Biomedical subjects

M Rothe

Publications and source records attributed to M Rothe.

At least 19 recordsLinked to original sources

PubMed2006

[Long-term results following denervation of the wrist in patients with stages II and III SLAC-/SNAC-wrist].

INTRODUCTION: Mid-carpal arthrodesis, proximal row carpectomy and wrist arthrodesis are commonly cited in the literature as successful methods for pain reduction in patients with advanced carpal collapse (SLAC-/SNAC-wrist stages II and III). However, studies of isolated wrist denervation in patients who still possess good wrist mobility appear to be an underrepresented topic, especially in the German literature. PURPOSE: Based on follow-up results, it was possible to establish to what extent patients benefit long-term from isolated wrist denervation, whilst retaining wrist mobility. PATIENTS AND METHOD: 46 patients (6 female, 40 male) with a diagnosis of SLAC-wrist (n = 10) or SNAC-wrist (n = 36) stages II and III underwent wrist denervation of points 1 - 4, 6, 9 and 10 according to Wilhelm, between 1990 and 2001, following a positive denervation test. No previous reconstructive wrist surgery had been performed on any of the patients, whose average age at the time of denervation was 47 years. In 29 cases the dominant hand was affected, and in 22 cases the symptoms could be classified as post-traumatic. Post-operative follow-up was possible in 32 out of 46 cases (70 %), and occurred on average 6.3 years post-denervation (range 2.3 to 14 years). RESULTS: Twelve out of 32 patients (32 %) reported to be pain-free at follow-up, with a post-operative period ranging from 2.3 to 11.4 years (average 6.2 years). Six patients (18.75 %) continued to experience pain when load-bearing, which remained unchanged over a period of several years. Three patients (9 %) reported pain after stress, five patients (15.6 %) the recurrence of severe pain, which developed on average 3.8 years post-operatively. Two patients (6.25 %) experienced no analgesic benefits following wrist denervation, and four patients underwent wrist arthrodesis on average 13.5 months post-denervation, due to remaining or additional symptoms. Nineteen out of 32 patients subjectively reported a significant improvement following wrist denervation. Reassessment of range of movement post-denervation showed a reduction of 8.1 % in wrist extension/flexion, and 20 % in radio/ulna-abduction. Compared to the unaffected side, this represented an average loss of 28 % wrist extension/flexion, and 33.5 % radio/ulna-abduction. An average 51 % increase in hand strength could be shown at follow-up, leaving an average 30 % deficit compared to the unaffected hand. Evaluation of these results using the Krimmer score showed a good - very good outcome in 20 of the 32 patients (62.5 %); six patients were found to have a satisfactory outcome, and six patients a poor outcome. Subjective measures of pain (obtained using visual analogue scales) showed a reduction from 68.13 pre-operatively to 25.63 post-operatively. The DASH assessment (parts A and B) attained an average post-operative value of 17.1. CONCLUSION: Denervation of the wrist in patients with SLAC-/SNAC-wrist stages II and III can achieve a long-term elimination or reduction of pain, whilst improving hand strength, and having only a minimal impact on wrist range of movement. Wrist denervation should therefore be given preference over wrist arthrodesis, midcarpal arthrodesis or proximal row carpectomy in patients with good wrist mobility.

Adult↗

PubMed2002

A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction.

Quantification of nucleic acids, especially of mRNA, is increasingly important in biomedical research. The recently developed quantitative real-time polymerase chain reaction (PCR) - a highly sensitive technology for the rapid, accurate and reproducible quantification of gene expression - offers major advantages over conventional quantitative PCR. Transcript quantification is performed in the exponential phase of the PCR reaction through extrapolation of fluorescence signals from a standard calibration curve which represents the initial copy number for a given fluorescence signal. We have developed a method for gene transcript quantification which is based on a LightCycler - assisted real-time PCR in combination with a simple and rapid approach for the construction of external cRNA standards with identical gene sequences as the target gene. Synthesis of cRNAs was performed by in vitro transcription with T7 RNA polymerase followed by reverse transcription and real-time PCR. We applied this approach for transcript quantification of eukaryotic initiation factor 3 p110 (EIF3S8) mRNA in normal testicular tissue. We also present a rapid and simple strategy for the construction of cRNA standards for use in real-time PCR.

