Biomedical subjects
Publications and source records attributed to M Berger.
Pooled data from 32 studies were reviewed to assess the timing of thrombolic complications after cardioversion of atrial fibrillation or flutter. We found that 98% of embolic episodes occurred within 10 days of cardioversion.
Neck pain after whiplash injury of the cervical spine often induces typical changes in head motion patterns (amplitude, velocity). These changes of kinematics may help to recognize malingerers. We investigated the hypothesis that malingerers are not able to reproduce their simulated head movement disturbances three times. The kinematics of head movements of 23 patients with neck pain after whiplash injury and of 22 healthy subjects trying to act as malingerers were compared. The healthy subjects were informed about the symptomatology of whiplash injury and were asked to simulate painful head movements. Two different kinds of head movements were registered and analyzed by Cervicomotography: (1) the slow free axial head rotation (yaw) and (2) the axial head rotation (yaw) tracking a moving visual target. Each experimental condition was presented three times, expecting the malingerers not to be able to produce as well as to reproduce the same head movement disturbances again and again. In patients, as a consequence of their distinct pain patterns, we expected less variance between the test repetitions. The statistical analysis showed significant differences of the calculated kinematic parameters between both groups and the inability of healthy subjects to simulate and to reproduce convincingly distinct pain patterns.
CD11b+ monocytic/macrophagic cells (Mo/Mph), which infiltrate into skin after UV irradiation, play an important role in UV-induced immunosuppression. Because in mice, blockade of CD11b (iC3b receptor) on monocytes and depletion of its ligand, iC3b, reverses UV-induced immunosuppression, we asked whether iC3b is deposited in human skin after UV, and whether iC3b can modulate the cytokine profile of Mo/Mph. Immunofluorescence studies revealed that iC3b was newly deposited in UV-exposed skin and was localized in apposition to infiltrating CD11b+ Mo/Mph. In addition, in situ hybridization studies showed that TNF-alpha mRNA was also induced in a similar microanatomic localization. To model the effects of these complex signals on infiltrating Mo/Mph following UV exposure, we then tested the effects of immobilized iC3b and TNF-alpha on resting blood monocytes. Both IL-10 mRNA synthesis and protein secretion were significantly induced by binding of iC3b in vitro and were synergistically increased by the presence of TNF-alpha. The effect was abrogated by a blocking Ab to CD11b, indicating CD11b-iC3b interaction. In contrast, iC3b binding resulted in suppression of IL-12 p40 mRNA and significantly inhibited the production of IL-12 p70 protein. Our studies thus define a novel mechanism for induction of tissue Mo/Mph into an IL-10high/IL-12low state via iC3b in combination with TNF-alpha.
We have previously demonstrated that human granulocyte-macrophage colony-stimulating factor fused to a truncated diphtheria toxin (DT388-GM-CSF) is toxic to patient acute myeloid leukemia progenitors bearing the GM-CSF receptor, but not normal marrow progenitors. We now report that exposure of mononuclear cells from five of seven (71%) juvenile myelomonocytic leukemia (JMML) patients and from 12 of 20 (60%) adult chronic myelomonocytic leukemia (CMML) patients to 10(-9) mol/L DT388-GM-CSF for 48 hours in culture reduces the number of cells capable of forming colonies in semisolid medium (colony-forming units-leukemia) 10-fold to 300-fold (1 to 2.5 log decrease). In contrast, normal myeloid progenitors (colony-forming unit-granulocyte-macrophage) from six different donors treated and assayed under identical conditions were consistently insensitive to the same fusion toxin even when treated as highly purified CD34(+) cells. The leukemic progenitors from the two other JMML patients showed intermediate sensitivity to DT388-GM-CSF and the leukemic progenitors from eight of the 20 (40%) CMML patients were not different from normal progenitors. Parallel measurements of the number and affinity of GM-CSF receptors on cells from the same samples showed no consistent differences between JMML, CMML, and normal light density or CD34(+) bone marrow cells. The increased sensitivity of leukemic progenitors from all JMML progenitors and some CMML patients to the fusion toxin is therefore not likely to be explained by an increased density of GM-CSF receptors on these cells. We also examined the DT388-GM-CSF sensitivity of two murine cell lines transfected with cDNAs encoding varying portions of the human GM-CSF receptor and/or beta chains. These studies showed that high-affinity ligand binding was sufficient for DT388-GM-CSF-induced toxicity, as this could occur even in the absence of functional signal transduction and that the background of the host cell had a major influence on the degree to which this decreased the toxicity of DT388-GM-CSF. The selective sensitivity to DT388-GM-CSF of leukemic progenitors from a majority of JMML and CMML patients suggests that this agent could have therapeutic potential for some patients with these diseases.
