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Enterococcus faecalis has emerged as an important cause of life-threatening multidrug-resistant bacterial infections in the hospital setting. The pathogenesis of enterococcal infections has remained a relatively neglected field despite their obvious clinical relevance. The objective of this study was to characterize the interactions between mast cells (MCs), an innate immune cell population abundant in the intestinal lamina propria, and E. faecalis. This study was conducted with primary bone marrow-derived murine MCs. The results demonstrated that MCs exerted an antimicrobial effect against E. faecalis that was mediated both by degranulation, with the concomitant discharge of the antimicrobial effectors contained in the granules, and by the release of extracellular traps, in which E. faecalis was snared and killed. In particular, the cathelicidin LL-37 released by the MCs had potent antimicrobial effect against E. faecalis. We also investigated the specific receptors involved in the recognition of E. faecalis by MCs. We found that Toll-like receptors (TLRs) are critically involved in the MC recognition of E. faecalis, since MCs deficient in the expression of MyD88, an adaptor molecule required for signaling by most TLRs, were significantly impaired in their capacity to degranulate, to reduce E. faecalis growth as well as to release tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) after encountering this pathogen. Furthermore, TLR2 was identified as the most prominent TLR involved in the recognition of E. faecalis by MCs. The results of this study indicate that MCs may be important contributors to the host innate immune defenses against E. faecalis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The Living with a Neurological Condition (LINC) study was part of the National Population Health Study of Neurological Conditions conducted in Canada. This article describes empirical original qualitative data collected in the third and final phase of this study and examines how individuals living with a neurological condition maintain continuity of their sense of self, with a particular focus on their strategies. Fifteen interviews were analysed for this paper. Emerging strategies for maintaining sense of self include: (1) avoidance and denial, (2) cognitive reframing, (3) articulation of the self through imagined positive identity, (4) strategies that reconnect to identity in the past, (5) adjusting and altering goals, (6) spiritual activities, (7) humour, (8) comparison with others: identity as shaped through social constructs, and (9) creating communities: a reciprocal reflection of self.

Histone deacetylase (HDAC) inhibitors have been associated primarily with an anti-proliferative effect in vitro and in vivo. Recent data provide evidence for an anti-inflammatory potency of HDAC inhibitors in models of experimental colitis. Because the balance of T cell subpopulations is critical for the balance of the mucosal immune system, this study explores the regulatory potency of HDAC inhibitors on T cell polarization as a mechanistic explanation for the observed anti-inflammatory effects. Although HDAC inhibition suppressed the polarization toward the pro-inflammatory T helper 17 (Th17) cells, it enhanced forkhead box P3 (FoxP3)(+) regulatory T cell polarization in vitro and in vivo at the site of inflammation in the lamina propria. This was paralleled by a down-regulation of the interleukin 6 receptor (IL-6R) on naïve CD4(+) T cells on the mRNA as well as on the protein level and changes in the chromatin acetylation at the IL6R gene and its promoter. Downstream of the IL-6R, HDAC inhibition was followed by a decrease in STAT3 phosphorylation as well as retinoic acid receptor-related orphan receptor γT (RORγT) expression, thus identifying the IL-6/STAT3/IL-17 pathway as an important target of HDAC inhibitors. These results directly translated to experimental colitis, where IL-6R expression was suppressed in naïve T cells, paralleled by a significant reduction of Th17 cells in the lamina propria of ITF2357-treated animals, resulting in the amelioration of disease. This study indicates that, in experimental colitis, inhibition of HDAC exerts an anti-inflammatory potency by directing T helper cell polarization via targeting the IL-6 pathway.

A device was built for matrix deposition in mass spectrometric imaging. This spray-type instrument requires no user interaction other than providing the spray solution and selecting the pre-defined or custom-built method. Robustness was achieved by utilizing a delta-robotics design in combination with a simple liquid system. All the information describing the systems is provided as open source and hardware and the design is therefore suitable for wide distribution and adaption by the scientific community.

Despite important progress in adjuvant and neoadjuvant therapies, metastatic disease often develops in breast cancer patients and remains the leading cause of their deaths. For patients with established metastatic disease, therapy is palliative, with few breaks and with mounting adverse effects. Many have hypothesized that a personalized or precision approach (the terms are used interchangeably) to cancer therapy, in which treatment is based on the individual characteristics of each patient, will provide better outcomes. Here, we discuss the molecular basis of breast cancer metastasis and the challenges in personalization of treatment. The instability of metastatic tumors remains a leading obstacle to personalization, because information from a patient's primary tumor may not accurately reflect the metastasis, and one metastasis may vary from another. Furthermore, the variable presence of tumor subpopulations, such as stem cells and dormant cells, may increase the complexity of the targeted treatments needed. Although molecular signatures and circulating biomarkers have been identified in breast cancer, there is lack of validated predictive molecular markers to optimize treatment choices for either prevention or treatment of metastatic disease. Finally, to maximize the information that can be obtained, increased attention to clinical trial design in the metastasis preventive setting is needed., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Inhibition of 17β-HSD2 is an attractive mechanism for the treatment of osteoporosis. We report here the optimization of human 17β-HSD2 inhibitors in the 2,5-thiophene amide class by varying the size of the linker (n equals 0 and 2) between the amide moiety and the phenyl group. While none of the phenethylamides (n = 2) were active, most of the anilides (n = 0) turned out to moderately or strongly inhibit 17β-HSD2. The four most active compounds showed an IC₅₀ of around 60 nM and a very good selectivity toward 17β-HSD1, 17β-HSD4, 17β-HSD5, 11β-HSD1, 11β-HSD2 and the estrogen receptors α and β. The investigated compounds inhibited monkey 17β-HSD2 moderately, and one of them showed good inhibitory activity on mouse 17β-HSD2. SAR studies allowed a first characterization of the human 17β-HSD2 active site, which is predicted to be considerably larger than that of 17β-HSD1.

