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Objective: Although the benefits of antenatal glucocorticoids are well known for infants who are born preterm, there is increasing evidence of adverse effects on brain development, which may relate to altered metabolic activity. We have determined the effect of maternal glucocorticoid administration at doses that are used clinically on cerebral substrate metabolism in the preterm ovine fetus., Study Design: Chronically instrumented pregnant sheep at 0.85 gestation received 2 intramuscular injections of betamethasone at 170 microg/kg maternal weight (n = 13) or saline (n = 10) 24 hours apart together with a continuous infusion of L-[1-(13)C] leucine to the fetus. Fetal cerebral substrate arteriovenous differences (O2, glucose, leucine, leucine enrichment) and blood flow (fluorescent microspheres) were measured at baseline, 24 hours after the first betamethasone/saline injection (late beta/saline 1), and 4 hours after the second betamethasone/saline injection (early beta/saline 2) to obtain substrate deliveries and fractional extractions., Results: Fetal pH, blood gases, and metabolites were little changed in either group over the course of the study, except for glucose values in the betamethasone animals, which increased 1.4- and 1.9-fold, measured late beta 1 and early beta 2, respectively (both P < .01). Cerebral blood flow, although little changed in the control group or at late beta 1, was decreased at early beta 2 by approximately 30% (P < .05). As such, early beta 2 animals showed a decrease in cerebral O2 delivery of approximately 20% (P = .06) and conversely an increase in cerebral glucose delivery of 1.4- and 1.3-fold at late beta 1 (P < .05) and early beta 2 (P = .08), respectively. Fractional extraction values for these substrates were not changed significantly, which resulted in corresponding decreases in estimated O2 uptake and increases in estimated glucose uptake, such that the glucose/oxygen quotient (as an index of glucose oxidative metabolism) measured 1.6 at early beta 2, which was considerably greater than baseline values at 1.1 (P < .05). Fractional extraction values for leucine and leucine enrichment averaged 2%-3%; although somewhat higher in the betamethasone animals, none of the between or within group differences were significant., Conclusion: Fetal cerebral metabolism in the preterm ovine fetus is altered by antenatal glucocorticoid administration, which is comparable with that used in human pregnancy, and includes an acute decrease in cerebral blood flow and a probable increase in anaerobic glucose metabolism. Although likely of short duration in conjunction with peak glucocorticoid levels, these metabolic effects may place the developing brain at added risk for superimposed hypoxic injury.

The role of the margin in leaf development has been debated over a number of years. To investigate the molecular basis of events in the margin, we performed an enhancer trap screen to identify genes specifically expressed in this tissue. Analysis of one of these lines revealed abnormal differentiation in the margin, accompanied by an abnormal leaf size and shape. Further analysis revealed that this phenotype was due to insertion of the trap into DWF4, which encodes a key enzyme in brassinolide biosynthesis. Transcripts for this gene accumulated in a specific and dynamic pattern in the epidermis of young leaf primordia. Targeted expression of DWF4 to a subset of these cells (the leaf margin) in a dwf4 mutant background led to both restoration of differentiation of a specific group of leaf cells (margin cells) and restoration of wild-type leaf shape (but not leaf size). Ablation of these cells led to abrogation of leaf development and the formation of small round leaves. These data support the hypothesis that events in the margin play an essential role in leaf morphogenesis, and implicate brassinolide in the margin as a key mediator in the control of leaf shape, separable from a general function of this growth factor in the control of organ size.

Background: The impact of infections with the human cytomegalovirus (HCMV) for the development of atherosclerosis and restenosis is still unclear. Both a clear correlation and no correlation at all have been reported in clinical, mostly serological studies. In our study we employed a human non-injury ex vivo organ culture model to investigate the effect of an in vitro permissive HCMV-infection on cell proliferation and neointimal hyperplasia for a period of 56 days., Results: During routine-nephrectomies parts of renal arteries from 71 patients were obtained and prepared as human organ cultures. Cell free HCMV infection was performed with the fibroblast adapted HCMV strain AD169, the endotheliotropic strain TB40E, and a clinical isolate (AN 365). After 3, 7, 14, 21, 28, 35, and 56 days in culture staining of HCMV-antigens was carried out and reactive cell proliferation and neointimal thickening were analysed. Successful HCMV-infection was accomplished with all three virus strains studied. During the first 21 days in organ culture no cell proliferation or neointimal hyperplasia was detected. At day 35 and day 56 moderate cell proliferation and neointimal hyperplasia was found both in HCMV-infected segments and mock infected controls. Neointimal hyperplasia in productively HCMV-infected segments was lower than in non infected at day 35 and day 56, but relatively higher after infection with the endotheliotropic TB40E in comparison with the two other strains., Conclusion: The data do not support the hypothesis that HCMV-infection triggers restenosis via a stimulatory effect on cell proliferation and neointimal hyperplasia in comparison to non infected controls. Interestingly however, even after lytic infection, a virus strain specific difference was observed.

Paralysis of one or both sides of the larynx musculature compromises breathing and speech function. Currently there is no surgical remedy to restore adequate function of the larynx. A plausible alternative solution is triggered electrical stimulation of the paralysed larynx site using a laryngeal pacemaker. Triggering of the pacemaker succeeds via constant EMG measurement of the muscle activity of the healthy larynx side. The EMG data analysis described in this work is one possible approach for regulating pacemaker triggering. In this study we used EMG data from the thenar muscles as a model to calculate a trigger point.