Polyunsaturated fatty acids (PUFAs) exert immunosuppressive effects, but the molecular alterations leading to T cell inhibition are not yet elucidated. Signal transduction seems to involve detergent-resistant membrane domains (DRMs) acting as functional rafts within the plasma membrane bilayer with Src family protein tyrosine kinases being attached to their cytoplasmic leaflet. Since DRMs include predominantly saturated fatty acyl moieties, we investigated whether PUFAs could affect T cell signaling by remodeling of DRMs. Jurkat T cells cultured in PUFA-supplemented medium showed a markedly diminished calcium response when stimulated via the transmembrane CD3 complex or glycosyl phosphatidylinositol (GPI)- anchored CD59. Immunofluorescence studies indicated that CD59 but not Src family protein tyrosine kinase Lck remained in a punctate pattern after PUFA enrichment. Analysis of DRMs revealed a marked displacement of Src family kinases (Lck, Fyn) from DRMs derived from PUFA-enriched T cells compared with controls, and the presence of Lck in DRMs strictly correlated with calcium signaling. In contrast, GPI-anchored proteins (CD59, CD48) and ganglioside GM1, both residing in the outer membrane leaflet, remained in the DRM fraction. In conclusion, PUFA enrichment selectively modifies the cytoplasmic layer of DRMs and this alteration could underlie the inhibition of T cell signal transduction by PUFAs.
We have assessed the functional effect of CD99 engagement on resting human peripheral blood (PB) T cells. CD99, as detected by the mAb 3B2/TA8, is constitutively expressed on all PB T cells and becomes further up-regulated upon cellular activation. In this study we demonstrate that cross-linking of the CD99 molecule with the agonistic mAb 3B2/TA8 cooperates with suboptimal TCR/CD3 signals, but not with phorbol ester, ionomycin, or CD28 mAb stimulation, to induce proliferation of resting PB T cells. Comparable stimulatory effects were observed with the CD99 mAb 12E7. Characterization of the signaling pathways involved revealed that CD99 engagement leads to the elevation of intracellular Ca2+, which is dependent on the cell surface expression of the TCR/CD3 complex. No CD99 mAb-induced calcium mobilization was observed on TCR/CD3-modulated or TCR/CD3-negative T cells. To examine the impact of CD99 stimulation on subsequent cytokine production by T cells, we cross-linked CD99 molecules in the presence of a suboptimal TCR/CD3 trigger followed by determination of intracellular cytokine levels. Significantly, T cell lines as well as Th1 and Th0 clones synthesized TNF-alpha and IFN-gamma after this treatment. In contrast, Th2 clones were unable to produce IL-4 or IFN-gamma when stimulated in a similar fashion. We conclude that CD99 is a receptor that mediates TCR/CD3-dependent activation of resting PB T cells and specifically induces Th1-type cytokine production in polyclonally activated T cell lines, Th1 and Th0 clones.