Colonization of the intestinal tract and dissemination into deeper tissues by the enteric pathogen Yersinia pseudotuberculosis demands expression of a special set of virulence factors important for the initiation and the persistence of the infection. In this study we demonstrate that many virulence-associated functions are coregulated with the carbohydrate metabolism. This link is mediated by the carbon storage regulator (Csr) system, including the regulatory RNAs CsrB and CsrC, and the cAMP receptor protein (Crp), which both control virulence gene expression in response to the nutrient composition of the medium. Here, we show that Crp regulates the synthesis of both Csr RNAs in an opposite manner. A loss of the crp gene resulted in a strong upregulation of CsrB synthesis, whereas CsrC levels were strongly reduced leading to downregulation of the virulence regulator RovA. Switching of the Csr RNA involves Crp-mediated repression of the response regulator UvrY which activates csrB transcription. To elucidate the regulatory links between virulence and carbon metabolism, we performed comparative metabolome, transcriptome, and phenotypic microarray analyses and found that Crp promotes oxidative catabolism of many different carbon sources, whereas fermentative patterns of metabolism are favored when crp is deleted. Mouse infection experiments further demonstrated that Crp is pivotal for a successful Y. pseudotuberculosis infection. In summary, placement of the Csr system and important virulence factors under control of Crp enables this pathogen to link its nutritional status to virulence in order to optimize biological fitness and infection efficiency through the infectious life cycle.

Aims: One of the first steps in optimizing future prevention and appropriately allocating resources to future diagnosis and treatment of Gestational diabetes (GDM) is to quantify its burden. The aim of this study was therefore to conduct a systematic review of current international literature on the prevalence of GDM with a focus on advanced economies., Methods: Based on literature searches with the database “PubMed” we included studies with a publication date between 2000 and 2011. All studies were classified independently by two authors. In accordance with the PRISMA Statement we identified 27 original studies., Results: The prevalence of GDM varies substantially between populations with a range of 1.7–11.6 %. Patient self-reports yield higher prevalence estimates than values that are based on medical diagnosis or clinical diagnostic tests. Prevalence seems to vary considerably within Europe with higher prevalence values being observed in Southern Europe than in Northern and Central Europe. Prevalence values from studies from Australia and North America were within this range., Conclusion: This review provides a comprehensive and sound summary of the current state of research on the prevalence of GDM in advances economies as a fi rst step in identifying starting points for future interventions.

E2 deficiency in elderly people has directly an effect on the skeleton and can lead to osteoporosis. As 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyses the conversion between active 17β-hydroxysteroid estradiol (E2) and testosterone (T) into their less active 17-ketosteroid and has been found in bones, 17β-HSD2 inhibitor may provide a new approach in the onset of osteoporosis. Bicyclic substituted hydroxyphenylmethanone derivatives were synthesised as steroidomimetics of the substrate E2 and were evaluated for their 17β-HSD2 inhibition and their selectivity toward 17β-HSD1, catalysing the reverse reaction the conversion of estrone (E1) into E2. Highly selective compounds (11, 12, 14, 21 and 22) have been identified, the most promising one (12) showing an IC(50) value in the low nanomolar range (101 nM) and a selectivity factor of 13 toward 17β-HSD1. These results make compound 12 an interesting candidate for further biological evaluation., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyzes the oxidation of the highly potent steroids: the estrogen estradiol (E2) and the androgen testosterone (T) to the less active estrone and androstenedione, respectively. Inhibition of this enzyme may help maintain the local E2 level in bone tissue when the circulating E2 level drops and is therefore a novel and promising approach for the treatment of osteoporosis. In this work, a series of new nonsteroidal and achiral 17β-HSD2 inhibitors, namely N-benzyl-diphenyl-3(or 4)-carboxamide and N-benzyl-5-phenyl-thiophene-2-carboxamide was designed and the compounds were synthesized in a two to three steps reaction. A small library was built applying parallel synthesis. Highly potent 17β-HSD2 inhibitors could be identified in the thiophene-2-carboxamide class with IC(50) in the low nanomolar range. These compounds also showed a good selectivity profile toward 17β-HSD1 and toward the estrogen receptors α and β. The most interesting 17β-HSD2 inhibitor identified in this study is the 5-(2-fluoro-3-methoxyphenyl)-N-(3-hydroxybenzyl)-N-methylthiophene-2-carboxamide 6w displaying an IC(50) of 61 nM and a selectivity factor of 73 toward 17β-HSD1., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Estrogen deficiency in postmenopausal women or elderly men is often associated with the skeletal disease osteoporosis. The supplementation of estradiol (E2) in osteoporotic patients is known to prevent bone fracture but cannot be administered because of adverse effect. As 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2) oxidizes E2 to its inactive form estrone (E1) and has been found in osteoblastic cells, it is an attractive target for the treatment of osteoporosis. Twenty-one novel, naphthalene-derived compounds have been synthesized and evaluated for their 17β-HSD2 inhibition and their selectivity toward 17β-HSD1 and the estrogen receptors (ERs) α and β. Compound 19 turned out to be the most potent and selective inhibitor of 17β-HSD2 in cell-free assays and had a very good cellular activity in MDA-MB-231 cells, expressing naturally 17β-HSD2. It also showed marked inhibition of the E1-formation by the rat and mouse orthologous enzymes and strong inhibition of monkey 17β-HSD2. It is thus an appropriate candidate to be further evaluated in a disease-oriented model.

