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J Gerdes

Publications and source records attributed to J Gerdes.

At least 19 recordsLinked to original sources
PubMed2005

Rare cancer cell analyzer for whole blood applications: automated nucleic acid purification in a microfluidic disposable card.

One current challenge facing point-of-care cancer detection is that existing methods make it difficult, time consuming and too costly to (1) collect relevant cell types directly from a patient sample, such as blood and (2) rapidly assay those cell types to determine the presence or absence of a particular type of cancer. We present a proof of principle method for an integrated, sample-to-result, point-of-care detection device that employs microfluidics technology, accepted assays, and a silica membrane for total RNA purification on a disposable, credit card sized laboratory-on-card ('lab card") device in which results are obtained in minutes. Both yield and quality of on-card purified total RNA, as determined by both LightCycler and standard reverse transcriptase amplification of G6PDH and BCR-ABL transcripts, were found to be better than or equal to accepted standard purification methods.

Fusion Proteins, bcr-abl↗
PubMed2005

Rare cancer cell analyzer for whole blood applications: microcytometer cell counting and sorting subcircuits.

We demonstrate sorting of rare cancer cells from blood using a thin ribbon monolayer of cells within a credit-card sized, microfluidic laboratory-on-a-card ("lab card") structure. This enables higher cell throughput per minute thereby speeding up cell interrogation. In this approach, multiple cells are viewed and sorted, not individually, but as a whole cell row or section of the ribbon at a time. Gated selection of only the cell rows containing a tagged rare cell provides enrichment of the rare cell relative to background blood cells. We also designed the cell injector for laminar flow antibody labeling within 20s. The approach combines rapid laminar flow cell labeling with monolayer cell sorting thereby enabling rare cell target detection at sensitivity levels 1000 to 10,000 times that of existing flow cytometers. Using this method, total cell labeling and data acquisition time on card may be reduced to a few minutes compared to 30-60 min for standard flow methods.

Antigens, CD34↗
PubMed2004

Expression of the proliferation marker Ki-67 during early mouse development.

In somatic tissues, the mouse Ki-67 protein (pKi-67) is expressed in proliferating cells only. Depending on the stage of the cell cycle, pKi-67 is associated with different nuclear domains: with euchromatin as part of the perichromosomal layer, with centromeric heterochromatin, and with the nucleolus. In gametes, sex-specific expression is evident. Mature MII oocytes contain pKi-67, whereas pKi-67 is not detectable in mature sperm. We investigated the re-establishment of the cell cycle-dependent distribution of pKi-67 during early mouse development. After fertilization, male and female pronuclei exhibited very little or no pKi-67, while polar bodies were pKi-67 positive. Towards the end of the first cell cycle, prophase chromosomes of male and female pronuclei simultaneously got decorated with pKi-67. In 2-cell embryos, the distribution pattern changed, presumably depending on the progress of development of the embryo, from a distribution all over the nucleus to a preferential location in the nucleolus precursor bodies (NPBs). From the 4-cell stage onwards, pKi-67 showed the regular nuclear relocations known from somatic tissues: during mitosis the protein was found covering the chromosome arms as a constituent of the perichromosomal layer, in early G1 it was distributed in the whole nucleus, and for the rest of the cell cycle it was associated with NPBs or with the nucleolus.

Animals↗
PubMed2001

The expression of Ki-67, MCM3, and p27 defines distinct subsets of proliferating, resting, and differentiated cells.