Human cytomegalovirus (HCMV) infection appears to be linked to the pathogenesis of atherosclerosis. An association between HCMV infection and an enhanced restenosis rate as well as the induction of vasculopathies after solid organ transplantation has been documented. Knowledge of the cellular and molecular basis of these findings is limited, however. By Northern blot and RT-PCR analysis of human foreskin fibroblasts (HFF) and human coronary artery smooth muscle cells (SMC), we identified extracellular matrix (ECM) genes that were downregulated after HCMV infection, including collagen type I and fibronectin. Quantitative immunoassays showed a significant reduction of soluble collagen type I and fibronectin proteins in supernatants of both cell types. This was shown to be a direct effect of HCMV infection and not due to a response to interferons released from infected cells, since neutralization of alpha and beta interferon activity could not block virus-induced downregulation of matrix proteins. As the amount of ECM depends on both synthesis and degradation, we also assessed the influence of HCMV on the activity of matrix metalloproteinases (MMP). Interestingly, a significant difference in virus-induced matrix degradation could be shown between the two cell types. HCMV upregulated MMP-2 protein and activity in SMC but not in HFF. Thus, HCMV infection of SMC reduces ECM dramatically by inducing two independent mechanisms that influence synthesis as well as degradation of ECM. These may represent molecular mechanisms for HCMV-induced pathogenesis of inflammatory vasculopathies and may facilitate dissemination of HCMV by promoting the detachment of infected cells in vivo.

Objective: The objective of this study was to explore risk behavior and routes of transmission in men having sex with men (MSM) with newly diagnosed sexually transmitted infections (STIs)., Methods: A questionnaire on clinical diagnosis and manifestation site for acute STIs was completed by physicians participating in a sentinel study. Patients contributed information on sexual risk behavior and the likely route of STI transmission., Results: Three hundred fifty-six diagnosis forms and 169 matching patient questionnaires could be analyzed. The most frequent diagnosis was syphilis (n = 147; 33% primary syphilis with ulcer localization 71% genital, 22% anorectal, and 8% oral; 67% secondary syphilis), followed by gonorrhea (n = 136; 59% genital, 34% rectal, 7% pharyngeal) and Chlamydia trachomatis infection (n = 51; 48% genital, 48% rectal, 4% pharyngeal). In 12 patients, more than one infection was diagnosed, and 2 or 3 sites were affected in 11 patients. Approximately 60% of infections were acquired by genital-oral and oral-anal practices. Unprotected anal intercourse (UAI) was reported more often by HIV-positive men (mostly receptive) and men with high partner numbers., Conclusion: High partner numbers, an important role of genital-oral sexual practices for the transmission of STIs, and relatively high frequencies of mostly receptive UAI in HIV-positive men are all contributing to increasing STI incidences among MSM.

In order to collect more convincing data on the aetiological agent of young pigeon disease syndrome (YPDS), a comprehensive study was performed on pigeons in German lofts with or without outbreaks of YPDS. The investigations included examination of histories, clinical signs and pathology, as well as parasitological and microbiological analysis. Pigeons in their 4th to 12th week of life exhibited clinical signs at higher frequency and with greater severity than pigeons of other ages. Greenish liquid in the crop, proventriculus and ventriculus, and yellow fluid in the small intestine were seen more often in YPDS-affected pigeons. Escherichia coli and Klebsiella pneumoniae were isolated more frequently from these birds. Depletion of splenic and bursal lymphocytes was only seen in pigeons with YPDS. Inclusion bodies were present in various organs, especially the bursa of Fabricius. The genome of pigeon circovirus was detected in lymphoid tissues from all pigeons with YPDS. The results of this study indicate that YPDS is a multifactorial disease in which pigeon circovirus might be a crucial factor, possibly by inducing immunosuppression in infected birds.

Background: Cytomegaloviruses have been shown to promote atherogenesis in animal models. In humans, several epidemiological and clinical studies suggest involvement of the human cytomegalovirus (HCMV) in the development of atherosclerosis. HCMV is suspected to be associated with an enhanced restenosis rate and the occurrence of vasculopathies after solid organ transplantation. However, knowledge about the cellular and molecular bases of these findings is very limited., Methods and Results: Human coronary artery smooth muscle cells (HCASMC) were successfully infected with HCMV in vitro. Infection of HCASMC with all HCMV strains analyzed resulted in a substantial upregulation of the beta-receptor of platelet-derived growth factor (PDGFR-beta) expression as demonstrated by immunohistochemistry, immunofluorescence, FACS, and Western blot analysis. The amount of PDGFR-beta protein present in HCASMC rapidly increased after 12 h of infection and this difference persisted for 72 h post-infection. We showed by quantitative FACS analysis that the extent of PDGFR-beta upregulation differed significantly between the HCMV strains TB40E, Toledo, and AD169. The expression of insulin-like growth factor receptors as well as hepatocyte growth factor receptors, however, was down-modulated in HCMV-infected HCASMC. Most importantly, the HCMV-associated upregulation of PDGFR-beta protein resulted in functionally intact receptors. A significantly higher increase of proliferative activity following stimulation with PDGF-BB was observed in HCMV-infected HCASMC compared to the uninfected control., Conclusions: Our data suggest that HCMV directly activates the PDGF system, which could promote atherogenesis and restenosis by activation of smooth muscle cell proliferation and neointima formation.