17β-Hydroxysteroid dehydrogenases (17β-HSDs) are oxidoreductases, which play a key role in estrogen and androgen steroid metabolism by catalyzing final steps of the steroid biosynthesis. Up to now, 14 different subtypes have been identified in mammals, which catalyze NAD(P)H or NAD(P)(+) dependent reductions/oxidations at the 17-position of the steroid. Depending on their reductive or oxidative activities, they modulate the intracellular concentration of inactive and active steroids. As the genomic mechanism of steroid action involves binding to a steroid nuclear receptor, 17β-HSDs act like pre-receptor molecular switches. 17β-HSDs are thus key enzymes implicated in the different functions of the reproductive tissues in both males and females. The crucial role of estrogens and androgens in the genesis and development of hormone dependent diseases is well recognized. Considering the pivotal role of 17β-HSDs in steroid hormone modulation and their substrate specificity, these proteins are promising therapeutic targets for diseases like breast cancer, endometriosis, osteoporosis, and prostate cancer. The selective inhibition of the concerned enzymes might provide an effective treatment and a good alternative to the existing endocrine therapies. Herein, we give an overview of functional and structural aspects for the different 17β-HSDs. We focus on steroidal and non-steroidal inhibitors recently published for each subtype and report on existing animal models for the different 17β-HSDs and the respective diseases. Article from the Special issue on Targeted Inhibitors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Inhibition of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is a novel and attractive approach to reduce the local levels of the active estrogen 17β-estradiol in patients with estrogen-dependent diseases like breast cancer or endometriosis. With the aim of optimizing the biological profile of 17β-HSD1 inhibitors from the hydroxyphenylnaphthol class, structural optimizations were performed at the 1-position of the naphthalene by introduction of different heteroaromatic rings as well as substituted phenyl groups. In the latter class of compounds, which were synthesized applying Suzuki-cross coupling, the 3-methanesulfonamide 15 turned out to be a highly potent 17β-HSD1 inhibitor (IC(50) = 15 nM in a cell-free assay). It was also very active in the cellular assay (T47D cells, IC(50) = 71 nM) and selective toward 17β-HSD2 and the estrogen receptors α and β. It showed a good membrane permeation and metabolic stability and was orally available in the rat.

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyses the conversion of active 17β-hydroxysteroids into the less active 17-ketosteroids thereby controlling the availability of biologically active estrogens (E2) and androgens (T) in the tissues. The skeletal disease osteoporosis occurs mainly in post-menopausal women and in elderly men when the levels of estrogens and androgens, respectively, decrease. Since 17β-HSD2 is present in osteoblasts, inhibition of this enzyme may provide a new and promising approach to prevent the onset of osteoporosis, keeping a certain level in estrogens and androgens in bone cells of ageing people. Hydroxynaphthyl, hydroxyphenyl and hydroxymethylphenyl-substituted moieties were synthesised as mimetics of the steroidal substrate. Compound 8 has been identified as promising scaffold for 17β-HSD2 inhibitors displaying high activity and good selectivity toward 17β-HSD1, ERα and ERβ., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Impaired ability to conduct daily activities is a diagnostic criterion for dementia and a determinant of healthcare services utilization and caregiver burden. What predicts decline in instrumental activities of daily living (IADLs) is not well understood. This study examined measures of episodic memory, executive function, and MRI brain volumes in relation to baseline IADLs and as predictors of rate of IADL change. Participants were 124 elderly persons with cognitive function between normal and moderate dementia both with and without significant small vessel cerebrovascular disease. Random effects modeling showed that baseline memory and executive function (EXEC) were associated with baseline IADL scores, but only EXEC was independently associated with rate of change in IADLs. Whereas hippocampal and cortical gray matter volumes were significantly associated with baseline IADL scores, only hippocampal volume was associated with IADL change. In a model including cognitive and neuroimaging predictors, only EXEC independently predicted rate of decline in IADL scores. These findings indicate that greater executive dysfunction at initial assessment is associated with more rapid decline in IADLs. Perhaps executive function is particularly important with respect to maintaining IADLs. Alternatively, executive dysfunction may be a sentinel event indicating widespread cortical involvement and poor prognosis.

Object: Trichostatin A (TSA) is a histone deacetylase inhibitor that causes growth inhibition of malignant cells. The authors' goal was to evaluate its effect on cell growth and cell cycle regulation in a large panel of glioma cell lines, as well as in human astrocytes, fibroblasts, and endothelial cells., Methods: Cell growth in response to TSA was evaluated using a tetrazolium colorimetric assay and a clonogenic assay. Cell cycle effects were examined using flow cytometry. A DNA fragmentation assay was used to evaluate induction of apoptosis. Histone acetylation status and the expression of p21WAF1, phosphorylated retinoblastoma protein (Rb), poly(adenosine diphosphate-ribose) polymerase (PARP), and caspase-3 were studied using Western blot analysis. In the glioma cell lines, there was significant inhibition of cell growth and detection of increased levels of acetylated histones after TSA treatment. The mechanisms underlying the growth inhibition include cell cycle arrest at the G2/M phase and apoptosis induction. The expression of p21WAF1 was activated, with a temporally related decrease in levels of phosphorylated Rb. Apoptosis was preceded by detection of cleaved PARP and activated caspase-3. The effects of TSA were less pronounced or absent in human astrocytes, fibroblasts, and endothelial cells., Conclusions: The TSA caused inhibition of glioma cell growth by both cell cycle arrest and apoptosis. Cell cycle arrest was associated with an increase in p21WAF1 expression and a decrease in phosphorylated Rb. Apoptosis was mediated at least partly through the activation of caspase-3. Because of the differential effects in glioma cells compared with nonneoplastic cells, TSA may provide a novel strategy for achieving tumor growth inhibition and cytotoxicity. Further investigation is warranted.

Matrix metalloproteinases (MMPs) are matrix-degrading enzymes involved in diverse homeostatic and pathological processes. Several MMPs are expressed within the CNS and serve important normal and pathological functions during development and adulthood. An early and major pathological effect of MMP activity after cerebral ischemia is opening of the blood-brain barrier (BBB). More recent work demonstrates emerging roles for MMPs and their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in the regulation of neuronal cell death. In addition, MMPs and TIMPs are likely to play important roles during the repair phases of cerebral ischemia, particularly during angiogenesis and reestablishment of cerebral blood flow. This review attempts to elucidate how MMPs and TIMPs may provide detrimental or beneficial actions during the injury and repair processes after cerebral ischemia. These processes will have important implications for therapies using MMP inhibitors in stroke., (Copyright 2005 Wiley-Liss, Inc.)