Feline odontoclastic resorptive lesions (FORL), previously known as 'neck lesions,' are commonly known in domestic, but also in non-domestic cats. They are characterized by odontoclastic resorptive processes, which take place at the dental root and at the periodontium. Chronic inflammation of gingiva and periodontium is believed to be an important etiological factor in the development of FORL. In this context, various feline viruses have been discussed to play a relevant role in the pathogenesis of these lesions. The aim of this project was to determine in a blinded study the incidence of FORL in 10 cats which were infected for several years with feline immunodeficiency virus (FIV), but were otherwise free of feline viral infections (feline leukemia virus, feline calicivirus, feline herpesvirus, feline parvovirus, feline coronavirus, feline syncytium-forming virus). Nine age-matched controls were kept under identical conditions, but free of FIV. Subgingival resorptive lesions were found in six of 10 FIV-positive cats, but in three of nine controls only. FIV-positive cats had significantly more often gingivae with an increased tendency for bleeding upon probing than FIV-negative cats (p=0.0055), and they had slightly more often hyperplastic gingivae (p=0.0867). In conclusion, signs characteristic of FORL such as subgingival lesions, granulomatous or hyperplastic gingivae with a tendency for bleeding, were found significantly more often in FIV-positive cats than in the controls (p=0.0198). Therefore, it was concluded that FIV infection is an important factor for the occurrence of FORL, possibly through immune suppression or changes of the (sub)gingival micro-environment. However, non-infected control cats also showed some evidence of FORL in the absence of all tested viral infections. Therefore, factors other than viral infections must also play a role in the development of FORL in cats.
We have isolated genomic recombinants containing the complete gene coding for the rabbit translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF) P23. The gene is organized into five introns and six exons and its total length amounts to 3819 nucleotides. All intron/exon boundaries are in accordance with the GT/AG rule. Transcription of the gene generates two mRNAs of 843 and 1163 nucleotides differing in the length of their 3'-untranslated regions. They are formed by alternative polyadenylation. The transcription initiation site has been determined by comparison of sequences of the gene and several processed TCTP pseudogenes. The full-length 5'-untranslated region comprises 116 nucleotides and starts with an oligopyrimidine tract important for translational regulation. Additionally 1.2 kb of the 5'-flanking promoter region has been sequenced. The promoter contains a TATA box at -30 and potential binding sites for transcription factors such as stimulating protein 1 (Sp1), nuclear factor 1 (NF1), activator protein 1 (AP1), c-Ets1, cAMP-response element (CP2), myeloid-specific zinc finger protein 1 (MZF1) and others. For functional analysis 5'-flanking sequences up to -918 were fused to the chloramphenicol acetyltransferase (CAT) gene and tested using a rabbit aortic smooth-muscle cell line by cell transfection and CAT assays. The results confirm that the analyzed gene is the actively transcribed TCTP gene.
Periodic limb movements during sleep (PLMS), with or without symptoms of a restless legs syndrome (RLS), may cause sleep disturbances. The pharmacologic treatments of choice are dopaminergic drugs. Their use, however, may be limited due to tolerance development or rebound phenomena. Anecdotal observations have shown that oral magnesium therapy may ameliorate symptoms in patients with moderate RLS. We report on an open clinical and polysomnographic study in 10 patients (mean age 57 +/- 9 years; 6 men, 4 women) suffering from insomnia related to PLMS (n = 4) or mild-to-moderate RLS (n = 6). Magnesium was administered orally at a dose of 12.4 mmol in the evening over a period of 4-6 weeks. Following magnesium treatment, PLMS associated with arousals (PLMS-A) decreased significantly (17 +/- 7 vs 7 +/- 7 events per hour of total sleep time, p < 0.05). PLMS without arousal were also moderately reduced (PLMS per hour of total sleep time 33 +/- 16 vs 21 +/- 23, p = 0.07). Sleep efficiency improved from 75 +/- 12% to 85 +/- 8% (p < 0.01). In the group of patients estimating their sleep and/or symptoms of RLS as improved after therapy (n = 7), the effects of magnesium on PLMS and PLMS-A were even more pronounced. Our study indicates that magnesium treatment may be a useful alternative therapy in patients with mild or moderate RLS-or PLMS-related insomnia. Further investigations regarding the role of magnesium in the pathophysiology of RLS and placebo-controlled studies need to be performed.