Adult↗

PubMed2002

[Treatment of bites to the hand and wrist--is the primary antibiotic prophylaxis necessary?].

Animal bites make up a large proportion of the injuries treated in an emergency department. Due to the type of injury, the variety of wounds and the contamination with aerobic and anaerobic organisms, they deserve special attention. In this study, we reviewed 98 patients (55 male/43 female) with bite wounds to the hand and wrist treated between 1995 and 2000. They were either treated conservatively (n = 65) or surgically (n = 33) depending on the clinical findings. In 18 of 33 cases, the reason for surgical treatment was an infection. A primary antibiotic prophylaxis, usually with cephalosporines, was administered in 47 of 98 cases. Results were analysed retrospectively. An infection developed in 32 patients. In six of these patients, an infection developed despite primary antibiotic prophylaxis. Operative treatment became necessary in four of these six cases. Twenty-six of 32 patients were treated without primary antibiotic prophylaxis. Surgical treatment was required in around half (n = 14) of these patients, while the other 12 patients were treated conservatively with antibiotic therapy.Twenty-one of 26 patients presented with bite wounds that were already infected. Microbiological examination revealed a variety of microbes, usually a mixed infection with Pasteurella multocida was found. All organisms were susceptible to treatment with second or third generation cephalosporines.A total of 15 patients had to be operated due to deeper injuries to the bone, extensive soft-tissue injury, or because of injury to a tendon and the tendon sheath. In most patients, a good to acceptable functional result was achieved. Primary antibiotic prophylaxis does not prevent the development of infection. Nevertheless, because of the inherently high infection risk associated with bite wounds to the hand and wrist, prophylaxis should be carried out. In case of severe damage to the soft tissue or signs of infection, early surgical therapy should be considered.

Adolescent↗

PubMed2001

Serological diagnosis of Epstein-Barr virus infection by novel ELISAs based on recombinant capsid antigens p23 and p18.

A new pair of Epstein-Barr virus ELISAs (Biotest Anti-EBV VCA IgG and VCA IgM ELISA) was evaluated for usefulness for routine diagnosis of acute EBV infections. The ELISAs are based on two viral capsid antigens (VCA), p23 (BLRF2, full-length) and p18 (BFRF3, carboxy-half), that are combined by autologous gene fusion. In total, 179 sera were tested in direct comparison with classical VCA immunofluorescence assays (IFA). With the help of clinical data and additional reference serology, i.e., heterophile antibodies, anti-EA IgG (IFA) and anti-EBNA-1 IgG (ELISA), the patients were divided into the following categories: seronegatives (46), acute primary infections (67), previous infections (39), suspected reactivations (20) and constellations with intermediate serological patterns (7). The VCA IgG and VCA IgM ELISAs showed overall agreement to IFA of 95.0% and 94.4%, respectively. The calculated analytical performance (sensitivity; specificity) of VCA IgG and VCA IgM was 94.0%; 97.8% and 97.1%; 96.5%, respectively. A certain delay in seroconversion of anti-p23-p18 IgG may account for a significant difference in sensitivity of the VCA IgG ELISA between primary (88.4%) and previous infections (100%). In summary, the new recombinant VCA ELISAs yielded good correlation to VCA IFA and in combination with EBNA-1 IgG allow rapid, sensitive, and specific diagnosis of infectious mononucleosis or EBV immune status in general.

Adolescent↗

PubMed2001

An antigen fragment encompassing the AD2 domains of glycoprotein B from two different strains is sufficient for differentiation of primary vs. recurrent human cytomegalovirus infection by ELISA.

