[Efficacy of zoledronate in treating persisting isolated tumor cells in bone marrow in patients with breast cancer. A phase II pilot study]. / Zoledronsäure bei persistierenden isolierten Tumorzellen im Knochenmark bei Mammakarzinom. Phase-II-Pilotstudie zur Evaluierung der therapeutischen Effektivität.

BACKGROUND AND OBJECTIVES: Recently the highest level of evidence indicated the prognostic value of isolated tumor cells (ITC) in bone marrow of patients with breast cancer, both at primary diagnosis and during recurrence-free follow-up. The aim of the present study was to investigate the therapeutic efficacy of zoledronate in reducing persistence of ITC in the bone marrow of patients with breast cancer after they had completed primary therapy. PATIENTS AND METHODS: In a non-randomized phase II pilot study, 4 mg of zoledronate were administered once every four weeks for six months, after a initial loading dose of 8 mg, to 31 patients with persisting ITC in bone marrow. All patients had completed surgery and adjuvant chemotherapy, if indicated, at least 6 months previously. The bone marrow was re-examined after 7.9 months (std 0,89). ITC were detected by immunocytochemical staining, using the monoclonal pan-cytokeratin antibody A45-B/B3 and the APAAP technique. Patients were followed-up prospectively for a median of 39 months after the first aspiration. RESULTS: ITC were detected in all 31 patients at the time of first bone marrow aspiration, but 27 of them (87 %) were free of ITC 6 months after the end of zoledronate therapy. The reduction in cell numbers between first and second aspiration were statistically significance (P < 0,0001). Ten of 12 patients without detection of ITC in bone marrow after treatment and who had undergone additional aspirations, still had no ITC a median time of 19 months (range 4.7 - 38.7 months) after the end of treatment. Zoledronate treatment was well tolerated, bone pain having been the most common side effect in 45 % of patients (n = 14). CONCLUSION: These results indicate a potential antineoplastic effect of zoledronate, a cell-cycle independent drug, on persisting ITC in a dormant state. Our data provide the basis for investigating the efficacy of zoledronate on ITC in treating primary breast cancer in prospectively randomized trials. ITC in bone marrow represents a useful marker in selecting patients at risk for recurrence and for monitoring therapeutic efficacy.

The sectional anatomy of the carpal tunnel and its related neurovascular structures studied by using plastination.

The aim of this study was to evaluate the morphology of the carpal tunnel and its related neurovascular structures. A slice anatomy study was performed on 12 right wrists of unfixed human cadavers by using the plastination technique. The measurements were performed at the level of the pisiform, hook of the hamate and in the middle between these structures. The diameters of the carpal tunnel and the median nerve were measured at the level of the hook of the hamate. The median nerve can be predicted to be 18 +/- 1.6 mm radial to the pisiform and the ulnar neurovascular bundle 6.8 +/- 1.4 mm radial to the pisiform. Between those structures there will be at least a 9-mm area, localized 8 mm radial to the pisiform, where the incision of the transverse carpal ligament could be performed risk-free. At the hamate hook the median nerve can be predicted at 9.24 +/- 1.18 mm and the ulnar artery lies usually 1.26 +/- 2.5 mm radial to the hook. An understanding of the contents and their positions, and relationships to each other allows an accurate identification of neurovascular structures in the carpal tunnel. Our findings can be used as anatomic landmarks of the carpal tunnel and could be helpful to physicians performing carpal tunnel investigations.

Potential of P40 plastination for morphometric hip measurements.

Total hip replacement has become one of the most successful surgical operations over the past 25 years. The duration of a total hip prosthesis depends on primary stability, and many studies have tried precisely to evaluate hip joint morphology to obtain excellent contact between bone and prosthetic component. This study performed a morphometric analysis of the human hip joint using, for the first time, the P40 plastination procedure. We cut 42 hip joint compounds into slices 3 mm thick; for exact distance measuring the sections were scanned into the computer. The following mean measurements for hip geometry were obtained: vertical diameter of acetabulum 4.894+/-0.274 cm, depth of acetabulum 1.643+/-0.245 cm, femoral head radius 2.268+/-0.149 cm, femoral neck length 4.3670+/-0.528 cm, acetabular perimeter 6.711+/-0.434 cm, vertical diameter of labrum acetabulare 4.759+/-0.476 cm, depth of labrum acetabulare 2.599+/-0.395 cm, sum of femoral head and neck lengths 6.759+/-0.550 cm, hip axis length 11.859+/-1.007 cm, femoral neck axis length 10.12+/-0.555 cm, and femoral neck diameter 3.349+/-0.276 cm. All of these data reveal a significant gender difference. Our aim was to indicate an unconventional and new method of gaining morphometric hip data by using plastination.

