First report of management and outcome of pregnancies associated with hereditary orotic aciduria

Two pregnancies in a 25-year-old woman with hereditary orotic aciduria who was managed prenatally on uridine therapy are described. The first pregnancy resulted in an infant with multiple congenital anomalies and a 47, xx, inv(4)(p12q25), + der(22)t(11;22)(p23;q11) karyotype. The proposita was found to be a carrier of a de novo 11;22 translocation and a pericentric inversion of chromosome 4. Subsequently, several carriers of orotic aciduria in this family were identified with the inverted chromosome 4. The second pregnancy resulted in a normal male with an inverted chromosome 4.     

Decreased perforin and granzyme B expression in senescent HIV-1-specific cytotoxic T lymphocytes

Cytotoxic T lymphocyte (CTL) senescence may be an important mechanism of immune failure in HIV-1 infection. We find that senescence of HIV-1-specific CTL clones causes loss of killing activity, preventable by transduction with telomerase. Furthermore, senescence is associated with reduced expression of the effector molecules granzyme and perforin, suggesting CTL "exhaustion" can result in hypofunction. These results agree with other studies showing that HIV-1-specific CTL exhibit abnormal phenotypes in vivo, and suggest the possibility that chronic turnover is an important mechanism of antiviral failure in HIV-1 infection.     

The cytotoxic effect of heterologous antisera to mouse IgG on mouse lymphocytes

Goat and rabbit anti-mouse IgG were significantly cytotoxic for adult mouse lymphocytes from spleen, lymph node, peripheral blood, and to a lesser extent, bone marrow. Newborn mouse spleen lymphocytes were not susceptible to this cytotoxic effect, but spleen lymphocytes from 1 week old and 1 month old donors were increasingly so. Absorption of anti-mouse IgG sera with mouse spleen cells or mouse IgG removed both IgG precipitins and lymphocytotoxins. Absorption with mouse red cells or foetal tissue, however, did not remove either cytotoxins or IgG precipitins. Antisera directed against human or rabbit IgG's were not cytotoxic for human or rabbit peripheral blood lymphocytes.     

Blood lymphocytes from ankylosing spondylitis patients fail to induce disease-specific cytotoxic T lymphocytes

The intriguing observation made by Geczy et al. (1) showing the possibility of generating specific ankylosing spondylitis--cytotoxic T lymphocytes by presenting HLA-B27+AS+ cells as antigen-specific stimulator cells prompted us (by using Geczy's approach) to identify cytotoxic T lymphocytes specific for this apparent B27+AS+ target structure. Peripheral blood mononuclear cells (PBMC) of 21 healthy B27+ individuals were stimulated in primary and in short-term cultures with PBMC of an HLA-identical sibling suffering from definite AS (n = 12). In addition, PBMC in vitro modified by "Geczy bacterial products" from two healthy B27+ individuals were used to stimulate B27+ AS- lymphocytes (either autologous or from a healthy HLA-identical sibling). Effector cells raised in primary AS- versus AS+ and AS- versus "modified B27" mixed lymphocyte culture combinations showed no proliferative nor cytotoxic activity at all. The scarcely observed cytotoxic reactivity of restimulated mixed lymphocyte culture was not restricted to AS+B27+ cells. These results demonstrate that PBMC from ankylosing spondylitis patients fail to induce disease-specific cytotoxic T lymphocytes and suggest that an ankylosing spondylitis--related "modified B27" structure does not exist, at least in the patient material tested.     

B- and T-lymphocyte markers on transformed lymphocytes from mitogen-stimulated cultures of normal and CLL lymphocytes and on tonsil blasts

Mitogen-transformed human peripheral blood lymphocytes and tonsil blasts were examined by rosette formation to detect the presence of membrane-bound immunoglobulin (Ig) and surface receptors for fixed IgG and fixed C₃. In addition, the capacity of these cells to rosette with sheep erythrocytes was evaluated as a reaction characteristic of T lymphocytes. In order for clear morphological recognition of the rosetting transformed lymphocytes and the rosetting tonsil blasts a cytocentrifuge technique was developed and used in conjunction with autoradiography and/or with Romanowsky stains. Using these techniques and the culture methods described in this paper phytohaemagglutinin, pokeweed mitogen, streptococcal filtrates and purified protein derivative stimulated predominantly T cells in the peripheral blood of man. A minority of the transformed cells in these mitogen-stimulated cultures (<24%) did rosette with B lymphocyte markers and presumably represent a B-cell response. No significant differences were found between the T- or B-cell specificity of the mitogens investigated. Lymphoid preparations from tonsils excised from normal donors with recurrent tonsillitis were found to contain 6–15% lymphoblasts and the large majority of these cells formed rosettes with the B-cell markers, less than 20% of these lymphoblasts formed spontaneous sheep erythrocyte rosettes. Using a mixed rosetting technique a small proportion (<5%) of PHA-transformed cells and tonsil lymphoblasts were found to have combined sheep Fc or combined sheep C₃ receptors. The investigation of B- and T-lymphocyte surface markers on mitogentransformed lymphocytes was extended to neoplastic lymphocyte populations and it was found that the majority of transformed cells (> 70%) present in chronic lymphocytic leukaemia cultures stimulated with PHA after 6 days incubation were transformed T lymphocytes.     

