Increased myelopoiesis during Leishmania major infection in mice: generation of 'safe targets', a possible way to evade the effector immune mechanism.

BALB/c mice are highly susceptible to Leishmania major infection and develop a disseminated lethal disease. Previous experiments indicate that during infection the spleen is heavily populated with large mononuclear cells containing amastigotes. Morphologically these cells resemble undifferentiated monocytes and granulocytes. In this study we examined myelopoiesis in BALB/c and C57BL/6 (resistant) mice during infection with L. major. The number of macrophage-granulocyte precursors in the spleen of infected BALB/c mice, determined by colony forming units in soft-agar cultures (cfu-c), increased steadily to a level of about 60 times that of normal sex- and age-matched controls. In C57BL/6 mice, spleen cfu-c peaked at about 1 month post-infection (four times that of normal controls) and declined thereafter to about two times normal levels. The number of cfu-c in the bone marrow did not change significantly in either strain during the infection. Colony stimulating activity (CSA) was found in supernates of cultures of adherent cells from the spleen of infected BALB/c mice. Under the same conditions, CSA was non-detectable in supernates of nonadherent spleen cells of infected mice, and those of adherent or nonadherent spleen cells of control animals. A possible role of undifferentiated macrophage-granulocytes in the exquisite susceptibility of BALB/c mice to L. major infection is discussed.

Regulation by muramyl dipeptide (MDP) of the lymphoproliferative responses and polyclonal activation of human peripheral blood mononuclear cells.

The ability of muramyl dipeptide (MDP), its adjuvant inactive stereoisomer, MDP(D-D), and the non-pyrogenic, adjuvant active analogue, MDP-butyl ester (MDP-BE), to induce in vitro proliferation and/or polyclonal activation (PA) of peripheral blood mononuclear cells (PBMNC) from normal volunteers, was studied. MDP, as well as its two analogues, were incapable of inducing 3H-thymidine uptake or immunoglobulin synthesis in PBMNC cultures from the majority of the individuals tested. However, these muramyl peptides were capable of regulating the in vitro proliferative responses of some individuals to concanavalin A and to soluble antigens of Candida albicans. At the same time, enhancement of the pokeweed mitogen-induced IgA and IgM but not IgG PA was observed with MDP, its adjuvant active analogue MDP-BE, but not with the adjuvant inactive stereoisomer MDP(D-D). Results are discussed with relation to a possible genetic restriction of the responsiveness to MDP.

Modulation of the growth of murine thymoma cell lines having different Lyt-phenotypes by MDP and MDP(D-D): macrophage-mediated inhibition of in vitro cell growth.

The ability of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and its adjuvant-inactive stereoisomer, N-acetylmuramyl-D-alanyl-D-isoglutamine, (MDP(D-D] to inhibit the in vitro growth of 3 murine ascitic thymoma lines, expressing different Lyt-phenotypes, was studied. MDP inhibited the growth of all 3 cell lines. MDP(D-D) inhibited the 2 cell lines expressing Lyt-1+2- or Lyt-1+2+ phenotypes, but not the third cell line which expressed the Lyt-1-2+ phenotype. The ability of MDP or MDP(D-D) to inhibit thymoma growth was lost when the ascitic cell populations were depleted of macrophages. MDP could be replaced by a supernatant derived from an ascitic Lyt-1+2- cell population exposed to MDP. The supernatant required the presence of macrophages for activity. The inhibition by MDP of the growth of the Lyt-1-2+ cell line was prostaglandin synthetase dependent: indomethacin antagonized the inhibitory activity of MDP. The inhibition by MDP of the Lyt-1+2- cell line was partially antagonized by indomethacin, and no antagonism was observed with the Lyt-1+2+ cell line.

Monoclonal antibodies to the synthetic adjuvant muramyl dipeptide: characterization of the specificity.

