TAK1 in brain endothelial cells mediates fever and lethargy

Systemic inflammation affects the brain, resulting in fever, anorexia, lethargy, and activation of the hypothalamus-pituitary-adrenal axis. How peripheral inflammatory signals reach the brain is still a matter of debate. One possibility is that, in response to inflammatory stimuli, brain endothelial cells in proximity to the thermoregulatory centers produce cyclooxygenase 2 (COX-2) and release prostaglandin E2, causing fever and sickness behavior. We show that expression of the MAP kinase kinase kinase TAK1 in brain endothelial cells is needed for interleukin 1β (IL-1β)-induced COX-2 production. Exploiting the selective expression of the thyroxine transporter Slco1c1 in brain endothelial cells, we generated a mouse line allowing inducible deletion of Tak1 specifically in brain endothelium. Mice lacking the Tak1 gene in brain endothelial cells showed a blunted fever response and reduced lethargy upon intravenous injection of the endogenous pyrogen IL-1β. In conclusion, we demonstrate that TAK1 in brain endothelial cells induces COX-2, most likely by activating p38 MAPK and c-Jun, and is necessary for fever and sickness behavior.     

The tumor suppressor CYLD interacts with TRIP and regulates negatively nuclear factor kappaB activation by tumor necrosis factor

Cylindromas are benign adnexal skin tumors caused by germline mutations in the CYLD gene. In most cases the second wild-type allele is lost in tumor tissue, suggesting that CYLD functions as tumor suppressor. CYLD is a protein of 956 amino acids harboring a functional deubiquitinating domain at the COOH-terminal end. To shed more light on the function of CYLD, we have performed a yeast two hybrid screen using an HaCaT cDNA library that identified the RING finger protein TRIP (TRAF-interacting protein) as interactor with full-length CYLD. Mapping of the interacting domains revealed that the central domain of CYLD binds to the COOH-terminal end of TRIP. Far Western analysis and coimmunoprecipitations in mammalian cells confirmed that full-length CYLD binds to the COOH-terminal domain of TRIP. Because TRIP is an inhibitor of nuclear factor (NF)-kappaB activation by tumor necrosis factor (TNF), the effect of CYLD on NF-kappaB activation was investigated in HeLa cells. The results established that CYLD down-regulates NF-kappaB activation by TNF-alpha. The inhibition by CYLD depends on the presence of the central domain interacting with TRIP and its deubiquitinating activity. These findings indicate that cylindromas arise through constitutive NF-kappaB activation leading to hyperproliferation and tumor growth.     

Encapsulation of doxorubicin into thermosensitive liposomes via complexation with the transition metal manganese.

In the present study, doxorubicin was encapsulated into two thermosensitive liposome formulations which were composed of DPPC/MSPC/DSPE-PEG(2000) (90/10/4 mole ratio) or DPPC/DSPE-PEG(2000) (95/5 mole ratio). Doxorubicin loading was achieved through the use of a pH gradient or a novel procedure that involved doxorubicin complexation with manganese. Regardless of the initial drug-to-lipid ratios (D:L), the final D:L reached a maximum of 0.05 (w/w) when doxorubicin was encapsulated via a pH gradient for both thermosensitive liposome formulations. In contrast, the final maximum D:L achieved through manganese complexation was 0.2 (w/w), and this loading method did not affect temperature-induced drug release, with 85% of drug released from MSPC-containing liposomes within 10 min at 42 degrees C but <5% released over 60 min at 37 degrees C. When the thermosensitive liposomes prepared via the two different loading methods were injected into mice, similar plasma elimination profiles were observed. Cryo-transmission electron microscopy analysis indicated the presence of doxorubicin fiber bundles in liposomes loaded via pH gradient, compared to a stippled and diffuse morphology in those loaded via manganese complexation. To investigate the effect of intraliposomal pH on drug precipitate morphology, the A23187 ionophore (mediates Mn(2+)/H(+) exchange) was added to liposomes loaded with doxorubicin-manganese complex, and the stippled and diffuse appearance could be converted to one exhibiting fiber bundles after acidification of the liposome core. This suggests that the formation of doxorubicin-manganese complex is favored when the intraliposomal pH is >6.5. During the conversion to the fiber bundle morphology, no doxorubicin release was observed when A23187 was added to liposomes exhibiting a 0.05 (w/w), whereas a significant release was noted when the initial D:L was 0.2 (w/w). Following acidification of the liposomal interior and establishment of an apparent new D:L equilibrium, the measured D:L ratio was 0.05 (w/w). In conclusion, the manganese complexation loading method increased the encapsulation efficiency of doxorubicin in thermosensitive liposomes with no major impact on temperature-triggered drug release or pharmacokinetics.     

