Behaviour of interleukin-2 serum levels in advanced non-small-cell lung cancer patients: relationship with response to therapy and survival

 Interleukin(IL)-2 is a T helper (Th) 1 type cytokine that has been shown to play an important role in antitumour immune responses. In this study, the prognostic significance of serum IL-2 levels was investigated in 60 advanced non-small-cell lung cancer (NSCLC) patients. IL-2 serum levels were determined before chemotherapy, at the end of chemotherapy and during follow-up, using a commercially available enzyme-linked immunoadsorbent assay kit. The results were analysed according to the response to therapy and were used to generate a model predicting overall survival and time to treatment failure. All 60 patients were shown to have higher IL-2 serum levels than controls (P < 0.0001). Stage IV patients had significantly lower IL-2 levels than stage III patients (P < 0.0001), although they were still significantly higher than controls (P < 0.0001). It is interesting that, when patients were divided into responders and non-responders according to the response to therapy, the former were shown to have significantly higher pre-chemotherapy levels than the latter (P < 0.0001). Moreover, a further significant increase in IL-2 serum levels (P=0.004) and a significant decrease (P < 0.0001) were shown in responders and non-responders, respectively at the end of the therapy. Using univariate and multivariate analyses, both overall survival and time to treatment failure were shown to be affected by the mean pathological levels of IL-2. Furthermore, the prognostic significance of the serum level of IL-2 was confirmed by the stepwise regression analysis. In conclusion, determination of pre-treatment IL-2 serum levels was shown to be of independent prognostic utility in patients with advanced NSCLC; therefore, its possible use for prediction of outcome is proposed. Received: 16 March 2000 / Accepted: 27 July 2000     

Serum anti-p53 autoantibodies in primary resected non-small-cell lung carcinoma

 Mutated p53 proteins accumulate in the nuclei of tumor cells, and anti-p53 autoantibodies are found in the sera of patients with non-small-cell lung carcinoma (NSCLC). We analyzed the correlation among serum anti-p53 autoantibodies, immunohistochemical staining for p53, and clinical features (age, gender, smoking history, histological type, differentiation, stage, T factor, tumor size, and N factor) in resected non-small-cell lung carcinomas. A total of 62 cases of resected NSCLC were studied (43 men and 19 women; 33 adenocarcinomas, 21 squamous cell carcinomas, 8 large-cell carcinomas). Preoperative serum titers of anti-p53 autoantibodies were detected in 13/62 cases (21.0%). A correlation between histological type and positive titers of serum p53 autoantibodies was seen (large-cell carcinoma versus squamous cell carcinoma and adenocarcinoma, P = 0.031, χ²-test). Out of 25 cases, 10 (40%) with positive immunohistochemical staining for p53 had positive titers, whereas 3 positive titers were found in 37 patients with negative immunohistochemical staining for p53 (P = 0.0025, χ²-test). Serum titers of anti-p53 autoantibodies were present in approximately 20% of the cases of NSCLC, and overexpression of p53 protein in tumor cells was detectable in approximately 40%. Serum anti-p53 autoantibodies may be a clinical parameter for the presence of p53 mutations and p53 overexpression in NSCLC patients. Received: 22 October 1997 / Accepted: 22 April 1998     

Coevolution of the mammalian chemokines and their receptors

 A phylogeny of mammalian chemokines revealed two major clusters, corresponding to the CC and CXC chemokines; the C chemokines appeared to be more closely related to the former. In a phylogeny of chemokine receptors, there were also two major clusters: one containing CC chemokine receptors plus other receptors of unknown function and another containing CXC receptors and other receptors of unknown function. However, within the CC receptors, there was not a close correspondence between the phylogenies of chemokines and their receptors. The CC chemokines contained two major subfamilies: (1) the MIP subfamily (including MIP-1α, MIP-1β, and RANTES); and (2) the MCP subfamily (including MCP-1,-2,-3, and -4 and eotaxin). Receptors having preferred ligands in the MCP subfamily did not constitute a monophyletic group but rather evolved twice independently. Reconstruction of ancestral amino acid sequences suggested that these two groups of MCP receptors did not convergently evolve any amino acid residues; rather, they convergently lost sequence features found in the third and fourth extracellular domains of known receptors for MIP-subfamily chemokines. Received: 1 May 1998 / Revised: 3 July 1998     