Monoclonal antibodies to MDP were prepared by hybridization of NSO myeloma cells with spleen cells of BALB/c mice immunized with MDP conjugated to methyl-BSA. Hybridomas secreting anti-MDP antibodies were selected by the binding activity of their supernates to MDP-A--L using a radioimmunoassay. After cloning in soft agar, the specificities of monoclonal anti-MDP antibodies were assayed by an inhibition of ELISA with various derivatives of MDP. Fine structural analysis of specificity for one such clone (2-4) is reported. This antibody recognizes the N-acetyl-muramic acid (N-Ac-Mur) linked to the dipeptide but not N-Ac-Mur or/and dipeptide alone. The N-Ac group on muramic acid is an important antigenic determinant and the glycopeptide linkage seems to be crucial in presenting the sugar moiety. Conservative substitution of L-Ala (i.e. by L-Ser or L-Val) had no effect on the binding ability to the antibody whereas a radical change, i.e. replacement of L-Ala by L-Pro or N-methyl-L-Ala completely abolished the antigenicity of the molecule. There was no clear correlation between biological activities of various derivatives of MDP and their ability to react with this antibody. Some possible hypotheses explaining this lack of correlation are presented.

[Demonstration of a bacterial structure in two human mediators: a sleep facilitating factor and a monokine]. / Mise en évidence d'une structure bactérienne dans deux médiateurs humains: un facteur favorisant le sommeil et une monokine.

A monoclonal anti-MDP antibody was found to bind to "Slow Wave Sleep" factor. This result confirms that this factor is a muramyl peptide and furthermore shows that it contains a structure characteristic of the synthetic adjuvant and of the bacterial cell wall, i.e. an acetylated muramic acid bound to L-alanine. This antibody was also shown to specifically inhibit a biological activity of a purified human monokine which induces fever. Because of these results and other recent observations we propose that a bacterial structure is present in certain mammalian mediators.

Absence of antibodies to muramyl dipeptide in patients with tuberculosis or leprosy.

The enzyme-linked immunosorbent assay (ELisa) was used to detect the presence of antibodies to muramyl dipeptide (MDP) in serum of patients with leprosy or tuberculosis. Using a conjugate of MDP-lysine to horse radish peroxidase, no such antibodies could be detected in sera of either patients or controls. Antibodies to a sonicate antigen of Mycobacterium tuberculosis were found in sera of all individuals tested and the binding of these antibodies to the M. tuberculosis antigen could not be inhibited by MDP. On the other hand, binding of MDP to anti-MDP antibodies, raised in rabbits, was largely inhibited by free MDP, slightly inhibited by M. tuberculosis antigen and was not inhibited by the patients' sera.

Use of the ELISA to screen for anti-thymocyte and anti-beta 2-microglobulin antibodies in leprosy and SLE.

A report is given of the use of the enzyme-linked immunosorbent assay to measure antibody to preparations of human thymocyte membranes (HTMA) and to beta 2-microglobulin. The assay described is simple and rapid, and requires only small quantities of an easily stored membrane preparation. The advantages of this technique over conventional methods involving cytotoxicity are discussed. Raised levels of IgM antibody to beta 2-microglobulin were detected in sera from SLE patients. Raised levels of IgG and IgM antibody to HTMA were found in sera from most active lepromatous cases. Two of eight sera from SLE patients showed raised IgG anti-HMTA, but not raised IgM. An attempt was made to study the subclass of the IgG antibodies found, but when checked against purified human IgG myeloma proteins, the available anti-subclass sera were found to lack the necessary degree of specificity in this assay.

Use of the ELISA to screen for anti-thymocyte and anti-beta 2-microglobulin antibodies in leprosy and SLE

A report is given of the use of the enzyme-linked immunosorbent assay to measure antibody to preparations of human thymocyte membranes (HTMA) and to beta 2-microglobulin. The assay described is simple and rapid, and requires only small quantities of an easily stored membrane preparation. The advantages of this technique over conventional methods involving cytotoxicity are discussed. Raised levels of IgM antibody to beta 2-microglobulin were detected in sera from SLE patients. Raised levels of IgG and IgM antibody to HTMA were found in sera from most active lepromatous cases. Two of eight sera from SLE patients showed raised IgG anti-HMTA, but not raised IgM. An attempt was made to study the subclass of the IgG antibodies found, but when checked against purified human IgG myeloma proteins, the available anti-subclass sera were found to lack the necessary degree of specificity in this assay.

A simplified immunoenzyme antigen binding technique as an approach for immunodiagnosis.

