Interactions between interferon γ and retinoic acid with transforming growth factor β in the induction of immune recognition molecules

The cell-surface expression of major histocompatibility (MHC) antigens and the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) is essential for target cell recognition by T lymphocytes. The expression of both classes of molecule is induced by various cytokines, notably interferon (IFN). Since transforming growth factor (TGF) has been recently reported to antagonise HLA-DR induction by IFN we have examined, using a number of murine and human cell lines, the effect of TGF on IFN-induced MHC class I and class II and ICAM-1 expression. All of the cell lines tested expressed elevated class I MHC following IFN treatment. Class II MHC induction was seen on most but not all of the cells, the exceptions being among a panel of human colorectal carcinoma cell lines. A striking difference between cells of different origin was noted in the response to TGF. TGF was found to antagonise IFN-induced class I and class II MHC expression on C3H 10T1/2 murine fibroblasts, early-passage BALB/c mouse embryo fibroblasts, a murine oligodendroglioma cell line, and on MRC5 human fibroblasts and two human glioblastoma cell lines. Class II MHC was much more strongly inhibited (sometimes completely) than class I MHC. TGF also inhibited induction of class I MHC expression by IFN. However, TGF did not inhibit class I or class II MHC induction by IFN in any of the nine colorectal carcinoma cell lines, although two of five of the lines tested were growth-inhibited by TGF. On the other hand, human ICAM-1 induction by IFN was not affected by simultaneous treatment with TGF in any of the cell lines. The down-regulation of IFN-induced MHC antigens by TGF is not, therefore, the result of a general antagonism of IFN. Retinoic acid has recently been reported to induce ICAM-1 expression on human tumour cells. We have confirmed this observation on MRC5, and the two human glioblastoma cell lines, however six colorectal carcinoma cell lines tested did not respond. In contrast to IFN-induced ICAM-1 expression, retinoic-acid-induced ICAM-1 expression was inhibited by TGF on two of the three responsive lines.     

Dynamics of cytokine production during coccidial infections in chickens: colony-stimulating factors and interferon

Abstract We assayed two classes of immunoregulatory cytokines, colony-stimulating factors (CSF) and interferon (IFN), during and immediately after a primary coccidial infection in chickens. Coccidial infection induces significant alterations in serum colony-stimulating activity (CSA) and these alterations immediately precede the characteristic biphasic leukocytosis. CSA rose sharply during the first 24 h post-inoculation (PI), but returned to control levels by 48 h PL. At this time, we detected an increase in peripheral blood leukocytes which peaked at 96 h PI. A second phase of CSA increase began 96 h PI and peaked at 120–144 h PI which again preceded the second phase of leukocytosis. We also examined the production of IFN during the first 20 days PI. Splenic T cells from Eimeria maxima -infected chickens produced significantly less IFN on day 5 PI compared to T cells from the coccidia-free controls. By days 10 and 15 PI, there was no significant difference in IFN production between the T cells of infected and non-infected chickens. However, by day 20 PI, IFN production by the T cells of the infected birds produced significantly more IFN than the control T cells. The results of our studies indicated the differential production of two different cytokines by chickens during and following a primary coccidial infection. Based on these experiments, CSF may be some of the first cytokines produced during an E. maxima -infection, while IFN may be one of the later cytokines produced.     

Identification and quantitative determination of 3-chloro-2-hydroxypropylmercapturic acid and α-chlorohydrin in urine of rats treated with epichlorohydrin

Epichlorohydrin (ECH) is used in many industrial processes. Different toxic effects of ECH were found in rodents. The metabolism of ECH was investigated before in rats using [¹⁴C]ECH. The aim of this investigation was the development of non-radioactive quantitative analytical methods for measuring two urinary metabolites of ECH, namely 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) and α-chlorohydrin (α-CH). The identity of CHPMA and α-CH excreted in urine of rats treated with 5 to 35 mg/kg ECH was confirmed by GC-MS. The quantitative analysis of CHPMA, involving ethyl acetate extraction from acidified urine and subsequent methylation and analysis by gas chromatography-flame photometric detection (GC-FPD), showed a method limit of detection of 2 μg/ml. The analysis of α-CH, based on ethyl acetate extraction and subsequent analysis by GC-ECD, showed a method limit of detection of 2 μg/ml. CHPMA and α-CH derivatives could be determined quantitatively down to concentrations of 0.5 and 0.4 μg/ml urine, respectively, by selected-ion monitoring GC-MS under EI conditions. Cumulative urinary excretion of CHPMA and α-CH by rats treated with ECH were found to be 31 ± 10 and 1.4 ± 0.6% (n = 13) of the ECH dose, respectively. For CHPMA, the dose-excretion relationship suggested partially saturated ECH metabolism. For α-CH, the dose-excretion relationship was linear. With fractionated urine collection it was found that approximately 74 and 84% of the total cumulative excretion of CHPMA and α-CH, respectively, took place within the first 6 h after administration of ECH. From these investigations it is concluded that the GC-FPD and GC-ECD based methods developed are sufficiently sensitive to measure urinary excretion of CHPMA and α-CH in urine from rats administered 5 to 35 mg/kg ECH. It is anticipated that the analysis of CHPMA and α-CH based on GC-MS may be sufficiently sensitive to investigate urinary excretion from humans occupationally exposed to ECH.     