Macrophage regulation of tumor angiogenesis: implications for cancer therapy

This article reviews the evidence for macrophages playing an important role in the regulation of tumor angiogenesis. Findings in mouse models show that macrophages promote angiogenesis in tumors both by producing excessive amounts of proangiogenic factors and by physically assisting sprouting blood vessels to augment the complexity of the intra-tumoral vascular network. Recent studies however suggest that macrophages may be dispensable for the initiation of angiogenesis in tumors. Rather, these cells express proangiogenic programs that enhance the complexity of the tumor-associated vasculature, leading to aberrant, plethoric and dysfunctional angiogenesis. Gene expression and cell depletion studies further indicate that tumor-associated macrophages (TAMs) comprise phenotypically and functionally distinct subsets. This may reflect “education” of the macrophage phenotype by signals in some areas of the tumor microenvironment and/or TAM subsets derived from distinct macrophage precursors. Among the better characterized TAM subsets are the proangiogenic (TIE2⁺) and the angiostatic/inflammatory (CD11c⁺) macrophages, which coexist in tumors. Such antagonizing TAM subsets occupy distinct niches in the tumor microenvironment and are present at ratios that vary according to the tumor type and grade. Specifically targeting TAMs or reprogramming them from a proangiogenic to an angiostatic function may “normalize” the tumor vasculature and improve the efficacy of various anticancer therapies, including radiotherapy, chemotherapy and vascular-disrupting agents.     

CD40 expression on human lung cancer correlates with metastatic spread

 Purpose: The poor prognosis associated with lung cancer is related to the high incidence of regional and distant metastasis. There is a crucial need to identify parameters that can predict a tendancy to metastatic spread to allow better prognostic evaluation and therapeutic approach. Methods: Using flow cytometry we evaluated 18 human lung cancer cell lines for the expression of different surface markers on lung cancers suggested to be possible prognostic parameters, including epidermal growth factor receptor (EGFR), intercellular adhesion molecule 1 (ICAM-1), Fas and CD40. Results: No correlation was found between tumor prognosis and EGFR, ICAM-1 or Fas. However, a statistically significant correlation was found between the surface expression of CD40 and the metastatic spread of the tumor. In this study, 14 of 18 lung cancer cell lines (78%) expressed CD40 on their surface. All of the 4 tumors that were CD40-negative, were stage I tumors, without any evidence of regional or distant metastasis. Of the 14 tumors that expressed CD40, all but 1 (93%) had either nodal or systemic metastasis at the time of diagnosis. Patients whose tumors were CD40-negative showed a significantly better N stage, overall stage at presentation and survival than those patients with CD40-positive patients. No significant differences between the two groups were observed in tumor size, gender, age, histology, differentiation or preoperative therapy. Conclusions: These results suggest that CD40 expression on lung cancer may play a role in metastatic spread, and also may serve as a prognostic marker and an indicator of advanced disease. Received: 30 August 1999 / Accepted: 22 December 1999     

In silico identification and expression analysis of 12 novel CC chemokines in catfish

Chemokines, a superfamily of chemotactic cytokines involved in recruitment, activation, and adhesion of a variety of leukocyte types to inflammatory foci, are a crucial component of the immune system of Sarcopterygiian vertebrates. Although all mammalian chemokines are believed to have been found, the status of these molecules in Actinopterygii was unknown until recently. The identification of chemokines in fish species has been complicated by low sequence conservation and confusion over expected numbers. Earlier discoveries of single fish chemokines coupled with rapidly expanding genetic resources in these species have recently provided a foundation for large-scale in silico discoveries of these important immune regulators. We report here the identification and expression analysis of 12 new CC chemokine sequences from catfish. When added to our previous report of 14 catfish CC chemokines, the number of CC chemokines in catfish now stands at 26, two more than known from humans. Establishing orthologous relationships among the majority of catfish CC chemokines, a newly available set of chicken CC chemokines, and their mammalian counterparts remain difficult, suggesting high levels of duplication and divergence within individual species.     