Primary human cytomegalovirus (HCMV) infection during pregnancy is a frequent cause of fatal damage in populations with low prevalence of HCMV. Differentiation of primary vs. recurrent HCMV infection is an important issue in prenatal counseling. Antibodies specific for viral glycoproteins become detectable only with considerable delay with relation to HCMV infection or IgG seroconversion. Thus, lack of glycoprotein specific (gp-specific) antibodies can serve as a convenient indicator to identify those pregnant women that bear an elevated risk for HCMV transplacental transmission and fetal sequelae. In the opposite case, presence of gp-specific antibodies virtually excludes HCMV primary infection several weeks before sampling. However, no standardized screening assay for HCMV gp-specific antibodies had been available thus far. For this reason, an ELISA based on procaryotically expressed fragments of HCMV glycoprotein B (gB; gpUL55) was developed. Small fragments of gB from two different laboratory strains, encompassing the antigenic domain 2 (AD2) sufficed for sensitive and specific detection of gp-specific antibodies. The gB-ELISA titers correlated with titers of virus neutralizing antibodies in serum samples from primary or recurrent HCMV infections. Seroconversion kinetics of the gB-ELISA in samples from patients with primary HCMV infection closely paralleled the delay in seroconversion of gp-specific antibodies as determined by neutralization assay. Thus this assay provides a diagnostic tool that is easy to perform and can significantly add to available methods for the timely identification of primary HCMV infection during pregnancy. In addition, the gB-ELISA may be helpful in other clinical settings for the differentiation of primary HCMV infection from diseases caused by other pathogens.

Acute Disease↗

PubMed2001

The Ets-1 transcription factor is up-regulated together with MMP 1 and MMP 9 in the stroma of pre-invasive breast cancer.

The first steps of stroma generation are of pivotal importance for carcinogenesis because at this stage are initiated both angiogenesis, the prerequisite for continuous tumour growth, and the proliferation of stromal fibroblasts. These developments contribute to the onset of tumour invasion by secreting several matrix-degrading proteases. Both angiogenesis and the production of proteases are tightly controlled at several levels; of significant importance is transcription. The Ets-1 transcription factor transactivates several genes encoding matrix-degrading proteases and is thought to be involved in both tumour vascularization and invasion. This study therefore investigated, by in situ hybridization and immunohistochemistry, the expression of Ets-1 and of two of its target genes, encoding matrix metalloproteinase (MMP) 1 and MMP 9, in order to demonstrate a topographical in vivo correlation between the expression of these three genes during breast cancer formation. All three genes were first expressed within both endothelial cells and stromal fibroblasts during the onset of stroma generation around intraductal and intralobular in situ carcinomas and they were significantly up-regulated in the stroma of invasive ductal and lobular cancers. The results of this study further support the suggested in vivo role of Ets-1 for both angiogenesis and tumour invasion, via matrix-degrading proteases which are already expressed during the early stages of breast carcinogenesis.

Breast Neoplasms↗

PubMed2001

Extraction of CO2 from air samples for isotopic analysis and limits to ultra high precision delta18O determination in CO2 gas.