Stabilization of beta-catenin by inhibiting the activity of glycogen synthase kinase-3beta has been shown to initiate axis formation or axial patterning processes in many bilaterians. In hydra, the head organizer is located in the hypostome, the apical portion of the head. Treatment of hydra with alsterpaullone, a specific inhibitor of glycogen synthase kinase-3beta, results in the body column acquiring characteristics of the head organizer, as measured by transplantation experiments, and by the expression of genes associated with the head organizer. Hence, the role of the canonical Wnt pathway for the initiation of axis formation was established early in metazoan evolution.

Human cytomegalovirus (HCMV) infection is known to modulate host gene expression and has been linked to the pathogenesis of vasculopathies; however, relevant pathomechanisms are still unclear. It was shown that HCMV infection leads to upregulation of vascular endothelial growth factor (VEGF) expression in human foreskin fibroblasts and coronary artery smooth muscle cells (SMC). Activation of VEGF transcription by HCMV infection was confirmed by transient-expression experiments, which revealed that a short promoter fragment, pLuc135 (-85 to +50), is sufficient for activation. Site-directed mutagenesis of Sp1-recognition sites within this fragment abolished the upregulation of transcription. Functional VEGF protein is released into the culture supernatant of infected SMC. Incubation of endothelial cells with supernatants from HCMV-infected SMC cultures induced upregulation of VEGF receptor-2 expression on endothelial cells, as well as a significant upregulation of DNA synthesis, implicating cell proliferation. The mean incline of DNA synthesis at 48 and 72 h post-infection was 148 and 197 %, respectively. Addition of neutralizing antibodies against VEGF completely abolished this effect. Supernatants from SMC cultures incubated with UV-inactivated virus induced a comparable effect. This virus-induced paracrine effect may represent a molecular mechanism for HCMV-induced pathogenesis, such as inflammatory vasculopathies, by inducing a proatherogenic phenotype in SMC.

Developmental gradients play a central role in axial patterning in hydra. As part of the effort towards elucidating the molecular basis of these gradients as well as investigating the evolution of the mechanisms underlying axial patterning, genes encoding signaling molecules are under investigation. We report the isolation and characterization of HyBMP5-8b, a BMP5-8 orthologue, from hydra. Processes governing axial patterning are continuously active in adult hydra. Expression patterns of HyBMP5-8b in normal animals and during bud formation, hydra's asexual form of reproduction, were examined. These patterns, coupled with changes in patterns of expression in manipulated tissues during head regeneration, foot regeneration as well as under conditions that alter the positional value gradient indicate that the gene is active in two different processes. The gene plays a role in tentacle formation and in patterning the lower end of the body axis.

Background: Current modalities of renal replacement therapy allow only a limited removal of larger, possibly toxic molecules, which accumulate in uraemia. Recently, a haemodiafilter has been made available with the new, high-flux, polyethersulfone-based membrane DIAPES HF800. We performed a study to compare DIAPES HF800 with two conventional high-flux membranes in on-line haemodiafiltration (HDF), with respect to the removal properties for the two marker proteins, beta(2)-microglobulin (beta(2)m, 11.8 kDa) and albumin (66.5 kDa)., Methods: In a prospective, controlled study 10 stable end-stage renal disease patients were randomly allocated to 30 sessions of post-dilutional on-line HDF with three types of steam-sterilized membranes: DIAPES HF800, polysulfone and polyamide. Blood flow rate was 250 ml/min and treatment time was 240 min. Pre-treatment beta(2)m and albumin plasma concentrations did not differ between the three groups. The concentration of the two proteins was determined before and after treatment in plasma as well as in the continuously collected haemodiafiltrate., Results: Tolerance of all treatments was very good, without any side-effects for all filters. The mean plasma reduction rate of beta(2)m was 77 +/- 1% for DIAPES HF800 and polysulfone whereas it was 71 +/- 1% for polyamide (P < 0.05). The mean beta(2)m amount removed and found in the haemodiafiltrate per session was 230 +/- 14 mg for DIAPES HF800, 186 +/- 13 mg for polysulfone and 147 +/- 13 mg for polyamide (P < 0.05 between each pair of membranes). The same ranking was obtained for albumin removed and found in haemodiafiltrate per session for the three membranes: 5.7 +/- 0.4, 3.5 +/- 0.4 and 1.0 +/- 0.4 g, respectively. Although DIAPES HF800 showed the highest value for albumin in haemodiafiltrate the mean post-treatment plasma albumin was higher after the treatment with DIAPES HF800 compared with the other membranes (P < 0.05)., Conclusions: On-line HDF has shown to achieve plasma reduction rates for beta(2)m of up to 77% for the DIAPES HF800 membrane and for polysulfone. The amounts of beta(2)m and albumin in haemodiafiltrate were much higher for DIAPES HF800 than for the other two membranes indicating a greater permeability for molecules up to a molecular weight of 66.5 kDa. This could, at least theoretically, offer the advantage also to remove uraemic toxins in the molecular weight range of albumin or of albumin-bound toxins. The future must show whether this will counterbalance the loss of albumin.

