Type 1 and type 2 tumor infiltrating effector cell subpopulations in progressive breast cancer

Effector T cells fall into two subpopulations based on cytokine-secretion. Type 1 cells secrete IFN-gamma, whereas type 2 cells secrete IL-4, IL-10, and GM-CSF. NKT cells represent a third subpopulation that secretes similar cytokines and have been associated with immunoregulation. Using the TS/A adenocarcinoma, we assessed the phenotype and kinetics of tumor-infiltrating lymphocytes (TIL) in mice challenged subcutaneously in the mammary region. Flow cytometric analysis shows that T cells do not infiltrate the primary tumor site until days 7-14 following tumor challenge. Both CD4 and CD8 TILs were predominantly CD44(High) and expressed CD25, CD69, and CD95 cell surface activation markers. Activated CD4/CD44(High) TIL numbers reached peak levels at day 21 that precipitously decreased by day 28 whereas corresponding CD8 cell numbers progressively increased, however, at lower levels and with later kinetics. Intracellular cytokine staining showed that greater numbers of IL-4-producing Th2 cells were elicited and with earlier kinetics than that of IFN-gamma-producing Th1 cells. T cells co-expressing DX5 (CD3(+)/DX5(+)) emerged (>21 days), suggesting a recruitment of NK-like T cells at later stages of tumor progression. Moreover, tumors selectively up-regulated TGF-beta, MIF, and IP-10 gene expression at times as early as day 4, with peak levels at day 7 in vivo. Such gene expression remained elevated and correlated with a continued progression in tumor growth suggesting that preferential effector cell recruitment and production of select factors during different stages of tumor maturation may aid in regulating effective endogenous antitumor responses in progressive breast cancer.     

Anti-proliferative effects of phosphodiester oligodeoxynucleotides

The immunostimulatory effects of cytosine-phosphate-guanosine (CpG)-containing oligodeoxynucleotides (ODNs) have been extensively documented. In this paper, we describe the inhibitory effects of ODNs that contain natural phosphodiester backbones (O-ODNs) on the immunostimulation caused by CpG-containing phosphorothioated ODNs (CpG-S). CpG-S stimulation of mouse splenocyte proliferation was reduced by the addition of O-ODNs that contained or lacked the CpG-motif (CpG-containing phosphodiester oligodeoxynucleotide, CpG-O or GpC-O). The total number of cultured splenocytes was up-regulated by CpG-S, whereas repetitive addition of O-ODNs to the cell cultures inhibited this effect. The frequency of T2-like B cells was found to be increased by CpG-S. The culture supernatants of CpG-S-treated splenocytes contained elevated levels of IL-10 and IL-6. However, IL-10 and IL-6 production was down-regulated significantly by the combination of CpG-S and either CpG-O or GpC-O. The O-ODN mediated inhibition of proliferation was less pronounced in IL-10-/- mice. Thus, the O-ODNs, irrespective of CpG content, exerted inhibitory activities on the proliferation of B cells. These anti-proliferative effects appear to be mediated both by the down-regulation of IL-10 production and increased apoptosis.     

Infrared spectroscopy: A new diagnostic tool in Alzheimer disease

Biological markers play an evolving role in the diagnosis of Alzheimer disease (AD). We compare conventional measurements of cerebrospinal fluid (CSF) tau and β-amyloid_1–42 proteins to a novel approach – Fourier transformed infrared (FT-IR) spectroscopy – a simple technique derived from chemical and physical sciences that characterizes intramolecular bonds. For automatic diagnostic analysis, we developed an artificial neural network (ANN). We examined 71 patients with a clinical diagnosis of AD and 66 controls. β-Amyloid_1–42 was decreased (sensitivity 80% and specificity 78%); tau was elevated (sensitivity 76% and specificity 88%) in CSF of AD patients. The combined tau/β-amyloid_1–42 quotient was able to distinguish healthy from diseased subjects with 99% sensitivity and 86% specificity. The ANN could separate FT-IR spectroscopy data with 88.5% sensitivity and 80% specificity. FT-IR spectroscopy proved to be cost-effective and simple to perform. Diagnostic sensitivity and specificity is in the range of CSF tau and β-amyloid_1–42 protein analysis. Larger sample numbers for ANN training and validation could increase diagnostic accuracy and thus prove to be a useful screening tool.     