致敏树突状细胞活化细胞毒性T细胞抗肿瘤作用的研究

目的：研究树突状细胞（DCs）激活的细胞毒性T细胞的抗肿瘤及预防肿瘤发生的作用。 方法： 细胞因子诱生人PBMC未成熟DCs，加入肿瘤细胞抗原提取物致敏DCs产生成熟DCs；通过细胞形态、表面标记鉴定成熟DCs，MTT法测成熟DCs活化的细胞毒性T细胞(CTL)的体外杀伤活性；裸鼠体内注射活化CTL观察其抑制移植瘤生长及发生的作用。 结果： 经过7 d培养，获得大量形态典型、具有强烈刺激增殖能力、高表达CD80(63.5%)、CD83（67.6%）和CD3/ HLA-DR(83.2%)的DCs。其活化的CTL在20∶1效靶比时对抗原来源细胞株自身的杀伤率达75%以上，对同系细胞株的杀伤活性为35%-45%，对其它种系肿瘤细胞仅有微弱杀伤力（P<0.01）。CTL对裸鼠结肠癌HT-29移植瘤有特异性的生长抑制和预防生成作用（P<0.05）。CTL治疗组肿瘤组织中PCNA表达水平显著低于对照组（P<0.05）。 结论： 肿瘤细胞抗原活化的DC诱导CTL对肿瘤有特异性的杀伤作用，体内应用可特异性抑制移植结肠癌的生长或预防小鼠结肠癌移植瘤的发生。     

Activation of secondary cytotoxic lymphocytes by cell-free factors from I-region-primed and D-region-primed lymphocytes.

Cell co-operation in the generation of secondary cytotoxic responses was studied by selectively sensitizing lymphocytes in mixed lymphocyte culture (MLC) across I or D region difference and by combining the primed lymphocytes in the secondary MLC. Secondary cytotoxic responses were induced in D-region-primed lymphocytes by restimulation with the original priming D-region antigens, by co-culturing with the I-region-primed lymphocytes in the presence of the priming I-region antigens, or by cell-free supernatants obtained 24 h after the restimulation of D-region-primed lymphocytes and I-region-primed lymphocytes, The active MLC supernatants produced by both I-region-primed and D-region-primed cells also induced accelerated proliferative responses in D-region-primed lymphocytes. Heat-treatment or ultraviolet irradiation of the stimulator cells eliminated the capacity of the cells to induce the production of CTL-helper factor in I-region-primed and D-region-primed lymphocytes. It was concluded that both I-region-primed and D-region-primed lymphocytes produce a cell-free factor which induces proliferation and secondary cytotoxicity in D-region-primed lymphocytes. The possible participation of D-region reactive helper T cells and D-region reactive cytotoxic T cells in the cytotoxic responses to D-region antigens in the absence of I-region difference is discussed.     

The cytotoxic activity of lymphocytes from human lymph in vitro

The in vitro cytotoxicity and DNA synthesis of thoracic duct and blood lymphocytes from four patients have been studied on the 1st day of drainage. Three patients were being drained as a pretreatment for kidney transplantation and one had myasthenia gravis. In one patient lymphocytes were obtained from a lymphatic fistula in the groin and from the blood 5 weeks after drainage began. Lysis of tissue culture cells (Chang cells) in the presence of PHA or antiserum to target cell antigens was quantitated by [⁵¹Cr]chromate release.

Lymphocytes from lymph were at best poorly cytotoxic to antibody-treated target cells under conditions where purified blood lymphocytes from the same donors had normal lytic activity. PHA-induced cytotoxicity by lymph-borne lymphocytes was noted but was considerably weaker than that of blood lymphocytes. In contrast, incorporation of [¹⁴C]thymidine into DNA of thoracic duct lymphocytes after stimulation with PHA was about 60% of that of the patients' blood lymphocytes. The DNA synthesis of thoracic duct lymphocytes induced by PPD or allogeneic lymphocytes was as good as that of blood lymphocytes. The mitotic response to PHA by lymphocytes from the lymph was reduced after two weeks drainage.

