was performed in order to reveal 'natural' antibodies to the common polysaccharide (rhamnan) and protein antigens of P. aeruginosa. Antibodies were detected by ELISA. Among blood donors from the Helsinki area high titres of antibodies to the protein antigens were detected in 42 active blood donors (24.4%) and very high titres in nine (5.3%) highly-active blood donors, whereas in the Moscow area in 15 (34.9%) and in one case (2.3%), respectively. Antibodies to the common polysaccharide antigen were determined in the Helsinki area in 23 active blood donors (13.4%) and in one (0.5%) highly active blood donor, whereas in the Moscow area in four active blood donors (8.6%). The plasma contained both polysaccharide and anti-protein antibodies. The level of antibodies to the polysaccharide antigen was lower than the level of antibodies to the protein antigens. There was no statistically significant difference between the corresponding values of blood donor groups from the Helsinki and Moscow areas.     

Difference in the distribution of e antigen among different ethnic groups in a population of blood donors.

Sensitive techniques were used to detect e antigen and the corresponding antibody (anti-e) among 368 voluntary blood donors positive for hepatitis B surface antigen in the Montreal area and 310 people living in close contact with them. Neither e nor anti-e was found in the absence of markers of hepatitis B virus (HBV). Among the blood donors e antigen was detected in 23 and anti-e in 313, and 32 were negative for both markers. Of the 368 blood donors 330 were of French origin and 38 from other ethnic groups. The 23 e-positive subjects were unequally distributed among the ethnic groups: only 14 (4.2%) were recruited among the French group while 9 (23.7%) were recruited among other ethnic groups (P less than 0.001). This differences among ethnic groups might be related to the vertical or horizontal mode of dissemination of HBV infection.     

Prevalence of antibodies to hepatitis B core antigen in blood donors in the middle West region of Brazil

The prevalence of antibodies to hepatitis B core antigen in 552 prime blood donors was of 9.4%. The majority (71.2%) has antibodies to hepatitis B surface antigen. The hepatitis B surface antigen was present in 0.7%, all of them antibodies to hepatitis B core antigen positive.     

In vitro modulation of cytokine production by lymphocytes in human coccidioidomycosis

The modulation of the cytokine response to coccidioidal antigen by lymphocytes from donors with coccidioidomycosis was examined. In initial experiments, samples from 13 healthy immune donors and seven donors with active coccidioidomycosis anergic to the coccidioidal antigen T27K were assessed for CD3 lymphocyte expression of intracellular IFN-gamma using whole blood analysis. Addition of 10 ng/ml of recombinant IL-12 significantly increased response to T27K among immune and anergic subjects (p<0.05), but the percent of cells expressing IFN-gamma was still significantly greater for immune subjects. Among immune donors, the percentage of CD3 lymphocytes expressing IFN-gamma was significantly reduced with the addition of 10 ng/ml of recombinant IL-4, IL-10, TGF-beta, or their combination (for all, p<0.05). Among anergic donors, addition of 10 ng/ml of anti-IL-10 significantly increased IFN-gamma production (p<0.05), but addition of anti-IL-4 or anti-TGF-beta did not. Among immune donors, the percent of both CD3 lymphocytes and NK cells expressing IFN-gamma after 24h of T27K was increased above control (p<0.05), while the percent of NK cells producing TNF-alpha in response to T27K was not greater than control. Depletion of NK cells from peripheral blood mononuclear cells resulted in significant increases in TNF-alpha and IL-10 (for both, p<0.05) but resulted in no significant decrease in IFN-gamma or IL-2. These data demonstrate a differential response to stimulation with the coccidioidal antigen T27K among donors with coccidioidomycosis that can be manipulated by cell type and cytokine environment.     

Membranes of EBV-carrying virus nonproducer cells inhibit leukocyte migration of EBV-seropositive but not seronegative donors

We have previously shown that the leukocytes of healthy EBV-seropositive (but not seronegative) donors respond with migration inhibition (LMI) when confronted with extracts of EBV-carrying (but not EBV negative) cells. In the present study, we have examined whether this EBV-specific LMI response is capable of detecting a membrane antigen on the surface of EBV-carrying virus nonproducer cells. Crude membranes from EBV-genome carrying and EBV-negative cell lines were used as antigen. Contamination with the EBV-determined nuclear antigen (EBNA) was ruled out. Membranes from EBV-genome carrying nonproducer cells inhibited the migration of leukocytes from healthy seropositive donors, whereas membranes from EBV-negative lines had no such effect. Seronegative donors did not show any LMI. The clear difference between the EBV-negative Ramos line and its EBV-converted sublines was particularly conclusive in showing that the membrane component is determined or induced by the viral genome.     

