Modulation of plasma complement by the initial dose of epirubicin/docetaxel therapy in breast cancer and its predictive value.

BACKGROUND: Despite the widespread use of neoadjuvant chemotherapy in breast cancer patients, prediction of individual response to treatment remains an unsolved clinical problem. Particularly, administration of an inefficient chemotherapeutic regimen should be avoided. Therefore, a better understanding of the molecular mechanisms underlying response to neoadjuvant chemotherapy is of particular clinical interest. Aim of the present study was to test whether neoadjuvant chemotherapy with epirubicin/docetaxel induces early changes in the plasma proteome of breast cancer patients and whether such changes correlate with response to therapy. METHODS: Plasma samples of 25 breast cancer patients obtained before and 24 h after initiation of epirubicin/docetaxel-based neoadjuvant chemotherapy were analysed using two-dimensional differential gel electrophoresis (2D-DIGE). Protein spots found to be differentially expressed were identified using mass spectrometry and then correlated with the pathological response after six cycles of therapy. Markers identified in a discovery set of patients (n=12) were confirmed in an independent validation set (n=13). RESULTS: 2D-DIGE revealed 33 protein spots to be differentially expressed in response to chemotherapy, including the complement factors C1, C3 and C4, inter-α-trypsin inhibitor, α-1-antichymotrypsin and α-2-Heremans-Schmid glycoprotein (AHSG). With respect to cytokines, only interleukin (IL)-6, IL-10 and soluble intracellular adgesion molecule 3 (sICAM3) were minimally modulated. Moreover, two protein spots within the complement component C3 significantly correlated with response to therapy. CONCLUSION: We have identified acute phase proteins and the complement system as part of the early host response to epirubicin/docetaxel chemotherapy. As complement C3 cleavage correlates with the efficacy of docetaxel/epirubicin-based chemotherapy, it has the potential as an easily accessible predictive biomarker.

Evaluation of soluble CD44v6 as a potential serum marker for head and neck squamous cell carcinoma.

In recent years, the measurement of soluble CD44 levels in the circulation of patients with malignant diseases has been introduced as a new and simple diagnostic tool for the detection of human cancer. The high CD44v6 expression in head and neck squamous cell carcinoma (HNSCC) would enable the use of soluble CD44v6 proteins present in the circulation of HNSCC patients as a marker of disease. In the present study, we determined CD44v6 plasma levels using a domain-specific ELISA in healthy volunteers, non-cancer patients, and HNSCC patients before and after surgical removal of the tumor. A difference between the CD44v6 plasma levels of HNSCC patients and controls could not be observed. Moreover, surgical removal of the tumor did not result in a reduction of the CD44v6 plasma level in the HNSCC patients. In addition, the spectrum of soluble v6-containing CD44 proteins present in the plasma of HNSCC patients and controls was determined by immunoprecipitation experiments, but again, tumor-related isoforms could not be distinguished in patient samples. Additional experiments to unravel the biological source of these circulating proteins indicated surprisingly that the v6-containing proteins present in the circulation of healthy individuals are only released in part, if at all, by activated lymphocytes or other nucleated blood cells. Most circulating CD44v6 proteins seem to be derived from the normal epithelial cell compartments, including breast cells, colon cells, and squamous cells. Taken together, these data do not support the use of soluble CD44v6 as a tumor marker in HNSCC or any other tumor type that has developed from tissues producing soluble isoforms.

Circulating adhesion molecules as prognostic factors for cutaneous melanoma.