The determination of delta18O values in CO2 at a precision level of +/-0.02 per thousand (delta-notation) has always been a challenging, if not impossible, analytical task. Here, we demonstrate that beyond the usually assumed major cause of uncertainty - water contamination - there are other, hitherto underestimated sources of contamination and processes which can alter the oxygen isotope composition of CO2. Active surfaces in the preparation line with which CO2 comes into contact, as well as traces of air in the sample, can alter the apparent delta18O value both temporarily and permanently. We investigated the effects of different surface materials including electropolished stainless steel, Duran glass, gold and quartz, the latter both untreated and silanized. CO2 frozen with liquid nitrogen showed a transient alteration of the 18O/16O ratio on all surfaces tested. The time to recover from the alteration as well as the size of the alteration varied with surface type. Quartz that had been ultrasonically cleaned for several hours with high purity water (0.05 microS) exhibited the smallest effect on the measured oxygen isotopic composition of CO2 before and after freezing. However, quartz proved to be mechanically unstable with time when subjected to repeated large temperature changes during operation. After several days of operation the gas released from the freezing step contained progressively increasing trace amounts of O2 probably originating from inclusions within the quartz, which precludes the use of quartz for cryogenically trapping CO2. Stainless steel or gold proved to be suitable materials after proper pre-treatment. To ensure a high trapping efficiency of CO2 from a flow of gas, a cold trap design was chosen comprising a thin wall 1/4" outer tube and a 1/8" inner tube, made respectively from electropolished stainless steel and gold. Due to a considerable 18O specific isotope effect during the release of CO2 from the cold surface, the thawing time had to be as long as 20 min for high precision delta18O measurements. The presence of traces of air in almost all CO2 gases that we analyzed was another major source of error. Nitrogen and oxygen in the ion source of our mass spectrometer (MAT 252, Finnigan MAT, Bremen, Germany) give rise to the production of NO2 at the hot tungsten filament. NO2+ is isobaric with C16O18O+ (m/z 46) and interferes with the delta18O measurement. Trace amounts of air are present in CO2 extracted cryogenically from air at -196 degrees C. This air, trapped at the cold surface, cannot be pumped away quantitatively. The amount of air present depends on the surface structure and, hence, the alteration of the measured delta18O value varies with the surface conditions. For automated high precision measurement of the isotopic composition of CO2 of air samples stored in glass flasks an extraction interface ('BGC-AirTrap') was developed which allows 18 analyses (including standards) per day to be made. For our reference CO2-in-air, stored in high pressure cylinders, the long term (>9 months) single sample precision was 0.012 per thousand for delta13C and 0.019 per thousand for delta18O.

Air Pollution↗

PubMed2001

[Treatment of Galeazzi's fracture - is the surgical revision of the distal radioulnar joint necessary?].

PURPOSE: Fractures of the radial shaft associated with disruption of the distal radioulnar joint (DRUJ) are termed as Galeazzi-fractures. In Galeazzi's fracture, the aim of treatment is restoring the congruency of the joint and the stability of the DRUJ, thus preventing a loss of pronation or supination. PATIENTS AND METHOD: In this study, we included 24 patients (m = 22, f = 2) with 25 Galeazzi fractures treated between 1980 and 1998. Surgical treatment and the duration of therapy were analyzed retrospectively. The clinical and radiological results of 15 patients were followed up. Two children were treated conservatively with immobilization in an above-the-elbow plaster. 19 patients were treated surgically by rigid internal fixation with a plate approximately one week after the accident. Four patients were treated initially in a different way. In 13 cases, the distal radioulnar joint was immobilized by pinning with Kirschner wires. In ten patients, the DRUJ showed no instability. Patients with DRUJ pinning received an above the elbow plaster for six weeks, the other patients received a forearm cast for the time of wound healing. RESULTS: Two patients developed a pseudarthrosis following Kirschner-wire or rush-pin osteosynthesis. The consolidation of remaining fractures was regular. In two patients, the DRUJ was not completely stable after temporary fixation. The remaining patients revealed a stable DRUJ. Eight patients showed a limited pro- or supination after temporary Kirschner wire fixation of the DRUJ. The other patients did not reveal a decrease in range of motion. CONCLUSION: A stable and optimal reduction and a rigid internal osteosynthesis are requisites for healing of the radius fracture. Open reduction of the DRUJ is only indicated when soft tissue interposition prevents exact reposition. Surgical revision of the distal radioulnar joint was not necessary in our patients. Patients after Kirschner-wire fixation showed a diminished pro- or supination. To prevent Kirschner-wire failure, postoperative cast immobilization is indicated. Due to the retrospective nature of the study it is not definitely clear if Kirschner wire fixation is superior to immobilization.

Adolescent↗

PubMed2001

Cross-reactivity of Epstein-Barr virus-specific immunoglobulin M antibodies with cytomegalovirus antigens containing glycine homopolymers.