Metalloproteinase activity at the cell surface influences cellular sensitivity to extrinsic death vs. survival signals in a variety of cell types, through proteolytic shedding of cell surface signalling molecules. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a unique natural metalloproteinase inhibitor that plays a pro-apoptotic role through its ability to inhibit metalloproteinases that proteolytically cleave death receptors and their ligands from the cell surface. To study the convergence of metalloproteinase activity and death receptor signalling in neurons, we established an in vitro model of neuronal apoptosis utilizing the chemotherapeutic drug, doxorubicin (Dox). Primary cultures established from embryonic rat cerebral cortices displayed robust and selective neuronal apoptosis in response to Dox, an effect that was dependent on the activation of the death receptor, Fas. We demonstrate that both TIMP-3 and matrix metalloproteinase-3 (MMP-3) are constitutively expressed by primary cortical neurons in culture, and selectively modulated Fas-mediated neuronal apoptosis induced by Dox. Metalloproteinase inhibition by TIMP-3 was found to be necessary for Dox-induced neuronal death, whereas addition of active MMP-3 markedly attenuated apoptosis and diminished Fas-Fas ligand interaction at the cell surface. These observations implicate a physiological role for the balance of TIMP-3 and MMP-3 activity at the neuronal surface in regulating death receptor sensitivity. The convergence of metalloproteinase activity and death receptor signalling at the cell surface may influence neuronal cell death vs. survival decisions.

Delayed complications associated with sublaminar and interspinous wiring in the pediatric cervical spine are rare. The authors present a case of delayed complication in which a cervical fusion wire migrated into the cerebellum, causing subsequent cerebellar abscess 2 years after posterior cervical arthrodesis. A craniotomy was required to remove the wire and drain the abscess. Despite their history of safety and successful fusion, procedures involving sublaminar and interspinous wiring carry a risk of neurological injury secondary to wire migration. A thorough neuroimaging evaluation is required in patients who have undergone fusion and who have neurological complaints to detect late instrumentation-related sequelae.

Keratinocytes represent the main constituents of the epidermis and have been found to play a regulatory role in a variety of inflammatory skin diseases. The functional activity of keratinocytes is highly heterogeneous, and depends on the cell localization in the epidermal architecture, and the maturation or differentiation state of the cells. Spontaneously proliferating HaCaT cells, showing several similarities to basal epidermal keratinocytes, were found to respond to external chemoattractants, including the chemokines RANTES (regulated on activation normal T cell expressed and secreted) and interleukin-8 and the mu-opioid agonist DAMGO ([d-ala2, N-Me-Phe4, Gly-ol5]enkephalin) in migration assays. The chemotactic responsiveness was highly dependent on the cell density of the monolayer, with greatest chemotactic activity at the highest cell density. Whereas RANTES was found to be the most potent chemoattractant, constitutive RANTES production was also detected in the HaCaT cultures. We found an inverse correlation between constitutive RANTES production and chemotactic responsiveness toward external RANTES, suggesting a possible functional down-modulation of the RANTES receptors, CC chemokine receptor 1 and CC chemokine receptor 5, during culture. Results from confocal laser scanning microscopy showed reduced CC chemokine receptor 1, but not CC chemokine receptor 5, expression by HaCaT cells at low cell densities, which was abolished in the presence of neutralizing antibodies against RANTES. The total CC chemokine receptor 1 pool (surface and intracellular receptors), however, showed no significant change during in vitro culture. Chemotactic responsiveness toward RANTES was directly correlated with the level of CC chemokine receptor 1 surface expression. Taken together these results show that with keratinocyte proliferation and the progressive increase in cell density there are dramatic alterations in keratinocyte function.

Chemokine receptors are subjected to heterologous desensitization by activation of formyl peptide receptors. We investigated the cross-talk between formyl peptide receptors and the chemokine receptor CCR5 in human monocyte-differentiated immature dendritic cells (iDC). Monocytes cultured with GM-CSF and IL-4 for 4 days exhibit markers characteristic of iDC and maintain the expression of both formyl peptide receptors FPR and FPRL1, as well as CCR5. Pretreatment of iDC with W peptide (WKYMVm), a potent agonist for FPR and FPRL1 but with preference for FPRL1, resulted in down-regulation of CCR5 from the cell surface and reduced cell response to the CCR5 ligands through a PKC-dependent pathway. Furthermore, W peptide induced a PKC-dependent phosphorylation of CCR5 and inhibited infection of iDC by R5 HIV-1. Our results indicate that the expression and functions of CCR5 in iDC can be attenuated by W peptide, which activates formyl peptide receptors, and suggest an approach to the design of novel anti-HIV-1 agents., (Copyright 2001 Academic Press.)

The G protein-coupled 7 transmembrane (STM) chemoattractant receptors can be inactivated by heterologous desensitization. Earlier work showed that formly peptide receptor-like 1 (FPRL1), an STM receptor with low affinity for the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalamine (fMLF), is activated by peptide domains derived from the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 and its activation results in desensitization and down-regulation of the chemokine receptors CCR5 and CXCR4 from monocyte surfaces. This study investigated the possibility of interfering with the function of CCR5 or CXCR4 as HIV-1 coreceptors by activating FPRL1. Cell lines were established expressing FPRL1 in combination with CD4/CXCR4 or CD4/CCR5 and the effect of a synthetic peptide, WKYMVm, a potent activator of formyl peptide receptors with preference for FPRL1 was determined. Both CXCR4 and CCR5 were desensitized by activation of the cells with WKYMVm via a staurosporine-sensitive pathway. This desensitization of CXCR4 and CCR5 also attenuated their capacity as the fusion cofactors for HIV-1 envelope glycoprotein and resulted in a significant inhibition of p24 production by cell lines infected with HIV-1 that use CCR5 or CXCR4 as coreceptors. Furthermore, WKYMVm inhibited the infection of human peripheral monocyte-derived macrophages and CD4(+) T lymphocytes by R5 or X4 strains of HIV-1, respectively. These results indicate that heterologous desensitization of CCR5 and CXCR4 by an FPRL1 agonist attenuates their major biologic functions and suggest an approach to the development of additional anti-HIV-1 agents. (Blood. 2001;97:2941-2947)

