Drug repositioning in SLE: crowd-sourcing, literature-mining and Big Data analysis.

Lupus patients are in need of modern drugs to treat specific manifestations of their disease effectively and safely. In the past half century, only one new treatment has been approved by the US Food and Drug Administration (FDA) for systemic lupus erythematosus (SLE). In 2014-2015, the FDA approved 71 new drugs, only one of which targeted a rheumatic disease and none of which was approved for use in SLE. Repositioning/repurposing drugs approved for other diseases using multiple approaches is one possible means to find new treatment options for lupus patients. "Big Data" analysis approaches this challenge from an unbiased standpoint whereas literature mining and crowd sourcing for candidates assessed by the CoLTs (Combined Lupus Treatment Scoring) system provide a hypothesis-based approach to rank potential therapeutic candidates for possible clinical application. Both approaches mitigate risk since the candidates assessed have largely been extensively tested in clinical trials for other indications. The usefulness of a multi-pronged approach to drug repositioning in lupus is highlighted by orthogonal confirmation of hypothesis-based drug repositioning predictions by "Big Data" analysis of differentially expressed genes from lupus patient samples. The goal is to identify novel therapies that have the potential to affect disease processes specifically. Involvement of SLE patients and the scientists that study this disease in thinking about new drugs that may be effective in lupus though crowd-sourcing sites such as LRxL-STAT (www.linkedin.com/in/lrxlstat) is important in stimulating the momentum needed to test these novel drug targets for efficacy in lupus rapidly in small, proof-of-concept trials conducted by LuCIN, the Lupus Clinical Investigators Network (www.linkedin.com/in/lucinstat).

Human peripheral blood dendritic cells and monocyte subsets display similar chemokine receptor expression profiles with differential migratory responses.

Human antigen presenting cells (APC) found in peripheral blood are considered to be precursors that have been released from the bone marrow and are in transit to the peripheral tissues. These APC populations include myeloid dendritic cells (mDC), plasmacytoid DC (pDC) and monocytes (Mo). To assign specialized functional roles and stages of development for APCs, CD33 expressing APC subsets were examined for their capacity to respond to chemokines. Three major CD33(+) subsets including CD33(bright)CD14(bright) Mo, CD33(bright)CD14(-) CD11c(+) mDC and CD33(dim)CD14(-) pDC were present. Dendritic cells subsets and Mo expressed low levels of CC and CXC receptors, but distinctive chemokine receptor expression profiles were not observed. The percentage of cells expressing a particular chemokine receptor varied from donor to donor and over time in the same donor. Myeloid DC and Mo but not pDC migrated toward CXCL12 in a concentration dependent manner. Monocytes and pDC, but not myeloid DC, were attracted by high concentrations of CXCL10. All CD33(+) subsets migrated in a concentration dependent manner toward CCL19, but responded less robustly to CCL21. CCL20 was not chemoattractant for any population. Despite the finding that APC did not exhibit unique surface chemokine receptor expression patterns, they exhibited differential migration to CXCL12, CXCL10 and CCL21 but not to CCL20 or CCL19.

Increased prevalence of activated CD70+CD4+ T cells in the periphery of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by loss of immune tolerance. A hallmark of SLE is the presence of autoantibodies resulting from B cell hyperactivity. Previous studies have shown that the presence of abnormal B cell subsets in the periphery, such as CD27highCD20- B cells, correlate with disease activity. We examined the relationship between the expression of CD70, the ligand for CD27 expressed by activated T cells, and indicators of disease activity. A significant increase in median CD70+CD4+ T cell frequencies and memory CD45RA-CD4+ T cell frequencies was observed in SLE samples as compared to healthy controls. The frequencies of CD70+CD4+ T cells correlated with disease duration but not age, treatment, or disease activity. Although a majority of CD70+CD4+ T cells appeared to be effector memory cells, mitogen-stimulated CD70+CD4+ T cells were capable of secreting a full repertoire of effector cytokines. Despite the presence of activated CD4+ T cells, no increase in immunosenescent CD4+ T cells, as defined by the loss of CD28 and/or the acquisition of CD57 was observed in samples from SLE patients. These studies indicate that increased CD70 expression might serve as a useful marker of abnormal T cell activity in SLE.