The mini-chromosome maintenance proteins (MCM), which are involved in the control of DNA replication, and the cyclin-dependent kinase inhibitors, such as p27/KIP1, represent two groups of proteins that are currently under investigation as diagnostic tumour markers. The expression of p27 and MCM3 was compared with the expression of the Ki-67 protein, an approved marker for proliferating cells, extensively used in histopathology and cancer research. The expression pattern of all three proteins was assessed on germinal centres and oral mucosa, which display a well-defined spatio-temporal organization. The expression of the p27 protein was closely related to differentiated cells, whereas MCM3 and Ki-67 were predominantly localized to the regions of proliferating cells. However, it is important to note that considerable numbers of cells that were growth-arrested, as confirmed by the absence of the Ki-67 protein, stained positive for the MCM3 protein. These results were verified in vitro using growth-arrested Swiss 3T3. The MCM3 protein is therefore expressed in cells that have ceased to proliferate, but are not terminally differentiated, according to the absence of p27 protein expression. In conclusion, a combined analysis of Ki-67, MCM3, and p27 protein expression may provide a more detailed insight into the cell proliferation and differentiation processes that determine individual tumour growth.

Animals↗
PubMed2001

Immunohistochemical detection of cell growth fraction in formalin-fixed and paraffin-embedded murine tissue.

Monoclonal antibody MIB-1 is a reliable tool for determining proliferating cells in human tissues, but does not react with the homologous mouse antigen and is therefore useless in experimental pathology using mice as model systems. Standard method for assessment of cellular proliferation in formalin-fixed, paraffin-embedded murine tissues is immunohistochemical detection of DNA synthesis using antibodies against exogenously injected 5-bromodeoxyuridine (BrdU), which is a tedious procedure and not useful for routine investigations. We tested monoclonal antibody MIB-5 and monoclonal and polyclonal anti-MCM3 antibodies as immunohistochemical proliferation markers for paraffin-embedded nonneoplastic and neoplastic tissues of wild-type and transgenic mice, compared to anti-BrdU immunostaining. Percentage of proliferating cells was determined with continuously decreasing antibody dilutions. Percentages of MIB-5 and anti-BrdU immunostained cells correlated strongly, as well as percentage of MIB-5-decorated cells and frequency of mitotic figures. Anti-MCM3 antibodies labeled significantly higher percentages of cells than anti-BrdU or MIB-5, and showed a linear decrease with increasing antibody dilutions. We conclude that MIB-5 detects reliably the cell growth fraction in formalin fixed, paraffin-embedded murine tissues, bypassing methodological drawbacks of BrdU. Anti-MCM3 antibodies are less useful for determination of proliferating cells although they might detect the fraction of cells remaining competent for proliferation.

Adenoma↗
PubMed2001

Reduced T-cell receptor CD3zeta-chain protein and sustained CD3epsilon expression at the site of mycobacterial infection.

Control of mycobacterial infection by the cellular immune system relies both on antigen-presenting cells and on T lymphocytes. The quality of an effective cellular immune response is dependent on functional signal transduction residing in the cytoplasmic tails of the T-cell receptor CD3 components. In order to investigate potential effects of mycobacteria on T-cell receptor signalling, we examined the protein expression of T-cell signal transduction molecules (CD3zeta, ZAP-70, p59fyn, p56lck). In Western blots of peripheral blood mononuclear cells of Mycobacterium tuberculosis infected patients, only the CD3zeta-chain showed a marked reduction in protein expression. To investigate the situation in situ, immunoenzymatic and immunofluorescence stainings for CD3epsilon and CD3zeta expression were performed on sections of normal lymphoid tissue, M. leprae infected and sarcoid tissue. CD3epsilon and CD3zeta expression were similar with respect to intensity, localization and the number of cells stained in normal lymphoid tissue and in sarcoid granulomas. In contrast, the granulomas of M. leprae infected tissues showed a significantly reduced expression of CD3zeta compared to CD3epsilon. Using double immunofluorescence analysis, virtually no CD3zeta expression could be detected in comparison to the CD3epsilon expression in the lesions. Apparently, mycobacteria are capable of significantly reducing CD3zeta-chain expression, which may be restored by cytokines. IL-2-enhanced zeta-chain expression and T-cell effector functions, defined by interferon-gamma release, in M. tuberculosis-specific and human leucocyte antigen-DR restricted CD4+ T cells isolated from granuloma lesions from patients with pulmonary tuberculosis. Because CD3zeta is essential for CD3 signalling and for eliciting T-cell effector functions, reduced CD3zeta protein expression could result in altered signal transduction and inefficient T-cell effector functions. Alternatively, reduced CD3zeta-chain expression may protect T cells from repetitive TCR stimulation associated with anergy or apoptosis.