Impact of medical and demographic factors on long-term quality of life and body image of breast cancer patients.

BACKGROUND: The impact of various medical and demographic factors on the quality of life (QoL) of breast cancer patients has been discussed controversially. We investigated the influence of six different factors on long-term QoL and body image of women with primary breast cancer. PATIENTS AND METHODS: Two-hundred and seventy-four breast cancer patients were administered the QoL questionnaire following a mean interval of 4.2 years after primary diagnosis. All women had been primarily treated for stage I to III breast cancer without evidence of distant metastases. QoL was evaluated by using the QLQ-C30 questionnaire Version 2.0. Supplementary scales included body image, satisfaction with surgical treatment, cosmetic result and fear of recurrence. We analyzed the impact of tumor stage, surgical treatment, adjuvant radiotherapy, adjuvant cytotoxic therapy, age and length of follow-up period on the examined outcome parameters. RESULTS: At the time of the follow-up examination, patients showed minor impairment of QoL (mean 67.8) and body image (mean 24.8), but more fear of recurrence (mean 60.7). None of the studied factors had a significant impact on overall QoL (P >0.05) according to the QLQ-C30 questionnaire. In contrast, with the exception of the factors 'cytotoxic therapy' and 'radiotherapy' all investigated variables influenced at least one of the additional psychological scales (P <0.05). The primary surgical treatment modality had the strongest impact and affected all four scales. Patients treated with breast conservation reported a more favorable body image, compared to those treated with mastectomy (17.2 versus 37.5, P <0.01), more satisfaction with surgical treatment (4.0 versus 10.7, P = 0.01), rated a better cosmetic result (75.5 versus 57.1, P <0.01), but presented more fear of recurrence (63.9 versus 55.3, P = 0.04). CONCLUSION: Current QoL questionnaires do not sufficiently cover all relevant aspects of QoL, but might be complemented by breast cancer specific aspects such as body image and fear.

[Progress in the early diagnosis of breast carcinoma during the years 1981-1990. Results of a longitudinal study]. / Fortschritte in der Früherkennung des Mammakarzinoms in den Jahren 1981-1990. Ergebnisse einer Longitudinalstudie.

BACKGROUND AND OBJECTIVE: Current meta-analyses have left in doubt whether general breast screening increases survival rate. This study investigated whether efforts at early diagnosis of cancer in the 1980s have had an effect on average tumor size at first diagnosis and on survival rate. PATIENTS AND METHODS: From 1981 to 1990, 1656 consecutive patients (average age 56.6 years) at the I. Women's Clinic at the Ludwig-Maximilian University of Munich and the Women's Clinic Berlin-Charlottenburg were operated on for primary breast cancer. In a retrospective analysis, average tumor size at the primary operation and survival rate were determined for two periods: 1981-1985 (n=849) and 1986-1990. Mean follow-up time was 63 months. RESULTS: There was no difference between the two cohorts regarding age (p = 0.77) and axillary node status (p = 0.14). During the follow-up period there was a gradual decrease in the tumor size at first diagnosis. (Pearson's correlation coefficient: -0.79, p < 0.001). Average tumor size in those operated on was 25 mm up to 1985, and 21 mm after 1986 (p < 0.001). Until 1985, the initial reason for mammography was the planned subsequent operation in 19% of patients (n = 164), and in 27% (n = 215; p < 0.001) since 1986. But there was no statistically significant rise in disease-specific survival rate (log rank, p=0.48). Multivariate analysis confirmed the conventional prognostic parameters, such as tumor size (relative risk 2.21) and axillary lymph node metastases (relative risk 3.57), but not the period of follow-up (p=0.90). CONCLUSION: During the stated periods of follow-up there was a significant decrease in average tumor size at initial diagnosis. But this did not result in any demonstrably better disease-specific survival rate.