Specific T Helper Cell Requirement for Optimal Induction of Cytotoxic T Lymphocytes against Major Histocompatibility Complex Class II Negative Tumors

This study shows that induction of tumor-specific CD4⁺ T cells by vaccination with a specific viral T helper epitope, contained within a synthetic peptide, results in protective immunity against major histocompatibility complex (MHC) class II negative, virus-induced tumor cells. Protection was also induced against sarcoma induction by acutely transforming retrovirus. In contrast, no protective immunity was induced by vaccination with an unrelated T helper epitope. By cytokine pattern analysis, the induced CD4⁺ T cells were of the T helper cell 1 type. The peptide-specific CD4⁺ T cells did not directly recognize the tumor cells, indicating involvement of cross-priming by tumor-associated antigen-presenting cells. The main effector cells responsible for tumor eradication were identified as CD8⁺ cytotoxic T cells that were found to recognize a recently described immunodominant viral gag-encoded cytotoxic T lymphocyte (CTL) epitope, which is unrelated to the viral env-encoded T helper peptide sequence. Simultaneous vaccination with the tumor-specific T helper and CTL epitopes resulted in strong synergistic protection. These results indicate the crucial role of T helper cells for optimal induction of protective immunity against MHC class II negative tumor cells. Protection is dependent on tumor-specific CTLs in this model system and requires cross-priming of tumor antigens by specialized antigen-presenting cells. Thus, tumor-specific T helper epitopes have to be included in the design of epitope-based vaccines.Adequate T helper cell activation is essential in the initiation of an immune reaction. The inability to control tumor outgrowth can be due to inadequate T helper responses underlying poor tumor-specific immunity. In the cellular immune response, specialized APCs process protein and present antigenic peptide fragments in MHC class II molecules to CD4⁺ T helper lymphocytes. These provide “help” to effector cells via the production of cytokines. Although tumor cells can directly present endogenously processed antigenic peptide in surface MHC class I molecules to CD8⁺ CTL precursors, initiation of tumor-specific CTL responses is likely to involve indirect presentation of tumor antigens by specialized APCs.Evidence for a role of T helper cell–mediated immunity comes from studies with genetically modified tumor cells. CD4⁺ cells can be directly activated by transfection of MHC class II α and β chain genes in mouse tumor cells (1–4). These cells become immunogenic, lose their tumorigenicity, and even induce protection against wild-type MHC class II negative tumors, indicating that direct MHC class II presentation of tumor expressed antigens can induce efficient anti–tumor responses.A central role of CD4⁺ T cells emerged from studies of immunity against FMR (Friend, Moloney, Rauscher)¹ murine leukemia virus (MuLV) type tumors by Greenberg (5). Transfer of purified polyclonal T cells from FBL (Friend MuLV-induced erythroleukemia cell line) vaccinated mice in naive animals can protect these mice against subsequent tumor challenge. Both purified CD4⁺ and CD8⁺ T cells transfer