It is assumed that the number of effector cells and/or supporting cells in antibody-induced cytotoxicity in thoracic duct lymph is too small to induce target cell lysis under the present experimental conditions. Moreover, our data indicate that PHA-induced and antibody-mediated cytotoxicity are at least partly mediated by different lymphocyte subpopulations.

     

Decreased T-colony formation by lymphocytes from patients with focal glomerular sclerosis

The T-colony-forming capacity was examined in 13 normal subjects and 8 patients with biopsy-proven focal glomerular sclerosis (FGS). Fifteen patients with chronic mesangial proliferative glomerulonephritis (CGN) without renal insufficiency were studied as a disease control. The two main assays used were the counting of T colonies formed by the patients' lymphocytes, and the measurement of T-colony-forming activity in conditioned medium from cultures of patients' lymphocytes. The levels of T-colony-forming cells (TCFC) and T-colony-stimulating factor (TCSF) were decreased in patients with FGS compared with those in normal controls and lower in FGS patients with the nephrotic syndrome (NS) than in those without NS. In contrast, these parameters in CGN patients with or without NS did not differ from normal subjects. TCSF activity for TCFC in both normal individuals and FGS patients was removed from media conditioned by phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PHA-LCM) with interleukin-2 (IL-2) receptor bearing cultured T cells. These in vitro findings suggest that IL-2 is the essential factor contained in PHA-LCM. Thus, depressed TCFC in FGS patients with NS may result in part from impaired generation of TCSF by lymphocytes.     

Characteristics of cytotoxic T lymphocytes eluted from allogeneic target cells

The increase in the specific cytotoxic effect of immune lymphocytes after their adsorption on the corresponding target cells (TC) and subsequent elution with pronase is due, not to increased cytotoxic activity of individual cells, but to a quantitative increase in the proportion of effector T cells in the population. The eluted and original immune lymphocytes are indistinguishable in the kinetics of their adsorption on TC. The fraction of eluted lymphocytes contains twice as many T cells and four to five times as many cells synthesizing DNA, on account of an increase in the percentage of medium-sized and large lymphocytes.Laboratory of Immunochemistry and Diagnosis of Tumors, 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 G. V. Vygodchikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 1, pp. 50–53, January, 1977.     

Cytoplasts from cytotoxic T lymphocytes are resistant to perforin-mediated lysis

Cytotoxic T lymphocytes (CTL) contain a potent cytolytic pore-forming protein (PFP, perforin or cytolysin) localized in their cytoplasmic granules. In the presence of calcium, perforin lyses a variety of target cells (TC) non-specifically. CTL, however, are generally resistant to the lytic effect of perforin. In this work, cytoplasts from CTL and susceptible TC were made by centrifuging cells on a Ficoll density gradient in the presence of cytochalasin B. Characterization by electron microscopy and a serine esterase assay established that both CTL and TC cytoplasts were completely devoid of nuclei and CTL cytoplasts contained essentially no granules. CTL cytoplasts are just as resistant to perforin-mediated lysis as the intact CTL, and both TC and their corresponding cytoplasts are very sensitive to lysis. Furthermore, CTL cytoplasts are less effective than TC cytoplasts in inhibiting hemolysis, a property shared by the respective intact cells. These results indicate that soluble granular components do not confer resistance on CTL, and suggest that the protective agent(s) acts by impeding perforin binding to the CTL membrane.     

