Ann Erlandsson

Epitope Specificity of 30 Monoclonal Antibodies against Cytokeratin Antigens: The ISOBM TD5-1 Workshop

The epitope specificities of 30 monoclonal antibodies (MAbs) against the most common human cytokeratins, i.e., Nos. 8, 18, and 19, in epithelial cells were investigated in the ISOBM TD-5 Workshop. Seven research groups from universities or companies participated independently in the evaluation of the antibody specificities. The complex assembly of cytokeratins in vivo, with obligatory heterologous dimeric combinations of different cytokeratins from each of the two major groups, comprising together more than 20 different individual cytokeratins, made analysis of the antibody reactivity patterns with isolated single cytokeratins necessary. The concordance of the evaluations was striking and independent of the technologies used. As antigens purified individual cytokeratins, chemically degraded purified cytokeratins, recombinant intact and truncated cytokeratins, as well as specific synthesized shorter peptides were used. In order to elucidate the epitope specificity, reactivity patterns in ELISA assays and immunoblots with partial enzymatic degradation of the antigens were performed. Competitive cross-inhibition experiments between antibodies using antigens and antibodies in all possible combinations were performed with radioimmunometric assays, BIAcore, and ELISA technology.All 30 antibodies could convincingly be classified with regard to target cytokeratin. One MAb (192) had to be deleted due to dual specificities in both isotype and epitope specificity against its target. Six antibodies bound selectively to cytokeratin 8, 14 to cytokeratin 18, and 10 to cytokeratin 19, as demonstrated by using native, recombinant, and synthesized antigens. The immunodominant part of the molecule for all three types of cytokeratins was located in the region of amino acid (aa) 270–400. Out of the six MAbs reactive with cytokeratin 8, four MAbs, i.e., 178, 199, 202, and 206, were reactive with a sequence in the interval aa 340–365, and MAb 191 reacted with a closely related epitope. The remaining antibody, 192, presented dual specificities. At least two closely related major immunogenic epitopes could be identified in cytokeratin 8. In cytokeratin 18 four distinct epitopes could be documented, again with the dominating sequence region 270–429 as target for 10 (181, 184, 186, 188, 189, 190, 193, 196, 198, and 200) out of 14 antibodies. Since MAb 193 is known to react with the M3 epitope, aa 322–342 in cytokeratin 18, this entire group is reactive in the region close to the charge shift, in the middle of the rod 2B region, as shown by competitive binding. The remaining four anticytokeratin 18 antibodies (180, 185, 203, and 205) displayed unique, noncompetitive binding to this filament. Cytokeratin 19, reactive with altogether ten antibodies, displayed two major epitopes, all of them also within the large immunodominant region. MAbs 179, 195, 197, and 204 were reactive with the peptides aa 311–335 also known as the KS 19.1 epitope, and MAbs 182, 183, 187, 194, and 201 bound to peptide aa 346–367, known as the BM 19.21 epitope. One antibody, 231, was selectively reactive with aa 356–370 in cytokeratin 19. A complex pattern of binding specificities comprising at least ten different, noncompetitive epitopes, mainly situated in the rod portion, 2A and 2B, situated close to the charge shift in the rod of all three cytokeratins was documented. Out of the 29 classifiable antibodies, altogether 22 were reactive in this very short region, i.e., from aa 311 to 370 in all cytokeratin filaments. The remaining seven antibodies displayed unique binding properties. The implications of the findings are of significance both for immunohistochemistry and for assaying circulating heterodimeric, partially degraded complexes in patients’ blood for tumor marker evaluation.

