Sequential chemotherapy with carboplatin followed by weekly paclitaxel in advanced ovarian cancer: results of a multicenter phase II study of the northeastern German society of gynecological oncology.

BACKGROUND: For the adjuvant setting of advanced ovarian cancer (AOC) after primary radical surgery the combination of paclitaxel and platinum in a 3-week schedule has emerged as the current standard. In preclinical studies additional anti-angiogenic effects of low dose paclitaxel infusion were demonstrated. A sequential schedule of carboplatin and paclitaxel has the potential to improve the therapeutic index. METHODS: In this multicenter phase II trial four cycles of carboplatin at a dose of AUC 5 (d1/q21d) followed by 12 cycles of weekly paclitaxel at a dose of 80 mg/m(2) (d1/q7d) were applied after primary radical surgery. Eligible were all optimally or sub-optimally debulked patients with FIGO IA-IV ovarian cancer. All patients with hemoglobin levels <12 mg/dl received erythropoietin additionally. RESULTS: Between July 2003 and May 2005, 105 patients from 27 institutions were enrolled. The median age was 60 years (range: 23-80 years). A median number of 16 courses (range 1-16) were applied. The incidence of non-hematological toxicities was very low. Only 41% of patients experienced alopecia (grade 1-2). Neurotoxicity (grade 3-4) was not observed. Grade 3-4 hematological toxicity (43% of all patients) included thrombocytopenia (17%), anemia (3%), leucopenia (23%), and neutropenic fever (0%). Ninety-seven percent received erythropoietin. Thromboembolic events (4%) were not increased in patients who received erythropoietin. After a median time of 23 months (range: 1-42 months) 32 patients had died, and the median overall survival was not reached. The progression-free survival was 25.4 months (95% CI: 18.8-40+). CONCLUSION: These results suggest that this sequential regimen using weekly paclitaxel represents an efficacious and well-tolerated regimen. A randomized study comparing this new schedule with the conventional 3-week protocol is warranted.

[Brachycephaly in dog and cat: a "human induced" obstruction of the upper airways]. / Brachyzephalie bei Hund und Katze: eine "menschengemachte" Obstruktion der oberen Atemwege.

Selective breeding for exaggerated features caused in many brachycephalic dog and cat breeds virtually a loss of the nose, with serious anatomical and functional consequences. In addition to respiratory and olfactory tasks, in dogs the nose is of vital importance for thermoregulation. As obligatory nose breathers, dogs suffer far more than humans when their nasal ventilation is restricted. An open discussion in the broad public has to motivate authorities and kennel clubs to recognize extreme brachycephalic breeding as seriously affecting animal health and welfare.

[Geometry and function of the dog nose: how does function change when form of the nose is changed?]. / Geometrie und Funktion der Hundenase: Wie ändert sich die Funktion, wenn die Form verändert wird?

Nasal airflow resistance in brachycephalic dogs is significantly elevated compared to normal dogs. LaserAssisted TurbinEctomy (LATE)-surgery as well as xylometazolin were shown to reduce pathologically increased intranasal airway resistance in brachycephalic dogs by approximately 50 %. Impulse oscillometry provides a reliable and sensitive method to examine intranasal stenoses in the canine nose. Acoustic rhinometry allows assessment of changes in cross sectional area and volume of the canine nasal cavity.

The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins.

The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length cDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated cDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen cDNA. The central part of the Ki-67 antigen cDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycle-associated nuclear nonhistone proteins.

[Health-related quality of life in children with spina bifida]. / Gesundheitsbezogene Lebensqualität bei Kindern mit Spina bifida.

BACKGROUND: There are no studies on the health-related quality of life (HRQoL) in German children with a myelomeningocele (MMC). This study aims to obtain generalizable epidemiological data on the HRQoL of such children. PATIENTS AND METHODS: KINDL-R questionnaires were filled out and clinical findings on typical MMC disabilities were also documented. Of the 115 families contacted, 70 MMC families responded (response rate 61%). Normative KINDL-R data from a sample of healthy children served as reference. RESULTS: No differences in clinical data were found when comparing responders and non-responders. KINDL total scores as well as scores across all scales were highly concordant for parental reporting and self-reporting. CONCLUSIONS: Despite the fact that children with MMC often suffer from severe physical limitations, their HRQoL is not necessarily lower than that of healthy children.

Tumor necrosis factor and lymphotoxin gene expression in human tumor cell lines.