Elevated interleukin-18 levels in bronchoalveolar lavage fluid of patients with eosinophilic pneumonia

BACKGROUND: Interleukin (IL)-18 can induce Th2 cytokine production particularly in collaboration with IL-2. Accumulation of Th2 cells and increased levels of Th2 cytokines are found in bronchoalveolar lavage fluid (BALF) from patients with eosinophilic pneumonia (EP). To evaluate the role of IL-18 in the pathogenesis of EP, we measured the concentration of IL-2, IL-12, IL-18, and Th2 cytokines in BALF from patients with EP. METHODS: The concentrations of interferon (IFN)-gamma, IL-2, IL-5, IL-10, IL-12, IL-13, and IL-18 in BALF were measured in patients with idiopathic acute eosinophilic pneumonia (AEP), with idiopathic chronic eosinophilic pneumonia (CEP), with sarcoidosis and healthy volunteers (HV). RESULTS: The BALF concentrations of Th2 cytokines, IL-5, IL-10, and IL-13, were higher in patients with EP than in sarcoidosis and control. The IL-2 level in BALF was higher in EP than in sarcoidosis and control. The IL-18 and IL-12 (p40 + p70) levels were higher in patients with EP than sarcoidosis, while the level of IL-12 (p70) was below the detection limit in patients with EP. There was a significant correlation between IL-2 level and both IL-5 and IL-13 in BALF of patients with EP. CONCLUSIONS: Our findings suggest that IL-18 may contribute to Th2 cytokine-dominant responses in patients with EP in collaboration with IL-2.     

Th1型细胞因子基因对结核分枝杆菌基因疫苗诱导BALB/c小鼠产生抗CFP10抗体水平的影响

目的：研究分别表达含IL—12和IL-18基因的质粒，对结核分枝杆菌(Mycobacterium tuberculosis，MTB)H37R1株CFP1O基因疫苗诱导免疫应答的影响。方法：从正常人外周血单个核细胞(PMBCs)中提取RNA，用RT—PCR扩增IL-18 cDNA，并克隆人载体pGEM—Teasy中。测序证实后，亚克隆至真核表达载体pcDNA3．1的BamH Ⅰ和EcoR Ⅰ酶切位点。将分别表达小鼠IL—12和人IL—18基因的真核表达质粒pcmlL12和pclL18，与MTB CFP10基因疫苗联合肌注免疫BALB／c小鼠，共免疫3次，每次间隔2wk。每次免疫后2wk采血、分离血清，用ELISA检测小鼠血清抗CFP10抗体的滴度。结果：用RT—PCR成功地从人PMBC的RNA中扩增出IL—18 cDNA，测序结果正确，用BamH Ⅰ和EcoR Ⅰ酶切鉴定证实，目的基因已插入载体pcDNA3．1中，阳性克隆命名为pcIL18。pcCFP10组第1次免疫后，血清抗CFP10抗体的平均滴度为1：600，末次免疫后的滴度为1：4000。pcIL18 pcCFP10组联合免疫后，血清抗CFP10抗体的滴度高于pcCFP10组，最终达1：8000。而pcmIL12 pcCFP10组联合免疫后滴度仅为1：200。结论：pcIL18与CFP10基因疫苗联合免疫，可增强CFP10抗原的特异性体液免疫应答；pcmIL12则可使CFP10基因疫苗产生的抗体水平降低。pcIL18 pcCFP10基因联合免疫是否具有增强CFP10抗原特异性细胞免疫的作用有待进一步研究。     

Opsonizing antibodies (IgG1) up-regulate monocyte proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and IL-6 but not anti-inflammatory cytokine IL-10 in mycobacterial antigen-stimulated monocytes-implications for pathogenesis

Cachexia is one of the prominent features of advanced tuberculosis (TB) seen in association with increased expression of the monokine TNF-alpha. Several mycobacterial proteins, including PPD, stimulate TNF-alpha secretion from monocytes. Host factors that may play a role in cytokine expression from monocytes remain largely unknown. One such factor is the opsonizing antibodies. Monocytes have high-affinity receptors (FcgammaI and FcgammaIII) for IgG1 and IgG3 antibodies that mediate antigen uptake. We have reported selective up-regulation of IgG1 (which bind to Fcgamma receptors) in advanced TB and have recently shown the ability of PPD-specific IgG1 antibodies to augment TNF-alpha expression in PPD-stimulated monocytes. These observations have now been extended to other cytokines with semipurified fractions from secreted antigens of Mycobacterium tuberculosis (containing 30 kD and 58 kD) that were devoid of lipids, glycolipids and carbohydrates. In the presence of heat-inactivated TB plasma containing known amounts of antigen-specific IgG1 antibodies, these fractions induced significantly increased TNF-alpha, IL-6 and IL-10 secretion. Absorption of IgG1 with Protein 'A' removed the augmenting activity for TNF-alpha and IL-6 secretion from the TB plasma samples. In the case of IL-10, removal of IgG1 resulted in increased rather than decreased IL-10 secretion. These results suggest a possible pathogenic role for antibodies in TB by enhancing proinflammatory and blocking down-regulatory cytokines such as IL-10 cytokines during the chronic phase of TB.     

