Immunization with recombinant surface antigen P50 of Babesia gibsoni expressed in insect cells induced parasite growth inhibition in dogs

This is a report of a vaccine trial directed against Babesia gibsoni infection in dogs with the use of the recombinant antigen P50. Dogs immunized with P50 showed partial protection manifested as a significantly low level of parasitemia. The results indicated that P50 is a primary vaccine candidate molecule against canine B. gibsoni infection.     

High-level expression of truncated surface antigen P50 of Babesia gibsoni in insect cells by baculovirus and evaluation of its immunogenicity and antigenicity

Previously, we identified an immunodominant antigen, P50 of Babesia gibsoni. In the present study, the gene encoding the truncated P50 (rP50t) without a C-terminal hydrophobic region (29 amino acids [aa]) was expressed in insect cells by a recombinant baculovirus. The highly hydrophobic C-terminal 20-aa regions seems to be a transmembrane region, which was evidenced by the fact that rP50t was effectively secreted into the supernatant of insect cells infected with the recombinant baculovirus. N-terminal amino acid sequence analysis of rP50t indicated that N-terminal 19 aa function as a signal peptide. The expression level of rP50t reached up to 2 mg per 10(8) cells infected with the recombinant baculovirus. The immunogenic property of rP50t was evaluated by an immunization test in mice. Mice immunized with rP50t induced a high-level antibody titer against the B. gibsoni merozoite. Monoclonal antibodies (MAbs) to rP50t were produced in mice to determine the immunogenic regions of P50. The epitope(s) recognized by all five MAbs were located between aa 190 and 273, suggesting that the central part of P50 is a highly immunogenic region. The diagnostic potential of rP50t was evaluated using an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate clearly (P < 0.0001) between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Our results indicated that the rP50t may provide a useful potential immunogenic reagent for use in diagnosis and as a subunit vaccine to control B. gibsoni infection in dogs.     

Inhibitory effect of allicin on the growth of Babesia and Theileria equi parasites

Allicin is an active ingredient of garlic that has antibacterial, antifungal, antiviral, and antiprotozoal activity. However, the inhibitory effects of allicin on Babesia parasites have not yet been examined. In the present study, allicin was tested as a potent inhibitor against the in vitro growth of bovine and equine Babesia parasites and the in vivo growth of Babesia microti in a mouse model. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia caballi, or Theileria equi was inhibited by allicin in a dose-dependent manner and had IC₅₀ values of 818, 675, 470, and 742 μM, respectively. Moreover, allicin significantly inhibited (P?<?0.001) invasion of B. bovis, B. bigemina, B. caballi, and T. equi into the host erythrocyte. Furthermore, mice treated with 30 mg/kg of allicin for 5 days significantly (P?<?0.05) reduced the parasitemia of B. microti over the period of the study. To further examine the potential synergism of allicin with diminazene aceturate, growth inhibitory assays were performed in vitro and in vivo. Interestingly, combinations of diminazene aceturate with allicin synergistically potentiated its inhibitory effects in vitro and in vivo. These results indicate that allicin might be beneficial for the treatment of babesiosis, particularly when used in combination with diminazene aceturate.     

Effect of antiserum against isologous aggregated immunoglobulins on delayed-type hypersensitivity reaction in mice immunized with sheep red blood cells

Injection of mouse antiserum against isologous aggregated immunoglobulins (MAAS) into mice previously receiving 10⁵ sheep red blood cells (SRBC) did not affect the intensity of the delayed-type hypersensitivity (HDT) reaction when tested at the peak of sensitization (4th day), but led to a marked increase in the intensity of the reaction in the later stages (6th day). MAAS completely abolished suppression of HDT observed after sensitization with 5·10⁷ SRBC. Transfer experiments showed that under the influence of MAAS the number of suppressor cells of HDT in the spleen of the sensitized mice was reduced. MAAS had no effect on proliferation of antibody-forming cells or on the intensity of hemagglutinin production, but reduced by 70% the number of rosette-forming cells (RFC) detectable in the spleen at the peak of the primary immune response. The results may be evidence that RFC take part in the suppression of HDT.Laboratory of Immunochemistry, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 11, pp. 578–580, November, 1979.     