Conditioned media from macrophages of M1, but not M2 phenotype, inhibit the proliferation of the colon cancer cell lines HT-29 and CACO-2

Solid tumors are infiltrated by stroma cells including macrophages and these cells can affect tumor growth, metastasis and angiogenesis. We have investigated the effects of conditioned media (CM) from different macrophages on the proliferation of the colon cancer cell lines HT-29 and CACO-2. CM from THP-1 macrophages and monocyte-derived human macrophages of the M1 phenotype, but not the M2 phenotype, inhibited proliferation of the tumor cells in a dose-dependent manner. Lipopolysaccaharide and interferon γ was used for differentiation of macrophages towards the M1 phenotype and CM were generated both during differentiation (M1DIFF) and after differentiation (M1). M1 and M1DIFF CM as well as THP-1 macrophage CM resulted in cell cycle arrest in HT-29 cells with a decrease of cells in S phase and an increase in G2/M phase. Treatment of HT-29 cells with M1DIFF, but not M1 or THP-1 macrophage CM, resulted in apoptosis of about 20% of the tumor cells and this was accompanied by lack of recovery of cell growth after removal of CM and subsequent culture in fresh media. A protein array was used to identify cytokines released from M1 and M2 macrophages. Among the cytokines released by M1 macrophages, tumor necrosis factor α and CXCL9 were tested by direct addition to HT-29 cells, but neither affected proliferation. Our results indicate that M1 macrophages inhibit colon cancer cell growth and have the potential of contributing to reducing tumor growth in vivo.

PCR Assay or Culture for Diagnosis of Bordetella pertussis in the Routine Diagnostic Laboratory

A nested PCR method was compared with culture for the detection of Bordetella pertussis in a routine clinical diagnostic laboratory. A total of 241 clinical nasopharyngeal aspirates were examined in parallel in the laboratory. Both methods were positive for 75 samples (31%), eight samples were positive by nested PCR only (3.3%), and one sample was positive by culture only (0.4%). The mean time actually required in the clinical laboratory (not operating with pertussis diagnosis during weekends) from the day of arrival to the diagnosis of a positive or negative sample by the nested PCR assay was 1.8 +/- 1.3 days (mean +/- SD), for positive culture 4.5 +/- 1.4 days and for negative culture 10.5 +/- 1.0 days. The hands-on time in the laboratory to perform the nested PCR was 2 h, for a positive culture 25 min, and for a negative culture 15 min. The cost analysis of the methods, when running one sample at a time, showed that the laboratory cost for PCR was six times higher than culture. When running four samples together the cost for PCR was three times higher than culture. In conclusion, the nested PCR is the more rapid and sensitive method compared to culture. With the present design, the PCR-protocol involves higher material expenditure and claims more hands-on time.

Immunohistochemical profiles of 30 monoclonal antibodies against cytokeratins 8, 18 and 19. Second report of the TD5 workshop.

In the first report of the TD5 workshop (TD5-1), the epitope specificities of 30 different monoclonal antibodies against cytokeratins 8, 18 and 19 were determined. This second report presents the immunohistochemical profiles of these antibodies using human appendix and normal skin for evaluation. Each antibody was tested by one or two different laboratories recruited from the Dutch Working Group on Immunohistochemistry and Cytochemistry. Eight different laboratories participated. The histological specimens were pretreated by the participants in three different ways for immunohistochemistry: microwave antigen retrieval in citrate buffer, enzymatic digestion to restore epitope exposure, no specific treatment (untreated paraffin-embedded samples), and tested blindly without knowledge of cytokeratin or epitope specificity of the antibodies at three different concentrations of 50, 10 and 1 μg/ml. Most of the tested antibodies (29/30) were useful in at least one pretreatment method, with microwave antigen retrieval being the most sensitive approach. For some antibodies, very high backgrounds were observed. Furthermore, it can be concluded that 11 MAbs performed well using all three staining protocols, including untreated paraffin-embedded sections. Interestingly, all the antibodies with documented selected specificity towards cytokeratin 8 (i.e. 178, 191, 199, 202 and 206) are reactive with an immunodominant region corresponding to amino acids 340–365 on cytokeratin 8, which evidently is well-suited as target for immunohistochemical interactions. Similarly, three antibodies with the same capacity to react with untreated samples had specificity against cytokeratin 19 (i.e. 179, 197 and 204) in the corresponding region in this filament, i.e. amino acids 311–335, or the KS 19.1 epitope. None of the six antibodies against the other major cytokeratin 19 epitope (BM 19.21) were found useful for immunohistochemistry on untreated samples. The overall conclusions from the present investigation are that all cytokeratin-8-specific antibodies with defined epitope specificities were very useful. Only one of the major two epitopes on cytokeratin 19 seems to be available for efficient immunohistochemistry. Cytokeratin 18 exposes some epitopes outside the immunodominant region reactive with the antibodies 190, 203 and 205 which can be used for untreated samples. The implications of these findings are of significance both for diagnostic histopathology and for the biology of tumor marker epitope expression in tissues.