It is now clear that opioid receptors participate in the function of the cells of the immune system, and evidence suggests that opioids modulate both innate and acquired immune responses. We review literature here which establishes that mu-, kappa-, and delta-opioid compounds alter resistance to a variety of infectious agents, including the Human Immunodeficiency Virus (HIV). The nature of the immunomodulatory activity of the opioids has been the subject of a great deal of research over the last ten years. There is increasing evidence that effects of opioids on the immune response are mediated at several levels. Modulation of the inflammatory response appears to be a target of these compounds, including effects on phagocytic activity, as well as the response of cells to various chemoattractant molecules. Moreover, findings from several laboratories have demonstrated the impact of opioid treatment on antibody responses, and the molecular basis for this effect is likely due, at least in part, to the modulation of both cytokine and cytokine receptor expression. Future research should provide a clearer understanding of the cellular and molecular targets of opioid action within the immune system.

Angiotensin-converting enzyme (ACE) inhibitors appear to possess unique cardioprotective benefits, even when used in patients without high blood pressure or left ventricular dysfunction (the traditional indications for ACE inhibitor therapy). The ACE inhibitors improve endothelial function and regress both left ventricular hypertrophy and arterial mass better than other antihypertensive agents that lower blood pressure equally as well. These agents promote collateral vessel development and improve prognosis in patients who have had a coronary revascularization procedure (i.e., percutaneous transluminal coronary angioplasty and coronary artery bypass graft surgery). Insulin resistance, present not only in type 2 diabetes but also commonly in patients with hypertension or coronary artery disease, or both, sensitizes the vasculature to the trophic effects of angiotensin II and aldosterone. This may partly explain the improvement in prognosis noted when patients who have atherosclerosis or diabetes are treated with an ACE inhibitor. Therapy with ACE inhibitors has also been shown, in two large, randomized trials, to reduce the incidence of new-onset type 2 diabetes through largely unknown mechanisms. The ACE inhibitors are safe, well tolerated and affordable medications. The data suggest that most people with atherosclerosis should be considered candidates for ACE inhibitor therapy, unless they are intolerant to the medication, or have systolic blood pressures consistently <100 mm Hg. Patients who show evidence of insulin resistance (with or without overt type 2 diabetes) should also be considered as candidates for prophylactic ACE inhibitor therapy. Although angiotensin receptor blockers should not be considered equivalent to ACE inhibitors for this indication, they may be a reasonable alternative for patients intolerant of ACE inhibitors.

Strong evidence for the direct modulation of the immune system by opioids is well documented. Mu-opioids have been shown to alter the release of cytokines important for both host defense and the inflammatory response. Proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-gamma-inducible protein-10 (IP-10) play crucial roles in cell-mediated immune responses, proinflammatory reactions, and viral infections. In this report, we show that [D-Ala(2),N:-Me-Phe(4),Gly-ol(5)]enkephalin (DAMGO), a mu-opioid-selective agonist, augments the expression in human PBMCs of MCP-1, RANTES, and IP-10 at both the mRNA and protein levels. Because of the proposed relationship between opioid abuse and HIV-1 infection, we also examined the impact of DAMGO on chemokine expression in HIV-infected cells. Our results show that DAMGO administration induces a significant increase in RANTES and IP-10 expression, while MCP-1 protein levels remain unaffected in PBMCs infected with the HIV-1 strain. In contrast, we show a dichotomous effect of DAMGO treatment on IP-10 protein levels expressed by T- and M-tropic HIV-infected PBMCs. The differential modulation of chemokine expression in T- and M-tropic HIV-1-infected PBMCs by opioids supports a detrimental role for opioids during HIV-1 infection. Modulation of chemokine expression may enhance trafficking of potential noninfected target cells to the site of active infection, thus directly contributing to HIV-1 replication and disease progression to AIDS.

Human interleukin 2 (IL-2; Proleukin) is an approved therapeutic for advanced-stage metastatic cancer; however, its use is restricted because of severe systemic toxicity. Its function as a central mediator of T-cell activation may contribute to its efficacy for cancer therapy. However, activation of natural killer (NK) cells by therapeutically administered IL-2 may mediate toxicity. Here we have used targeted mutagenesis of human IL-2 to generate a mutein with approximately 3,000-fold in vitro selectivity for T cells over NK cells relative to wild-type IL-2. We compared the variant, termed BAY 50-4798, with human IL-2 (Proleukin) in a therapeutic dosing regimen in chimpanzees, and found that although the T-cell mobilization and activation properties of BAY 50-4798 were comparable to human IL-2, BAY 50-4798 was better tolerated in the chimpanzee. BAY 50-4798 was also shown to inhibit metastasis in a mouse tumor model. These results indicate that BAY 50-4798 may exhibit a greater therapeutic index than IL-2 in humans in the treatment of cancer and AIDS.

Interactions between cell surface receptors are important regulatory elements in the complex host responses to infections. In this study, it is shown that a classic chemotactic factor, the bacterial chemotactic peptide N-formyl-methionyl-leucylphenyl-alanine (fMLF), rapidly induced a protein-kinase-C-mediated serine phosphorylation and down-regulation of the chemokine receptor CCR5, which serves as a major human immunodeficiency virus (HIV)-1 coreceptor. The fMLF binding to its receptor, formyl peptide receptor (FPR), resulted in significant attenuation of cell responses to CCR5 ligands and in inhibition of HIV-1-envelope-glycoprotein-mediated fusion and infection of cells expressing CD4, CCR5, and FPR. The finding that the expression and function of CCR5 can be regulated by peptides that use an unrelated receptor may provide a novel approach to the design of anti-inflamatory and antiretroviral agents. (Blood. 2000;96:2887-2894)

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.