We have previously demonstrated that human granulocyte-macrophage colony-stimulating factor (GM-CSF) fused to a truncated diphtheria toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines bearing the GM-CSF receptor. We now report that exposure of malignant cells from 50 different patients with AML for 48 hours in culture to DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the number of leukemic cells capable of forming colonies in semisolid media (leukemic colony-forming cells [CFU-L]) with a median IC50 of 3 x 10(-12) mol/L (range, 5 to >4,000 x 10(-12) mol/L). Furthermore, the cell kill is dependent on the presence of high-affinity GM-CSF receptors on leukemic blasts, because CFU-L from 27 of 28 AML samples expressing > or = 35 GM-CSF receptors per cell were inhibited by the toxin, whereas the colony growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less than 35 receptors per cell was unaffected by the drug. Sensitivity of CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at near control numbers after exposure to much higher toxin concentrations (4 x 10(-9) mol/L) than those required to kill CFU-L from most patients. Thus, leukemic progenitors isolated directly from the peripheral blood of most AML patients show the same sensitivity to DT388-GMCSF as previously demonstrated for AML cell lines. Under the same conditions of exposure, normal hematopoietic progenitors are relatively unaffected by DT388-GMCSF, suggesting its potential as a therapeutic agent in AML.
Chemokine receptors play a crucial role in the recruitment of immune cells to sites of inflammation. Although chronic diseases of the brain are often accompanied by inflammatory events, there is presently no information about the occurrence and regulation of these receptors in the central nervous system (CNS). Moreover, one CC-chemokine receptor, CKR5, has recently been identified as coreceptor for HIV-1 entry into macrophages. HIV-1 target cells in brain are macrophage-related microglia, which suggests that they are infected by the same mechanism (He et al.,: Nature 385:645-649, 1997). Although rats are not susceptible to HIV-1 infection, they can be used to study chemokine receptor regulation in a variety of brain pathologies. After cloning CC-CKR5 and establishing reverse transcriptase polymerase chain reaction (RT-PCR) for its ligands macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated on activation, normal T cell-expressed and secreted (RANTES), we studied expression of these four mRNAs in purified microglia and compared it with their expression in rat brain. Lipopolysaccharide (LPS)-treated microglia showed transiently increased mRNA levels of both CKR5 and its ligands. Similar data were obtained from brains of LPS-injected rats. In middle cerebral artery occluded (MCAO)-animals, RANTES mRNA was unaffected, whereas CKR5 mRNA showed a sustained rise until 96 hr after surgery. MIPs exogenously added to microglial cultures markedly reduced CKR5 mRNA expression, whereas RANTES did not. MIP mRNAs, in contrast to RANTES and CKR5 mRNAs, were undetectable in normal brain. RANTES appears to play a role distinct from MIPs in brain. In summary, upregulation of CC-chemokines and CKR5 in the CNS upon bacterial infection or in ischemia may impact on microglial activation stage and result in increased risk of HIV-1 infection.
Polymorphonuclear neutrophils (PMN) contain multiple distinct secretory compartments that are sequentially mobilized during cell activation. Complement receptor type 1 (CR1) is a marker for a readily mobilizable secretory vesicle compartment, which can undergo exocytic fusion with the plasma membrane independently of secretion of traditional granule contents. The basis for the formation of these distinct compartments is incompletely understood. Primary and secondary granules are generated directly from the Golgi complex during different stages of development of the cell, obviating the need for sorting signals for proper packaging of their constituents. To determine whether the secretory vesicles are formed in a similar manner, we studied a stable rat basophilic leukemia cell line (RBL-CR1) transfected with a plasmid containing the cDNA of human CR1 driven by a viral promoter. The CR1 was present primarily intracellularly in small vesicles resembling the CR1 storage pools in resting PMN. Activation of RBL-CR1 resulted in translocation of intracellular CR1 to the plasma membrane, with mobilization requirements different from those of the classical RBL granules. Thus, in RBL-CR1, continuously synthesized CR1 is stored and upregulated in much the same way as in PMN. This suggests that differential timing of gene expression is not essential for proper storage of CR1 and that other sorting mechanisms are involved, which can be studied in RBL-transfectants.