BACKGROUND: Overexpression of adhesion molecules in tissues of human neoplasms, including malignant melanoma, has been reported to be clinically relevant, but the predictive value of circulating adhesion molecules for clinical outcome and life expectancy in patients with primary malignant melanoma (PMM) and metastases of primary malignant melanoma (MMM) remains undetermined. OBJECTIVE: Our purpose was to examine the prognostic relevance of circulating adhesion molecules, namely circulating CD44 standard (cCD44std), and the isoforms CD44v5 (cv5), CD44v6 (cv6), and CD44v10(cv10), circulating intercellular adhesion molecule-1 (cICAM-1), and circulating platelet/endothelial cell adhesion molecule-1 (cPECAM-1, CD31). METHODS: Levels of cCD44std, cv5, cv6, cv10, cICAM-1, and PECAM-1 were measured by enzyme-linked immunosorbent assays in 119 patients with PMM and MMM, in 12 persons with dysplastic nevi (Clark's nevi), and in 28 patients with inflammatory cutaneous diseases. RESULTS: Patients with PMM, MMM, and inflammatory cutaneous diseases showed an elevation in levels of cCD44std and cICAM-1 compared with normal blood donors, but these levels were not significantly increased. Levels of cv5, cv6, and cv10 were not increased, and cPECAM-1 was only marginally elevated. Even in patients with clinically provable systemic or cutaneous metastases and in five patients who died of MMM, levels did not differ significantly compared with normal blood donors; this was also independent of the mode of therapy. CONCLUSION: Circulating CD44std and the isoforms cv5, cv6, and cv10, cICAM-1, and cPECAM-1 were detectable in persons with dysplastic nevi and in patients with PMM and MMM. None of the measured adhesion molecules was significantly elevated and of prognostic relevance in any of the subgroups studied. However, some of the patients with PMM and MMM showed high levels of cCD44std and cICAM-1; that finding should prompt us to examine these patients in more detail.

Significant elevation of tumour-associated isoforms of soluble CD44 in serum of normal individuals caused by cigarette smoking.

While performing a prospective study on sCD44 variant isoforms as tumour markers in certain malignancies, we detected relevant differences in the control group between non-smokers and smokers. For a detailed evaluation of these findings, serum levels of sCD44 variant proteins, including sequences encoded by exon v5 and exon v6, respectively, were adjusted to sex, age and smoking habit. We were able to demonstrate a significant elevation of serum levels of sCD44v5 and sCD44v6 in normal individuals due to cigarette smoking (non-smokers to smokers: sCD44v5: 33 +/- 11 microg/l to 62 +/- 30 microg/l; sCD44v6: 142 +/- 34 microg/l to 232 +/- 86 microg/l). Stepwise multiple linear regression analysis of the concentrations of sCD44v5 and sCD44v6 on the possible influence factors sex, age and smoking habit revealed cigarette smoking as the only factor influencing these isoforms (both p << 0.001). Further investigations have to elucidate a possible clinical importance of these findings in smokers. However, in patients with suspected or proven malignancy the diagnostic specifity of sCD44v5 and sCD44v6 is diminished due to this observation.

Serum levels of soluble CD44 variant isoforms are elevated in rheumatoid arthritis.

Serum levels of soluble CD44 variant proteins including sequences encoded by exon v5 and exon v6 (sCD44v5, sCD44v6) were determined in patients with inflammatory rheumatic diseases: 56 with rheumatoid arthritis (RA+) and 31 with miscellaneous inflammatory rheumatic diseases (MIRD). There were very significantly higher serum levels of sCD44v5 and sCD44v6 in patients with RA+ than in those with MIRD (RA+ to MIRD: sCD44v5: 81 +/- 54 ng/ml to 33 +/- 13 ng/ml; sCD44v6: 237 +/- 124 ng/ml to 166 +/- 53 ng/ml; both P << 0.001). In RA+ elevated serum levels of sCD44v5 were correlated with the inflammatory activity of disease. In 17 patients with RA+ three or four follow-up measurements of sCD44v5 were performed within 6 months. The development of sCD44v5 serum levels reflected the clinical course of disease in the patients investigated.

Evaluation of a soluble E-selectin enzyme-linked immunosorbent assay under posttraumatic conditions.

The up-regulation of E-selectin, one of the adhesion molecules on the endothelium, is an important event in the mediation of the inflammatory response. Because the presence of E-selectin cannot be determined directly in vivo except by invasive biopsy techniques, the only available information concerning its activity is the serum level of the soluble form. Therefore we tried to measure soluble E-selectin levels in trauma and sepsis situations, where endothelial activation is supposed to occur. We have investigated the soluble E-selectin levels in a group of patients undergoing the trauma associated with cardiac surgery and the use of extracorporeal circulation, some of whom developed a systemic inflammatory response syndrome (SIRS). We have also confirmed that our enzyme-linked immunosorbent assay (ELISA) will detect the levels of soluble E-selectin that are produced as a result of the exposure of cultured human umbilical endothelial cells to even low levels of endotoxin. The data presented in this paper indicate that in patients with SIRS after extracorporeal circulation, the levels of circulating soluble E-selectin are numerically higher but---at least in this group of patients---not statistically significantly different from the levels in patients who have undergone the surgery. These results suggest that the measurement of serum levels of soluble E-selectin is not a reliable method for monitoring the onset of SIRS in patients having undergone surgical trauma, although we have confirmed that our ELISA will detect the levels of soluble E-selectin that are produced as a result of the exposure of cultured human endothelial cells to even low levels of endotoxin.