Optic nerve compression analyzed by using plastination.

Plastination is an excellent tool for studying different anatomical and clinical questions. The E12 technique provides transparent slices which show the anatomical structures in their initial position, especially those of the muscular, vascular and interstitial tissue. Here we demonstrate on a plastinated specimen optic nerve compression due to hypertrophy and degeneration of the extraocular muscles near the apex of the orbit. The paper aims to demonstrate the possibilities offered by the E12 plastination technique in analyzing anatomical structures. The plastinated slices obtained were scanned and then analyzed. In a further step the plastinated slices were cut into thin slices (150 microm), stained and finally examined histologically. We measured the intraorbital length (A-B), which had an average value of 25,93+/-0,03 mm. Dividing the optic nerve into uncompressed (UP) and compressed (CP) parts, we obtained the following values for optic nerve width: UP=4192+/-0,455 mm, CP=3215+/- 0,411 mm. Using the E12 plastination method we were able to take morphometric measurements on plastinated orbital slices with coincidentally detected optic nerve compression. This allowed the determination of different parameters of the optic nerve, as well as histological examination by hematoxylin and eosin staining up to a magnification of x40.

[The influence of the hemoglobin concentration on the hematogenous tumor cell dissemination at the time of the initial diagnosis of breast cancer]. / Der Einfluss des Hämoglobinwertes auf die hämatogene Tumorzelldisseminierung zum Zeitpunkt der Primärdiagnose des Mammakarzinoms.

BACKGROUND AND OBJECTIVE: In the last few years special attention has been paid to the serum concentration of haemoglobin in patients with cancer. It was the aim of this study to ascertain whether at the time of the initial diagnosis low haemoglobin levels in patients with breast cancer denote a higher risk of primary haematogenous dissemination of tumour cells in bone marrow than that in those with higher levels. PATIENTS AND METHODS: Between March 1994 and March 2000 bone marrow aspirates were performed and serum haemoglobin concentrations (g/dl) measured before primary surgical treatment in 360 consecutive patients (mean age 57.5 years) with primary breast cancer. Evidence of isolated tumour cells in bone marrow was obtained with the pancytokeratin antibody A45-B/B3. The cohort was divided into two groups on the basis of mean haemoglobin values, and the patients underwent follow-up examination a mean of 30.7 months after the initial diagnosis. Patients with metastases at first diagnosis or those who had received nonsurgical treatment at that time were excluded. RESULTS: The mean pre-treatment haemoglobin concentration of the cohort was 13.8 g/dl (median 13.9 g/dl, S.D.1.2). There was no statistically significant difference in the frequency of cytokeratin-positive bone marrow findings between the two groups. While disseminated tumour cells were demonstrated in the bone marrow of 48 (28%) patients with a pre-treatment haemoglobin of 13.9 g/dl. There was also no difference between the two groups regarding median survival time (67.9 vs. 65.8 months; p = 0.46). However, there was a significant difference in probability of survival between patients with or without isolated tumour cells in the bone-marrow (59.7 vs. 69.2 months; p < 0.0001). CONCLUSION: There is no evidence at present that the preoperative haemoglobin concentration is of prognostic value regarding the haematogenous dissemination of tumour cells and the survival time of patients with primary breast cancer.

[Incidence and prognostic significance of disseminated tumor cells in patients with cervical cancer]. / Inzidenz und prognostische Signifikanz disseminierter Tumorzellen bei Patientinnen mit Zervixkarzinom.

The clinical course of cervical carcinoma is widely determined by locoregional recurrence. There is increasing data, however, that haematogenic micrometastases occur early during the disease and might result in distant recurrence during follow-up. These occult disseminated tumor cells in blood, lymph nodes and bone marrow escape conventional tumor staging. Therefore, molecular and immunoytochemical techniques based on markers against human papilloma virus or cytokeratins (CK) have been applied. At present, there is only one study available on the prognostic relevance of disseminated tumor cells in bone marrow. No correlation between the bone marrow status and overall survival was observed. Still, there was a strong trend towards shorter distant disease free survival in patients with a positive bone marrow status. In view of the data on disseminated tumor cells in other tumor entities, these early results might offer new options for refined tumor staging and improved treatment options.