Quantification of Bordetella pertussis in clinical samples by colorimetric detection of competitive PCR products

Quantification of microorganisms is an important part of the normal diagnostic work of a clinical microbiology laboratory. Traditionally the diagnosis of pertussis is subject to a yes or no approach with no quantitative dimension. This can, however, be of interest as a factor when judging the risk of a patient spreading the bacterium and as a research tool. The aim of the present study was to develop a PCR‐based quantitative assay for Bordetella pertussis DNA in clinical nasopharyngeal aspirates by combining a quantitative PCR with a colorimetric detection principle, DIANA (detection of immobilised amplified nucleic acid). A competitor to the PCR target sequence in IS‐481, containing a lac‐operator, was constructed and calibrated, and a test protocol prepared. A total of 46 clinical nasopharyngeal aspirates, previously diagnosed using a standard nested PCR assay and quantified by culture, were analysed by the quantitative PCR. The method showed acceptable precision and accuracy considering that it estimates the total number of bacterial genomes while culture detects viable bacteria. Recognised advantages were the simple colorimetric detection, the inborn indication of a working PCR assay, and the possibility of obtaining results even when partial inhibition of the PCR assay was seen. In addition, the quantitative PCR result can be obtained within one day compared to 3–10 days for culture. The present results and the qualities of the quantitative PCR suggest that this assay will be a useful complement in routine diagnostics and in research.

Effect of macrophages on breast cancer cell proliferation, and on expression of hormone receptors, uPAR and HER-2

Malignant tumors, including breast cancers, are frequently infiltrated with innate immune cells and tumor-associated macrophages (TAMs) represent the major inflammatory component in stroma of many tumors. In this study, we examined the immunoreactivity of the macrophage markers CD68 and CD163 as well as the hormone receptors estrogen receptor α (ERα), progesterone receptor (PR), estrogen receptor β1 (ERβ1), human epidermal growth factor receptor 2 (HER-2), matrix metalloproteinase 9 (MMP-9), urokinase-type plasminogen activator receptor (uPAR) and the proliferations marker Ki67 in 17 breast cancer biopsies. The quantitative score for CD68+ and CD163+ strongly indicate M2 phenotype dominance in the currently investigated biopsies. We found that an increasing level of macrophages was negatively associated with ERα or PR, whereas a positive association was observed for Ki-67 or uPAR. No significant association could be seen between the level of macrophage and HER-2, ERβ1 or MMP-9 expression. Effect of conditioned media (CM) generated from cultured human M1 and M2 macrophage phenotypes were investigated on the proliferation and expression of selected markers in the T47D breast cancer cell line. We found that in contrast to the in vivo situation, in particularly the CM from M1 macrophages decreased the growth and Ki67 expression in T47D, and significantly increased ERβ1 mRNA levels. Moreover, in accordance to the in vivo situation the CM from the macrophages decreased the expression of ERα protein as well as ERα or PR mRNA. In conclusion our results show that macrophages alone have the capability to decrease the tumor cell expression of ERα and PR in vitro. In the tumor environment in vivo macrophages also contribute to an increase in tumor cell expression of uPAR and Ki67, suggesting that macrophages are involved in impairing the prognosis for breast cancer patients.