Leukemic cells arise from cloned cytotoxic lymphocytes during cell culture

In spite of many promising attempts to apply T cell clones to questions of in vitro and in vivo function of T cells it is still unclear to what extent continuous propagation of T lymphocytes in vitro effects their original properties. This study describes the appearance of malignant cells from long-term cultured C57BL/6 (B6) cytotoxic T lymphocytes (CTL). Four out of five T cell lines (CTLL.1,3,4,5) representing distinct stages of development of T effector cells in vitro were repeatedly cloned and all five CTLL were tested for various cellular parameters. It is shown that transformation of H-Y-specific CTLL into malignant cells in vitro was accompanied by alterations in growth characteristics, successive loss of specificity and cytolytic function and by quantitative changes in the expression of cell surface markers. Whereas growth of the H-Y-specific CTLL (CTLL.1) was dependent on antigen and concanavalin A (Con A) supernatant (Con ASN) the CTLL variants could be either maintained in Con ASN alone (CTLL.3) or in the absence of both antigen and lymphokine sources (CTLL.4,5). CTLL.1 was cytolytic for male B6 target cells and lysed P815 tumor targets in the presence but not the absence of lectin. In contrast, CTLL.3 lost its original specificity but lysed P815 cells in the absence or presence of lectin. CTLL.2 representing an intermediary stage showed cytolytic activity on both male B6 and P815 target cells. In contrast, CTLL.4 and CTLL.5 lost the ability to lyse any of the indicated target cells. Although all CTLL expressed the surface markers Thy-1, Lyt-2, Kb, Db and interleukin 2 receptor (IL 2 R), Thy-1 and Lyt-2 markers were drastically reduced and Kb/Db and IL 2 R structures significantly increased on CTLL.4 and CTLL.5 compared to CTLL.1,2,3. In addition, multiple karyotypic alterations including the appearance of metacentric chromosomes were observed in long-term cultured CTLL. Investigations on the expression of the alpha-, beta-, and gamma-chains of the T cell antigen receptor in CTLL.1-5 indicate that all three chains were expressed as mRNA irrespective of whether the lymphocytes expressed their original specificity and/or function. However, distinct beta variable chain genes were used by H-Y-specific CTLL and its long-term culture variants CTLL.2 and CTLL.3 suggesting that the expression of the new specificity was accompanied by the rearrangement of a new beta-chain gene in T effector cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Competition for receptors for immunoglobulin on cytotoxic lymphocytes

Target cell killing by lymphocytes can be induced by appropriate antibody complexed to target cell antigens. In this paper it is shown that this form of lymphocyte mediated cytotoxicity is susceptible to inhibition by third party immune complexes which compete with target cell bound antibody for receptors for immunoglobulin on the cytotoxic lymphocytes. The physical state of the complexes is investigated in relation to their inhibitory efficiency. Evidence is presented to show that soluble complexes which exist in antigen–antibody equilibrium or slight antigen excess are the most effective inhibitors.

No evidence could be obtained to support the hypothesis that soluble immune complexes can induce indiscriminate cytotoxic activity in lymphocytes. The biological significance of this effect is discussed in relation to chronic inflammatory diseases.

     

Effect of soluble H-2 antigens on the cytotoxic effect and adsorption of immune lymphocytes

Serologically active preparations of soluble H-2 antigens were obtained by extraction with 3 M KCl from ascites cells of leukemia L1210 (H-2^d) and sarcoma MCh-11 (H-2^b). These preparations had no specific effect on the cytotoxic action of immune lymphocytes on target cells in vitro and did not inhibit adsorption of lymphocytes on a monolayer of the corresponding target cells.Laboratory of Immunochemistry and Diagnosis of Tumors, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. Central Research Laboratory, Saratov Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 9, pp. 333–336, September, 1977.     

Effect of pre-existing cytotoxic T lymphocytes on therapeutic vaccines

Therapeutic vaccines which aim to induce CD8(+) cytotoxic T lymphocyte (CTL) responses will often be required to perform in the presence of pre-existing CTL which recognize epitopes within the vaccine. Here we explore the ability of a viral vaccine vector presenting several co-dominant CTL epitopes to prime CTL responses in animals that have a pre-existing CTL response to one of the epitopes in the vaccine. The vaccine was usually capable of inducing multiple new responses, suggesting that immunodomination effects of pre-existing CTL may generally be minimal following vaccination. However, when large numbers of pre-existing CTL were present, a novel type of immune modulation was observed whereby (1) the vaccine failed to prime efficiently new CTL responses that were restricted by the same MHC gene as the pre-existing responses, and (2) vaccine-induced CTL responses restricted by other MHC genes were enhanced. These results may have implications for therapeutic multi-epitope vaccines for diseases like HIV and melanoma, which aim to broaden CTL responses.     

Studies on the cytotoxic effect of in vivo and in vitro immunized lymphocytes on liver target cells.

Lymph node and spleen cells from mice immunized in vivo to allogeneic of syngeneic liver antigen are cytotoxic for syngeneic liver cells, but not for syngeneic fibroblasts or established liver cell cultures of allogeneic origin. The cytotoxic activity is mainly dependent on T-cell activity, but a non-T-cell-mediated cytotoxicity may also play a role. Lymphocytotoxicity is inhibited by preincubation of the lymphocytes with syngeneic liver antigen, but not with syngeneic kidney homogenate. The liver-specific lymphocytotoxicity corresponds to the in vivo function of lymphocytes in the development of experimental hepatitis. In vitro immunization of lymphocytes in a Mishell-Dutton culture system also induces liver-specific cytotoxicity. The results indicate that the natural tolerance to self antigens can be lost after invivo as well as in vitro immunization. The induction of self-reactivity of lymphocytes in these experiments may be attributed to regulatory mechanisms of the immune reaction at a cellular level.     