A completely foreign receptor can mediate an interferon-gamma-like response.

A tripartite receptor comprising the external region of the erythropoietin (Epo) receptor, the transmembrane and JAK-binding domains of the gp130 subunit of the interleukin-6 (IL-6) receptor, and a seven amino acid STAT1 recruitment motif (Y440) from the interferon (IFN)-gamma receptor, efficiently mediates an IFN-gamma-like response. An analogous completely foreign chimeric receptor in which the Y440 motif is replaced with the Y905 motif from gp130 also mediates an IFN-gamma-like response, but less efficiently. The IFNGR1 signal-transducing subunit of the IFN-gamma receptor is tyrosine phosphorylated through the chimeric receptors and the endogenous IL-6 and OSM receptors. Cross phosphorylation of IFNGR1 is not, however, required for the IFN-gamma-like response through the chimeric receptors, nor does it mediate an IFN-gamma-like response to IL-6 or OSM. The data argue strongly for modular JAK/STAT signalling and against any rigid structural organization for the "pathways" involved. They emphasize the likely high degree of overlap between the signals generated from disparate JAK-receptor complexes and show that relatively minor changes in such complexes can profoundly affect the response.

Expression of cytokeratin-18-related tissue polypeptide-specific (TPS) antigen in Wilms tumor.

BACKGROUND: So far, there is no approved tumour marker for diagnosis or follow-up in Wilms tumour (WT). Tissue polypeptide-specific antigen (TPS), a cytokeratin 18 proteolytic fragment, has been suggested to be of value in the clinical management of WT patients. Cytokeratin 18 fragments are an early indicator of apoptosis and cytokeratin 18 might influence tumour cell behaviour. We investigated TPS expression in specimens of WT and other paediatric renal malignancies PROCEDURE: Immunoreactivity of WT sections (n = 9), clear cell sarcomas (CCSK, n = 3), and a renal cell carcinoma (RCC), and two pediatric kidney tumour cell lines (WT: SK-NEP-1 and rhabdoid tumour of the kidney: G-401) were investigated using the monoclonal antibody M3. Additionally, immunoblotting and RT-PCR analysis were performed. Cell culture supernatants were evaluated for TPS release. Serum TPS was measured in five patients at diagnosis, during chemotherapy and after surgical resection. RESULTS: Moderate to strong immunoreactivity for TPS was found in tubular and blastemal components of nearly all (8/9) WT specimens. This was confirmed by Western-blotting. Cystic and epithelial-like portions of CCSKs and RCC showed distinct reactivity (3/3). The supernatant of G-401 but not of SK-NEP-1 showed a time- and cell number-dependent increase of TPS release. Interestingly, TPS synthesis was demonstrated in SK-NEP-1 cells. Median preoperative serum TPS was elevated (293 U/l) compared to healthy children and lowest after surgical resection (49.5 U/l). CONCLUSIONS: This is the first study demonstrating the synthesis and release of TPS by WTs and other paediatric renal malignancies. Considering the elevated levels of TPS in serum of these patients, a further investigation of this marker by larger clinical trials seems to be justified.

A region encompassing the FERM domain of Jak1 is necessary for binding to the cytokine receptor gp130.

The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (Four-point-one, Ezrin, Radixin, Moesin) homology domain comprising 19 conserved hydrophobic regions. To determine the role of this domain in governing recruitment of Jak1, but not Jak3, to the gp130 subunit of the interleukin-6 family of cytokine receptors, the interaction of three Jak1/Jak3 chimeras with gp130 was investigated. Chimeras 1, 2 and 3 (Jak1 FERM regions 1-19, 1-18 and 1-8/Jak3, respectively) were all enzymically active. Chimeras 1 and 2 interacted with the cytoplasmic domain of gp130, although less efficiently than Jak1. Only chimera 2, however, restored gp130 signalling in Jak1-negative cells. The data are consistent with recruitment of Jak1 to gp130 through the Jak1 FERM domain, but also emphasise the likely requirement for precise Jak/receptor orientation to sustain function.