RT－PCR联合一步法扩增G蛋白α亚基基因的开放阅读框

报道逆转录－多聚酶链式反应（ＲＴ－ＰＣＲ）联合一步程序,扩增大于１ｋｂ的拟南芥菜Ｇ蛋白α亚基基因的开放阅读框,该程序中,当ＲＮＡ模板和引物变性,并在同一管中加入ＰＣＲ缓冲液,４种ｄＮＴＰ底物,逆转录酶和ＴａｑＤＮＡ聚合酶之后,就不需其它手工操作步骤,因而可同时进行大批量样品的操作,实验结果证明ＡＭＶ（鸟类骨髓性白血病病毒）逆转录酶对ＴａｑＤＮＡ聚合酶的活性没有抑制作用,这种ＲＴ－ＰＣＲ联合一步法可     

人胸腺素α原cDNA的克隆及在大肠杆菌中的表达

采用基因工程方法制取人胸腺素α原获得成功。用２０ｕｇ／ｍｌ植物血球凝集素（ＰＨＡ）和５００Ｕ／ｍｌ重组人白细胞介素２（ＩＬ－２）联合刺激人胎儿胸腺细胞,从中提取总ＲＮＡ,经反转录ＰＣＲ获得了人胸腺素α原ｃＤＮＡ;将之克隆入ｐＵＣ１９中,序列测定表明与已报道序列一致,进一步将之亚克隆入原核表达载体ｐＢＶ２２０,转化大肠杆菌ＤＨ５ａ．观察到在不改变氨基酸编码的前提下,增加胸腺素ａ原上游引物中Ａ、Ｔ含量,可以明显提高胸腺素α原的表达量,同时,不同培养基对它的表达也有影响。胸腺素α原在大肠杆菌中以可溶形式表达,不需复性。初步活性测定显示,它可明显刺激人外周血淋巴细胞Ｅ－玫瑰花结形成率。重组人胸腺素α原在大肠杆菌中表达,为其临床应用及基础研究奠定了基础。     

Identification and partial characterization of α2-macroglobulin from the tuatara (Sphenodon punctatus)

α₂-Macroglobulin (α₂-M), a large molecular mass proteinase-binding protein, was identified in plasma from tuatara (Sphenodon), a rare reptile endemic to New Zealand. In this genus, α₂-M constitutes 11–13% of total plasma protein (∼2.2–3.9 mg/ml). Analysis of blood samples collected at approximately monthly intervals from individual tuatara indicated that the plasma level of α₂-M remains fairly constant. The subunits of tuatara α₂-M have an apparent molecular mass of ∼160 kDa as determined by SDS-polyacrylamide gel electrophoresis and the intact protein is an oligomer that contains inter-chain disulfide bonds. N-terminal sequence analyses of tuatara α₂-M revealed a distinct similarity to α-macroglobulins of other vertebrates and that at least two types of α₂-M subunits are present in plasma of tuatara.     

Examination of lymphokines induced in mice following immunization with recombinant simian virus 40 large tumor antigen

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was used to immunize BALB/c mice to examine the lymphokines produced following immunization. Specifically, we examined production of interleukin-2 (IL-2), IL-4, IL-5 and interferon (IFN) from immune lymphocytes cultured with decreasing concentrations of recombinant SV40 T-Ag. We identified elevated levels of IFN and IL-2 by enzyme-linked immunosorbent assay and a murine CTLL-2 proliferation biossay respectively. We were unable to detect either IL-4 or IL-5. These data indicate the previously reported tumor immunity induced by recombinant SV40 T-Ag immunization most likely reflects a TH1-like immune response based on thein vitro production of both IFN and IL-2 by immune lymphocytes.     

Combination of highly purified human leukocyte interferon α and 13-cis-retinoic acid for the treatment of metastiatic melanoma

 The effect of 13-cis-retinoic acid and highly purified human leukocyte interferon α (Alphaferon) therapy for metastatic melanoma was studied. A group of 17 patients with disseminated malignant melanoma were treated over a 6-month period. They received 60 mg 13-cis-retinoic acid/day continuously and ten cycles of interferon α (IFNα). IFN was administered by subcutaneous injection, at a daily dose of 6×10⁶ IU Alphaferon. The 5-day treatment period was followed by an IFN-free interval of 2 weeks. We were able to observe an overall response rate of 30% with 12% complete responses (2 out of 17 patients). Sites of response included the skin, lung, liver and lymph nodes. All responses have now lasted over 6 months. Therapy was generally well tolerated and could be performed on an outpatient basis. Side-effects of this combination therapy did not exceed the established side-effects of the two substances. We also studied 2′-5′-oligoadenylate synthetase, β2-microglobulin and neopterin levels during the whole treatment course. All patients were within the normal range before treatment and a sharp rise occurred during each IFN cycle. The maximum being observed 24 h after the third injection. This indicates a high biological activity of IFNα administered cyclicly during the whole treatment course. This finding also corresponds well with the absence of neutralizing antibodies before and after the whole treatment period. Received: 8 December 1994 / Accepted: 10 January 1995     

Potentiation of antitumor effects of tumor necrosis factor α and interferon γ by macrophage-colony-stimulating factor in a MmB16 melanoma model in mice

The efficacy of systemic infusion of recombinant human macrophage-colony-stimulating factor (M-CSF) in combination with local treatment with human recombinant tumor necrosis factor (TNF) and mouse recombinant interferon (IFN) was studied in vivo on a subclone of B16 melanoma (MmB16) in mice. Short-term intravenous administration of M-CSF at a dose of 10⁶ units daily had no antitumor effect in vivo. Similarly, local treatment of tumor with TNF (5 g daily) did not produce any therapeutic effect. However, simultaneous administration of the same dose of TNF with IFN (1000 units daily) resulted in a synergistic effects manifested by the retardation of tumor growth. Addition of systemic infusion of M-CSF to the local therapy with TNF and IFN induced further augmentation of antitumor efficacy and delayed progression of MmB16 melanoma. The strengthened antitumor effect of combination therapy including M-CSF, TNF and IFN was most probably due to the increased release of monocytes from the bone marrow, their recruitment into the site of tumor growth and subsequent local stimulation of their antitumor activity.