In rabbit reticulocytes an arachidonic acid 15-lipoxygenase (15-LOX) is expressed at high yield. Rescreening a rabbit reticulocyte cDNA library for alternative 15-LOX transcripts, a full length cDNA which encodes a novel lipoxygenase was isolated. The predicted amino acid sequence of this enzyme shared a high degree (99%) of identity with the reticulocyte-type 15-lipoxygenase. Among the six amino acid residues different in both enzymes a Phe-Leu exchange was detected at position 353. Recently, site-directed mutagenesis studies have revealed that this amino acid exchange converts a 15-lipoxygenase to a 12-lipoxygenase. In fact, when the novel enzyme was expressed in Escherichia coli, mainly 12-lipoxygenation of arachidonic acid was observed. The recombinant enzyme exhibited a rather broad substrate specificity. Various C-18 and C-20 polyenoic fatty acids and even complex substrates such as biomembranes were effectively oxygenated. Thus, the novel enzyme may be classified as leukocyte-type 12-lipoxygenase. Genomic polymerase chain reaction of the 3' region of the leukocyte-type 12-lipoxygenase gene indicated that introns 10 to 13 differed to about 10% from the corresponding sequences of the 15-lipoxygenase gene although their size and the intron-exon organization were very similar. In the 3'-untranslated region of the novel mRNA a C+U-rich, 20-fold repetitive element was found which appears to be highly related to the differentiation control element of the 15-lipoxygenase mRNA. Activity assays with a variety of cells and tissues prepared from normal rabbits suggested that only peripheral monocytes abundantly express the enzyme, suggesting a tissue-specific regulation of gene expression. These data indicate for the first time the co-expression of two separate genes for a reticulocyte-type 15-lipoxygenase and for a leukocyte-type 12-lipoxygenase in one species. This is of importance for the implication of both enzymes in red blood cell development and atherogenesis.
During development, mice with mutations of stem cell factor (SCF) or its receptor c-kit exhibit defects in melanogenesis, as well as hematopoiesis and gonadogenesis. Consequently, accumulating evidence suggests that the c-kit/SCF system plays a crucial role in all of these processes and in tumors which derive from them. Especially in neuroblastoma (infant tumors of neuroectoderm crest derivation such as melanocytes) it would appear that an autocrine loop exists between c-kit and SCF, and that the functional block of the c-kit receptors with monoclonal antibodies (MoAbs) results in a significant decrease in cellular proliferation. We studied the expression and role of c-kit and SCF in cell lines of soft tissue sarcoma of neuroectodermic origin, such as Ewing's sarcoma (ES) and peripheral neuro-ectodermal tumors (PNET). Using flow cytometry with MoAb CD117 PE, c-kit expression was highlighted in all six of the cell lines examined. This receptor was specifically and functionally activated by SCF, as shown by the binding experiments and the intracellular phosphotyrosine and immunoprecipitation studies that were performed. Using reverse transcriptase polymerase chain reaction analysis, five of the six cellular lines expressed the mRNA of SCF. In the medium measured by using an enzyme- linked immunosorbent assay, low concentrations of SCF were found: only the TC32 cellular line produced significantly higher levels (32 pg) than control. In serum-free culture the addition of SCF reduced the percentage of apoptotic cells from 25% to 90% in five out of the six cellular lines. This observation was confirmed by (1) the functional block of c-kit with MoAb: after 7 days of culture more than 30% of the cells were apoptotic (range 31.5% to 100%) in five out of six cell lines and there was also a decrease in the percentage of cells in phase S, and (2) c-kit antisense oligonucleotides: in the cellular lines treated with oligonucleotides (in relation to the untreated lines) there was a notable reduction (P < .001) both in the absolute number of cells and the 3H-thymidine uptake. These results indicate that ES and PNET express c-kit and its ligand SCF and that SCF is capable of protecting the tumor cells against apoptosis. Furthermore, the reverse transcriptase-polymerase chain reaction performed on the biopsies revealed the presence of mRNA both of SCF and c-kit in practically all of the samples studied. Our in vitro data lead us to assume that SCF may also inhibit tumor cell apoptosis in vivo.