OBJECTIVE: Cavernous malformations are angiographically occult cerebrovascular malformations found in approximately 0.5% of the population. To help further understand the natural history of these lesions, we prospectively followed 68 patients harboring cavernous malformations. METHODS: The 68 patients in this study were all diagnosed radiographically (67 patients) or surgically (1 patient) and were entered into a patient database. Age, sex, clinical symptoms, seizure frequency, focal neurological deficits, and presence or absence of extralesional hemorrhage were all recorded at presentation. Patients were then followed prospectively to determine the rate of hemorrhage and new-onset seizures. RESULTS: The mean follow-up per patient was 5.2 years, and the total follow-up was 352.9 patient-years. There was an average of 3.4 lesions per patient. Thirteen of the patients (19%) had familial cavernous malformations. Patients with familial disease were more likely to have multiple lesions than patients with sporadic disease (85% versus 25%, respectively [P = 0.001]). Initial presentation included headache (65%), seizures (49%), and focal neurological deficit (46%). Eleven symptomatic, radiologically proven, extralesional hemorrhages occurred during the 352.9 patient-years of follow-up for an overall hemorrhage rate of 3.1% per patient-year. Female patients had a significantly higher prospective hemorrhage rate (4.2% per patient-year versus 0.9% per patient-year [P = 0.04]). A history of hemorrhage at presentation was not a risk factor for subsequent hemorrhage during follow-up. The rate of new-onset seizures was 2.4% per patient-year. CONCLUSION: The clinical presentation and prospective hemorrhage rate reported here agree well with findings of other prospective studies. This information, combined with our new-onset seizure rate, should aid clinicians caring for patients with cavernous malformations.

Context: With the public's increasing use of complementary and alternative medicine, medical schools must consider the challenge of educating physicians about these therapies., Objectives: To document the prevalence, scope, and diversity of medical school education in complementary and alternative therapy topics and to obtain information about the organizational and academic features of these courses., Design: Mail survey and follow-up letter and telephone survey conducted in 1997-1998., Participants: Academic or curriculum deans and faculty at each of the 125 US medical schools., Main Outcome Measures: Courses taught at US medical schools and administrative and educational characteristics of these courses., Results: Replies were received from 117 (94%) of the 125 US medical schools. Of schools that replied, 75 (64%) reported offering elective courses in complementary or alternative medicine or including these topics in required courses. Of the 123 courses reported, 84 (68%) were stand-alone electives, 38 (31%) were part of required courses, and one (1%) was part of an elective. Thirty-eight courses (31%) were offered by departments of family practice and 14 (11%) by departments of medicine or internal medicine. Educational formats included lectures, practitioner lecture and/or demonstration, and patient presentations. Common topics included chiropractic, acupuncture, homeopathy, herbal therapies, and mind-body techniques., Conclusions: There is tremendous heterogeneity and diversity in content, format, and requirements among courses in complementary and alternative medicine at US medical schools.

Interleukin 4 (IL-4) is a pleiotropic cytokine. Of the cell types responsive to IL-4, T cells express one IL-4 receptor (IL-4R) type, IL-4Ralpha/IL-2Rgamma (class I IL-4R), whereas endothelial cells express another type, IL-4Ralpha/IL-13Ralpha (class II IL-4R). It was hypothesized that IL-4 variants could be generated that would be selective for cell types expressing the different IL-4Rs. A series of IL-4 muteins were generated that were substituted in the region of IL-4 implicated in interactions with IL-2Rgamma. These muteins were evaluated in T cell and endothelial cell assays. One of these muteins, containing the mutation Arg-121 to Glu (IL-4/R121E), exhibited complete biological selectivity for T cells, B cells, and monocytes, but showed no activity on endothelial cells. Receptor binding studies indicated that IL-4/R121E retained physical interaction with IL-2Rgamma but not IL-13Ralpha; consistent with this observation, IL-4/R121E was an antagonist of IL-4-induced activity on endothelial cells. IL-4/R121E exhibits a spectrum of activities in vitro that suggest utility in the treatment of certain autoimmune diseases.

The dopaminergic phenotype of neurons in human substantia nigra deteriorates during normal aging, and loss of these neurons is prominent in Parkinson's disease. These degenerative processes are hypothesized to involve oxidative stress. To compare oxidative stress in the nigra and related regions, we measured carbonyl modifications of soluble proteins in postmortem samples of substantia nigra, basal ganglia, and prefrontal cortex from neurologically normal subjects, using an improved 2,4-dinitrophenylhydrazine assay. The protein carbonyl content was found to be about twofold higher in substantia nigra pars compacta than in the other regions. To further analyze this oxidative damage, the distribution of carbonyl groups on soluble proteins was determined by western immunoblot analysis. This method revealed that carbonyl content of the major proteins in each region was linearly dependent on molecular weight. This distribution raises the possibility that protein carbonyl content is controlled by a size-dependent mechanism in vivo. Our results suggest that oxidative stress is elevated in human substantia nigra pars compacta in comparison with other regions and that oxidative damage is higher within the dopaminergic neurons. Elevated oxidative damage may contribute to the degeneration of nigral dopaminergic neurons in aging and in Parkinson's disease.

The effective training of facilitator/ tutors is crucial to the success of a problem-based learning approach to medical education. New skills are needed to accomplish the change from giver of information to facilitator of learning, coach, and guide. This paper outlines considerations for the design of a tutor training programme and describes the Harvard Medical School programme in detail.