Soluble adhesion molecules in sera of patients with leprosy: levels of soluble intercellular adhesion molecule-1 (sICAM-1) rapidly decrease during multi-drug therapy.

The clinicopathological spectrum of leprosy is associated with an altered immunological reaction. The expression of adhesion molecules on endothelial cells directs the cellular traffic to sites of local skin and nerve inflammation. Soluble forms of adhesion molecules, which are released upon cytokine activation, can be detected in the circulation and may reflect ongoing tissue inflammation. We determined the serum levels of sICAM-1, sE-selectin and sL-selectin in 74 patients with leprosy (tuberculoid form, n = 23; lepromatous form, n = 36; acute leprous reaction, n = 16) and 15 healthy age- and sex-matched control donors. Patients with lepromatous leprosy had significantly higher levels of sICAM-1 (564 +/- 174 versus 450 +/- 92 versus 334 +/- 57 ng/ml) and E-selectin (90 +/- 31 versus 74 +/- 29 versus 50 +/- 10 ng/ml) than patients with tuberculoid leprosy and normal donors (P < 0.01). No differences between groups were detected for L-selectin. Patients with leprous reactions had similar high levels to lepromatous patients. Twenty lepromatous patients were re-examined after 4 weeks of therapy. A significant decrease in sICAM-1 serum levels was observed after 1 month of anti-mycobacterial treatment, which was accompanied by a reduction of mycobacteria in skin biopsies (P < 0.01). Patients with leprous reactions (n = 13) also demonstrated a drop in sICAM-1 after anti-inflammatory therapy. sE-selectin and sL-selectin serum values decreased only in lepromatous patients after therapy. It can be concluded that soluble adhesion molecules like sICAM-1 and sE-selectin are promising activity markers in patients with leprosy, which may be useful for treatment monitoring.

Levels of circulating intercellular adhesion molecule-1 and -3 but not circulating endothelial leucocyte adhesion molecule are increased in patients with rheumatoid vasculitis.

The aim of this study was to investigate whether levels of circulating adhesion molecules reflect vascular inflammation in rheumatoid vasculitis (RV). Levels of circulating intercellular adhesion molecule-1 (cICAM-1), c-ICAM-3 and circulating endothelial leucocyte adhesion molecule (cE-selectin) were determined in 14 patients with RV and compared to 47 patients with rheumatoid arthritis (RA) and 100 healthy donors (HD). Enzyme-linked immunosorbent assays were used to quantify cICAM-1, cICAM-3 and cE-selectin. We found that in RV significantly (P < 0.0001) elevated levels of cICAM-1 and cICAM3, but not cE-selectin, were found when compared with RA patients. Levels > 2 S.D. above the mean level of HD were present for cICAM-1, cICAM-3 and cE-selectin in 57, 71 and 21%, respectively of patients with RV and 2, 21 and 44%, respectively of the RA patients. Increased levels of both cICAM-1 and cICAM-3 were found in 43% of the RV patients and in none of the RA patients. Comparison of the serum levels of patients studied in an active and inactive phase of RV revealed significantly lower levels of cICAM-3 levels in the inactive phase. In conclusion we find that determination of cICAM-1 and cICAM-3 may be useful as a marker of vascular inflammation in patients with RV.

Circulating forms of ICAM-3 (cICAM-3). Elevated levels in autoimmune diseases and lack of association with cICAM-1.