Relationship between secretion of the Anton blood group antigen in saliva and adherence of Haemophilus influenzae to oropharynx epithelial cells

Abstract Inhibition of adherence of bacteria to epithelial cells contributes to a reduction of infections by these bacteria. We have shown that the Anton blood group antigen, the erythrocyte receptor for Haemophilus influenzae (van Alphen et al. 1986, FEMS Microbiol. Lett. 37, 69–71), occurs in saliva, that the occurrence is not related to the secretor state of the donor of the saliva and that saliva containing Anton antigen could not inhibit the adherence of H. influenzae to oropharynx epithelial cells.
Anton antigen was detected in saliva samples of 14 donors by immunoblotting with two different anti-Anton sera. The amount of Anton antigen correlated with the ability of H. influenzae to adhere to the epithelial cells of the donor of the saliva: 4.1 ± 0.1 Anton antigen units for donors with more than 50 H. influenzae per cell and 1.6 ± 0.5 units for donors with less adhering epithelial cells. No correlation between the amount of Anton antigen in saliva and secretor status of the donor was observed. Adherence of H. influenzae to epithelial cells was not inhibited by saliva of secretors ( N = 11) or non-secretors ( N = 3). The same saliva did not inhibit the interaction of the bacteria with Anton antigen bearing erythrocytes as measured by haemagglutination inhibition. This indicates that the amount of Anton antigen in saliva is probably too low to interfere with the interaction of H. influenzae with oropharynx epithelial cells and erythrocytes.     

Relationship between secretion of the Anton blood group antigen in saliva and adherence of Haemophilus influenzae to oropharynx epithelial cells

Inhibition of adherence of bacteria to epithelial cells contributes to a reduction of infections by these bacteria. We have shown that the Anton blood group antigen, the erythrocyte receptor for Haemophilus influenzae (van Alphen et al. 1986, FEMS Microbiol. Lett. 37, 69-71), occurs in saliva, that the occurrence is not related to the secretor state of the donor of the saliva and that saliva containing Anton antigen could not inhibit the adherence of H. influenzae to oropharynx epithelial cells. Anton antigen was detected in saliva samples of 14 donors by immunoblotting with two different anti-Anton sera. The amount of Anton antigen correlated with the ability of H. influenzae to adhere to the epithelial cells of the donor of the saliva: 4.1 +/- 0.1 Anton antigen units for donors with more than 50 H. influenzae per cell and 1.6 +/- 0.5 units for donors with less adhering epithelial cells. No correlation between the amount of Anton antigen in saliva and secretor status of the donor was observed. Adherence of H. influenzae to epithelial cells was not inhibited by saliva of secretors (N = 11) or non-secretors (N = 3). The same saliva did not inhibit the interaction of the bacteria with Anton antigen bearing erythrocytes as measured by haemagglutination inhibition. This indicates that the amount of Anton antigen in saliva is probably too low to interfere with the interaction of H. influenzae with oropharynx epithelial cells and erythrocytes.     

Phenotype frequency of LKE in the Danish population

A panel of 897 randomly chosen Danish donors of blood groups O and A was tested for the LKE antigen using a human anti-LKE. In this sample, 78.7% were LKE positive, 20.6% were weakly positive, whereas 0.7% were LKE negative. These phenotype frequencies are similar to those obtained by the first discovered human alloserum and by the monoclonal anti-SSEA-4 in previous studies. The previously reported lower LKE antigen strength in group A versus group O donors, and the association of LKE to the P system was confirmed.     

Antigen modulation of the secondary immune response. VI. Contribution of cells recruited from precursor pool to expression of memory.

The adoptive transfer system has been used extensively to study the ability of antigen triggered memory cells to become antibody forming cells and/or to proliferate and expand the memory cell population. Selective antigen triggering of the memory cells for low and high affinity antibody formation has also been studied in this way. One of the main counter-arguments to the interpretation of these data is that the presence of antigen in the adoptive host may lead to recruitment of new memory cells from either a host or donor precursor population. In this paper we examined the contribution of both host and donor precursor cells to the total antibody response in adoptive secondary recipients. The following donor-host combinations were used in which the recipients were given 1 mg fluid antigen intravenously: (A) normal (non-immune) donors to normal irradiated recipients; (B) normal donors to carrier primed irradiated recipients; (C) carrier primed donors to normal irradiated recipients; (D) normal donors to carrier primed recipients with challenge and subsequent transfer to additional carrier primed recipients; (E) carrier primed donor to normal recipients to carrier primed recipients; (F) repeat of B and C above with multiple antigen administration; (G) purified immune (DNP-BGG) donor T cells mixed with normal B cells transferred to normal irradiated recipients. In most cases recruitment was seen but this represented less than 4% of the responses seen with immune cells. Thus we conclude that this level of recruitment does not compromise the use of the adoptive transfer system for studying selective antigen triggering of memory cells.     