In this paper we show that eight of 17 tumor cell lines of various tissue origin constitutively express tumor necrosis factor (TNF) mRNA. Five of these eight cell lines concomitantly contained lymphotoxin (LT) mRNA. Of the remaining nine cell lines that lacked detectable TNF or LT gene expression, five could be induced by phorbol ester and/or cytokines to accumulate the respective mRNAs. While TNF mRNA was found not only in neoplastic hematopoietic cells, but also in cell lines derived from carcinomas, LT gene expression seemed to be restricted to lymphoid tumor cells. Tumor cells that expressed LT mRNA also secreted LT protein and proved to be resistant to the cytostatic/cytotoxic effects of their own protein product as well as to exogenous recombinant TNF and recombinant LT. In contrast, most of the cell lines containing TNF mRNA did not release TNF protein into the supernatants, indicating that TNF gene expression may be controlled predominantly at a post-transcriptional level. Thus, the presence of TNF mRNA does not necessarily reflect a TNF-resistant phenotype. Our findings demonstrate that TNF and/or LT mRNA expression is a rather common phenomenon in long-term cultured tumor cell lines and reveal that ectopic TNF and/or LT production by tumor cells may be involved in certain paraneoplastic syndromes as well as in tumorigenesis.

Evidence for a hepatocyte membrane fatty acid transport protein using rat liver mRNA expression in Xenopus laevis oocytes.

The aim of the present study was to directly demonstrate that hepatocellular uptake of long-chain fatty acids represents a non-diffusional uptake mechanism. Xenopus laevis oocytes were used for expression of rat liver mRNA to identify the liver fatty acid uptake system. Injection of total rat liver poly(A)+ RNA into oocytes resulted in a dose-dependent increase in fatty acid uptake. The most active mRNA was found in the 1.1-2.1 kb subfraction. In contrast, expression of the liver cytosolic fatty acid binding protein (L-FABP) or the previously suggested candidate carrier protein, mitochondrial aspartate aminotransferase (mGOT), did not induce fatty acid uptake. It is concluded that in rat liver, fatty acid transport represents a protein-mediated transport system.

Ventricular tachycardia during follow-up in patients resuscitated from ventricular fibrillation: experience from stored electrograms of implantable cardioverter-defibrillators.

OBJECTIVE: The purpose of this study was to use the electrogram storage capabilities of the implantable cardioverter-defibrillator (ICD) to categorize any arrhythmic event during follow-up in a group of patients who had survived an episode of ventricular fibrillation (VF) and to possibly identify clinical predictors of future arrhythmic events. BACKGROUND: Little is known about the electrophysiologic characteristics of ventricular arrhythmias recurring during follow-up in survivors of VF as the sole documented arrhythmia at the time of resuscitation. METHODS: Forty patients (58+/-10 years; 73% men; left ventricular ejection fraction 42+/-18%; 70% with coronary artery disease) who had survived an episode of VF and subsequently received an ICD capable of intracardiac electrogram recording and storage were followed for 23+/-11 months. In all patients, the arrhythmogenic substrate was investigated by means of programmed electrical stimulation (PES). RESULTS: Among the 40 patients, 41 episodes of ventricular arrhythmias were documented in 13 patients (33%): 36 episodes of ventricular tachycardias (VT) were recorded in 11 patients (28%) and 5 episodes of VF were recorded in the remaining 2 patients (5%). Age, gender, cardiac disease and left ventricular ejection fraction failed to distinguish between patients with clinical recurrences and patients without. The sensitivity, specificity and positive accuracy of PES were 29%, 63% and 46%, respectively, for prediction of ventricular arrhythmia recurrence; 45%, 70% and 36%, respectively, for prediction of VT; and 50%, 98% and 50%, respectively, for prediction of VF during follow-up. CONCLUSIONS: In survivors of VF receiving ICD therapy, VT is the most common ventricular arrhythmia recorded on device-incorporated electrograms during follow-up. This finding, associated with the relatively well-preserved ventricular function, may account for the ability of these patients to survive at time of the index arrhythmia; the use of antitachycardia pacing as a modality to treat arrhythmia recurrences may contribute to reduce the incidence of shock during follow-up in these patients.

The CXC receptor 2 is overexpressed in psoriatic epidermis.