Macrophages of M1 phenotype have properties that influence lung cancer cell progression

Stromal macrophages of different phenotypes can contribute to the expression of proteins that affects metastasis such as urokinase-type plasminogen activator (uPA), its receptor uPAR, and plasminogen activator inhibitor-1 (PAI-1), but knowledge of how essential their contribution is in comparison to the cancer cells in small cell lung cancer (SCLC) and lung squamous cell carcinoma (SCC) is lacking. The expression of uPA, uPAR, and PAI-1 and of the matrix metalloproteinases (MMP)-2 and MMP-9 were studied in human macrophages of M1 and M2 phenotype and compared to a lung SCC (NCI-H520) and a SCLC (NCI-H69) cell line. Effects of treatment with conditioned media (CM) from M1 and M2 macrophages on the expression of these genes in H520 and H69 cells as well as effects on the cell growth were investigated. In addition, data on the stromal macrophages immunoreactivity of uPAR, MMP-2, and MMP-9 in a few SCC and SCLC biopsies was included. uPAR, MMP-2, and MMP-9 were confirmed in stromal cells including macrophages in the SCC and SCLC biopsies. In vitro, both macrophage phenotypes expressed considerably higher mRNA levels of uPA, uPAR, PAI-1, and MMP-9 compared to the cancer cell lines, and regarding uPAR, the highest level was found in the M1 macrophage phenotype. Furthermore, M1 CM treatment not only induced an upregulation of PAI-1 in both H520 and H69 cells but also inhibited cell growth in both cell lines, giving M1 macrophages both tumor-promoting and tumor-killing potential.

Conditioned media from human macrophages of M1 phenotype attenuate the cytotoxic effect of 5‑fluorouracil on the HT‑29 colon cancer cell line.

Resistance of tumor cells to chemotherapy, such as 5-fluorouracil (5-FU), is an obstacle for successful treatment of cancer. As a follow-up of a previous study we have investigated the effect of conditioned media (CM) from macrophages of M1 or M2 phenotypes on 5-FU cytotoxicity on the colon cancer cell lines HT-29 and CACO-2. HT-29 cells, but not CACO-2 cells, having been treated with a combination of M1 CM and 5-FU recovered their cell growth to a much larger extent compared to cells having been treated with 5-FU alone when further cultured for 7 days in fresh media. M1 CM treatment of HT-29, but not CACO-2 cells, induced cell cycle arrest in the G0/G1 and G2/M phases. 5-FU treatment induced accumulation of cells in S-phase in both HT-29 and CACO-2 cells. This accumulation of cells in S-phase was attenuated by combined M1 CM and 5-FU treatment in HT-29 cells, but not in CACO-2 cells. The mRNA expression of cell cycle regulatory proteins and 5-FU metabolic enzymes were analyzed in an attempt to find possible mechanisms for the M1 CM induced attenuation of 5-FU cytotoxicity in HT-29. Thymidylate synthetase (TS) and thymidine phosphorylase (TP) were found to be substantially downregulated and upregulated, respectively, in HT-29 cells treated with M1 CM, making them unlikely as mediators of reduced 5-FU cytotoxicity. Among cell cycle regulating proteins, p21 was induced in HT-29 cells, but not in CACO-2 cells, in response to M1 CM treatment. However, small interfering RNA (siRNA) knockdown of p21 had no effect on the M1 CM induced cell cycle arrest seen in HT-29 and neither did it change the growth recovery after combined treatment of HT-29 cells with M1 CM and 5-FU. In conclusion, treatment of HT-29 cells with M1 CM reduces the cytotoxic effect of 5-FU and this is mediated by a M1 CM induced cell cycle arrest in the G0/G1 and G2/M phases. So far, we lack an explanation why this action is absent in the CACO-2 cells. The current findings may be important for optimization of chemotherapy in colon cancer.

