Post lung-stage schistosomula of Schistosoma mansoni exhibit transient susceptibility to macrophage-mediated cytotoxicity in vitro that may relate to late phase killing in vivo

Studies of protective immunity against Schistosoma mansoni in immunized mice suggest that a proportion of challenge parasites may be eliminated after they have passed through the lungs of the host several days after infection; however, no potential immune effector mechanism of resistance against this stage of the parasite has yet been identified, since schistosomes have been shown to rapidly become resistant to antibody-dependent killing mechanisms. In this study, different development stages of S. mansoni were examined for their susceptibility to in vitro cytotoxicity by lymphokine-activated macrophages. As previously shown, newly transformed larvae were readily killed by lymphokine-treated peritoneal macrophages or the macrophage cell line IC-21 (80% mortality over 48 h in vitro), whereas 7 and 10 day old lung-stage parasites had become refractory to macrophage effects. However, after 2 to 2 1/2 weeks of development in vivo, juvenile parasites recovered from the liver were again susceptible to activated macrophage-mediated cytotoxicity (25-65% mortality). Ultrastructural studies of 2 1/2 week old parasites co-cultured with activated IC-21 cells revealed that damage was largely restricted to the areas beneath the parasite surface and gut syncitia; surface membrane disruption was not evident. This late stage of susceptibility was transient and by 4 to 6 weeks liver-stage worms had again become refractory to macrophage killing. The interaction of post lung-stage parasites with activated macrophages was antibody independent. Furthermore, schistosomes isolated from the portal circulation 2 1/2 weeks after infection showed no evidence of surface-bound immunoglobulin in a quantitative immunofluorescence assay, nor did antisera from chronically infected mice (CIS) or mice vaccinated with irradiated cercariae (VS) react with the surface of these parasites in vitro, making the possibility of direct antibody-dependent killing mechanisms unlikely. However, both CIS and VS did recognize excretory/secretory proteins synthesized by 2 1/2 week old liver-stage schistosomes, including a major antigen of approximate Mr (X 10(-3] 220 (220K). It is therefore possible that such antigens might participate in protective immunity, for example via immune complex formation or activation of sensitized T cells. These observations support the role of macrophages as immune effector cells in mice immunized against Schistosoma mansoni, and provide the first physiologically relevant mechanism whereby the immune system might recognize and kill post-lung stage schistosomes.     

Signal transduction via phosphorylated adhesion molecule, LFA-1{beta} (CD18), is increased by culture of natural killer cells with IL-2 in the generation of lymphokine-activated killer cells

It is well known that IL-2 stimulates natural killer (NK) cellsto express lymphokine activated killer (LAK) activity and thatthis stimulation prompts the acquisition of the ability to lysepreviously insensitive target cells. The possible role of adhesionmolecules in the IL-2 activation process was probed by focussingon a lymphocyte function-associated antigen (LFA)-1-dependentmodel system. A mAb to the LFA-1ß chain abrogatedLAK activity, but only moderately suppressed NK activity, suggestinga differential role for LFA-1ß In LAK compared withNK mediated lysis. Orthophosphate labeling demonstrated thatthe LFA-1ß chain was strongly phosphorylated in LAKbut not NK cells; in contrast, the chain was phosphorylatedsimilarlyin both effector cell types. At least a portion ofthe phosphorylation of the ß chain was on tyrosineresidues, as shown by Western blotting with anti-phosphotyrosineantibody of LFA-1ß immunoprecipitates. Crosslinkingof the LFA-1ß chain with plastic-adhered antibodystimulated Ca²⁺-dependent release of cytoplasmic lytic granulesand induced phosphatidyl inositol turnover in LAK but not NKcells. We conclude that the IL-2-induced phosphorylation oftheß chain of the LFA-1 adhesion molecule in LAK cellsand associated alteration in signal transduction may be importantin the stimulation of LAK cell activity in NK cells.     

COS/TPC同源重组法高效制备人B7(CD80)重组腺病毒

本研究探讨了FN对LAK细胞活性的影响.Extracellular Matrix Protein(ECM)中的一种成分FN是基底膜、细胞间质及细胞表面的重要组成成分.本文发现FN能调节LAK细胞的功能.首先、LAK细胞和FN的粘付能力被增强.第二、经过与FN共同培养的LAK细胞活性比正常要高15～20%左右.FN对LAK细胞活性增强机理是通过激活LAK细胞表面的LFA-1的活性(不是LFA-1数量的增加)来增强LAK细胞和肿瘤细胞的结合功能、提高LAK细胞的杀伤肿瘤细胞的能力.本文的结果表明FN-LAK可能会改善肿瘤治疗效果、并间接提示肿瘤细胞表面及肿瘤组织中的FN成分的多少可直接影响LAK细胞的治疗效果.     