Timely and reliable detection of acute primary human cytomegalovirus (HCMV) infection is important in prenatal screening programs and for differential diagnosis of infectious mononucleosis-like disease. Enzyme-linked immunosorbent assays (ELISAs) based on HCMV proteins enable the sensitive detection of immunoglobulin M (IgM) antibodies during primary infection. However, concerns have been raised about possible cross-reactivities of the HCMV antigens used for the design of such ELISAs with IgM antibodies induced by Epstein-Barr Virus (EBV). In this study we investigated whether IgM antibodies generated during acute EBV infection reacted with recombinant HCMV antigens. Serum samples from patients with primary EBV infection frequently scored positive when tested in different HCMV IgM ELISAs, irrespective of whether conventional or recombinant antigens were used for the design of the HCMV IgM assays. Such cross-reactive IgM antibodies were found to be directed against short glycine-rich motifs contained within the nonstructural HCMV proteins pUL44 and pUL57. Further analyses revealed that these glycine-rich motifs were major antigenic domains for IgM antibodies induced during HCMV infection. Their deletion from recombinant proteins abrogated reactivity with IgM synthesized during HCMV infection. EBV-induced IgM antibodies that reacted with HCMV antigens showed similar kinetics of reactivity in HCMV- or EBV-specific assays in the course of primary EBV infection, indicating that the two populations of antibodies were highly overlapping. The results demonstrate that primary EBV infection leads to the induction of IgM antibodies that specifically bind to widely used diagnostic antigens of HCMV. This has to be considered in the interpretation of HCMV-specific IgM assays.

Amino Acid Motifs↗

PubMed2001

Invasive properties of serous human epithelial ovarian tumors are related to Ets-1, MMP-1 and MMP-9 expression.

The invasive potential of serous epithelial ovarian tumors is the main factor determining their biological behaviour. In contrast to invasive serous ovarian carcinomas serous borderline tumors generally present without stromal invasion and without or non-invading peritoneal implants. Little is known about the reasons underlying these differences. In the present study we found that two matrix-degrading metalloproteinases, collagenases 1 and 4 (MMPs 1 and 9), as well as the Ets-1 transcription factor are expressed at very low levels in serous benign cystadenomas, upregulated in the fibroblastic stroma, but not in the epithelium of borderline tumors and most strongly expressed in both stromal and epithelial tumor cells of serous invasive carcinomas. Since expression of Ets-1 and of MMPs 1 and 9 are topographically related, a transcriptional regulation of both proteases by Ets-1 is suggested. Upregulation of MMPs 1 and 9 within fibroblastic stromal cells of borderline tumors might be related to matrix remodelling and additional expression of both enzymes by the neoplastic cells of invasive carcinomas could then allow invasive propagation. The different expression patterns might supports the view, that no transition of serous borderline tumors into invasive carcinomas occurs.

3T3 Cells↗

PubMed2000

Eukaryotic initiation factor 3 p110 mRNA is overexpressed in testicular seminomas.

Testicular germ cell tumors are important neoplasms and seminoma accounts for 40 to 50% of these tumors. Little is known concerning the molecular events underlying the development of these malignancies. In the present study we used a modified differential display approach to compare gene expression between seminoma and normal testicular parenchyma, both of which are mixed tissues. We first analyzed mRNA (cDNA) expression by differential display and then directly used differentially expressed cDNAs for the synthesis of radiolabeled riboprobes to attribute differential expression to specific cell types in tissue sections by in situ hybridization. Using this approach along with real-time quantitative reverse transcriptase-polymerase chain reaction analysis we found an overexpression of eukaryotic initiation factor 3 p110 mRNA (EIF3S8) in seminoma cells compared to normal germ cells of testicular tubules. The elF3-p110 subunit may promote seminoma development by generally increasing translation leading to enhanced cellular growth and division rates.

Adult↗

PubMed2000

Generation of monoclonal antibodies directed against the immunogenic glycoprotein K8.1 of human herpesvirus 8.