CD3 Complex↗
PubMed2001

A human in vitro granuloma model for the investigation of multinucleated giant cell and granuloma formation.

A method for the in vitro generation of granulomas and its use in the analysis of the human granulomatous response is summarized. As a target for the cellular response L3 larvae of Nippostrongylus brasiliensis are coincubated with human mononuclear blood cells, and within seven to fourteen days the development of blood monocytes to mature macrophages and to epithelioid cells and multinucleated giant cells (MGC) as typical constituents of granulomas clustered around the nematode is observed. The following review describes the uses and applications of this model for phenotyping, functional, formation and modulating studies of granulomas and MGCs, taking into account its unique features compared to other in vitro models. With respect to MGC formation, procedures are described and examples are given which allow the phenotyping of these cells using immunofluorescence and immunohistological techniques. In addition, the potential of this model for illuminating functional aspects of MGC is described applying an isolation protocol for MGC and a subsequent reverse-transcriptase polymerase chain reaction method for the analysis of single cells. Moreover, the significance and relevance of using this granuloma model is discussed in the follow up analysis of in vivo findings of interleukin-6 expression in MGC of granulomas of patients with sarcoidosis. These in vivo results implicated a role for interleukin-6 in granuloma and MGC development. The in vitro granuloma model was used to investigate potential modulatory effects of this cytokine by analysing the cell numbers and the number of MGC per in vitro granuloma, the size of the MGC formed, the fusion index and the morphology of the in vitro granuloma. The results demonstrated significant modulatory effects of interleukin-6 on the cell number per in vitro granuloma and on the morphology of the cells involved. Conceivably, elevated interleukin-6 levels may modulate granuloma formation with respect to the number of cells involved and in influencing distinct cell populations involved in granuloma formation.

Animals↗
PubMed2001

The fate of bacillus Calmette-Guerin after intravesical instillation.

PURPOSE: Long-term activation of immunocompetent cells of the bladder wall as well as case reports of systemic infections some months or years after intravesical bacillus Calmette-Guerin (BCG) therapy imply that mycobacteria may persist in the body. Therefore. we investigated the fate of BCG in patients after uncomplicated intravesical instillation therapy. MATERIAL AND METHODS: A total of 49 patients were included in the study, from whom various numbers of specimens were used for mycobacterial culture and molecular biological detection techniques. In 23 patients who received a total of 128 instillations urine, sputum, venous blood and bladder biopsies were screened for BCG by acid-fast staining and culture at different times before and after instillation. From 16 of the 23 patients and from an additional 26 a total of 180 bladder biopsies obtained at intervals 3 to 30 months after instillation were screened for mycobacterial 16S ribosomal DNA by a nested polymerase chain reaction protocol. RESULTS: No viable BCG was found in venous blood or in 127 of 128 sputum specimens before and 2 hours after instillation. Two of 56 bladder biopsies were culture positive. In urine BCG was detected in 96.4% of the specimens after 2 hours and in 67.9% after 24 hours after instillation. The number of positive specimens decreased and it was 27.1% on day 7 immediately before the next instillation. In 14 of 44 bladder biopsies (31.8%) mycobacterial ribosomal DNA was found within 1 week after the sixth instillation. A positive polymerase chain reaction was evident up to 24 months in between 4.2% and 37.5% of the investigated biopsies. After 30 months no ribosomal DNA was evident in the 6 samples available for testing. CONCLUSIONS: Nontraumatic intravesical instillation of BCG is not accompanied by systemic mycobacterial spread. Local persistence during the instillation course is evident since viable BCG is commonly found in the urine. Long lasting and persistent BCG DNA in the bladder wall may account for long-term immuno-activation. However, the remaining BCG may be a possible source of late systemic infections.