自体LAK细胞制备及其在临床上的应用

以基因重组白细胞介素Ⅱ（ｒＩＬ－２）诱导患者自体ＬＡＫ细胞。使用ＬＡＫ／ｒＩＬ－２治疗３例经病理或临床确诊为妇科晚期恶性肿瘤患者。ＬＡＫ细胞（２．２～３．１）×１０９／ｒＩＬ－２１．６×１０６单位治疗一个疗程。治疗前后用Ｂ超、Ｘ线平片观察肿块变化。结果显示，１例癌肿块明显缩小（ＰＲ），１例肿块缩小（ＭＲ），１例肿块无改变（ＳＤ）。患者主观症状改善，生活质量提高。除１例病人在治疗时出现一过性发热外，未见其他毒副作用。     

淋巴因子诱导的细胞毒细胞的诱导及杀瘤活性的实验研究

本文研究了用重组白细胞介素２（ｒＩＬ－２）和含有细胞毒性细胞分化因子（ＣｙｔｏｔｏｘｉｃＣｅｌｌＤｉｆｆｅｒｅｎｔｉａｔｉｏｎＦａｃｔｏｒ，ＣＣＤＦ）的条件培养液，培养正常小鼠脾细胞获得的ＬＩＣＣ在体外的细胞毒作用及其培养条件。结果表明ＬＩＣＣ对体外传代的肿瘤细胞系有一定杀伤活性，对新鲜分离的同基因型肿瘤细胞也有杀瘤效应，对同基因型淋巴母细胞则无杀伤力。细胞的培养活化时间短，需ｒＩＬ－２量较少，诱导产量较高，活性较强。在诱导活化过程中需要Ｍ分泌的ＣＣＤＦ参与，这对于进一步探讨免疫细胞间的相互作用有一定的意义。     

T560: an (H-2b x H-2a) F1 hybrid, phosphorylcholine (PC)-binding, murine B cell lymphoma that bears receptors for IgA and IgG, presents antigen and secretes IL-4.

We describe T560, a tissue culture-adapted B lymphoma derived from the gut-associated lymphoid tissue (GALT) of a (B10 x B10.H-2a H-4b)F1 hybrid mouse. This lymphoma is interesting and useful not only because it bears an unusual IgA receptor, fully described elsewhere, but also because it is potentially capable of presenting antigen to T cells restricted by the MHC of either parent. Here we document that T560 cells are IgG2a kappa +, Ia+, B220+, J11d.2+, CD3-, CD4-, CD5-, Mac 1-, Mac 2-, non-specific esterase-. They bind bromelain-treated mouse RBC (BrMRBC) in a PC chloride-inhibitable manner but do not bind SRBC, ox RBC (ORBC) or TNP-ORBC. Two lines, T560.1 and T560.2, and several clones are available. T560.1 and its clones contain low numbers of IgA rosette-forming cells (RFC), intermediate numbers of IgG2a RFC and moderately high numbers of IgG2b RFC; T560.2 and its clones contain moderately high numbers of IgA RFC and low numbers of both IgG2a and IgG2b RFC. Both lines stimulate both B10 and B10.A cells in mixed lymphocyte reactions (MLR) and present keyhole limpet hemocyanin (KLH) to KLH-reactive T cells. T560.2 populations are, however, more efficient possibly because they have somewhat higher proportions of brightly fluorescent Ia+ cells and secrete larger quantities of lymphokine than T560.1 cells. They present PC-conjugated KLH (PC-KLH) approximately 20 times more efficiently than unconjugated KLH, suggesting that their PC binding receptors function in antigen uptake. They constitutively produce IL-1, IL-4 and IL-6, but not IL-2, IL-5 or TGF beta. Neither their IgA nor their IgG receptor expression is affected by IL-4 or by IFNs-alpha, -beta, or -gamma. In their ability to bind BrMRBC and secrete IL-4, they resemble the CH12 lymphoma but differ from it in that they are of F1 hybrid origin, are CD5-, bear IgG2a rather than IgM, do not bind sheep erythrocytes and have a receptor for IgA not present on CH12.     