Long-term CD4+ and CD8+ memory T cells developed in severe combined immunodeficiency mice during homoeostasis exhibit differences in sensitivity to antigen

T cells transferred in small numbers to lymphopenic hosts proliferate spontaneously, and naïve T cells turn into memory cells without complete cellular reconstitution of the lymphoid compartment. In this study, neonatal severe combined immunodeficiency mice were treated with peripheral CD4⁺ or CD8⁺ T cells purified from the spleen of syngeneic C.B-17 mice. At 2 weeks and more pronounced at 10 weeks post treatment, a majority of the residing donor T cells showed memory phenotype, with high expression of CD44 and an early onset of proliferation and cytokine production upon stimulation. These memory type of donor cells were sustained in numbers for at least 1.5 years post treatment in a homoeostatic fashion, recognized by normal CD4/CD8 ratio and no bias towards type 1 or type 2 immune response. Furthermore, amongst the memory type of cells, there was a striking difference in their response, where the CD8⁺ donor cells had higher threshold for stimulation than the CD4⁺ donor cells.     

The effect of pertussis vaccine on the immune response of mice to sheep red blood cells

Bordetella pertussis is an adjuvant when given to mice immunized with sheep RBC. The adjuvant activity of pertussis is reflected in an increase in the number of cells producing antibody as measured by the localized haemolysis in gel technique. γG-PFC have a more marked response to pertussis than do γM-PFC, and in general γG-PFC are more sensitive than γM to variations in dose or injection schedule of pertussis organisms. Pertussis increases the response to doses of antigen which previously were considered to be maximal. The distribution of PFC between spleen, blood and lymph nodes is altered by pertussis injections.     

Effect of mouse antiserum against isologous aggregated immunoglobulins on accumulation of rosette-forming and antibody-forming cells in mice immunized with sheep's red blood cells

Data are given on the effect of mouse antiserum against isologous aggregated immunoglobulins (MAAS) on the kinetics of rosette-forming (RFC) and antibody-forming cells (AFC) in mice immunized with sheep's red blood cells (SRBC). The effect of MAAS in the experimentsin vivo was assessed by injecting this serum for 5 days into CBA mice, the first injecting being combined with injection of 5.10⁷ SRBC. Injection of MAAS into mice immunized with SRBC was shown to cause a marked decrease in the number of RFC in the spleen on the 5th and 9th days after immunization. MAAS has no appreciable effect at these same times on proliferation of AFC producing IgM hemagglutinins. Meanwhile MAAS intensified proliferation of IgG-AFC during the period when the number of these cells of the spleen in the immunized mice was maximal. After adsorption of MAAS with immune complexes formed by mouse IgG antibodies this serum was shown to lose much of its ability to block RFCin vivo. It is postulated on the basis of these results that the property of MAAS of influencing the accumulation of RFC and AFC producing IgG hemagglutinins is due to a factor which reacts with the immune complex formed by mouse IgG antibodies. This factor may perhaps be antibodies against aggregated immunoglobulins of this class.Laboratory of Immunochemistry, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 5, pp. 557–560, May, 1978.     

Separate application of adjuvant and antigen: the effect of a water-in-oil emulsion on the splenic plaque-forming cell response to sheep red blood cells in mice

The effect of a non-immunogenic adjuvant on the murine splenic plaque-forming cell (PFC) response against sheep red blood cells (SRBC) was studied. The adjuvant, a stable water-in-oil (W/O) emulsion, was injected intraperitoneally at the same time as or prior to the intravenous (i.v.) injection of SRBC. Enhancement of the SRBC-specific IgM-, but not IgG- and IgA-responses was observed. The stimulatory effect depended on the dose of both adjuvant and antigen and on the interval between their application. The minimal dose of adjuvant needed to induce maximal stimulation increased with the interval between the injections. Administration of an optimal adjuvant dose one week before antigen application still resulted in a clear stimulation of the response to the antigen. In adjuvant-treated animals, the primary PFC response did not exceed the maximum level reached after i.v. injection of a high dose of SRBC. Adjuvant therapy also resulted in polyclonal B cell-activation, since the number of spontaneous Ig-secreting cells in the spleen was increased. The kinetics and isotype distribution of the SRBC-specific and polyclonal responses, however, were different. Therefore, the observed stimulatory effect on the SRBC-specific PFC-response cannot be explained by the polyclonal activation of the immune system. From this study it appears that injection of a W/O emulsion provokes an active stimulation of the immune system, which demonstrates that the adjuvant effect of W/O emulsions is not only passively obtained by prolonged antigen presentation by depot formation.     