Quantification of the excess mortality from all causes associated with obesity remains controversial. In this paper, 6,193 obese patients, those with a body mass index (weight (kg)/height (m)2 (BMI)) range of 25-74 kg/m2, recruited from 1961 to 1994 in Düsseldorf, Germany, were followed for a mean time of 14 (standard deviation, 8.2) years, yielding 87,179 observed patient-years. During the study period, 1,028 patients (16.6%) died. The entire cohort was grouped into approximate quartiles according to BMI: group 1, BMI from 25 to < 32; group 2, BMI from 32 to < 36; group 3, BMI from 36 to < 40; group 4, BMI > or = 40 kg/m2. The following risk ratios were estimated by means of Cox proportional hazards models using the lowest BMI group as reference category: group 2 for men, 1.02 (95% confidence interval 0.76-1.37); for women, 1.23 (95% confidence interval 0.96-1.58); group 3 for men, 1.50 (95% confidence interval 1.09-2.06); for women, 1.33 (95% confidence interval 1.03-1.73); and group 4 for men, 2.10 (95% confidence interval 1.53-2.88); for women, 2.25 (95% confidence interval 1.78-2.84). The following standardized mortality ratios were calculated by using the respective geographic area (the Federal State of North Rhine Westphalia) as reference population: group 1 for men, 1.26 (95% confidence interval 0.98-1.61); for women, 1.00 (95% confidence interval 0.81-1.23); group 2 for men, 1.31 (95% confidence interval 1.09-1.57); for women, 1.20 (95% confidence interval 1.02-1.40); group 3 for men, 1.92 (95% confidence interval 1.53-2.38); for women, 1.27 (95% confidence interval 1.07-1.50); and group 4 for men, 3.05 (95% confidence interval 2.47-3.73); for women, 2.31 (95% confidence interval 2.04-2.60). In addition to age, sex, and BMI, Cox proportional hazards models revealed systolic blood pressure, glucose intolerance, diabetes, and smoking as significant independent mortality risk factors, whereas cholesterol was not significant. In this prospective study of a large cohort of obese persons, morbid obesity (BMI of > or = 40 kg/m2) was a strong predictor of premature death. Excess mortality risks associated with gross obesity (BMI from 32 to < 40 kg/m2) were considerably lower than hitherto assumed; moderate degrees of obesity (BMI from 25 to < 32 kg/m2) were not significantly associated with excess mortality.
BACKGROUND: Autologous peripheral blood stem cell (PBSCs) are frequently used to reconstitute hematopoiesis following administration of megatherapy in children with advanced stage IV neuroblastoma. Some centers prefer the use of autografts enriched for CD34+ progenitor cells because the positive selection procedure is believed to reduce indirectly tumor cell contamination. PROCEDURES: In this study, we monitored the efficiency of tumor cell purging following CD34 selection in PBSCs from seven patients with advanced neuroblastoma by using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, Amplification of tissues-specific mRNA transcript of tyrosine hydroxylase gene with nested primers enabled the detection of residual neuroblastoma cells with a sensitivity of one malignant-cell per 10(6) normals. RESULTS: Using this method, contaminating tumor cells were detected in seven of nine leukapheresis products of the patients. After positive immunoselection of CD34+ cells on Ceprate column, only one of nine enriched stem cell fraction still contained tumor cells detectable by RT-PCR. In six cases, PCR positive PBSCs became PCR negative after selection. CONCLUSIONS: We conclude that tumor cell contamination may be frequently detected in PBSC harvests of stage IV neuroblastoma patients by sensitive molecular analysis. The load of contaminating malignant cells might be reduced following CD34 selection.
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Quality-assurance activities will become more important in psychiatry during the next few years. In relation to other medical disciplines, some special aspects concerning structure, process and outcome quality as well as practical realization and methodologic aspects must be considered. These specific issues were the focus of a study dealing with the treatment of depressed inpatients. The experiences and results as well as considerations concerning future quality-assurance projects are discussed.