The intercellular adhesion molecule-3 (ICAM-3) has been identified as the third LFA-1 ligand in addition to ICAM-1 and ICAM-2. In this report we have identified circulating forms of ICAM-3 (cICAM-3) in human serum. Using a sandwich ELISA with two monoclonal anti-ICAM-3 Abs, we detected cICAM-3 in concentrations between 40 and 360 ng/ml in all of 112 healthy controls. An analysis of patient sera from 10 different immune-mediated diseases revealed a distinct pattern of expression. Significantly elevated cICAM-3 levels were found in rheumatoid arthritis, systemic lupus erythematosus, Guillain-Barré syndrome, and multiple sclerosis, but not in type I diabetes, Grave's disease, chronic autoimmune thyroiditis, ulcerative colitis, or Crohn's disease. cICAM-3 levels were significantly higher in systemic lupus erythematosus patients with active compared with nonactive disease. Despite their binding to the same integrin receptor, serum levels of cICAM-3 did not correlate with cICAM-1 concentrations in either normal persons or in patients. The majority of patients had either elevated cICAM-3 or cICAM-1 levels but not both. In conclusion, a circulating form of ICAM-3 is present in human sera. cICAM-3 expression is elevated in certain immune-mediated diseases but occurs independently of cICAM-1.

Serial analysis of circulating adhesion molecules and TNF receptor in serum from patients with multiple sclerosis: cICAM-1 is an indicator for relapse.

We determined the serum levels for circulating adhesion molecules (circulating intercellular adhesion molecule-1 [cICAM-1], circulating endothelial leukocyte adhesion molecule-1 [cELAM-1], and circulating L-selectin [cL-selectin]) and circulating tumor necrosis factor receptor (cTNF-R) p60 in 29 patients with relapsing-remitting MS serially over a period of 12 months. During this period there were 27 relapses in 14 patients (48%). There was progression of disease activity in 12/25 patients (48%), as assessed by the occurrence of new lesions on nonenhancing, T2-weighted MRIs of the head. Clinically active patients with relapse or disease progression on MRI (n = 18) had frequent fluctuations in their serum levels for cICAM-1 if compared to patients with stable MS (n = 11). There were significant differences in the cumulative cICAM-1 production between the two groups (502 +/- 218 ng/ml in active versus 225 +/- 82 ng/ml in stable MS patients; p < 0.001). cTNF-R p60 serum levels were higher in patients with stable compared to active disease (2.3 +/- 0.5 ng/ml versus 1.5 +/- 0.6 ng/ml; p < 0.005). A significant increase in cICAM-1 levels was present at the time of a relapse (799 +/- 263 ng/ml versus 449 +/- 95 ng/ml; p < 0.001), whereas the highest serum levels for cTNF-R p60 occurred 4 weeks after the onset of a relapse (1.8 +/- 0.5 ng/ml at relapse versus 2.3 +/- 0.6 ng/ml 4 weeks after a relapse; p < 0.01). Interestingly, the cL-selectin serum levels in all MS patients were significantly higher than in healthy donors, whereas there were no differences for cELAM-1. These results reflect distinct changes of inflammatory variables in serum of patients with MS and revealed that cICAM-1 is an indicator for disease activity and that high serum levels for cTNF-R p60 are associated with remission.

Scanning mutagenesis of the arginine-rich region of the human immunodeficiency virus type 1 Rev trans activator.

The structural proteins of human immunodeficiency virus type 1, for example, Gag and Env, are encoded by unspliced and incompletely spliced viral transcripts. The expression of these mRNAs in the cytoplasm, along with their commensurate translation, is absolutely dependent on the virally encoded Rev trans activator. Previous studies have demonstrated that Rev binds directly to its substrate mRNAs via an arginine-rich element that also serves as its nuclear localization sequence. In an attempt to define the specific amino acid residues that are important for in vivo activity, we have constructed a series of missense mutations that scan across this region. Our data demonstrate that all eight arginine residues within this element can, individually, be substituted for either leucine or lysine with no apparent loss of function. Importantly, these findings suggest that no single amino acid within the arginine-rich domain of Rev is, by itself, essential for activity and that considerable functional redundancy is therefore likely to exist within this region. Interestingly, one mutant in which a tryptophan had been substituted for a serine failed to accumulate exclusively in the nucleus but still bound RNA in a manner that was indistinguishable from that of the wild-type protein. This observation indicates that features of the arginine-rich region that are additional to those required for RNA binding are important for Rev's correct accumulation in the nucleus.

Amniotic fluid interleukin-8 as a marker for intraamniotic infection.

We measured the amniotic fluid Interleukin-8 (AF IL-8) levels of 80 women to see whether or not AF IL-8 levels were of value in the diagnosis of intraamniotic infection. Of twelve patients developing conventional signs of infection, 9 had an AF IL-8 concentration above 10,000 pg/ml serum. In two patients, whose baby had a serious neonatal infection, AF IL-8 concentration also exceeded 10,000 pg/ml. Only one out of 66 apparently uninfected patients had an AF IL-8 level above 10,000 pg/ml. We therefore suggest that measuring the AF IL-8 levels is of value in cases of suspected intraamniotic infection.