Inhibition of CD26 in human cord blood CD34+ cells enhances their engraftment of nonobese diabetic/severe combined immunodeficiency mice

CD26, a surface serine dipeptidylpeptidase IV (DPPIV) expressed on different cell types, cleaves the amino-terminal dipeptide from some chemokines, including stromal-derived factor-1 (SDF-1/CXCL12). SDF-1/CXCL12 plays important roles in hematopoietic stem cell (HSC) homing, engraftment, and mobilization. Inhibition of CD26 peptidase activity enhances homing, engraftment, and competitive repopulation in congenic mouse bone marrow cell transplants. Our studies evaluated a role for CD26 in in vivo engraftment of HSCs from human umbilical cord blood (CB) into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Pretreating purified CD34(+) human CB cells with Diprotin A, a DPPIV inhibitor, for 15 min significantly enhanced engraftment. Treatment did not affect differentiation of CD34(+) cells in vivo, as measured phenotypically by human markers CD33, CD38, CD19, and CD34. We found that the percentage of CD26(+) cells within the more immature cells (CD34(+)CD38()) was significantly higher than in the more mature population (CD34(+)CD38(+)). These results suggest that inhibition of CD26 may be one way to enhance engraftment of limiting numbers of stem cells during CB transplantation.     

Inhibitory effect of Toxoplasma lysate antigen on the multiplication of transplanted tumor in mice

Tachyzoites of the RH strain of Toxoplasma gondii were pretreated by a process of freezing and thawing followed by ultrasonication. After ultracentrifugation at 144,000 g for 120 minutes, the resulting supernatant contained a protein (TLA 144), which consisted mainly of a component of protozoan origin with a molecular weight of 10,000-20,000, and additional sugars, peptides, and amino acids. TLA 144 was soluble in water and of very low toxicity. Mice that had been inoculated with allogeneic (S-180) and syngeneic (Meth A) transplantable tumor cells, were injected intramuscularly with 100 micrograms of TLA 144 once a week for some time beginning one week after transplantation. It was found that after TLA treatment the multiplication of tumor cells was more intensely inhibited than following administration of OK-432, one of the biological response modifiers (BRMs).     

The effect of antigen given intravenously on specific antigen-sensitive lymphocytes of peripheral blood in man

In the course of fibrinolytic therapy with the streptococcal antigen streptokinase the effect of antigen intravenously on specific antigen-sensitive lymphocytes in the peripheral blood was studied.

Seven out of ten patients had streptokinase-sensitive lymphocytes in the peripheral blood before the beginning of therapy. Stimulation by streptokinase was temporarily rendered negative in all seven cases during the course of treatment. Some time after the termination of therapy specifically sensitive lymphocytes reappeared in the circulation. Lymphocytes of three patients were stimulated by streptokinase only after the treatment period.

Stimulation of lymphocytes by phytohaemagglutinin or by tuberculin in sensitized individuals, was not disturbed. Thus streptokinase given intravenously involves selectively streptokinase-sensitive cells. It is assumed that after interaction with homologous antigen these lymphocytes are directed to lymphoid tissues where proliferation and differentiation takes place.

     

Formation of hypersensitivity of delayed type to sheep's red blood cells after separate and combined injection of antigen and cyclophosphamide

Ability to form hypersensitivity of delayed type (HDT) to the corresponding antigen was investigated in mice receiving an injection of a massive dose of sheep's red blood cells and cyclophosphamide at various times before the experiments. Depression of HDT formation as a result of injection of either the cytostatic alone or the massive dose of antigen alone was studied at the same times. Combined treatment was shown to lead to the formation of tolerance as reflected in various tests for HDT (skin test and inhibition of macrophage migration). This form of tolerance is based on a true deficiency of the corresponding clone of T cells. Injection of cyclophosphamide alone leads to some degree of depression of HDT. Injection of the massive dose of antigen alone leads to a different form of areactivity, due to suppressor cells, the nature of which is not yet clear.Laboratory of Immunologic Tolerance, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 5, pp. 449–452, May, 1979.     