Studies with animal cytomegaloviruses, epidemiological data from humans as well as in vitro studies suggest the involvement of the human cytomegalovirus (HCMV) in the development of atherosclerosis. Cell culture systems are insufficient for examination of the entire pathogenetic process and a satisfactory animal model for HCMV is not available. An organ culture model was established for HCMV infection of human renal arteries in vitro. After infection with three representative HCMV strains, infectious virus was recovered from supernatants until 144 days post-infection with a peak around day 30 due to a long-lasting productive HCMV infection in still vital cells. Differences in cell tropism and kinetics of infection were identified between the HCMV strains. Specifically, differences in infecting endothelial cells and virus penetration into the lamina media were observed. In infected artery segments, but also in some non-infected arteries from seropositive donors, HCMV DNA could be localized by in situ PCR. Nevertheless, HCMV early antigen was detected by immunohistochemistry exclusively in artery segments infected in vitro. The new organ culture model will permit the study of functional and molecular consequences of HCMV infection in a more physiological micro-environment.

Whether or not infectious agents are involved in the pathogenesis of atherosclerosis has been a matter of discussion for the past 2 decades. Although there is no definite proof of a causal role of human cytomegalovirus in atherogenesis, a body of knowledge supports the concept that this virus is involved in the development of atherosclerotic lesions. This review assesses the most important data published in support of this hypothesis.

Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.

Background: Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells, stimulates angiogenesis, and increases vascular permeability. Increased VEGF expression has been associated with poor clinical outcomes in many malignancies. Several recent reports have documented over expression of VEGF in papillary thyroid cancer. We hypothesized that increased expression of VEGF would be associated with either an increased risk of recurrence or a decreased recurrence-free survival in papillary thyroid cancer., Methods: Immunohistochemistry was used to detect VEGF expression in archival paraffin-embedded surgical thyroid specimens from 96 subjects with papillary thyroid cancer., Results: VEGF expression was detected in 98% (94/96) of the samples, predominantly of slight-to-moderate intensity in the majority of malignant cells. However, the specific finding of a diffuse pattern of intense immunostaining for VEGF was detected significantly more often than less intense, patchy immunostaining patterns in subjects with distant metastasis at diagnosis (63% versus 15%, P =.005), local recurrence (58% versus 13%, P =.001), and distant recurrence (83% versus 14%, P =.001). Furthermore, this specific pattern of diffuse, intense VEGF expression was associated with a significantly shorter recurrence-free survival than other staining patterns (P =.007)., Conclusions: These data demonstrate that the immunohistochemical pattern of VEGF staining in the initial surgical specimen is strongly associated with the incidence of local and distant metastasis in papillary thyroid cancer.

A spatially localized photochemical reaction induced by near-field femtosecond laser pulses is demonstrated on a nanometer scale and used for high-density optical data storage. Recorded domains down to 120 and 70 nm are obtained with one-photon and two-photon excitation, respectively. It is shown that the local-field confinement that is due to the quadratic dependence of two-photon excitation on light intensity has the potential to increase the near-field optical storage density.

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF withdrawal results in progressive ES cell differentiation. Here we show that during this differentiation process, part of the cells undergo apoptosis concomitant with an activation of the p38 MAP kinase. To gain insight into events mediated by LIF in ES cells, the expression of potential candidate genes was analyzed in the absence or presence of this cytokine by using a semiquantitative RT-PCR assay. We focused on early response genes and on a new type of cytokine repressors (the Socs proteins), some of which exhibit anti-apoptotic properties. We found that expression of c-Fos, c-Jun, and JunB was induced upon LIF treatment whereas that of JunD, the tyrosine phosphatase ESP, and the components of the LIF receptor remained unaffected. Expression of Socs-3, but not Socs-1 or Socs-2, was stimulated in the presence of LIF. Finally, uncontrolled overexpression of Socs-1 and Socs-3 led to repression of LIF-dependent transcription and severely reduced cell viability, suggesting that the disturbance of a well balanced Socs protein content has adverse effects on cell survival.

The influence of human cytomegalovirus (HCMV) on the transcription of 11 selected, representative extracellular matrix genes was investigated in cell culture. Northern blot hybridization indicated the downregulation of all mRNAs investigated. Based on our results and the known repression of other extracellular matrix transcripts and the beta-actin transcription during HCMV infection, we suggest that one molecular mechanism contributing to the cytopathic effect may be the transcriptional downregulation of genes encoding proteins involved in cell structure and intercellular connection. To further study the biological relevance of this and other pathogenetic mechanisms, we established a human renal artery organ culture system and characterized this new infection model for HCMV. Our model is a new suitable system for the investigation of molecular as well as functional consequences of HCMV infection in a more physiological microenvironment., (Copyright 2000 S. Karger AG, Basel.)

To monitor early and late events of immune system activation after primary and secondary flavivirus infection, 17 healthy persons were vaccinated with the standard 17D vaccine virus strain of yellow fever (YF). Twelve of these persons had not received YF vaccine previously and 5 had been vaccinated once at least 10 years before. Viremia and various parameters of humoral and cellular immune activation were followed daily for 7 days and weekly thereafter. Viremia was detected by reverse transcriptase-polymerase chain reaction in all 12 first-time vaccinees beginning from the second to the sixth day after vaccination; most tested positive between the fourth and sixth day. Infectious 17D virus was detected using a plaque forming assay in the serum of 7 of the 12 first-time vaccinees. As first parameters of immune activation, neopterin and beta2-microglobulin markedly increased between day 2 and day 6 postvaccination. In parallel to the viremia, circulating CD8+ T-cells significantly increased, with peak levels at day 5 after primary vaccination, indicating an activation of the cellular immune system. Neither viremia nor significant changes of these activation markers were observed in the five revaccinated persons. Neutralizing antibodies directed against the 17D vaccine strain developed in all persons within 2 weeks after vaccination. No correlation was found between the extent of viremia and the titer of neutralizing antibodies. Revaccination was followed by a minor and transient increase of neutralizing antibodies. High titers of neutralizing antibodies persisted for at least 10 years after primary vaccination.