慢性肾病患者细胞因子测定的临床意义

目的：探讨了慢性肾病患者血清IL-6、IL-8、IL-10和IL-18水平的变化及意义。方法：分别应用放射免疫分析和酶联法对32例慢性肾病患者进行了血清IL-6、IL-8、IL-10和IL-18测定,并与35名正常健康人作比较。结果：慢性肾病患者血清IL-6、IL-8、IL-10和IL-18水平显著地高于正常人组（P〈0.01）,经治疗6个月后与正常人组比较仍有差异（P〈0.05）。结论：检测慢性肾病患者血清IL-6、IL-8、IL-10和IL-18水平的变化对疾病的预后观察具有重要的临床价值。     

人缺血脑组织中IP-10和IFN-γ的表达

目的：探讨趋化因子IP-10和细胞因子IFN-γ是否参与人缺血脑损伤过程。方法：将21例脑梗死死亡病例按发病持续时间分为〈7天、7～14天和15～21天3组，以非缺血侧半球做对照，用HE染色法观察炎性细胞浸润情况；通过免疫组织化学方法检测缺血半球脑组织与非缺血半球脑组织中趋化因子IP-10和细胞因子IFN-γ的表达。结果：在〈7天组和7～14天组中，缺血脑组织可见大量炎性细胞浸润。在〈7天组、7～14天组和15～2l天组中，趋化因子IP-10在缺血半球脑组织中的表达高于非缺血半球（分别是1．74倍增高，P〈0．01；1．41倍增高，P〈0．05和1．52倍增高，P〈0．01）。在对细胞因子IFN-γ的检测中发现〈7天和7～14天组中，IFN-γ在缺血半球脑组织中的表达高于非缺血半球（分别是1．65倍增高，P〈0．05和1．32倍增高，P〈0．05）；在15～21天组中，IFN-γ在缺血半球与非缺血半球中的表达没有显著差异（P〉0．05）。结论：在人缺血脑组织中观察到IP-10和IFN-γ的表达，提示IP-10和IFN-γ参与了炎症反应对脑组织的损伤过程。同时也提示IP-10可能参与后期对损伤脑组织的修复。     

Pentoxifylline attenuates the development of hyperalgesia in a rat model of neuropathic pain

Pentoxifylline, a non-specific cytokine inhibitor, has shown to be beneficial in inflammatory pain in both experimental and clinical studies. The present study demonstrates for the first time, to our knowledge, the antihyperalgesic effect of pentoxifylline in the neuropathic pain using L5 spinal nerve transection rat model. In a preventive paradigm, pentoxifylline (12.5, 25, 50, or 100 mg/kg intraperitoneally) was administered systemically daily, beginning 1 h prior to nerve transection. Pentoxifylline (50, or 100 mg/kg i.p.) produced significant decrease in the mechanical and thermal hyperalgesia. However, pentoxifylline (100 mg/kg i.p.) did not influence the paw pressure thresholds and paw withdrawal latency in sham-operated rats. In order to understand the possible antinocicieptive effect of pentoxifylline in neuropathic pain, we examined the level of TNFα, IL-1β, IL-6 and IL-10 protein in the contralateral brain on day 7 post-transection. Pentoxifylline administration resulted in a dose-dependent reduction of the production of proinflammatory cytokines like TNFα, IL-1β and IL-6, and enhancement of IL-10. Furthermore, we investigated the activity of nuclear factor kappa B (NF-κB) in the contralateral brain on days 7 after surgery. In accordance with the change of proinflammatory cytokines, Pentoxifylline (50 or 100 mg/kg) significantly inhibited the activation of NF-κB in the brain. This research supports a growing body of literature emphasizing the importance of neuroinflammation and neuroimmune activation in the development of neuropathic pain states, and the potential preventive value of pentoxifylline in the treatment of neuropathic pain.     

Biosynthesis of two stage-specific membrane proteins during transformation of Plasmodium gallinaceum zygotes into ookinetes

We have studied the synthesis and expression of surface proteins in zygotes of Plasmodium gallinaceum during their transformation to mature ookinetes. The cells were biosynthetically labelled in vitro using [35S]methionine and proteins were immunoprecipitated with rabbit anti-ookinete serum or monoclonal antibodies. Early zygotes (approx. 2 h post-gametogenesis and fertilization) synthesized and expressed on their surface a protein of Mr 26 000 as observed under reducing conditions on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (31 000 under non-reducing conditions) and continued to do so for 8-10 h; thereafter synthesis of the Mr 26 000 protein declined and little or none was synthesized in the mature ookinetes (greater than 20 h post-gametogenesis). Between 3-5 h post-gametogenesis, zygotes also began to synthesize a protein of Mr 28 000 (34 000 under non-reducing conditions). Synthesis and expression of this surface protein continued throughout development; and the Mr 28 000 protein was the predominant surface protein synthesized by the mature ookinete. Mr 26 000 and Mr 28 000 proteins have been designated earlier as PgO-1 and PgO-2 respectively (Carter and Kaushal, Mol. Biochem. Parasitol. (1984) 13, 235-241). Neither protein was synthesized in the gametocytes prior to gametogenesis. Both proteins could be labelled with [3H]glucosamine or [3H]mannose. When zygotes were incubated with [3H]palmitic acid both PgO-1 and PgO-2 bound fatty acids in covalent linkage. The two proteins do not otherwise appear to be structurally related. They were differentially immunoprecipitated by different monoclonal antibodies and gave rise to distinct patterns of peptides following digestion with proteases such as Staphylococcus aureus V-8, trypsin and chymotrypsin.