Purpose: To determine if soluble binding proteins (BP) for fatty acids (FA), particularly docosahexaenoic acid (DHA), could be identified in the cytosol of rat and bovine retinas under in vitro and in vivo conditions., Methods: In vitro, cytosol fractions from normal bovine and rat retinas were delipidated and incubated with [14C]-DHA with or without a number of competing fatty acids. After crosslinking bound FA to BP, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioactivity determined in each fraction. For in vivo experiments, rats received [14C]-DHA by gavage. At selected periods after ingestion, retinas were collected and cytosolic fractions were prepared from each. These were crosslinked and subjected to SDS-PAGE, and radioactivity was determined in each fraction., Results: In vitro, several peaks of radioactivity (approximately 13, 20, 32, 43-45, 50, 63, and 94-105 kd) were found that exhibited [14C]-DHA binding. Relative specificity of binding was assessed by blocking of the [14C]-DHA binding with unlabeled DHA and by competition experiments with other FAs. After in vivo ingestion of [14C]-DHA, a large peak of radioactivity was observed at 43 kd by 4 hours. At 6 hours, this peak decreased and, after 24 hours, it approached baseline., Conclusions: These findings demonstrate that there is a discrete grouping of proteins in retinal cytosol capable of binding DHA and other FA. Although the identities of these proteins have yet to be determined, the group may include a member of the 12- to 15-kd group of small fatty acid binding proteins (FABP). In particular, a unique 43-kd binding peak could play a major role in the uptake, binding, or both of DHA by the retina in vivo.

Transport of opsin from photoreceptor inner to outer segments has been assumed to occur via the connecting cilium, the only permanent structural connection between these two regions. However, in prior work, little or no immunoreactive opsin has been detected in the cilium, despite the high rate of transport of this protein. This suggests that immune epitopes are masked during passage through the cilium or that opsin is transported via an extra-ciliary route. In this study, we stained the photoreceptors of Xenopus laevis with well-characterized monoclonal antibodies directed at the N-terminal, C-terminal, and 5-6 loop regions of bovine opsin. This was done on isolated retinas incubated in vitro under conditions that support rapid disc assembly, to insure that opsin transport to forming discs was occurring at the time of fixation. Five MAbs that gave robust staining of Xenopus rod inner segment/rod outer segment preparations with the light microscope were utilized for electron microscopic studies on LR White embedded or cryo-ultrathin sections. Four of these stained outer segment discs and inner segment vesicles and plasma membrane. However, no significant staining of the connecting cilium was found. Furthermore, freeze-fractured mouse photoreceptors prepared by the 'fracture-label' technique showed extensive labelling of membrane compartments but lacked staining of the connecting cilium. Isolated retinas incubated under conditions that support robust rod disc synthesis contained many finger-like and vesicular projections of the apical inner segment plasma membrane and inner segment vesicles extending into them. Rod outer segment nascent discs usually made close contact with the inner segment. Both the vesicular profiles associated with the inner segment plasma membrane and the basal discs extending to the inner segment were heavily stained with all four anti-opsin antibodies. This suggests an alternate route for bulk transport of opsin to newly forming discs that involves direct transfer from apical inner segment plasma membrane to nascent discs.

Objective: This study attempted to determine how often psychiatric residents are exposed to violence, the types of violence they encounter, and what institutional changes might increase their safety., Methods: Safety conditions at two private general hospitals and one state hospital that served as training sites for a psychiatric residency program were assessed through a survey of psychiatric residents and site visits to the hospitals. The survey asked residents to quantify violent incidents occurring in the emergency rooms, wards, and clinics at each site. The site visits focused on safety issues related to staff training, physical layout, staffing patterns, current policies, and compliance with policies., Results: All 47 residents in the training program responded to the survey. None reported serious injury, although as many as 56 percent had been physically assaulted on the wards of one hospital, and 54 percent of residents had encountered a weapon in one emergency room. Almost all residents had been verbally threatened or had witnessed violence to others. A paradoxical finding of the survey was that the residents felt safest in the hospital that had the highest rate of violence. The site visits revealed that deficiencies in the safety procedures were allowing weapons to be brought into patient care areas., Conclusions: Psychiatric residents are often exposed to dangerous situations, although serious injury is rare. Residents' beliefs about their level of safety seem to be influenced more by how competent they perceive the staff to be than by the frequency of violence. The findings from the site visits pointed to two steps to increase safety: creating a weapon-free environment by searching all patients and finding ways to improve compliance with existing safety measures.

Patients developing neuroleptic-induced akathisia have been reported to show higher levels of psychopathology. We sought to replicate this finding and determine its symptom specificity. We confirmed a significant relationship between ratings of akathisia and total score on the Brief Psychiatric Rating Scale (BPRS) during both acute and maintenance neuroleptic treatment. Using stepwise regression models, BPRS anxious-depressive subscale scores were the strongest predictors of akathisia during both treatment conditions. Paranoid subscale scores predicted akathisia only during maintenance treatment. These results suggest that neuroleptic-induced dysphoria largely explains the relationship between akathisia and residual psychopathology during both acute and maintenance neuroleptic treatment.

Study of the dynamics of membrane protein and phospholipid transport from the inner to the outer segment of vertebrate photoreceptors has shown an interesting dissociation of the two components under a number of experimental treatments which inhibit protein synthesis or transport. Under conditions which block the addition of opsin to outer segments, various lipids continue to be synthesized and transported to the outer segment in the presence of monensin, puromycin, brefeldin A, tunicamycin and several general metabolic inhibitors. In the current study, isolated retinas from adult Xenopus laevis were incubated with or without 20 micrograms mg-1 of tunicamycin in total darkness or light for 2-12 h in the presence of [3H]choline to study the dependence of phosphatidylcholine synthesis and transport on protein transport to the outer segment. Phosphatidylcholine is a major bulk lipid of outer segments, comprising close to one half of the phospholipid of outer segment phospholipids, and blocking choline uptake in retinas is known to cause photoreceptor degeneration. Biochemical analysis demonstrates that tunicamycin does not block the synthesis of phosphatidylcholine in photoreceptor inner segments or transport of radiolabelled phosphatidylcholine to outer segments during 6 h incubations with [3H]choline in light or total darkness. Light and electron microscopic autoradiography and morphometric analysis show that [3H]choline radiolabelled phospholipid does not accumulate in a band of newly formed basal discs in the outer segment or in the tubulo-vesicular structures which accumulate in the intersegmental space of tunicamycin-treated retinas. We conclude that transport of phosphatidylcholine can occur independently of opsin transport to the outer segment but whether this represents two separable components of a single pathway or involves two distinct routes of transport to the outer segment is still unresolved.