Hepatic veno-occlusive disease (VOD) is a frequent and severe complication after bone marrow transplantation (BMT). We previously have described plasminogen activator inhibitor-1 (PAI-1) as a possible marker of VOD. To confirm the significance of this finding, we now determined PAI-1 levels in 31 of 186 consecutive patients undergoing BMT who developed hyperbilirubinemia greater than 3 mg/dL for various reasons. Diagnoses were made by clinical criteria and confirmed by biopsy in 23 of 31 patients. They included VOD (n = 7), acute graft-versus-host disease (GVHD) of the liver (n = 7), and other hepatic injury (n = 17). PAI-1 (mean +/- SD) was significantly (P < .001) elevated in patients with VOD (321.6 +/- 161.2 ng/mL) as compared with patients with GVHD (22.8 +/- 8.4 ng/mL) or other hepatic damage (32.8 +/- 30.8 ng/mL) at the timepoint of bilirubin increase. At the peak bilirubin concentration, the corresponding PAI-1 levels were 426.1 +/- 230.0 ng/mL in patients with VOD, 41.0 +/- 20.6 ng/ mL in patients with GVHD, and 44.6 +/- 32.9 ng/mL in patients with other hepatic injury (P < .001 VOD v GVHD/other hepatic injury). Our results underline the relevance of PAI-1 in the differential diagnosis of hyperbilirubinemia after BMT and its significance as a sensitive and specific marker of severe VOD.

Veno-occlusive disease (VOD) of the liver is one of the most frequent fatal complications after bone marrow transplantation (BMT). A decrease of natural anticoagulants, in particular protein C (PC), has been assumed to be involved in the pathogenesis of the disease. We determined PC and antithrombin III (AT III) levels in two patients undergoing BMT and subsequent liver transplantation due to VOD. Additionally, in one of the patients protein S (PS) levels were also measured. Normal baseline (day-8) PC levels (86 and 89%) were markedly reduced in both patients at the time of VOD manifestation on day 20 and 40, respectively (26 and 31%). PS levels lay within the normal range from day-8 (before myeloablative chemotherapy) until one week after clinical onset of VOD when substitution therapy with fresh frozen plasma (FFP) was initiated. AT III levels decreased moderately during the second and third posttransplant week, but were normal in the patient with a late clinical manifestation of VOD. In both patients PC and PS levels lay within the normal range after liver transplantation which was performed on day 41 and 79, respectively. AT III was substituted several times. Both patients died due to infectious complications on day 141 and 101, respectively. The data confirm previous reports that a decrease of PC is observed in BMT recipients and can be associated with hepatic vein occlusion. Whereas the relevance of AT III is uncertain, PS does not seem to be involved in the pathogenesis of VOD. Liver transplantation lead to normalization of PC levels, but its significance remains to be discussed in terms of ethical justifiability, medical feasibility and costs.

Tumor necrosis factor-alpha (TNF alpha) is a central mediator in the pathogenesis of sepsis. It also interferes with the hemostatic system and exerts and a net procoagulant effect. Since TNF alpha may contribute to thrombotic complications in sepsis patients, we determined markers of thrombin activation, parameters of the fibrinolytic system (D-dimer, tissue plasminogen activator antigen (tPA) urinary type plasminogen activator antigen (uPA), plasminogen activator inhibitor antigen (PAI-1) and von Willebrand factor antigen (vWF) in 30 patients with sepsis or septic shock. All patients were treated with standard therapy, but 14 patients were treated additionally with an anti-TNF alpha monoclonal antibody (MAK 195F); 16 patients served as historical controls. No significant effect of the antibody on the parameters of the hemostatic system could be determined. Our data speak against a modulation of coagulation or the fibrinolytic system by the monoclonal anti-TNF alpha antibody MAK 195F in this cohort of sepsis patients.

Capillary leak syndrome (CLS) is a severe complication after bone marrow transplantation (BMT). To investigate whether there is a pathogenetic role of the complement system, we monitored the levels of the terminal complement complex C5b-9 (TCC) and C3a-desArg as indicators of an activation of the complement system and the inhibitor of the classical pathway of the complement cascade, C1 inhibitor (C1-INH), in 48 bone marrow transplant recipients from 1 week before to 5 weeks after transplantation. Capillary leak syndrome developed in 7 out of 48 patients between days 1 and 12 after BMT. Complement activation as indicated by TCC levels was more pronounced in patients with CLS (n = 7) from day -8 to +28 (p < 0.05; day -1) and the elevation of TCC levels lasted longer in CLS patients (peak day 21) than in patients without this complication (peak day 7). Mean C3a-desArg levels were highest in patients with CLS reaching a peak at day 7. During the early posttransplant period a significant elevation of C1-INH levels (p < 0.01 and p < 0.05 respectively) compared with baseline levels (day -8) was found in patients with and without CLS, which was more pronounced in those patients with CLS (p < 0.05). Although we could not observe an absolute C1-INH deficiency as compared to healthy individuals our data support the presence of a relative deficiency of the inhibitor which might explain the reported beneficial effects of C1-INH substitution in BMT related CLS.