Binding of TS1, an anti-keratin 8 antibody, in small-cell lung cancer after 177Lu-DOTA-Tyr3-octreotate treatment: a histological study in xenografted mice

BackgroundSmall-cell lung carcinoma (SCLC) is an aggressive malignancy characterised by an early relapse, a tendency towards drug resistance, and a high incidence of metastasis. SCLC cells are of neuroendocrine origin and express high levels of somatostatin receptors; therefore, future treatment might involve targeting tumours with radiolabelled somatostatin analogues. This therapy induces abundant necrotic patches that contain exposed keratins; thus, keratin 8, which is one of the most abundant cytoskeletal proteins may represent an interesting secondary target for SCLC. This study aimed to investigate the effects of177Lu-DOTA-Tyr3-octerotate and the binding of the monoclonal anti-keratin 8 antibody, TS1, in vitro in treated SCLC- and midgut-xenografted mouse models.MethodsNCI-H69- and GOT1-xenotransplanted mice were treated with three doses of 30 MBq177Lu-DOTA-Tyr3-octreotate administered 24 h apart. Mice xenotransplanted with NCI-H69 were sacrificed 1, 5, 12, 20 and 150 days post-injection or when the tumour had regrown to its original size. GOT1-xenotransplanted mice were sacrificed 3 days post-injection. Immunohistochemistry was performed to evaluate TS1 staining in tumours and in seven human biopsies of primary SCLC from pulmonary bronchi. Central cell density and nucleus size were determined in NCI-H69 sections.ResultsTwelve days after177Lu-DOTA-Tyr3-octerotate treatment, the SCLC xenograft response was extensive. Twenty days after treatment, one of three analysed tumours displayed complete remission. The other two tumours showed 1/4 the cell density of untreated controls and cell nuclei were about three times larger than those of untreated controls. At 150 days after treatment, one of four mice exhibited complete remission. Treated tumours displayed increased TS1 antibody accumulation and high TS1 binding in necrotic patches. All seven human SCLC biopsies displayed necrotic areas with TS1 staining.ConclusionsRadiation treatment with three injections of 30 MBq177Lu-DOTA-Tyr3-octreotate had pronounced effects on tumour cell density and cell nuclei, which indicated mitotic catastrophe. Despite these anti-tumour effects, two of three SCLC tumours recurred. Further studies should investigate the nature of tumour cell survival and develop more effective treatments. High TS1 accumulation in tumour sections in vitro after177Lu-DOTA-Tyr3-octerotate treatment indicated that TS1 might represent a promising secondary therapeutic strategy.

Stability and Immunoreactivity of the Monoclonal Anticytokeratin Antibody TS1 after Different Degrees of Iodination

The immunoreactivity, stability and in vivo kinetics of an anticytokeratin 8 monoclonal antibody, TS1, were investigated following different degrees of labeling with 125I (0.2, 1 and 2-3 125I/TS1 MAb). By testing with ELISA, it was demonstrated that a high degree of iodination, i.e. > 2 125I/TS1, caused a rapid decrease in immunoreactivity to almost zero within 10 days. Furthermore, a complete degradation to low molecular weight fragments and free iodine was seen, as shown by SDS PAGE and autoradiography. The differently labeled radionuclide conjugates were injected into nude mice inoculated with HeLa Hep2 cells and tumor doses (estimated by MIRD formalism), tumor:non-tumor dose ratios, % I.D./gram tissue, Gy/MBq and in vivo kinetics of the differently labeled MAbs were determined. Despite the in vitro instability of the highest iodinated radionuclide conjugate, it was possible to deliver high doses to the tumors if the conjugate was injected into the animal immediately after completion of the iodination procedure. Increases from 1.4 Gy to 15.2 Gy delivered tumor dose were obtained with a tenfold increase in the specific activity, without alterations in the tumor:non-tumor tissue dose ratios. There is room for significant improvements in efficacy at radioimmunotherapy, which can be gained by optimizing the degree of iodination. For therapeutical applications a high degree of iodination may be an advantage.