Adjuvants, Immunologic↗
PubMed2001

Evidence for disease phenotype associated haplotypes (DR.TNF) in sarcoidosis.

BACKGROUND AND AIM OF THE WORK: In a previous study, gene polymorphisms of the MHC locus (-308 TNFalpha promotor, HLA-DR) in patients with pulmonary sarcoidosis had been investigated and significant correlations with the presentation of the disease as defined by the presence of Löfgren syndrome had been found. Since genotyping of both loci was necessary to reveal a significant difference on the genetic level between the two patient groups Löfgren and non-Löfgren, a working hypothesis was derived in which a disease course associated haplotype, rather than single specific genes, was considered to play a role in the pathogenesis of sarcoidosis. METHODS: A first step to test this hypothesis was taken by calculating virtual haplotypes from these previous data. Statistical analysis of disease phenotype association and relative risk of these virtual haplotypes was performed to assess correlations with sarcoidosis and the two disease phenotypes. RESULTS: The results not only substantiated the previous findings, but also showed that the virtual haplotype DR3.TNFalpha2 was significantly associated with Löfgren syndrome and that the virtual haplotype DR2.TNFalpha1 was noticeably although not significantly associated with the non-Löfgren patients. CONCLUSIONS: Using theoretical calculations based on actual data, haplotypes, rather than single genes interacting with each other, were found to be a highly likely explanation for the previously published observations. In addition, the results allow the conclusion to be drawn that disease course associated haplotypes in sarcoidosis are highly probable and that further investigation of polymorphisms in the MHC gene region holds the potential of defining prognostic markers.

HLA-DR Antigens↗
PubMed2000

The proliferation-specific human Ki-67 protein is a constituent of compact chromatin.

The human nuclear Ki-67 protein (Ki-67p) is expressed in proliferating, but not in quiescent, cells and is therefore widely used as a proliferation marker in histopathological research and practice. However, information regarding its intranuclear location is scarce and controversial. Here we describe the results of cell fractionation and nuclease digestion experiments using nuclei isolated from human HeLa cells in interphase. Ki-67p dissociates at 0.3-0.4 M NaCl from its nuclear binding sites, and gradient centrifugations indicate that the released Ki-67p is most likely a single molecular entity and not complexed to other proteins. In nuclei, prepared under physiological salt conditions, the binding sites are largely resistant against micrococcal nuclease. However, when prepared at very low ionic strengths, chromatin regions with associated Ki-67p become accessible to micococcal-nuclease-producing chromatin fragments that carry bound Ki-67p. We conclude that Ki-67p is a chromatin protein and resides at densely packed regions, probably heterochromatin. Our data provide a useful basis for further biochemical research on this human nuclear protein.

Biomarkers↗
PubMed2000

The Ki-67 protein: fascinating forms and an unknown function.

The Ki-67 protein is a nuclear and nucleolar protein, which is tightly associated with somatic cell proliferation. Antibodies raised against the human Ki-67 protein paved the way for the immunohistological assessment of cell proliferation, particularly useful in numerous studies on the prognostic value of cell growth in clinical samples of human neoplasms. The primary structure revealed potential phosphorylation site for a range of essential kinases, PEST sequences, and a forkhead-associated domain, which are features present in a variety of cell-cycle-regulating proteins, but information about the position of the Ki-67 protein within the protein network that drives the cell cycle remained scarce. There is now evidence that posttranslational modifications based on phosphorylation by cdc2 kinase and PKC accompany the remarkable redistribution of the Ki-67 protein from the interior of the nucleus to the perichromosomal layer during mitosis and vice versa. The discovery of Ki-67 equivalents in other species is advantageous for a precise and cross-species investigation of the structural requirements for its yet unknown function. The recently published data add new pieces to the challenging puzzle of this multifaceted protein, which are waiting to be put together.