protection to FBL tumors (6). FBL cells do not express MHC class II molecules, but CD4⁺ T cells can protect mice even in the absence of CD8⁺ T cells. In this case, macrophages seem to play an important effector role. CD8⁺ T cells can only be effective if CD4⁺ T cells are present or if exogenous IL-2 is administered. Neither B cells nor NK cells seem to exert a significant role in the FBL sytem. These data suggest involvement of APCs, presenting tumor antigens, and a crucial regulatory role of Ths, which was strongly supported by experiments performed in Friend MuLV env-transgenic mice (7). These mice were rendered tolerant for env-specific Th responses and it was not possible to protect these mice against FBL tumors by vaccination.Immune responsiveness to MuLV is classically regulated by the genes of the H-2 (MHC) complex (8). In particular, the H-2^b haplotype confers resistance, and studies using H-2 recombinant and H-2 mutant mouse strains have mapped the protective effects to the class II I-A^b locus (9, 10). This MHC class II association indicates an important role of T helper cells influencing both CTL activity as well as class switching of antiviral antibodies from IgM to IgG. The H-2 I-A^b phenotype protects against early lymphomagenesis. The identification of two Friend MuLV env-derived epitopes, presented by MHC class II, I-A^b and I-E^b/d, respectively, indicated that tumor-directed T helper immunity is virus specific (11). The few lymphomas that arise in H-2^b mice have abrogated viral antigen or (more rarely) MHC class I expression (12), indicating that CTLs also play a crucial role. CTLs have been proven to recognize viral antigens, both gag and env proteins encode CTL epitopes (13). We have identified a K^b-presented, env-derived Moloney and Rauscher CTL epitope that is subdominant in C57BL/6 mice making use of the D^b mutant BM13 mouse strain (14). The D^b-presented gag-leader (gag-L) derived immunodominant CTL epitope for the FMR type of MuLV has been identified only recently (15).Vaccination with a synthetic peptide comprising a relevant T cell epitope is a powerful method to induce highly specific T cells. Protective vaccination using CTL peptide epitopes has been achieved in pathogenic viral models (16, 17) and tumor models (18–20). Peptide vaccination in IFA led to measurable specific CTL induction and protective immunity against virulent virus or tumor cells. Importantly, peptide vaccination can also be applied succesfully for therapy of established tumors by presenting the peptide in IFA, on RMA-S cells, or on activated dendritic cells (21, 22).We now report the induction of tumor-protective immunity by a single vaccination with a tumor-specific MuLV env-encoded T helper peptide. Strong protection can be achieved against highly aggressive tumor cells that lack MHC class II expression. This indicates the requirement of cross-priming of tumor antigens by local APCs. We show that CD8⁺ T cells, recognizing the gag-L–encoded CTL epitope, are crucial effector cells that are efficiently activated with help from peptide-primed tumor-specific CD4⁺ T cells. Vaccination with a mixture of the T helper peptide and the immunodominant CTL epitope resulted in synergistic, long-term tumor protection.     