The CXC chemokines interleukin-8 and GRO/melanoma growth-stimulatory activity (GRO-alpha) are potent activators of neutrophils and lymphocytes, but also stimulate growth and differentiation of nonhematopoietic cells like keratinocytes, fibroblasts, and melanocytes. High mRNA and protein levels have been detected in psoriatic epidermis. Chemokine activation of target cells is mediated by specific receptors and two CXC receptors have been described with similar affinity for interleukin-8 but different affinities for GRO-alpha. In this study, we examined the expression of both CXCR1 and CXCR2 in psoriatic tissue, identifying the target cells of chemokine activation in psoriasis. By immunohistochemistry and in situ hybridization, as confirmed by northern blot analysis and reverse transcriptase polymerase chain reaction, we could detect expression of the CXCR2 in suprabasal lesional psoriatic keratinocytes but not in healthy skin. The CXCR1 could not be localized in psoriatic keratinocytes with immunohistochemistry and in situ hybridization, but infiltrating cells in the dermal compartment expressed both types of receptors. These data suggest that in addition to neutrophil activation by both CXCR1 and CXCR2, activation of keratinocytes mediated by CXCR2 could contribute to the characteristic epidermal changes observed in psoriasis.

Molecular characterization of the gene locus of the human cell proliferation-associated nuclear protein defined by monoclonal antibody Ki-67.

The human antigen defined by the monoclonal antibody Ki-67, the 'Ki-67 protein', is an ubiquitously expressed human nuclear protein strictly associated with cell proliferation and is widely used in routine pathology as a 'proliferation marker' to measure the growth fraction in human tumours. In immunoblots of proteins from proliferating cells, Ki-67 detects two bands with the apparent molecular weights of 345 and 395 kDa. Recently we reported on the cloning and sequencing of the complete cDNA of the Ki-67 protein. We found two isoforms of cDNA with full lengths of 11.4 and 12.5 kb, respectively, likely formed by the alternative splicing of exon 7. The remarkable exon 13 at the 'centre' of this gene contains 16 homologous segments of 366 bp (Ki-67 repeats), each including a highly conserved new motif of 66 bp (Ki-67 motif). Computer analyses confirmed that the cDNAs encode for a new class of nuclear proteins. The complete gene locus of the Ki-67 protein, comprising a 74 bp 5' region and a 264 bp 3' region, has been sequenced and aligned to a continuous sequence of 29,965 bp length located on chromosome 10q25-ter. The gene is organized in 15 exons with sizes from 67 to 6845 bp and in 14 introns with sizes from 87 to 3569 bp. Three introns contain homologue copies of 'Alu-repeats'. Interestingly, the introns flanking the alternative spliced exon 7 are free of any consensus donor and acceptor splicing signal. All other intron-exon transitions contained a potential branch site. The complete 5' region including the first two exons represents a CpG-rich island. We found the transcription initiation site in exon two adjacent to the consensus sequence of a cap site. Upstream of this cap site no TATA- or CCAAT-box could be located, but downstream we found two remarkable directly repeated elements of 24 bp lengths each containing a TATA box in inverse orientation.

The use of reverse transcription polymerase chain reaction to analyse large numbers of mRNA species from a single cell.

A PCR method is described for determining the expression of multiple heterogeneous mRNAs from single cells. The total mRNA pool of a single selected cell is subjected to reverse transcription and subsequent tailing with poly(dA). This cDNA is preamplified by a sequence non-specific PCR protocol using oligo(dT)-containing primers. The single cell cDNA library obtained permits the analysis of virtually unlimited numbers of mRNA species per cell using sequence-specific PCR. This procedure of multiple mRNA analysis enables phenotyping of any cell for its mRNA composition and could be used to study the cytokine mRNA expression of individual human T cells ex vivo. The method should greatly facilitate the analysis of combinatorial expression of known genes in any cell.

An examination of the immune system of the duck (Anas platyrhynchos) for factors resembling some defined mammalian cytokines.

Duck lymphoblasts generated by phytohaemagglutinin (PHA) did not respond to recombinant or Jurkat cell line human interleukin (IL)-2 or possess surface antigens resembling mammalian IL-2 receptors or IL-1 beta. Supernatant fluids from normal and PHA-stimulated duck lymphocyte cultures, and normal and lipopolysaccharide (LPS)-stimulated monocytes, gave negative results in a range of assays for biological activity and immunochemical presence of factors resembling mammalian IL-1 and IL-2. However, supernatant fluids from LPS-stimulated duck monocytes contained IL-6-like activity (up to 35 units/mL) assessed on the 7TD-1 murine cell line. We were unable to demonstrate mRNA that would hybridize to cDNA probes for human IL-1 beta, IL-6, and tumour necrosis factor (TNF) in extracts of blood and lymphoid organs from normal and antigen-stimulated ducks. Because homologous serum or plasma is essential for duck lymphocytes and macrophages to respond to mitogens in vitro, we asked whether this growth-factor-like activity might be caused by substances resembling mammalian cytokines. Serum and plasma were examined for activity consistent with IL-1 and IL-6 on mammalian target cells. None was detected. Instead, both serum and plasma contained inhibitors of human IL-1 beta and IL-6, detected at dilutions up to 1:100. Inhibition by serum was heat (56 degrees C, 30 min) labile but inhibition by plasma was heat stable. The identities and biological functions of these inhibitors remain to be defined.