Animals↗
PubMed2000

The Ki-67 protein: from the known and the unknown.

The expression of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G(1), S, G(2), and mitosis), but is absent from resting cells (G(0)), makes it an excellent marker for determining the so-called growth fraction of a given cell population. In the first part of this study, the term proliferation marker is discussed and examples of the applications of anti-Ki-67 protein antibodies in diagnostics of human tumors are given. The fraction of Ki-67-positive tumor cells (the Ki-67 labeling index) is often correlated with the clinical course of the disease. The best-studied examples in this context are carcinomas of the prostate and the breast. For these types of tumors, the prognostic value for survival and tumor recurrence has repeatedly been proven in uni- and multivariate analysis. The preparation of new monoclonal antibodies that react with the Ki-67 equivalent protein from rodents now extends the use of the Ki-67 protein as a proliferation marker to laboratory animals that are routinely used in basic research. The second part of this review focuses on the biology of the Ki-67 protein. Our current knowledge of the Ki-67 gene and protein structure, mRNA splicing, expression, and cellular localization during the cell-division cycle is summarized and discussed. Although the Ki-67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear. There are indications, however, that Ki-67 protein expression is an absolute requirement for progression through the cell-division cycle.

Biomarkers, Tumor↗
PubMed2000

Posttranslational modifications of the KI-67 protein coincide with two major checkpoints during mitosis.

Ki-67 is a nuclear protein present in all proliferating cells that are in the active part of the cell division, but not in resting cells. This feature is extensively used in tumor diagnostics to estimate the growth fraction of a given cell population. We now demonstrate that the spatial and temporal regulation of the Ki-67 protein during the cell cycle is associated with mitosis-specific phosphorylation. These posttranslational modifications of the Ki-67 protein are accompanied by a characteristic redistribution of the protein from the interior of the nucleus to the periphery of the condensed chromosomes and vice versa. Phosphorylation could be suppressed by activating cell-cycle checkpoints that control the entry into mitosis through the activity of the cyclin B/cdc2 complex. In vitro experiments confirm that the presence of the cdc2 kinase and its regulatory subunit cyclin B is required for the phosphorylation of the Ki-67 protein. We further demonstrated that the Ki-67 protein is a new member of the family of MPM-2 reactive phosphoproteins, which includes both structural and functional proteins that are necessary for the control and timing of mitosis. Phosphorylation and dephosphorylation of the Ki-67 protein are therefore controlled by key regulatory structures of the cell cycle and occur at two hallmark events within the cell cycle: the breakdown and the reorganization of the nucleus during mitosis.

Adenosine Triphosphate↗
PubMed2000

Molecular genetic characterization and subcellular localization of Theileria annulata mitochondrial heat-shock protein 70.

A Theileria annulata mitochondrial heat-shock protein of the 70-kDa family (Tamthsp70) was isolated by screening of the cDNA library of a T. annulata-infected bovine lymphoblastoid cell line with an antibody raised against T. annulata schizonts. The Tamthsp70 coding sequence was found to be most closely related to a previously reported mitochondrial hsp70 gene of Eimeria tenella exhibiting a similarity of 67% with mitochondrial hsp70 genes of eukaryotic plants (Pisum sativum, Phaseolus vulgaris) and with dnaK proteins of prokaryotes (Rhizobium meliloti, Agrobacterium tumefaciens). The Tamthsp70 mRNA is expressed within the sporozoite, schizont, and merozoite stages of the parasite, which suggests that it is constitutively transcribed throughout the life cycle. The gene encodes a polypeptide of 681 amino acids and exhibits a mitochondrial targeting sequence and several sequence motifs common to mitochondrial hsp70 and prokaryotic dnaK proteins. The protein level of the Tamthsp70 protein after heat shock decreased slightly during the exposure of infected cells to a temperature of 42 degrees C in comparison with cells cultured at 37 degrees C. By immunofluorescence the protein was located in the area in which the schizonts reside within infected cells. Immunoelectron microscopy showed that the hsp70 protein was predominantly localized in the mitochondria of the parasites. However, it was also found in small amounts in the cytoplasm of the parasite and host cell. This indicates (1) that Tamthsp70 is very probably translated in the parasite cytoplasm and then transported across the mitochondrial membrane into the mitochondrial matrix and (2) that it is transported across the parasite membrane into the host-cell cytoplasm.