Diprotin A infusion into nonobese diabetic/severe combined immunodeficiency mice markedly enhances engraftment of human mobilized CD34+ peripheral blood cells

Hematopoietic stem cell (HSC) graft cell dose impacts significantly on allogeneic transplant. Similarly, HSC gene therapy outcome is affected by loss of repopulating cells during culture required for ex vivo retrovirus transduction. Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a central role in marrow trafficking of HSCs, and maneuvers that enhance CXCR4 activation might positively impact outcome in settings of limiting graft dose. CD26/dipeptidyl peptidase IV (DPP-IV) is an ectoenzyme protease that cleaves SDF-1, thus reducing CXCR4 activation. We show that injection of irradiated nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with >or=2 micromol Diprotin A (a tripeptide specific inhibitor of CD26 protease activity) at the time of transplant of human granulocyte colony-stimulating factor (G-CSF) mobilized CD34(+) peripheral blood cells (CD34(+) PBCs) results in a >3.4-fold enhancement of engraftment of human cells. We also show that CD26 on residual stromal cells in the irradiated recipient marrow milieu, and not any CD26 activity in the human CD34(+) PBC graft itself, plays the critical role in regulating receptivity of this environment for the incoming graft. Human marrow stromal cells also express CD26, raising the possibility that Diprotin A treatment could significantly enhance engraftment of HSCs in humans in settings of limiting graft dose just as we observed in the NOD/SCID mouse human xenograft model.     

Obtention of a human primary humoral response against schistosome protective antigens in severe combined immunodeficiency mice after the transfer of human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) from healthy donors were injected into C.B.-17 severe combined immunodeficiency (scid) mice which were subsequently immunized with crude Schistosoma mansoni adult worm antigenic preparation (SWAP) or with recombinant S. mansoni 28-kDa glutathione transferase (r-Sm-28-GST) antigen. PBMC from a S. mansoni-infected patient were also transferred. The specific human anti-SWAP and anti-Sm-28-GST antibody responses were monitored. The presence in both cases of human specific antibodies in scid mouse sera was determined by enzyme-linked immunosorbent assay and Western blotting techniques using anti-human immunoglobulin reagents. No antibodies were detected in these sera using anti-mouse immunoglobulin antisera. These antibodies were functional since a cytotoxic activity against schistosomula was observed when monocytes were incubated with scid mouse sera positive for anti-Sm-28-GST antibodies.     

肉桂酸锗不同剂量组诱导小鼠子宫颈癌(U_(14))细胞凋亡的实验研究

目的探讨肉桂酸锗不同剂量组(1.5mg/ml、3mg/ml、6mg/ml)对小鼠子宫颈癌(U14)细胞的抑制效应及其作用机制。方法抗肿瘤活性实验采用以下方法:(1)药理试验;(2)HE染色观察;(3)免疫组化检测;(4)特殊染色观察;(5)流式细胞仪检测。结果 (1)肉桂酸锗组(3mg/ml)及环磷酰胺阳性对照组瘤重明显减轻,其抑瘤率分别为46.65%和54.27%;(2)流式细胞仪证实有凋亡细胞,并检测到肉桂酸锗组(3mg/ml)及环磷酰胺阳性对照组诱导U14细胞的凋亡率分别为22.65%和20.61%;(3)在DNA直方图上可见肉桂酸锗组(3mg/ml)在G0-G1期细胞前出现"亚G1"峰(凋亡峰),生理盐水阴性对照组无明显凋亡峰;另外,肉桂酸锗组(3mg/ml)对瘤细胞周期有明显影响,它使瘤细胞G2-M期减少、细胞周期延长、对瘤细胞增殖的抑制作用增强。结论肉桂酸锗组(3mg/ml)对小鼠子宫颈癌(U14)细胞的生长有一定抑制作用,诱导瘤细胞凋亡可能是其抑制U14细胞生长的主要作用方式。     

Inhibitory effect of sheep erythrocyte fragments on rosette formation of human T lymphocytes with sheep red blood cells.

The effect of sheep red blood cells (SRBC) fragments on rosette formation of human peripheral T lymphocytes with SRBC was evaluated on the active and total T-rosette tests. The rosetting capacity of active rosette-forming cells was selectively and nearly completely inhibited by the pretreatment of lymphocytes with SRBC fragments. The decrease in total rosettes by blocking with SRBC fragments was almost parallel to that of active rosettes. SRBC fragments had no inhibitory effect on the rosetting capacity of a lymphocyte population in which active rosette-forming cells were removed by gradient centrifugation. These results suggested that active rosette-forming cells in human T lymphocytes have the receptors of high affinity for SRBC and these receptors readily bind SRBC fragments, resulting in block of rosette formation.