Three-photon-absorption-induced frequency-upconversion f luorescence emission has been observed in a solution of 2,5-benzothiazole 3,4-didecyloxy thiophene (BBTDOT) in tetrahydrofuran, pumped with 10-ns Q-switched 1.06-microm laser pulses. The spectral peak of the observed fluorescence emission is located in the 450-480-nm range, and the intensity dependence of the visible emission on the IR excitation obeys the cubic law. At a higher solute concentration (0.18 M/L) and moderate IR excitation intensity levels (50-200 MW/cm(2)), obvious optical limiting behavior has been observed as a result of three-photon absorption. The measured nonlinear absorption coeff icient and the corresponding molecular three-photon-absorption cross section are gamma= 2.7 x 10(-18) cm(3)/W(2) and sigma'(3) = 8.8 x 10(-76) cm(6) s(2), respectively.

Veno-occlusive disease (VOD) represents one of the more frequent and most severe complications after BMT. The pathophysiology of VOD is poorly understood. To investigate a possible link between endothelial cell damage and VOD, tissue plasminogen activator (tPA) and its inhibitor (PAI-1) were measured in 32 patients as endothelial cell-derived parameters of the fibrinolytic system. A nearly fivefold increase (mean 103.9 ng/ml, range 22.6-582.4 ng/ml, p < 0.05) in PAI-1 levels was found in the four patients who developed VOD compared with patients without this complication (mean 22.2 ng/ml, range 1.4-131.6 ng/ml). No significant difference was found in tPA levels between patient groups with or without VOD or other complications following BMT, indicating a shift of the fibrinolytic balance towards hypofibrinolysis particularly in patients with VOD. We conclude that alterations of the fibrinolytic system occur in patients undergoing BMT. Hypofibrinolysis seems to be at least one factor in the pathogenesis of VOD and the determination of PAI-1 might be helpful for diagnosing the disease. Our data also may explain the reported successful treatment of VOD by recombinant tPA.

Background: Veno-occlusive disease (VOD) leads to obliteration of small intrahepatic venules and is one of three most important complications with fatal outcome after bone marrow transplantation (BMT). The etiology of VOD is not completely understood. Endothelial cell injury induced by the conditioning myeloablative radiochemotherapy with subsequent activation of the coagulation cascade seems to be a crucial step in the pathogenesis of the disease., Patients and Methods: We investigated tissue plasminogen activator (tPA), its main inhibitor (PAI-1) and the natural anticoagulants protein C and S by enzymimmunoassay prospectively in 32 bone marrow transplant recipients., Results: VOD developed in four patients. They presented with extremely elevated levels of PAI-1 after BMT whereas tPA levels remained low. Additionally a transient decrease of protein S was found one week after BMT which was more pronounced in VOD patients. No protein C deficiency was observed., Conclusion: Our data suggest that hypofibrinolysis due to an excess of PAI-1 may be involved in the pathogenesis of VOD. The determination of PAI-1 may be useful to recognize the development of VOD and facilitate the decision for thrombolytic therapy with rtPA. A decrease of protein S may play a role as a cofactor in the early phase after BMT.

In these studies, neonatal C.B-17 severe combined immunodeficient (nSCID) mice were reconstituted with human cord blood leukocytes (hu-CBLs). The resulting hu-CBL-nSCID mice contained readily detectable human CD3+ T lymphocytes and CD20+ human B cells, and produced substantial levels of human IgM and IgG (including all subclasses). Human cells persisted in lymphoid organs and peripheral blood for at least 8 weeks, and CD4+ T cells outnumbered CD8+ T cells. Engraftment of human cells in peripheral lymphoid organs and blood was much greater than that seen in adult SCID mice grafted with adult peripheral blood leukocytes (PBLs). Hu-CBL-nSCID mice were susceptible to infection with laboratory-adapted and fresh clinical human immunodeficiency virus type 1 (HIV-1) isolates. Following infection with HIV-1, virus could be recovered by the coculture of spleen, lymph node, peritoneal cavity, liver, and plasma samples from hu-CBL-nSCID mice with fresh human peripheral blood mononuclear cells, and proviral copies were detectable following amplification using the polymerase chain reaction (PCR). HIV p24 core antigen levels in hu-CBL-nSCID mouse plasma were consistent with ongoing viral replication and high viral burdens. Rapid CD4+ T cell depletion occurred following infection with laboratory isolates of HIV-1 or a syncytium-inducing clinical isolate, but a non-syncytium-inducing clinical isolate caused expansion of CD8+ T cells, leading to an inversion of the CD4:CD8 ratio with only a transient decrease in CD4+ T cells. These results suggest that the hu-CBL-nSCID mouse system has unique features that mimic certain aspects of pediatric HIV infection, and distinguish it from other animal models of HIV infection, including the related hu-PBL-SCID model.