Inductive influence of macrophages on cytotoxic properties of lymphocytes.

Normal rat lymphocytes preincubated over syngeneic peritoneal macrophages derived from rats sensitized to tuberculous bacilli and then cultured with PPD showed a cytotoxic effect on sheep red blood cells coated with PPD. This effect was antigen specific and did not involve the nonspecific influence of macrophages.     

Specific separation of cytotoxic T lymphocytes on immunoadsorptive films.

A method for the specific separation of cytotoxic T lymphocytes (CTL) on immunoadsorptive films is described.The films were prepared by polymerizing a mixture of gelatine and polyethyleneimine with glutaraldehyde on the bottom of plastic tissue culture flasks. By using an excess of glutaraldehyde unsaturated aldehyde groups on the film surface can react with other chemical groups predominantly with aliphatic amino groups. In this study detergent solubilized H-2 antigens were conjugated covalently to the films CTL, prepared by filtering spleen cells from alloimmunized mice through nylon wool columns, were incubated on immunoadsorptive films and the adsorbed cells were quantitatively recovered by mechanical detachment. The cells were functionally intact and tested in a microcytotoxicity assay using embryonic fibroblasts as target cells. Specificity adsorbed cells were enriched in specific cytotoxic cells up to 100 fold compared to the original fraction. Depending on the cell density per film area the non-adsorbed cells could be quantitatively depleted of CTL. The immunoadsorptive films are stabile at 37 degrees C and can be reused.     

Discrimination of human cytotoxic lymphocytes from regulatory and B-lymphocytes by orthogonal light scattering

Light scattering properties of human lymphocyte subpopulations selected by immunofluorescence were studied with a flow cytometer. Regulatory and B-lymphocytes showed a low orthogonal light scatter signal, whereas cytotoxic lymphocytes identified with leu-7, leu-11 and leu-15 revealed a large orthogonal light scatter signal. Two populations in light scatter histograms could be observed with monoclonal antibodies directed against determinants present on both regulatory and cytotoxic lymphocytes. By analysis of the lymphocytes of 16 individuals we found a linear relation between the number of cells with a large orthogonal light scattering and the number of cytotoxic lymphocytes identified with leu-7, leu-11 and leu-15. These observations demonstrate physical differences between cytotoxic lymphocytes and regulatory and B lymphocytes. Moreover, the results suggest a method to estimate the amount of cytotoxic lymphocytes without using monoclonal antibodies.     

Prolonged cytotoxic effect of colchicine released from biodegradable microspheres

One the main problems of cancer chemotherapy is the unwanted damage to normal cells caused by the high toxicities of anticancer drugs. Any system of controlled drug delivery that would reduce the total amount of drug required, and thus reduce the side effects, would potentially help to improve chemotherapy. In this respect, biodegradable gelatin microspheres were prepared by water/oil emulsion polymerization and by crosslinking with glutaraldehyde (GTA) as the drug-carrier system. Microspheres were loaded with colchicine, a model antimitotic drug, which was frequently used as an antimitotic agent in cancer research involving cell cultures. Microsphere sizes, swelling and degradation properties, drug-release kinetics, and cytotoxities were studied. Swelling characteristics of microspheres changed upon changing GTA concentration. A decrease in swelling values was recorded as GTA crosslink density was increased. In vitro drug release in PBS (0.01M, pH 7.4) showed rapid colchicine release up to approximately 83% (at t = 92 h) for microspheres with low GTA (0.05% v/v), whereas a slower release profile (only approximately 39%) was obtained for microspheres with high GTA (0.50% v/v) content, for the same period. Cytotoxicity tests with MCF-7, HeLa and H-82 cancer cell lines showed that free colchicine was very toxic, showing an approximately 100% lethal effect in both HeLa and H-82 cell lines and more than 50% decrease in viability in MCF-7 cells in 4 days. Indeed, entrapped colchicine indicated similar initial high toxic effect on cell viability in MCF-7 cell line and this effect became more dominant as colchicine continued to be released from microspheres in the same period. In conclusion, the control of the release rate of colchicine from gelatin microspheres was achieved under in vitro conditions by gelatin through the alteration of crosslinking conditions. Indeed, the results suggested the potential application of gelatin microspheres crosslinked with GTA as a sustained drug-delivery system for anticancer drugs for local chemotherapy administrations.