Noncytocidal mechanisms of action of tumor necrosis factor-alpha on human tumor cells: enhancement of HLA gene expression synergistic with interferon-gamma.

In this report, we present evidence that tumor necrosis factor alpha (TNF-alpha) exerts regulatory activity on the expression of HLA genes in human tumor cell lines at the level of mRNA expression and at a posttranscriptional level. These mechanisms are independent of direct cytotoxic/cytostatic effects, as enhancement of HLA antigen expression was observed in both sensitive cell lines and in cell lines resistant to TNF-mediated growth inhibition. In a priori HLA-negative cells, a strong TNF-alpha-mediated enhancement of the Interferon gamma (IFN-gamma) induced expression of HLA genes was revealed by Northern blot and immunofluorescence analysis. A distinct mechanism of TNF-alpha action is suggested from enhancement of constitutively expressed HLA genes. In this case, TNF-alpha raised HLA antigen density without apparent enhancement of cytoplasmic mRNA levels. This indicates that TNF-alpha influences HLA expression also at the level of translation or at a posttranslational level. TNF-alpha treatment of HLA-negative cells did not by itself result in HLA gene induction, suggesting that TNF-alpha acts as an enhancer and not as an inducer of HLA gene expression.

Detection of Mycobacterium leprae by three-primer PCR.

Recently, polymerase chain reaction has been introduced for the species-specific assessment of Mycobacterium leprae (1). To avoid Southern blotting techniques using radioactively labelled oligonucleotide probes, the aim of this study was to establish a three primer-based single-step PCR technique. Using primers designed for this purpose we amplified a part of the gene encoding for the 16S ribosomal RNA of slowly growing mycobacteria. Due to the species-specific antisense primer a second, smaller fragment specific for M. leprae was amplified. Our results show that the employment of a second antisense primer in the PCR may be a substitution for Southern blot hybridization.

Mycobacterium leprae DNA content, cellular and cytokine patterns in skin lesions of leprosy patients undergoing multidrug therapy (MDT).

Skin biopsies from untreated and MDT-treated patients were examined for infiltrating cells and cells producing the cytokines TNF-alpha, IFN-gamma, and IL-1 beta using immunohistochemistry. Biopsy specimens from untreated tuberculoid leprosy patients were characterized by the presence of cells producing TNF-alpha, IFN-gamma, and IL-1 beta and of subepidermal Langerhans cells. These cells were rarely found or completely absent in biopsies of untreated lepromatous leprosy patients, but tended to increase under MDT. In a short-term therapy trial for three months with brodimoprim, dapsone, and rifampicin, 12 patients were monitored by follow-up biopsies. Semiquantitative PCR for mycobacterial DNA revealed two groups of patients: one group in which mycobacterial DNA in follow-up biopsies remained constant and a second group in which a decrease of mycobacterial DNA during therapy was noted. Immunophenotyping in these follow-up biopsies revealed that in the latter group IFN-gamma-positive cells and Langerhans cells were present and gamma delta T cell receptor-positive cells tended to decrease during therapy. In contrast, in patients whose mycobacterial DNA did not change during therapy, these phenotypical manifestations were not observed. We therefore, conclude that assessment of mycobacterial DNA in combination with phenotyping of infiltrating cells and determination of cytokine patterns may be useful tools in establishing criteria for the effectiveness and duration of MDT in patients with leprosy.

New antiserum against Ki-67 antigen suitable for double immunostaining of paraffin wax sections.