Human Herpesvirus 8 (HHV-8) is clearly associated with Kaposi's sarcoma (KS), body cavity-based lymphomas (BCBL), and certain forms of multifocal Castleman's disease (MCD). It appears to be the sexually transmissible agent involved in the development of AIDS-associated KS. HHV-8 genomes are invariably present in BCBL-derived cell lines where lytic replication of the virus can be induced by phorbol esters (PE). First-generation HHV-8 serological assays were based on these cell lines. More recently, several genes encoding HHV-8 antigens have been identified. One of the most reactive antigens is encoded by HHV-8 open reading frame K8.1. Although K8.1 does not exhibit overt sequence homology to any other known gene, it is likely to be analogous to gp220/350 of Epstein-Barr or gp150 of murine herpesvirus-68, virion-envelope glycoproteins involved in target cell recognition. Mice were immunized with purified GST-K8.1 fusion protein expressed in E. coli. After fusion of murine plasma cells with the myeloma cell line P3-X63-Ag8. monoclonal antibodies (MAbs) were generated, which are specifically directed against K8.1 protein. The binding site for each MAb was identified by deletion mutant analysis using recombinant GST-K8.1 mutants and K8.1-specific peptides. Without exception, the epitopes recognized by these MAbs were located within the N-terminal part of the protein [amino acids (aa) 29 to 80], thus identifying a highly immunogenic region. These antibodies will not only be useful tools for HHV-8 diagnostics, but will also facilitate the analysis of K8.1 function.

Animals↗

PubMed2000

Evaluation of the measurement of leukotriene B4 concentrations in exhaled condensate as a noninvasive method for assessing mediators of inflammation in the lungs of calves.

OBJECTIVE: To determine whether measurement of an inflammatory mediator in exhaled condensate could provide a noninvasive method for evaluating lungs of calves. ANIMALS: 84 calves < or = 2 months old. PROCEDURE: Concentration of leukotriene B4 (LTB4) was evaluated in the exhaled condensate of healthy calves and calves with experimentally induced respiratory tract infections. For collection of samples of exhaled condensate, the total amount of exhaled air was directed into a cooled double-jacketed tube. Each tube was sealed and stored at -80 C. The LTB4 concentration was measured, using an ELISA. RESULTS: In exhaled condensates of clinically healthy calves, normally distributed and highly reproducible LTB4 concentrations (mean +/- SD, 116.1 +/- 55.4 pg/ml) were measured. After experimentally induced infection with Pasteurella multocida serovar D, LTB4 in exhaled condensate increased significantly (mean, 179% increase), compared with basal concentrations before infection; this increase in LTB4 was significantly correlated with deterioration in lung function. In 2 of 4 calves experimentally infected with bovine respiratory syncytial virus, the LTB4 concentration in exhaled condensate increased (300 to 400% increase), compared with baseline values, which was associated with development of bronchial hyperresponsiveness after infection. CONCLUSIONS AND CLINICAL RELEVANCE: Collection of exhaled condensate is tolerated well by calves and is an acceptable method for obtaining fluid from exhaled air originating from the lungs. This method provides alternatives for diagnosing and evaluating treatment of naturally acquired and experimentally induced diseases of the lungs and airways in calves.

Animals↗

PubMed1999

Comparison of the immunoglobulin-G-specific seroreactivity of different recombinant antigens of the human herpesvirus 8.

The open reading frames ORF 52, ORF 65, K12, and K8.1 of the human herpesvirus 8 (HHV8) were expressed as glutathione-S-transferase (GST) fusion proteins and analysed by Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). The open reading frame (ORF) 65 and K8.1 antigens gave the highest reactivity (71%) in sera from HIV-dependent Kaposi's sarcoma (KS) patients. Therefore both antigens appear to be essential for HHV8 diagnostics, whereas ORF K12 and ORF 52 were of minor importance. Using polymerase chain reaction (PCR) out of the peripheral blood of these KS patients, 48% were detected as positive. By testing an N-terminal-deleted construct (amino acid 80-171) of ORF 65, we could show that the N-terminal region of this protein is essential to mediate full immunogenic reactivity. By analysing different deletion mutants of ORF K8.1, the major epitope was found to be located between aa 29 and 101. The prevalence of antibodies directed against the different antigens was determined for healthy blood donors to be 3-6%. The different antibody patterns obtained in HIV-patients with and without KS support the hypothesis that different antibody profiles develop during the course of KS.