Escherichia coli JM103 carrying the expression plasmid pMTC48, repressor plasmid pRK248, and protection plasmid pEcoR4 was grown in a 60-L working volume airlift tower loop reactor on M9 minimal medium. Production of fusion protein SpA::EcoRI was induced by a temperature shift from 30 to 38 (optimum), 40, or 42 degrees C. The following parameters were monitored: cell mass concentration (X), total cell counts (TCC), number of colony-forming units (CFU), concentrations of glucose, acetate, ethanol, pyruvate, lactate, succinate, amino acids, and ammonia, and soluble and total protein content, as well as product concentration (enzyme activity of the fusion protein), dissolved oxygen concentration, oxygen utilization rate (OUR), CO2 production rate (CPR), respiration quotient (RQ), and volumetric mass-transfer coefficients (kLa). Product formation by temperature shift was only observed if LB concentrate was added to the culture at the same time the aeration rate was increased to avoid oxygen-transfer limitation. No product accumulation was observed with glucose and ammonia supplementation. During gene expression, X and TCC increased, CFU decreased, acetate increased, and the primary metabolite (ethanol, pyruvate, lactate) concentrations as well as OUR and CPR passed a maximum while RQ changed only slightly. These facts indicate that, during gene expression, the metabolic activity of the cell passes a maximum, and after that it decreases. With increasing aeration rate, the volumetric productivity increased, but the specific productivity with respect to the cell concentration decreased.

There are currently no accepted criteria for positive Western blots in Lyme disease. In a retrospective analysis of 225 case and control subjects, the best discriminatory ability of test criteria was obtained by requiring at least 2 of the 8 most common IgM bands in early disease (18, 21, 28, 37, 41, 45, 58, and 93 kDa) and by requiring at least 5 of the 10 most frequent IgG bands after the first weeks of infection (18, 21, 28, 30, 39, 41, 45, 58, 66, and 93 kDa). When these definitions were tested in a prospective study of all 237 patients seen in a diagnostic Lyme disease clinic during a 1-year period and in 74 patients with erythema migrans or summer flu-like illnesses, the IgM blot in early disease had a sensitivity of 32% and a specificity of 100%; the IgG blot after the first weeks of infection had a sensitivity of 83% and a specificity of 95%. Among patients with indeterminate IgG responses by ELISA, 6 of 9 patients with active Lyme disease had positive blots compared with 2 of 34 patients with other illnesses (P < .001). Thus, Western blotting can be used to increase the specificity of serologic testing in Lyme disease.

Single and multisensor field effect transistors (FET) with a pH-sensitive Si/SiO2/Si3N4/Ta2O5-gate and reference electrode (for single sensor) were developed and used for manufacturing the following biological (Bio)-FETs: for glucose analysis, glucose oxidase-FET (GOD-FET); for urea analysis, urease-FET; and for cephalosporin C analysis, cephalosporinase-FET. The GOD-FETs were integrated into flow injection analysis (FIA) of the Eppendorf variables analyser (EVA) system and used for monitoring the glucose concentration in microbial cultivation and production processes with recombinant Escherichia coli K12 MF, recombinant E. coli JM103, Saccharomyces cerevisiae H620, and Candida boidinii. Urease-FET-FIA was used to monitor the urea concentration in a simulated cultivation of Cephalosporium acremonium and urease-FET-FIA and GOD-FET-FIA for the monitoring of urea and glucose concentrations in simulated S. cerevisiae cultivations.

Among 6 patients with prolonged episodes of Lyme arthritis, the mean response of peripheral blood lymphocytes (PBL) to all Borrelia burgdorferi antigens (stimulation index [SI] 46) was greater than that among 5 patients with brief attacks of Lyme arthritis (SI 13; P less than 0.1), as well as that among 7 control patients with rheumatoid arthritis and among 6 normal control subjects (in both instances SI 3; P less than 0.05). In individual patients with brief episodes of Lyme arthritis, PBL had similar low levels of reactivity with the 20-kd, 31-kd, 34-kd, 41-kd, 55/58-kd, and 66-kd spirochetal polypeptides. In individual patients with prolonged arthritis, PBL usually had similar marked responsiveness to the 34-kd, 41-kd, 55/58-kd, and 66-kd polypeptides, but they had greater reactivity with the 34-kd outer surface protein B than with the 31-kd outer surface protein A (P less than 0.05). In the 2 patients tested, paired samples of synovial fluid lymphocytes and PBL had a similar pattern of reactivity, but the response was 2-100-fold greater in synovial fluid lymphocytes. We conclude that patients with prolonged Lyme arthritis have T cell responses to multiple spirochetal polypeptides.

The kinetics of chlorophyll fluorescence were measured during the state 1-state 2 and the state 2-state 1 transition in some members of the Florideae (Halymenia latifolia, Phycodrys rubens and Delesseria sanguinea) and in Porphyra umbilicalis as a member of the Bangiales. With the members of the Florideae it was possible to induce 70-80% of the maximum transition in direction to state 1 (obtained by us in longtime experiments) by an illumination of only a few (2-5) seconds with medium intensities of light 1. A complete transition back could be induced by a similar short illumination with light 2. The transition process itself is slow with values of tau1/2 in the range of 10 to 20 s and occurs in the dark with nearly the same speed as in continuous light 1 or light 2. The state 1-state 2 transient of chlorophyll fluorescence is kinetically clearly different from the dark-light transient phenomenon. In dark times longer than 1 min Halymenia (starting from state 1) slowly approaches state 2. This decay of state 1 is slower by a factor of about five in Halymenia than in Porphyra. This may explain, why in Halymenia, but not in Porphyra, the whole transition process can be resolved experimentally into a fast light reaction and a slow dark reaction. The results are discussed on the basis of a model which assumes the rearrangement of charges in the plane of the thylakoid membrane as a primary inducing event in the state 1-state 2 transition. This may lead to a redistribution of excitation energy among Photosystems I and II by a slow dark process, which may include a conformation change of some protein(s).