AIMS: To prepare a rabbit antiserum equivalent to MIB 1 to permit the simultaneous assessment of cell proliferation and other markers of interest using double labelling studies. METHODS: Rabbits were immunised with a synthetic peptide deduced from the cDNA sequence coding for the Ki-67 antigen. Serum samples were tested for immunoreactivity using different immunobiochemical methods. RESULTS: A polyclonal antiserum was derived which detects the native as well as recombinant parts of the Ki-67 antigen in different test systems. Furthermore, the antiserum stains the Ki-67 antigen in routinely processed, paraffin wax embedded material. CONCLUSIONS: After antigen unmasking by microwave treatment the antiserum described here represents a powerful tool for the determination of growth fractions even in archival material. It is especially suitable for double staining experiments in combination with monoclonal antibodies.

Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein).

AIMS--To elucidate the fine specificities of the antibodies MIB 1 and MIB 3 and of additional monoclonal antibodies which also recognise the Ki-67 protein (MIB 5, IND.64, JG-67-2a). METHODS--Different parts of the Ki-67 protein cDNA were expressed in Escherichia coli. Bacterial lysates were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on to nitrocellulose. Additionally different peptides were synthesised on a membrane support (SPOT-Blot). The immunoreactivity of the antibodies with the recombinant proteins and the immobilised synthetic peptides, respectively, was analysed. A competition enzyme linked immunosorbent assay (ELISA) using a soluble synthetic peptide was also performed. RESULTS--The epitopes of all antibodies tested were contained within the same region of seven amino acids. The antibodies MIB 1 and MIB 3 required the five amino acid sequence FKELF for binding, whereas Ki-67, JG-67-2a, MIB 5 and IND.64 detected the sequence FKEL. CONCLUSIONS--It is concluded that the amino acid sequence FKELF represents an immunodominant area of the Ki-67 protein and that there is no correlation between the ability to detect the Ki-67 protein in paraffin wax sections irradiated with microwaves and the epitopes recognised by the antibodies.

Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins.

A decade of studies on the human nuclear antigen defined by monoclonal antibody Ki-67 (the "Ki-67 protein") has made it abundantly clear that this structure is strictly associated with human cell proliferation and that the expression of this protein can be used to assess the growth fraction of a given cell population. Until recently the Ki-67 protein was described as a nonhistone protein that is highly susceptible to protease treatment. We have isolated and sequenced cDNAs encoding for this antigen and found two isoforms of the full length cDNA of 11.5 and 12.5 kb, respectively, sequence and structure of which are thus far unique. The gene encoding the Ki-67 protein is organized in 15 exons and is localized on chromosome 10. The center of this gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp ("Ki-67 repeats"), each containing a highly conserved new motif of 66 bp ("Ki-67 motif"). The deduced peptide sequence of this central exon possess 10 ProGluSerThr (PEST) motifs which are associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors, etc. Like the latter proteins the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit 3H-thymidine incorporation in permanent human tumor cell lines in a dose-dependent manner.

Cytokines in mycobacterial infections: in vitro and ex vivo studies.

Different species of mycobacteria differ in their capacity to induce the production of tumor necrosis factor-alpha (TNF-alpha) by human monocytes in vitro. Whereas M. tuberculosis is a potent inducer of TNF-alpha, M. leprae is much less potent. TNF-alpha production is found to be associated with the availability of H2O2 generated by activated monocytes, as superoxide enhancing H2O2 concentration increases and catalase degrading H2O2 decreases TNF-alpha production. Furthermore, M. kansasii with high intrinsic catalase induce less TNF-alpha than mycobacteria with low intrinsic catalase. In vitro infection of monocytes with M. tuberculosis leads to an impairment of the antigen-presenting capacity, as determined by a reduction of antigen-induced T cell proliferation and interferon gamma (IFN-gamma) production. Of crucial importance is this impairment is the M. tuberculosis-induced down-modulation of MHC class II antigens. The role of TNF-alpha in vivo is reflected in patients with various forms of leprosy. In skin lesions of lepromatous leprosy patients TNF-alpha, interleukin 1 beta (IL-1 beta), and INF-gamma production are found to be rare, whereas these cytokines are well expressed in skin lesions of patients with tuberculoid leprosy. After multidrug chemotherapy an increase of local cytokine production is found. Taken together, these findings suggest that components of mycobacteria may interfere with local cell-mediated immune reactions in vivo. The molecular mechanisms involved in these local responses need to be defined.

"Very delayed" graft function in a patient after living related kidney transplantation: a case report.

We report the case of a patient who experienced anuric renal transplant failure for 44 days after living related kidney transplantation. Immunosuppressive and other therapies were carefully adapted to the findings of frequent renal transplant biopsies, which ultimately led to excellent graft function.