Amino Acid Sequence↗
PubMed1999

Extracellular matrix regulates steady-state mRNA levels of the proliferation associated protein Ki-67 in endometrial cancer cells.

We investigated whether components of the extracellular matrix have the potential to regulate the proliferative activity of endometrial adenocarcinoma cells. Culturing of cells on the reconstituted basement membrane matrigel down-regulated the steady-state mRNA levels of the proliferation associated protein, Ki-67, in the endometrial adenocarcinoma cell lines HEC 1B(L) and Ishikawa after 48-96 h of culture on the matrix substrate. Proliferation of Ishikawa was stimulated again if cells were cultured on matrigel and challenged by proteins representing functional domains of tenascin-C, a mesenchymal glycoprotein. The fibronectin-type-III-like repeats 6-8 of tenascin-C were found to be the most potent. In summary, evidence is provided that components of both epithelial and stromal extracellular matrices can function as regulators of cell growth.

Adenocarcinoma↗
PubMed1999

Follicular dendritic cells and T cells: nurses and executioners in the germinal centre reaction.

The humoral immune response constitutes an efficient system to protect the organism against diseases caused by invading pathogens. To guarantee a highly efficient defence, the humoral immune system has to be tightly regulated. Two cell subsets in particular, T cells and follicular dendritic cells (FDCs), contribute to the success of these regulation processes. Whereas the particular role of T cells is the elimination of autoreactive clones, the main role of FDCs is to present unprocessed antigen and check B-cell clones for higher affinity. B-cell clones unsuited for improved humoral immune response will be specifically killed. Involvement of Fas-mediated apoptosis might be an additional tool not only in T-cell-mediated regulation, but also in FDC-B cell interaction in the germinal centre.

Antibody Formation↗
PubMed1999

Polymorphisms at position -308 in the promoter region of the TNF-alpha and in the first intron of the TNF-beta genes and spontaneous and lipopolysaccharide-induced TNF-alpha release in sarcoidosis.

TNF-alpha is a potent pro-inflammatory cytokine. Previous studies have proved that biallelic polymorphisms in the TNF-alpha (-308, TNFA) and TNF-beta genes (intron 1, TNFB) influence TNF-alpha production. In sarcoidosis, a chronic granulomatous disease, as a result of an unknown in vivo activation bronchoalveolar lavage (BAL) cells release high amounts of TNF-alpha, spontaneously and after in vitro stimulation. Thus, sarcoidosis could serve as a model to test the in vivo effect of TNF gene polymorphisms. We determined the TNFA and TNFB polymorphisms of 44 patients with sarcoidosis and found the following allele frequencies: 0.80, 0.20, 0.38 and 0.62 for TNFA1, TNFA2, TNFB1 and TNFB2, respectively. To examine the in vivo effect of the named polymorphisms on the TNF-alpha production, the spontaneous and LPS-induced TNF-alpha release of BAL cells and peripheral blood mononuclear cells were also determined in patients with sarcoidosis. Statistical analysis did not reveal any significant difference between sarcoidosis patients with different genotypes. The results show that TNFA and TNFB polymorphisms do not determine the level of TNF-alpha release of mononuclear cells activated during the course of sarcoid inflammation.

Alleles↗