To evaluate the immediate effect of exposure to a high level of noise on the sympatho-adrenal and pituitary-adrenal systems, measurements were made of circulating catecholamines, growth hormone, ACTH, and cortisol in seven normal male subjects. They were studied on two random experimental days: a control day and a noise-exposure day with an intermittent noise alternating between 99 dB (A) and 45 dB (A) for 2 h. Analysis did not reveal any variation in the plasma levels of norepinephrine (NE), epinephrine (E) or dopamine (D), measured every 20 min, which might have been related to noise exposure. Similarly, analysis of the 2-h urine samples collected from 8 a.m. to 2 p.m. revealed no significant changes in urinary catecholamine excretion. Plasma levels of GH and ACTH did not differ significantly from those for control days, but cortisol showed a brief, significant levelling-off in its pattern during the exposure period. The data demonstrate that exposure to a high level of noise, although considered as "unpleasant", does not induce any important endocrinological changes in man. These conclusions differ from those for studies on animals where reactions to noise may be related to a more general stressing situation.

The effect of 90-min heat exposure (46 degrees C, 35 mbar) on plasma aldosterone (PA) patterns was studied and the respective roles of plasma renin activity (PRA), adrenocorticotropin (ACTH), Na+ and K+ concentrations in the control of PA response were in investigated in eight subjects on a low sodium diet and in five subjects on a high sodium diet. In all subjects, transitory PA increases of varying importance were observed, which were not related to sweat losses (less than 1% bodyweight) or to rectal temperature rise. In sodium-repleted subjects, basal PA and PRA levels as well as heat-induced rises were low (mean PA peak level = 12.62 +/- 1.15 ng/100 ml). They were enhanced by sodium depletion and PA reached a mean peak level of 34.07 +/- 2.73 ng/100 ml. But, in both conditions, the heat-induced PA peaks were 3-times higher than the initial levels. PA correlated with PRA in all but one of the sodium-repleted subjects and in 6 of the 8 sodium-depleted subjects. ACTH release, as measured by plasma cortisol (PC) levels, occurred in those subjects who noted an increased feeling of annoyance and discomfort. Thus, PA correlated positively with PC in 4 sodium-depleted subjects. A high sodium intake improved heat-tolerance. Plasma K+ and Na+ concentrations were not significantly modified by exposure to heat. PA increases can occur without concomitant changes in PRA, PC, K+ or Na+, which suggests that an additional factor may play a role in aldosterone regulation during acute heat exposure.

Specific associations of spectrin with Bands 2.1 and 4.1 have been examined by measuring the binding of purified 125I-Band 2.1 and 125I-Band 4.1 to [32P]spectrin in solution. Binding of Bands 2.1 and 4.1 to spectrin was measured as 125I radioactivity precipitated by an anti-spectrin. Staphylococcus aureus complex. The association between spectrin and Band 2.1 is characterized by relatively high affinity (Kd congruent to 10(-7) M at pH 7.6) and saturation of available binding sites at a molar ratio of 1:1 (Band 2.1/spectrin heterodimer). Band 4.1 binding to spectrin is characterized by a similar affinity (Kd congruent to 10(-7) M at pH 7.6) with saturation of available sites occurring at a stoichiometric ration of 2:1 (Band 4.1/spectrin heterodimer). Scatchard plots of Band 4.1 binding to spectrin are curvilinear and consistent with a positively cooperative interation. Bands 2.1 and 4.1 bind to different sites on the spectrin molecule: unlabeled Band 4.1 does not competitively displace 125 I-Band 2.1 from spectrin in solution, and low angle rotary-shadowed platinum-carbon replicas of these polypeptides reveal two discrete binding sites.

A study was carried out in 11 children, ranging in age from 7 to 12 years, to determine the clinical effect of tamitinol, in addition to psychotherapy, in alleviating disturbances of learning and behaviour. Tamitinol was administered as one 100 mg tablet per 10 kg body weight daily for 8 weeks. Assessments were made of the patients' mental condition using a 7-point rating scale, before and during treatment. Global response to treatment and tolerance were evaluated at the end of the trial by the clinician and the patients (or parents). Significant improvement was noted in 7 of the 10 children who completed the study. Six of the 13 psychiatric symptoms evaluated by the clinician were significantly improved (p less than 0.01). The drug was well tolerated by all patients.

We have used electron microscopy to examine purified intact variable surface glycoproteins (VSGs) from clones derived from two distinct stocks of Trypanosoma brucei. The VSG molecule from MITat 1.2 has a large elongated domain consistent with the shape of the dimeric N-terminal domain determined by X-ray analysis (see preceding paper), and a heretofore unseen short, thin fibrous tail presumed to be the C-terminal domain. Electron microscopy on DiTat 1.3, however, indicates a morphology quite distinct from that of MITat 1.2. Analysis of four VSG amino acid sequences reveals 7-fold periodicities (heptad repeats) which indicate that alpha-helical coiled-coil secondary structure elements occur in all of these VSGs, consistent with the observation of helical bundles in one VSG. These results suggest the possibility that VSG antigenic diversity may be related to a diversity in length and disposition of alpha-helical bundles and coiled-coil domains.