The fate and prognostic value of occult metastatic cells in the bone marrow of patients with breast carcinoma between primary treatment and recurrence.

BACKGROUND: The current study examines the fate of occult metastatic cells detected in bone marrow (BM) at primary diagnosis and evaluates whether persistently positive findings support the prognostic influence of these cells in patients with Stage I--III (International Union Against Cancer) breast carcinoma. METHODS: The authors analyzed BM aspirates, at the time of primary diagnosis and after a median interval of 19 months (range, 7--67 months), from 89 patients who were free of recurrence. The presence of cytokeratin (CK) positive cells was assessed with the monoclonal anti-CK antibody A45-B/B3. Patients were observed prospectively for a median of 41 (range, 12--78) months after the first aspiration. RESULTS: At the time of primary diagnosis, 24 of 89 patients (27%) presented with occult metastatic cells in the BM. Of the same 89 patients, 25 (28%) had a positive BM finding at the time of the second BM analysis. Among those patients with an initially negative BM finding, 15 patients (17%) had occult metastatic cells at time of the second BM aspiration, whereas 10 patients (11%) had a persistently positive BM finding. Patients with a persistently negative BM status (n = 50) had a significantly better overall survival than patients with a positive BM status at the time of the second BM aspiration (n = 25), both by univariate analysis (P = 0.045, log-rank) and multivariate analysis (P = 0.034, Cox regression). CONCLUSIONS: In many patients with primary breast carcinoma, minimal residual disease can be detected by follow-up examination of the BM. This finding is prognostically relevant and provides reason to include BM monitoring in future clinical trials.

Quality of life influenced by primary surgical treatment for stage I-III breast cancer-long-term follow-up of a matched-pair analysis.

BACKGROUND: Breast-conserving therapy has been demonstrated to be just as safe and a less disruptive experience compared with mastectomy for surgically manageable breast cancer. There is, however, no agreement in the literature about the impact of these procedures on several important aspects of quality of life (QOL). The purpose of the present study is to compare the long-term impact of these two surgical approaches on QOL in patients with identical tumor stages and to suggest possible shortcomings of the standard QOL questionnaires. METHOD: Between August 1999 and May 2000, QOL questionnaires were answered by 152 pair-matched patients at the I. Frauenklinik, Ludwig-Maximilians University Munich, as part of routine follow-up examinations. The pairs of patients, each consisting of one patient after mastectomy and one after breast conservation, were selected according to the highest degree of equivalence in tumor stage. All patients had been initially treated for stage I-III breast cancer without evidence of distant metastases. The QOL was evaluated by using the QLQ-C30 questionnaire version 2.0 of the EORTC Study Group on Quality of Life. We formulated seven additional questions about the patients' satisfaction with the primary surgical treatment modality as viewed from their current perspective. The QOL questionnaires were answered after a median interval of 46 months following primary treatment. RESULTS: Tumor stage, prognostic factors, and adjuvant systemic treatment were well balanced between the two groups. No differences between the two groups were observed in terms of all QOL items measured by the QLQ-C30. Our additional questions, however, revealed that patients in the mastectomy group were less satisfied with the cosmetic result of their primary operation (P < .0001), were more likely to feel basic changes in their appearance (P < .0001), and were more likely to be emotionally stressed by these facts (P < .0001). From their perspective at the time of completing the questionnaires, 11 patients in the mastectomy group (15%) would decide differently about the surgical treatment modality, compared with only 3 patients (4%) in the breast conservation group (P = .025). CONCLUSION: While the primary surgical treatment modality seems to have no long-term impact on general QOL, certain body-image-related problems may be caused by mastectomy. Standard measuring instruments for QOL may fail to detect differences in satisfaction and adaptation with the primary surgical treatment modality.

Organization and regulation of the rat Cx31 gene. Implication for a crucial role of the intron region.