A simple immunoenzyme technique is described in which the enzyme beta-galactosidase of E. coli (Z) is used as a marker. The principle of the test is based on the ability of the cells to bind the antigen. Conditions of the assay using Z both as the antigen and marker are described. As a screening technique, conditions are defined at limited antigen concentrations when binding is not directly proportional to the number of cells present. Hence, drops of blood can be used directly for detection of an immune response without counting the cells. Furthermore, treatment of whole blood with ethanol was shown to (a) increase the binding capacity and (b) allow the storage of specimens for weeks without any loss of activity. With human hydatid fluid (HHF) antigens conjugated to Z as a model, it was possible to detect the immune response of rabbits injected with HHF. The method is simple, requires no sophisticated equipment and can be used in large scale epidemiological surveys.

Histocompatibility antigens in patients with leprosy from Azarbaijan, Iran.

The distribution of 24 histocompatibility antigens in 88 Azerbaijani patients with leprosy was determined and compared with those of 125 normal, ethnically matched individuals. HLA-BW35 was increased in frequency among the Kurdish patients as compared to the controls; HLA-A1, however, displayed decreased frequency in patients with the lepromatous form of the disease. Among the Turks, diminished frequency of HLA-BW15 was noted in the total patient population. None of these comparisons, however, reached statistical significance when corrected for the number of antigens tested. Across the two ethnic groups, differences in the frequencies of HLA antigens between the patients and the controls were only marginal.

Esophageal cancer studies in the Caspian Littoral of Iran: introductive assessment of the HLA profile in patients and controls.

The possibility of an association between HLA profile and the probability of having esophageal cancer was explored among patients from different ethnic groups residing in the Caspian Littoral of Iran. A total of 151 esophageal cancer patients and 214 normal controls with ethnic affiliations similar to those of the patients were typed for HLA antigens. No statistically significant differences were found between patients and controls with regard to HLA antigens.

Generalized infection and lack of delayed hypersensitivity in BALB/c mice infected with Leishmania tropica major.

The susceptibility of a few strains of mice to a subcutaneous injection of Leishmania tropica major, the causative agent of cutaneous leishmaniasis in humans, was studied. The infection in six strains (CBA, AKR/J, AKR/cu, C57BL/6, A/J, and C3H) remained cutaneous, and the animals recovered within 3 to 4 months. In contast, the infection in BALB/c became generalized and killed 1005 of infected animals. Intraperitoneal injection of infected liver of BALB/c to A/J and syngeneic mice produced a lethal disease in BALB/c but no infection in A/J mice. Lower doses of the parasite produced a lethal infection in BALB/c but no apparent disease in A/J. Hence, the host rather than the parasite is responsible for the outcome of the disease. The peak antibody titer of BALB/c mice was not significantly higher than that of A/J mice. However, BALB/c failed to show any delayed hypersensitivity to leishmania tested by footpad reaction, whereas A/J mice showed a strong response.

Binding of antigen-enzyme complex by antigen-binding cells as an approach for immunogdiagnosis.

An immunoenzyme method capable of detecting specific antigen-binding cells is developed. The method is based on the chemical coupling of the antigen to beta-galactosidase and binding of the conjugate to the corresponding antigen-binding cells. Human hydatid fluid was used as an antigen model, and cells derived from rabbits immunized with human hydatid fluid showed specific binding to human hydatid fluid-beta-galactosidase conjugate. The method is specific and reproducible and could be used as an immunodiagnostic test for various diseases in which little or no antibody is produced.

Effect of cyclophosphamide on Toxoplasma gondii infection: reversal of the effect by passive immunization.

Treatment of mice with cyclophosphamide (CY) at a dose of 250 mg/kg body weight 24 hr prior to infection with an avirulent strain of Toxoplasma gondii delays the appearance of antibody by about one week and results in 70% mortality. To discount other effects of CY besides inhibition of antibody synthesis, CY-treated infected mice were passively immunized with a pooled specific serum collected from chronically infected syngeneic animals. Passive immunization reversed the effect of CY treatment if the titre of antibody in recipients reached 1 : 512 or more, as measured by the indirect immunofluorescence technique (IFT). It is therefore suggested that antibody plays an important role in establishing an infection-immunity (premunition) in this system.