Antigens, Viral↗

PubMed1999

Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2.

The life-threatening complications of sepsis in humans are elicited by infection with Gram-negative as well as Gram-positive bacteria. Recently, lipopolysaccharide (LPS), a major biologically active agent of Gram-negative bacteria, was shown to mediate cellular activation by a member of the human Toll-like receptor family, Toll-like receptor (TLR) 2. Here we investigate the mechanism of cellular activation by soluble peptidoglycan (sPGN) and lipoteichoic acid (LTA), main stimulatory components of Gram-positive bacteria. Like LPS, sPGN and LTA bind to the glycosylphosphatidylinositol-anchored membrane protein CD14 and induce activation of the transcription factor NF-kappaB in host cells like macrophages. We show that whole Gram-positive bacteria, sPGN and LTA induce the activation of NF-kappaB in HEK293 cells expressing TLR2 but not in cells expressing TLR1 or TLR4. The sPGN- and LTA-induced NF-kappaB activation was not inhibited by polymyxin B, an antibiotic that binds and neutralizes LPS. Coexpression together with membrane CD14 enhances sPGN signal transmission through TLR2. In contrast to LPS signaling, activation of TLR2 by sPGN and LTA does not require serum. These findings identify TLR2 as a signal transducer for sPGN and LTA in addition to LPS.

Cell Line↗

PubMed1999

Cutting edge: cells that carry A null allele for toll-like receptor 2 are capable of responding to endotoxin.

Toll-like receptor (TLR) 2 and TLR4 have been implicated in the responses of cells to LPS (endotoxin). CD14-transfected Chinese hamster ovary (CHO)-K1 fibroblasts (CHO/CD14) are exquisitely sensitive to endotoxin. Sequence analysis of CHO-TLR2, compared with human and mouse TLR2, revealed a single base pair deletion. This frameshift mutation resulted in an alternative stop codon, encoding a protein devoid of transmembrane and intracellular domains. CHO-TLR2 cDNA failed to enable LPS signaling upon transient transfection into human epithelial kidney 293 cells. Site-directed mutagenesis of CHO-TLR2 enabled expression of a presumed full-length hamster TLR2 that conferred LPS responsiveness in human epithelial kidney 293 cells. Genomic TLR2 DNA from primary hamster macrophages also contained the frameshift mutation found in CHO fibroblasts. Nevertheless, hamster peritoneal macrophages were found to respond normally to LPS, as evidenced by the induction of cytokines. These results imply that expression of TLR2 is sufficient but not essential for mammalian responses to endotoxin.

Alleles↗

PubMed1999

Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes.

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Loss of heterozygosity, differentiation, and clonality in microdissected male germ cell tumours.

Testicular germ cell tumours (TGCTs) are heterogeneous neoplasms with different histological patterns and malignant potential. The aim of this study was to determine whether the main TGCT subtypes (seminoma, embryonal carcinoma, yolk sac tumour, choriocarcinoma, and mature teratoma) are distinguished by their loss of heterozygosity (LOH) patterns and whether LOH typing can help to distinguish between clonal and multifocal development of different components in mixed TGCTs. In 76 tumours analysed for allelic losses at 25 chromosomal loci, different LOH patterns were found in distinct histological subtypes. A region around D18S543 frequently lost in yolk sac tumours could harbour one or more tumour suppressor genes. In 20 microdissected mixed tumours, losses of identical alleles in different histological components in 11 of 20 cases (over 50 per cent) were found, which is in favour of current histogenetic models of clonal TGCT development. Clonal losses were most often found at D13S317 (6 of 20 tumours). Two classes of allelic losses may therefore occur during TGCT development: clonal losses which are involved in early transformational events and others related to TGCT differentiation along different lines.

Adult↗