The role of CXC chemokines in the transition of chronic inflammation to esophageal and gastric cancer

Chronic inflammation may increase the risk to develop cancer, for instance esophagitis or gastritis may lead to development of esophageal or gastric cancer, respectively. The key molecules attracting leukocytes to local inflammatory sites are chemokines. We here provide a systematic review on the impact of CXC chemokines (binding the receptors CXCR1, CXCR2, CXCR3 and CXCR4) on the transition of chronic inflammation in the upper gastrointestinal tract to neoplasia. CXCR2 ligands, including GRO-α,β,γ/CXCL1,2,3, ENA-78/CXCL5 and IL-8/CXCL8 chemoattract pro-tumoral neutrophils. In addition, angiogenic CXCR2 ligands stimulate the formation of new blood vessels, facilitating tumor progression. The CXCR4 ligand SDF-1/CXCL12 also promotes tumor development by stimulating angiogenesis and by favoring metastasis of CXCR4-positive tumor cells to distant organs producing SDF-1/CXCL12. Furthermore, these angiogenic chemokines also directly enhance tumor cell survival and proliferation. In contrast, the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 are angiostatic and attract anti-tumoral T lymphocytes and may therefore mediate tumor growth retardation and regression. Thus, chemokines exert diverging, sometimes dual roles in tumor biology as described for esophageal and gastric cancer. Therefore extensive research is needed to completely unravel the complex chemokine code in specific cancers. Possibly, chemokine-targeted cancer therapy will have to be adapted to the individual's chemokine profile.     

Characteristics of genetic variation in the progenies of protoplast-derived plants of rice, Oryza sativa cv Nipponbare

Genetic variation in protoplast-derived rice (Oryza sativa L.) plants was characterized using first and second generation selfed progenies. A total of 133 regenerated plants were obtained from ten protoplasts of the japonica rice cultivar Nipponbare. Sixty two regenerated plants which set enough seeds for the subsequent field tests at the next generation and were derived from five protoplasts were selected, and their selfed seeds were used as the first selfed-seed progeny generation). Fifteen plants were selected from each of the 15 lines, and their selfed seeds were used for tests at the generation. Thirty seven lines (60%) segregated plants with detrimental mutant characters of yellow-green phenotype, dwarf stature, dense and short panicle, or low seed fertility. According to the segregation patterns in the lines having mutated plants among those originated from the same protoplasts, the stages of mutation induction were estimated. Additionally, five quantitative traits were changed in almost all and lines. Varied quantitative traits of heading date, number of spikelets per panicle, and seed fertility, were in a heterozygous state. However, culm and panicle lengths showed high uniformity, whereas reduced culm and panicle lengths were caused by mutational changes in polygenes and/or multiple genes. Received: 20 March 1996 / Accepted: 21 June 1996     

Expression of CXC and CC type of chemokines and its receptors in tuberculous and non-tuberculous effusions

Chemokines mediate their biological functions by transmigration of various immune cells to the site of infection. Tuberculous pleurisy provides an effective model to study the role of chemokines in the recruitment of immune cells to the pleura. Our aim was to understand the cumulative effect of chemokines (IP-10, MIG, IL-8, MCP-1, MIP-1α and RANTES) and its receptors (CXCR2, CXCR3, CCR1, CCR2, CCR5 and CCR7) in the recruitment of CD4⁺ T cells obtained from blood (BL) and pleural fluid (PF) of tuberculous (TB) and non-tuberculous (NTB) patients. We observed significant increase in CD4⁺ T cells in TB PF indicating lymphocytic rich effusion. All chemokines except RANTES were significantly high in PF compared to BL in TB group, whereas IL-8 and MCP-1 showed significant increase only in NTB PF. The significantly high levels of IFN-γ and ΤΝF-α in TB PF and their positive correlation with IP-10 and MIP-1α indicated their synergistic action to elicit a strong protective Th1 response. In spite of high levels of Th1 cytokines and chemokines in TB PF, significantly lower levels of RANTES indicated its limited role at the site. The CXC receptors in PF of both the groups and CC receptors except CCR5 in TB PF were significantly high compared to BL. Only CXCR2, CCR5 and CCR7 showed significant increase in TB compared to NTB. Thus a selective concentration of chemokines, cytokines and abundant expression of chemokine receptors confirm the accumulation of activated and memory T cells at the site of infection and help in polarizing Th1 immune response.     

Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule

 The generation of cytotoxic effector T cells requires delivery of two signals, one derived from a specific antigenic epitope and one from a costimulatory molecule. A phase I clinical trial was conducted with a non-replicating canarypoxvirus (ALVAC) constructed to express both human carcinoembryonic antigen (CEA) and the B7.1 costimulatory molecule. This was the first study in cancer patients to determine if the delivery of costimulation with a tumor vaccine was feasible and improved immune responses. Three cohorts of six patients, each with advanced CEA-expressing adenocarcinomas, were treated with increasing doses of an ALVAC-CEA-B7.1 vaccine (4.5 × 10⁶, 4.5 × 10⁷, and 4.5 × 10⁸ plaque-forming units, PFU). Patients were vaccinated by intramuscular injection every 4 weeks for 3 months and monitored for side-effects, tumor growth and anti-CEA immune responses. ALVAC-CEA- B7.1 at doses up to 4.5 × 10⁸ PFU was given without evidence of significant toxicity or autoimmune reactions. Three patients experienced clinically stable disease that correlated with increasing CEA-specific precursor T cells, as shown by in vitro interferon-γ enzyme-linked immunoassay spot tests (ELISPOT). These three patients underwent repeated vaccination resulting in augmented CEA-specific T cell responses. This study represents the first use of costimulation to enhance antitumor vaccines in cancer patients. This approach resulted in CEA-specific immunity associated with stable diseases in three patients. This study also demonstrated that CEA-specific T cell responses could be sustained by repeated vaccinations. Although the number of patients was small, the addition of B7.1 to virus-based vaccines may improve immunological and stable diseases to vaccination against tumor-associated antigens with tolerable toxicity. Received: 6 May 2000 / Accepted: 13 July 2000     

Epigenetic silencing of human T (brachyury homologue) gene in non-small-cell lung cancer

Early detection of lung cancer is challenging due to a lack of adequate biomarkers. To discover novel tumor suppressor genes (TSGs) silenced by aberrant promoter methylation, we analyzed the gene expression profiles of two lung adenocarcinoma cell lines using pharmacologic-unmasking and subsequent microarray-analysis. Among 617 genes upregulated, we selected 30 genes and investigated the methylation status of their promoters by bisulfite sequencing analysis. Aberrant methylation was detected in four genes (CRABP2, NOEY2, T, MAP2K3) in at least one lung adenocarcinoma cell lines. Furthermore, the T promoter was methylated in 60% of primary lung adenocarcinomas versus 13% of non-malignant lung tissues. Conversely, RT-PCR analysis revealed T expression was low in lung tumors, while high in normal tissues. In addition, no non-synonymous mutations related to gene silencing were found. While further analysis is warranted, our results suggest that T has the potential to be a novel candidate TSG in lung cancer.     

Multiple CC chemokines in channel catfish and blue catfish as revealed by analysis of expressed sequence tags

Chemokines represent a superfamily of chemotactic cytokines involved in recruitment, activation and adhesion of a variety of leukocyte types to inflammatory foci, as well as in the organization and maintenance of lymphoid organ architecture and in normal developmental processes. Nearly all chemokines have been identified in human and mouse, but only a handful of fish chemokines have been identified. Here we describe 14 distinct chemokines from channel catfish and blue catfish identified by analysis of 30,000 expressed sequence tags. Based on sequence analysis, sequence similarity, and the arrangement of the conserved cysteine residues, all 14 chemokines were identified as members of the CC subfamily. Phylogenetic analysis did not reveal clear evidence of orthology of the catfish and human or mouse chemokines. Similarity analysis indicated that nine of the 14 CC chemokines were identified for the first time in fish. The availability of this pool of catfish CC chemokines should facilitate rapid identification and phylogenetic analysis of CC chemokines from other fish and related species.     