The connexin31 (Cx31) gene, a member of the connexin multigene family, is expressed in a characteristic spatiotemporal pattern during placental development in rodents. To elucidate the trophoblast-specific regulation of Cx31, we have isolated the rat Cx31 gene and performed structural and functional promoter analysis. The isolated Cx31 gene contains two exons separated by an intron of 2.6 kb. The first exon of the Cx31 gene is preceded by a TATA-less promoter region. Transcription is initiated in exon 1 from two transcription start sites producting transcripts of 105 and 139 bp. The 935 bp of the 5' flanking region of exon 1 comprises five putative binding sites for the GATA transcription factors as well as a NF-kappa B element, a CAAT-box and E-box/E-box-related sequences. For functional promoter analysis, the rat choriocarcinoma cell line Rcho-1 and the mouse keratinocyte cell line Hel37, which both express Cx31, were chosen. Only constructs including exon 1 and the complete intron showed high activity in transient transfection experiments in both cell lines. All deletion fragments of the putative promoter region, but which contain the entire intron sequence, did not reveal any obvious changes in luciferase activity. However, deletion of 1.1 kb of the intron sequence downstream of the splice donor site resulted in the loss of promoter activity. The intron exhibits no enhancer activity for the gene; however, the mRNA stability was increased in the presence of the intron sequence. These results indicate that parts of the intron sequence are critical for basic promoter function of the Cx31 gene.

[Chemotherapy-associated myelosuppression in gynecological oncology]. / Chemotherapieassoziierte Myelosuppression in der gynäkologischen Onkologie.

INTRODUCTION AND OBJECTIVE: A clinically important myelosuppression due to adjuvant chemotherapy is seen more frequently as dosage is intensified and new drugs are used. The assessment of the cytopenia expected is frequently hampered by a lack of directly comparable data. The aim of this study was to compare - in our own patient population - the chemotherapy-associated myelosuppression of four chemotherapeutic regimens used in gynecological oncology. METHODS: 79 patients with primary breast cancer and 26 patients with epithelial ovarian carcinoma underwent cytostatic treatment, and the associated myelosuppression was evaluated by the determination of cytopenia and the need for supportive therapy. The chemotherapy regimens investigated were CMF (cyclophosphamide 600 mg/m(2), methotrexate 40 mg/m(2), 5-fluorouracil 600 mg/m(2), 6xq3w), EC/CMF (epirubicin 90 mg/m(2), cyclophosphamide 600 mg/m(2), 4xq3w, followed by CMF, 3xq3w), DE (docetaxel 75 mg/m(2), epirubicin 90 mg/m(2), 6xq3w) and CC (cyclophosphamide 600 mg/m(2), carboplatin AUC 6, 6xq3w). RESULTS: The EC/CMF and DE regimens were used significantly more frequently for more advanced tumor stages, but there were no differences concerning tumor-dependent prechemotherapeutic myelosuppression. Hemopoiesis was most impaired in the CC group with a mean drop of serum hemoglobin of 1.5 g/dl to the end of the cytostatic treatment; correspondingly, most transfusions of concentrated erythrocytes were needed in this group. The strongest suppression of leukopoiesis was found in the DE group, with a mean drop in leukocyte counts of 6.2 x 10(3)/microliter per cycle; in this group, a mean of 7.6 ready-made syringes with 263 microgram Lenogastrim was used to stimulate leukopoiesis. The severest drop in the mean thrombocyte count, i.e. 171.7 x 10(3)/microliter, was found in the CC group. CONCLUSIONS: The CC regimen impairs thrombo- and erythropoiesis most, whereas the DE regimen causes marked leukopenia. The regimen with the smallest myelosuppression was CMF. No severe cytopenia-associated complications were detected in any of the cases investigated.

Induction of recombinant gene expression in stably transfected cell lines using attenuated vaccinia virus MVA expressing T7 RNA polymerase with a nuclear localisation signal.