Different individual immune responses elicited by in vitro immunization

We have previously demonstrated that the addition of muramyl dipeptide (MDP), interleukin-2 (IL-2) and IL-4 effectively raises antibody production from L-Leucyl-L-leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBLs) against specific soluble antigen when immunized in vitro. However, PBLs from individual donors were separate optimal conditions regarding concentrations for IL-2 and IL-4, which in turn required us to optimize each individual PBLs to effectively produce antigen specific human antibody by in vitro immunization. These individual differences in the requirement for IL-2 and IL-4 reflects the differences in individual immune responses against a specific soluble antigen, which can be elicited by in vitro immunization. In the present study, we investigated these individual differences in the requirement for IL-2 and IL-4 to induce antibody productionin vitro in the PBLs of 12 volunteers (9 healthy donors and 3 allergenic patients). IL-2 requirements for antibody production varied dependent upon each donor, while higher amounts of IL-4 inhibited IgM and IgG production in all of the healthy donors. However, some of the characteristic features for PBLs donated from allergenic included lowered IgM production compared to PBLs derived from healthy donors, and very high IgE production in the absence of cytokines and allergen. These results demonstrate that the sensitivity of PBLs against antigen sensitization differs between healthy donors and atopic patients, which suggests that the frequency of antigen sensitization might be reflected in differing activation states and/or differing subpopulations of lymphocytes in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

The expression of hepatitis-C virus antigen by immunohistochemical stain and polymerase chain reaction in corneas of seropositive corneal donors

Background: Of the donor corneas rejected for transplantation, the largest group is that from donors testing seropositive for hepatitis C virus (HCV). In situations of severe shortage in supply of donor corneal tissue, we may consider the use of seropositive donors for transplantation if we can prove with high certainty the absence of HCV RNA in the donor corneal tissue. Polymerase chain reaction (PCR) is a highly sensitive and specific technique for direct detection of HCV RNA and can be used for this purpose. Nevertheless, it is not applicable for routine clinical use in most eye departments due to its unavailability and cost effectiveness.Purpose: To study the possible use of immunohistochemical method for detection of HCV antigen in corneal tissue of seropositive donors and correlate the results with those of PCR. Immunohistochemical methods have not yet been studied in donor corneal tissue.Materials and methods: Eight corneas of 4 seropositive and 8 corneas of 4 seronegative corneal donors were studied by immunohistochemical and PCR methods for the presence of HCV antigen in their corneal tissue and sera.Results: HCV RNA was not detected in the sera and corneal tissue of all seropositive and seronegative corneal donors by either PCR and immunohistochemical methods.Conclusion: Although the study is too small for conclusive results, the correlation between the immunohistochemical and PCR studies for direct detection of HCV antigen in corneal tissue of seropositive donors may raise the possibility of using the immunohistochemical method for screening of donor corneas for the detection of HCV antigen. A larger prospective study investigating the sensitivity, specificity and clinical applicability of the immunohistochemical method is warranted. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differences in HLA antigen recognition by human influenza virus-immune cytotoxic T cells.

The specificity of in vitro induced human influenza-immune cytotoxic effector cells was analyzed with respect to recognition of HLA-A and -B-linked gene products. The influenza-immune cytotoxic activity observed on panels of virus-infected targets demonstrated that virus-immune effectors preferentially lyse targets with which they share HLA-A or -B specificities. Virus-immune effectors from certain donors recognized virus in conjunction with some, but not all, of their self HLA-A and -B antigens. Among donors who share a given HLA antigen (such as A2 or B7), there are differences in the ability of their virus-immune T cells to recognize the shared antigen. Virus-infected target cells from HLA-A2 or -B7 "nonresponder" donors could be lysed by virus-immune T cells obtained from other donors who shared only the HLA-A2 or -B7 antigen with these target cells. These observations suggest that the absence of cytotoxic T cell responses by some donors to influenza virus in conjunction with HLA-A2 or -B7 is not due to control by the structural genes that code for these HLA antigens, but rather may result from control by regulatory genes that act at the level of the responder and/or stimulator cell. The results are discussed in the context of Ir gene regulation of human T cell responses.     

Occurrence of HBs antigen and anti-HBs antibodies in analytical laboratory workers in Warsaw

The study aimed at answering the question whether markers of the viral hepatitis, namely HBs antigen and anti-HBs antibodies, are significantly more frequent in the personnel of the analytical laboratories than in blood donors of the City Blood Donation Centre. Together 1,284 persons employed at 88 analytical laboratories were examined. These persons were divided into the groups according to the occupation, age and duration of the employment. HBs antigen was detected with EIA technique in 13 subjects making 1,025% of all examined individuals whereas anti-HBs antibodies were detected with EIP technique in 20 subjects, i.e. 1,560%. Detectability of HBs antigen and anti-HBS antibodies in blood donors was 0.443% and 0.04% respectively. The obtained results indicate significantly more frequent occurrence of both markers in the employees of the analytical laboratories.     

Possibility of detecting human embryonal leukemia antigen]

A possibility of detecting embryonic leukemic antigen on human leukemic blast cells in an acute human leukemia cytotoxicity test with the sera and 7S and 19S serum immunoglobulins of the placental blood was studied. The presence on the blast cells of patients suffering from acute leukemia of an antigen detectable by antibodies of placental blood (of parturients) was demonstrated; this antigen was absent on the leukocytes of healthy donors.