Specifying the introgressed regions from H. argophyllus in cultivated sunflower (Helianthus annuus L.) to mark Phomopsis resistance genes

A method based upon targetting of intro-gressed markers in a Phomopsis-resistant line (R) of cultivated sunflower, issuing from a H. argophyllus cross was used to mark the Phomopsis resistance regions. Our study was based upon 203 families derived from a cross between an inbred line susceptible to Phomopsis (S1) and the introgressed resistant line (R). Families were checked for Phomopsis resistance level in a design with replicated plots and natural infection was re-inforced by pieces of contaminated stems. Thirty four primers were employed for RAPD analysis. Out of 102 polymorphic fragments between (S1) and H. argophyllus, seven were still present in (R) suggesting that they marked introgressions of H. argophyllus into (R). The plants were scored for the presence or absence of 19 fragments obtained from five primers, and the relationships between the presence/absence of fragments in plants and Phomopsis resistance/susceptiblity in the progenies was determined by using an analysis of variance. We found that at least two introgressed regions, as well as favourable factors from sunflower, contributed to the level of Phomopsis resistance in cultivated sunflower. Received: 28 June 1996 / Accepted: 5 July 1996     

Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line

A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with chromosomes was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes. Received: 24 April 1996 / Accepted: 24 May 1996     

Antimetastatic effect of recombinant human macrophage-colony-stimulating factor against lung and liver metastatic B16 melanoma

 We studied the effect of recombinant human macrophage-colony-stimulating factor (rhM-CSF) on the formation of lung and liver metastases following the i.v. injection of the B16 melanoma subline (B16 LiLu) into mice. When rhM-CSF was administered before the B16 inoculation, the number of tumor metastases decreased in the lung and liver. However, the administration of rhM-CSF after B16 inoculation did not produce an antimetastatic effect in the lung, but did in the liver. B16 cells labeled with 5-[¹²⁵I]-iodo-2′-deoxyuridine (¹²⁵I-dUrd) were injected and the arrest of tumor cell emboli was examined in the capillary beds of the lung and liver of mice treated with either vehicle or rhM-CSF. In both groups, there were the same numbers of B16 cells in both the lung and the liver 3 minutes after the B16 injection, and almost all tumor cells died within 24 h. However, the number of cells surviving in the lung was decreased in mice injected with rhM-CSF (37%). There was no difference in the number of cells in the livers of mice treated either with vehicle or rhM-CSF in the first 24 h after tumor cell injection. The administration of rhM-CSF increased NK 1.1⁺ cells in the mouse spleen and facilitated NK activity in vivo. At the same time, the administration of an anti-NK 1.1 antibody blocked the antimetastatic effect of rhM-CSF in the lung but not in the liver. The antibody was effective only when it was injected before the B16 inoculation. These results suggest that the antimetastatic effect of rhM-CSF in the lung was mediated by NK 1.1⁺ cells within 24 h of B16 injection. In contrast, the antimetastatic effect of rhM-CSF in the liver was mediated not only by NK 1.1⁺ cells but also by other antimetastatic systems such as macrophages. Received: 8 April 1996 / Accepted: 26 November 1996     

Evolution of CC chemokines in teleost fish: a case study in gene duplication and implications for immune diversity