There are major drawbacks using vaccinia virus (VV) expressing T7 polymerase for eukaryotic expression. VV is infectious for humans and due to cytosolic replication of Poxviridae, transient transfection of T7 promoter containing plasmids is necessary, which varies in efficiency. Several improvements have been introduced to this system to enhance expression of herpes viral glycoproteins. Stably transfected cell lines were generated with an EBV-based episomal plasmid vector which can be pushed to increasing copy numbers under selective pressure. The avirulent vaccine MVA strain was adopted to generate a safe laboratory vector for inserting the bacteriophage T7 RNA polymerase gene with (+) or without (-) a nuclear localisation signal. Constructs were designed for recombination into the VV haemagglutinin gene as recombinants could not be isolated successfully when inserting into the MVA thymidine kinase locus. Both T7 MVA recombinants induced foreign protein expression in transiently transfected cells but only the T7-/+ MVA induced target protein expression in stably transfected cells. The level of protein expression by this induction mechanism was comparable to, or superior to levels obtained with VV recombinants expressing the gene under control of the VV 11 k IE promoter. The results suggests that the T7+ MVA virus can be used to induce gene expression in stable recombinant cell lines and offers an attractive and safe alternative to other inducible eucaryotic expression systems.

Use of apathogenic vaccinia virus MVA expressing EHV-1 gC as basis of a combined recombinant MVA/DNA vaccination scheme.

The nonreplicating chicken adapted vaccinia virus strain MVA was used in a combined vaccine scheme. Using the equine herpesvirus type 1 (EHV-1) encoded complement-receptor glycoprotein C as antigen, only poor antibody response was induced by exclusive vaccination with DNA plasmids. The administration of recombinant MVA followed by plasmid immunization elicited both humoral and cellular immune responses in hamster comparable to EHV-1 full virus vaccines. Our results indicate that recombinant constructs based on MVA represent a safe and efficient way to overcome problems of poor immunogenicity of certain antigens upon intramuscular DNA vaccination, thus replacing sophisticated adjuvants or application methods, which are not readily applicable in routine practice.

The hemagglutinin-esterase of mouse hepatitis virus strain S is a sialate-4-O-acetylesterase.

By comparative analysis of the hemagglutinin-esterase (HE) protein of mouse hepatitis virus strain S (MHV-S) and the HE protein of influenza C virus, we found major differences in substrate specificities. In striking contrast to the influenza C virus enzyme, the MHV-S esterase was unable to release acetate from bovine submandibulary gland mucin. Furthermore, MHV-S could not remove influenza C virus receptors from erythrocytes. Analysis with free sialic acid derivatives revealed that the MHV-S HE protein specifically de-O-acetylates 5-N-acetyl-4-O-acetyl sialic acid (Neu4, 5Ac2) but not 5-N-acetyl-9-O-acetyl sialic acid (Neu5,9Ac2), which is the major substrate for esterases of influenza C virus and bovine coronaviruses. In addition, the MHV-S esterase converted glycosidically bound Neu4,5Ac2 of guinea pig serum glycoproteins to Neu5Ac. By expression of the MHV esterase with recombinant vaccinia virus and incubation with guinea pig serum, we demonstrated that the viral HE possesses sialate-4-O-acetylesterase activity. In addition to observed enzymatic activity, MHV-S exhibited affinity to guinea pig and horse serum glycoproteins. Binding required sialate-4-O-acetyl groups and was abolished by chemical de-O-acetylation. Since Neu4,5Ac2 has not been identified in mice, the nature of potential substrates and/or secondary receptors for MHV-S in the natural host remains to be determined. The esterase of MHV-S is the first example of a viral enzyme with high specificity and affinity toward 4-O-acetylated sialic acids.

Identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza C virus and bovine coronavirus.

We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substrates p-nitrophenyl acetate, alpha-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse alpha1 macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.

Structural organization and chromosomal localization of Hyal2, a gene encoding a lysosomal hyaluronidase.

The human HYAL2 gene encodes a lysosomal hyaluronidase that is related to the testicular PH-20 hyaluronidase. Regions conserved in these proteins have been used to design PCR primers suitable for the isolation of a fragment of the murine Hyal2 gene. This fragment was used to isolate the Hyal2 cDNA from a cDNA library. The cloned cDNA has an open reading frame of 473 codons and a 3'-untranslated region of 302 bases plus a poly(A) tail. Using this cDNA, the corresponding genomic DNA was characterized from 129SVJ mice. The murine Hyal2 gene is approximately 3.5 kb, contains the coding sequence for the mRNA on four exons, and is localized on chromosome 9 between the microsatellite markers D9Mit183 and D9Mit17 near the genes for dystroglycan and transferrin. The gene is expressed ubiquitously, the sole exception being adult brain.