Value of Scintigraphy for Assessing Delayed Gastric Emptying After Pancreatic Surgery

Background

Delayed gastric emptying (DGE) occurs frequently after pancreatic surgery. Recently a consensus definition of DGE was introduced, and this grading system is currently widely used. The aim of this study was to compare results of gastric emptying scintigraphy with the grade of DGE after pancreatic surgery.

Methods

In 44 patients undergoing exploration for a pancreatic head or periampullary tumor, 28 pancreatoduodenectomies (PDs) and 16 double-bypass procedures were performed. All patients underwent preoperative and postoperative gastric emptying scintigraphy. We investigated whether the incidence of DGE was correlated with the results of gastric emptying scintigraphy.

Results

DGE occurred in 19 (43 %) patients. Clinically relevant DGE (grades B and C) prevailed in the PD group. Median postoperative residual activity at t = 2 h (%RA120) in these groups was 36 % (no DGE), 75 % (grade A), 93 % (grade B), and 95 % (grade C). DGE grade B or C was found in 7 of 10 patients with %RA120 of ≥94 % on postoperative day (POD) 7.

Conclusions

Postoperative %RA120 on scintigraphy is positively associated with severity of DGE. Gastric emptying scintigraphy on POD 7 can predict the severity of DGE. When postoperative gastric emptying scintigraphy shows high residual radioactivity, the likelihood of further progression to grade B or C DGE is high and warrants investigation for underlying causes.     

Duplex sonographic criteria for measuring carotid stenoses

PURPOSE: The aim of this retrospective study was to determine optimal duplex sonographic criteria for use in our institution for diagnosing severe carotid stenoses and to correlate those findings with angiographic measurements obtained by the European Carotid Surgery Trial (ECST), North American Symptomatic Carotid Endarterectomy Trial (NASCET), and Common Carotid (CC) methods of grading carotid stenoses. METHODS: We analyzed the angiographic data using the ECST, NASCET, and CC methods and compared the results with the duplex sonographic findings. We then calculated the sensitivity, specificity, positive and negative predictive values, and accuracy of the duplex sonographic method. Taking these parameters into account, the optimal intrastenotic peak systolic velocity (PSV) and end diastolic velocity (EDV) were derived for diagnosing severe stenoses according to the 3 angiographic methods. RESULTS: Optimal PSV and EDV values for diagnosing a 70% or greater stenosis in our laboratory were as follows: with the NASCET method of angiographic grading of stenoses, PSV 220 cm/second or greater and EDV 80 cm/second or greater, and with the ECST and CC methods, PSV 190 cm/second or greater, and EDV 65 cm/second or greater. The optimal PSV and EDV for diagnosing a stenosis of 80% or greater with the ECST grading method were 215 cm/second or greater and 90 cm/second or greater, respectively. CONCLUSIONS: Duplex sonography is a sensitive and accurate tool for evaluating severe carotid stenoses. Optimal PSVs and EDVs vary according to the angiographic method used to grade the stenosis. They are similar for stenoses 70% or greater with the NASCET method and for stenoses 80% or greater with the ECST method.     

Characteristics of bifocal pacing: right ventricular apex versus outflow tract. An interim analysis

Bifocal RIGHT ventricular stimulation (BRIGHT) is an ongoing, randomized, single-blind, crossover study of atrial synchronized bi-right ventricular (RV) pacing in patients in New York Heart Association heart failure functional class III, a left ventricular ejection fraction <35%, left bundle branch block and QRS complexes >/=120 ms. This analysis compared the electrical and handling characteristics, and the complications of pacing at the RV apex (Ap) with passive, versus RV outflow tract (OT) with active fixation leads. A mean of 1.6 +/- 0.9 and 2.2 +/- 2.0 attempts were needed to position the Ap and OT leads, respectively (ns). R-wave amplitudes at Ap versus OT were 23 +/- 13 mV versus 14 +/- 8 mV (n = 36, P < 0.001). R-wave amplitudes at the Ap remained stable between implant and M7. R-wave amplitudes at the OT could not be measured after implantation. In two patients, atrioventricular block occurred during active fixation at the OT. Conduction recovered spontaneously within 4 months. Ventricular fibrillation was induced in one patient during manipulation of an Ap lead in the RV. Marked differences were found between leads positioned in the OT versus Ap, partly related to the difference in lead design. Mean R-wave amplitude was higher at the Ap that at the OT. Ease and success rate of lead implant was similar in both positions.     

Is technology the best medicine? Three practice theoretical perspectives on medication administration technologies in nursing

Even though it is often presumed that the use of technology like medication administration technology is both safer and more effective, the importance of nurses' know‐how is not to be underestimated. In this article, we accordingly try to argue that nurses' labor, including their different forms of knowledge, must play a crucial role in the development, implementation and use of medication administration technology. Using three different theoretical perspectives (‘heuristic lenses') and integrating this with our own ethnographic research, we will explore how nursing practices change through the use of medication technology. Ultimately, we will argue that ignoring (institutional) complexity and the various types of important knowledge that nurses have, will seriously complicate the implementation of medication administration technology.