Chemokines are a superfamily of cytokines responsible for regulating cell migration under both inflammatory and physiological conditions. CC chemokines are the largest subfamily of chemokines, with 28 members in humans. A subject of intense study in mammalian species, the known functional roles of CC chemokines ligands in both developmental and disease conditions continue to expand. They are also an important family for the study of gene copy number variation and tandem duplication in mammalian species. However, little is known regarding the evolutionary origin and status of these ligands in primitive vertebrates such as teleost fish. In this paper, we review the evolution of the teleost fish CC chemokine gene family, noting evidence of widespread tandem gene duplications and examining the implications of this phenomenon on immune diversity. Through extensive phylogenetic analysis of the CC chemokine sets of four teleost species, zebrafish, catfish, rainbow trout, and Atlantic salmon, we identified seven large groups of CC chemokines. It appeared that several major groups of CC chemokines are highly related including the CCL19/21/25 group, the CCL20 group, CCL27/28 group, and the fish-specific group. In the three remaining groups that contained the largest number of members, the CCL17/22 group, the MIP group, and the MCP group, similarities among species members were obscured by rapid, tandem duplications that may contribute to immune diversity.     

The role of wingless in the development of multibranched crustacean limbs

 Arthropods are the most diverse and speciose group of organisms on earth. A key feature in their successful radiation is the ease with which various appendages become readily adapted to new functions in novel environments. Arthropod limbs differ radically in form and function, from unbranched walking legs to multibranched swimming paddles. To uncover the developmental and genetic mechanisms underlying this diversification in form, we ask whether a three-signal model of limb growth based on Drosophila experiments is used in the development of arthropod limbs with variant shape. We cloned a Wnt-1 ortholog (Tlwnt-1) from Triops longicaudatus, a basal crustacean with a multibranched limb. We examined the mRNA in situ hybridization pattern during larval development to determine whether changes in wg expression are correlated with innovation in limb form. During larval growth and segmentation Tlwnt-1 is expressed in a segmentally reiterated pattern in the trunk. Unexpectedly, this pattern is restricted to the ventral portion of the epidermis. During early limb formation the single continuous stripe of Tlwnt-1 expression in each segment becomes ventrolaterally restricted into a series of shorter stripes. Some but not all of these shorter stripes correspond to what becomes the ventral side of a developing limb branch. We conclude that the Drosophila model of limb development cannot explain all types of arthropod proximodistal outgrowths, and that the multibranched limb of Triops develops from an early reorganization of the ventral body wall. In Triops, Tlwnt-1 plays a semiconservative role similar to that played by Drosophila wingless in segmentation and limb formation, and morphological innovation in limb form arises in part through an early modulation in the expression of the Tlwnt-1 gene. Received: 22 September 1998 / Accepted: 12 January 1999     

Intradermal administration of lipopolysaccharide in treatment of human cancer

 Lipopolysaccharide (LPS) has been recognized as a potent antitumor agent in animal tumor models; however, its use in human cancer therapy has been limited to only one trial, in which LPS from Salmonella was given intravenously. It was not very successful because of poor tumor response and was also toxic. We originally developed LPS prepared from Pantoea agglomerans (LPSp), and this was a well-purified, small-molecular-mass (5 kDa) agent. We chose intradermal rather than intravenous administration in the hope that the former would release LPS slowly into the bloodstream, and thus be less toxic while preserving antitumor activity. In our animal tumor models, intradermal administration was indeed less toxic and more beneficial for tumor regression than intravenous administration. We made a pilot study with intradermal administration of LPSp on the treatment of ten advanced cancer patients. Five of them had evaluable tumor, which had failed earlier to respond to conventional chemotherapy. Cyclophosphamide was also administered in this trial, in anticipation of its synergistic effect with LPSp. In this study LPSp was injected intradermally into each patient twice a week, starting with an initial dose of 0.4 ng/kg, and raising it to 600 or 1800 ng/kg. A 400-mg/m² dose of cyclophosphamide was given intravenously every 2 weeks. After completion of the dose escalation, the treatment was continued for at least 4 months, and it was found that 1800 ng/kg LPSp was well tolerated. A significant level of cytokines was observed in the sera for at least 8 h. These results indicate higher tolerable doses and remarkably more continuous induction of the cytokines than were reported in a previous study by others using intravenous administration. Three of the five evaluable tumors showed a significant response to our combined therapy. Intradermally administered, LPS was less toxic and elicited a tumor response in combination with cyclophosphamide; it can thus can be applied to cancer treatment even in humans. Received: 3 August 1995 / Accepted: 2 April 1996