Objective: To evaluate the relative contribution of affective and cognitive skills to ratings of students' clinical performance by their supervisors., Methods: Each students' nonverbal behavior was analyzed by examining 33 nonverbal behavioral characteristics in videotape of each of 36 students interviewing a parent or patient three times during a 4-week pediatric clerkship. These ratings were then compared with the student's formal academic evaluation., Results: The 33 nonverbal behavioral characteristics rated for each student were reduced to five composite variables. Three of these correlated significantly with the final grade, providing an affective profile of the highly rated student. Regression analysis of the five composite variables revealed that affective skills accounted for at least 46% of the variance in the students' final grades (multiple R = .68, P = .0015)., Conclusions: Ratings of students' affective characteristics were highly related to clinical evaluations in this pediatric setting. Students who were evaluated highly had a specific affective profile that could be described by analysis of their nonverbal behaviors.

The DSM-III-R Checklist is an efficient method for screening psychiatric patients for major psychiatric disorders while amassing a data base that can be used for later clinical and research activities. It yields valid diagnoses and key adjunctive symptoms. Patients enjoyed the interview, demonstrating an interest in the computer as well as a feeling of confidence in the complete review of symptoms. The psychiatry resident users in this project found the Checklist to be a valuable teaching instrument, a way to develop their thinking about symptoms and diagnostic rules, an efficient means of collecting an automated clinical data base, and a stimulus to pursue further research activities. Finally, the Checklist is an ideal diagnostic tool to amass a research data base and is currently being used in numerous studies by clinicians and nonclinicians.

Tunicamycin inhibits the dolichol pathway for N-linked glycosylation of proteins, including photoreceptor opsin, and causes a buildup of tubulo-vesicular profiles in the intersegmental space between photoreceptor rod inner and outer segments associated with disruption of new disc assembly. We tested the hypothesis that a tunicamycin lesion in photoreceptors would block lipid transport into the outer segment. Adult Xenopus retinas were preincubated in dim red light with 20 micrograms ml-1 of tunicamycin for one hour followed by incubation in the light for 2-6 h with tunicamycin plus either [3H]mannose, [3H]leucine, [2-(3)H]glycerol or [3H]myo-inositol. Tunicamycin caused accumulation of tubulo-vesicular membranes in the intersegmental space and significantly reduced both [3H]leucine and [3H]mannose incorporation into the basal region of rod outer segments. However, tunicamycin had no effect on [3H]glycerol incorporation into the rod outer segment phospholipids. After 5 h incubation with [3H]glycerol, radiolabel in outer segment fractions was associated primarily with phosphatidylinositol in both control and tunicamycin treated retinas. Quantitative light microscope autoradiography of both [3H]glycerol and [3H]inositol labelled retinas showed diffuse labelling over the entire rod outer segment in both control and tunicamycin treated retinas with no accumulation of radioactivity in the basal discs of control retinas or in the tubulo-vesicular structures in the intersegmental space of tunicamycin treated retinas. Our results indicate that despite the morphological disruption and inhibition of glycoprotein transport to outer segments after tunicamycin treatment, transport of labelled phosphatidylinositol occurs normally. These data add to a growing body of evidence separating the lipid and protein transport pathways to the outer segment.

This study was undertaken to determine the mode of transport of the essential (n-3) fatty acids docosahexaenoic acid 22:6(n-3) and linolenic acid 18:3(n-3). Male weanling Sprague-Dawley rats received a mixture of corn oil and [14C]18:3(n-3) or [14C]22:6(n-3) by gavage. At periods of 1 to 4 days after the injection, four rats per time point were killed and samples of blood were taken via heart puncture and the livers and retinas were collected. Blood lipoproteins and plasma proteins were separated by ultracentrifugation and analyzed by HPLC. Lipids were extracted and saponified and the fatty acids were converted to phenacyl esters for separation of individual fatty acids. After 1 and 2 h, radioactivity from 18:3(n-3) and 22:6(n-3) was observed primarily in the chylomicron/very low density lipoprotein fraction. By 4 h, radioactivity in the lipoprotein fraction was greatly decreased, with a small amount of radioactivity associated with albumin in the soluble protein fraction. After 24 h, the total amount of radioactivity associated with lipoprotein was further reduced, with more than half of the remaining label occurring in association with albumin and another unidentified protein. In the liver, 22:6(n-3) was concentrated in triacylglycerols (40.7%) and phospholipids (51.1%), with a maximum specific activity at 4 h. In the rod outer segments (ROS), the specific activity of [14C]22:6(n-3) increased to a maximum at 24 h and maintained a high level even at 4 days. These data suggest that after injection, 18:3(n-3) and 22:6(n-3) are esterified to triglyceride and phospholipid by the intestinal absorptive cells and transported in chylomicrons to the liver. After conversion of 18:3(n-3) to 22:6(n-3) in the liver, the retina accumulates 22:6(n-3) which may be transported from the liver via albumin and another unidentified protein, and is retained by the rod outer segments.

There is increasing evidence for structural differences in the brains of patients with affective disorders. Recent magnetic resonance imaging (MRI) studies have reported focal signal hyperintensities in the deep white matter of bipolar patients. These previous reports had focused on adult patients with prior episodes of illness. In this case report, the authors discuss a young adolescent patient during her first episode of mania and the finding of subcortical focal signal hyperintensities on brain MRI. The etiology, pathophysiology, and clinical correlates of these lesions will be reviewed.