Dominant-negative mutants are clustered in a domain of the human T-cell leukemia virus type I Rex protein: implications for trans dominance.

The 27-kDa Rex trans-acting protein appears to be essential for replication of human T-cell leukemia virus type I. Mutations introduced outside of the Rex RNA-binding domain-nucleolar localization signal display either wild-type activity or, conversely, yield dominant-negative proteins. We generated missense mutations in a particular domain of the Rex protein (amino acid residues 54 to 69) which is characterized by a cluster of dominant-negative mutants. Our results indicate that amino acids 57 to 67 are critically important for Rex function mediated through the RxRE cis-acting RNA sequence. Within this domain, only amino acids 61 to 63 could be mutated without loss of function. All other missense and deletion mutants yielded dominant-negative proteins. In vitro RNA-binding studies performed with glutathione S-transferase-Rex fusion proteins demonstrated that all of the mutant Rex proteins interacted specifically with RxRE RNA. Analysis of chimeric Rex-Rev proteins suggests that this Rex domain is important for oligomerization.

Definition of the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex protein activation domain by functional exchange.

The human retroviruses human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) are characterized by complex regulation of gene expression. Each virus encodes a posttranscriptional regulator, the 19-kDa HIV-1 Rev protein and the 27-kDa HTLV-I Rex protein, which is required for viral replication. Expression of these trans activators results in the cytoplasmic accumulation of unspliced or singly spliced viral mRNA which encode the gag, pol, and env gene products. The finding that the HTLV-I Rex protein is able to functionally substitute for the Rev protein of HIV-1 indicates that HIV-1 Rev and HTLV-I Rex may interact with the same component of a cellular pathway involved in either mRNA splicing or transport. In this study, we have generated functional Rev/Rex hybrid proteins by domain exchange. We have defined, using in vivo and in vitro analyses, the activation domains of Rev and Rex which are the putative targets of a common host cell factor(s) required for Rev and Rex function.

Mutational analysis of the human T-cell leukemia virus type I trans-acting rex gene product.

Expression of the human T-cell leukemia virus type I (HTLV-I) rex gene is a prerequisite for the expression of the retroviral structural proteins. We have generated internal deletion mutants of this 27-kDa nucleolar trans-acting gene product to define functional domains in the Rex protein. The phenotype of the various mutant proteins was tested on the homologous HTLV-I rex response element sequence and the heterologous human immunodeficiency virus type 1 (HIV-1) rev response element sequence. Our results indicate that a region between amino acid residues 55 and 132 in the 189-amino-acid Rex protein is required for Rex-mediated trans activation on both retroviral response element sequences. In addition, substitution of the Rex nuclear localization signal by a sequence of the HIV-1 rev gene product targets the Rex protein to the correct subcellular compartment required for Rex function.

Bidirectional promoter activity of the 5' flanking region of the mouse thymidine kinase gene.

The 5' flanking region of the gene coding for cytoplasmic thymidine kinase (TK) in the mouse (a total of 490 bp upstream of the initiation codon) was tested for promoter activity using the chloramphenicol acetyltransferase gene as reporter. It was found that the region can be divided into two parts, one of which carries promoter activity in the direction of TK, whereas the 5'-half has promoter activity in the opposite direction. A fragment of 140 bp was sufficient for growth-dependent promoter activity in the direction of TK, although about 100 bp further upstream, enhanced the activity. Expression from the divergent promoter was independent of cell growth.

Optimal replication of plasmids carrying polyomavirus origin regions requires two high-affinity binding sites for large T antigen.

The efficiency of replication of plasmids containing the control region of polyomavirus DNA including one, two, or all three of the strong binding sites for large T antigen was measured in COP 8 cells which provide polyomavirus T antigen in trans. It was found that plasmids carrying only binding site A (the one closest to the origin core region) exhibited only 10% of the replication competence of plasmids with binding sites A and B or A and C. Plasmids containing all three binding sites, A, B, and C, did not replicate more efficiently than those with only two strong T-antigen-binding sites. We conclude, therefore, that optimal T-antigen-dependent replication of polyomavirus DNA requires two high-affinity T-antigen-binding sites.