Bioassay of interleukins

Summary Sensitive microassay methodologies are described for the bioassay of interleukins 1, 2, 3, and 4 in both serum-free and serum-containing culture supernatants. Interleukins 2, 3, and 4 are measured directly by their growth-promoting activities on the CT6, FDC-P1, and HT-2 indicator cell lines, respectively. Interleukin 1 is assayed indirectly by its ability to stimulate interleukin 2 production by LBRM-33 1A5 lymphoma cells in the presence of phytohemagglutinin. Quantitation is based on measurement of [³H]thymidine incorporation into cellular DNA. The bioassays are performed in microcultures and are semiautomated so that panel testing of small volumes of interleukin-containing culture supernatants can be readily accomplished within 3 d.     

Cellular regulation of immune interferon production

Spleen cells obtained from mice injected 24–72 h previously with a T-cell mitogen were poor producers of immune interferon (IFNγ) when restimulated with the same mitogen in vitro. The reduced response appeared to be due to a suppressor cell population, since virgin spleen cells also gave a reduced response when cultured with the cells from injected mice. Through the use of different concentrations of monoclonal anti-Thy-1 antibody, the mitogen-induced suppressor cell population was shown to contain a relatively high density of the Thy-1 antigen. The IFN-producing cell population contained a relatively low density of the Thy-1 antigen, and either evolved from cells with a high density of Thy-1 antigen or required such cells as helpers in production of IFNγ.A model is proposed according to which the induction of T cells in mice for IFNγ production then involves a cell population with a relatively high density of Thy-1 antigen. The induced cells appear to evolve into IFN-producing cells of low density of Thy-1 antigen. IFN production by the cells with low density of Thy-1 antigen is suppressed or regulated by another stimulated cell population that, like the virgin T cells, possesses a high density of Thy-1 antigen. The IFN-producing cells with low density of Thy-1 antigen appear to represent a small portion of the total T-cell population.     

Practical in vitro assay systems for the measurement of hematopoietic growth factors

Summary A tetrazolium salt (MTT) was used to develop rapid, practical colorimetric assays for the measurement and differential identification of picomolar levels of certain hematopoietic growth factors (GM-CSF, CSF-1, and Multi-CSF/IL-3). The signal generated is directly proportional to the number of viable target cells present after 2 d of culture. Thus, this method can be used to quantitate proliferation or survival of all types of factor responsive cells in vitro. Assays using MTT reduction or [³H]thymidine as read-outs give comparable results; however, MTT reduction is easier, faster, and less expensive. The use of two target cell lines, each with well-characterized growth factor responses, and with differing responsiveness to the myeloid growth factors CSF-1, GM-CSF, and Multi-CSF/IL-3, is described. We further detail steps necessary to adapt other target cells to this convenient assay system.     

The development of anti-interleukin-2 antibodies in patients treated with recombinant human interleukin-2 (IL-2)

Approximately 65% (11/17) of cancer patients participating in an ongoing Phase I clinical trial with recombinant interleukin-2 developed nonneutralizing serum IgG anti-interleukin-2 antibodies within 1 month of initiating therapy. These antibodies could be detected using any of several standard techniques including immunoblots and enzyme-linked immunosorbent assays. Western blot analysis and retention experiments with protein A-Sepharose indicate that the antibodies are specific for interleukin-2. The interleukin-2 mutein utilized in this clinical trial (des-ala-ser₁₂₅ r-IL-2) differs from the major species of the human T cell-derived lymphokine in that it lacks the N-terminal alanine of the native molecule, is not glycosylated, and possesses a serine-cysteine substitution at position 125. Another recombinant interleukin-2, identical to the mutein except that it retains the cysteine at position 125 (des-ala-cys₁₂₅ r-IL-2), strongly competes with the mutein in competitive enzyme-linked immunosorbent assays, suggesting that the amino acid substitution is not responsible for the recognition of the molecule by serum antibodies. Conversely, nonrecombinant T cell-derived interleukin-2 fails to compete in these assays and is not retained by protein A-Sepharose columns when mixed with high-titer antiserum. These results suggest that the anti-interleukin-2 serum antibodies generated in the course of treatment do not react with the nonrecombinant lymphokine but recognize epitopes peculiar to recombinant forms which are not dependent on the amino acid substitution at position 125. The failure of the antibodies to neutralize the biological activity of recombinant interleukin-2 (IL-2) in lymphocyte proliferation assays and to bind to the native lymphokine suggests that they may not affect IL-2-dependent cellular immune functionsin vivo.