Musculoskeletal outcomes in multiple body regions and work effects among nurses: the effects of stressful and stimulating working conditions.

This study investigated the various stressors encountered by the nursing profession. In particular, the following hypotheses were tested: (1) working conditions of nurses significantly affect perceived risk of injury and illness, work dissatisfaction, work satisfaction, energy state at the end of workday, the effort exerted by the registered nurse (RN), psychosomatic outcomes, and musculoskeletal symptoms (in multiple body regions); (2) both intermediate work effects (i.e., effort, perceived risk of injury/illness, work satisfaction/dissatisfaction, energy state at end of workday) and psychosomatic outcomes significantly affect musculoskeletal outcomes (in multiple body regions); (3) both working conditions and effects significantly affect musculoskeletal outcomes. In a preliminary study conducted on 34 registered nurses, results show that: (1) stressful working conditions affect musculoskeletal outcomes in multiple body regions, and (2) physical maladies such as lower back problems are not only associated with physical factors but also with a complex interaction of working conditions. Further research is warranted to obtain a better understanding of the complex interaction and the synergistic effects of the various nursing working conditions.

Expansion of activated human naïve T-cells precedes effector function.

Naïve T-cells divide and mature, both functionally and phenotypically, upon stimulation through the T-cell receptor. Although much is known about the overall changes that occur in naïve cells upon TCR stimulation, and the different memory/effector populations that arise following stimulation, the relationship between cell division and functional and phenotypical changes that occur after activation is poorly understood. Here, we examine the early stages of human naïve and antigen-experienced T-cell activation, and the relationship between cell division and acquisition of effector function during the transition from resting antigen-experienced or naïve T-cells into effector cells. Stimulated naïve T-cells proliferate prior to acquisition of effector function, as measured by cytokine production and expression of effector-associated cell surface molecules. Additionally, we show that interlukin-7 (IL-7) can drive proliferation of naïve T-cells without TCR:MHC peptide interactions. IL-7 alone does not, however, drive the proliferation of antigen-experienced T-cells. Memory T-cells will divide in response to exogenous IL-7 but only in the presence of naïve T-cells and IL-2. This study contributes to the current understanding of the mechanistic differences between naïve and memory T-cell responses by defining the functional and phenotypic changes that occur to T-cells after stimulation.

Cytochrome P450 CYP2J9, a new mouse arachidonic acid omega-1 hydroxylase predominantly expressed in brain.

A cDNA encoding a new cytochrome P450 was isolated from a mouse brain library. Sequence analysis reveals that this 1,958-base pair cDNA encodes a 57-58-kDa 502-amino acid polypeptide that is 70-91% identical to CYP2J subfamily P450s and is designated CYP2J9. Recombinant CYP2J9 was co-expressed with NADPH-cytochrome P450 oxidoreductase (CYPOR) in Sf9 cells using a baculovirus system. Microsomes of CYP2J9/CYPOR-transfected cells metabolize arachidonic acid to 19-hydroxyeicosatetraenoic acid (HETE) thus CYP2J9 is enzymologically distinct from other P450s. Northern analysis reveals that CYP2J9 transcripts are present at high levels in mouse brain. Mouse brain microsomes biosynthesize 19-HETE. RNA polymerase chain reaction analysis demonstrates that CYP2J9 mRNAs are widely distributed in brain and most abundant in the cerebellum. Immunoblotting using an antibody raised against human CYP2J2 that cross-reacts with CYP2J9 detects a 56-kDa protein band that is expressed in cerebellum and other brain segments and is regulated during postnatal development. In situ hybridization of mouse brain sections with a CYP2J9-specific riboprobe and immunohistochemical staining with the anti-human CYP2J2 IgG reveals abundant CYP2J9 mRNA and protein in cerebellar Purkinje cells. Importantly, 19-HETE inhibits the activity of recombinant P/Q-type Ca(2+) channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release. Based on these data, we conclude that CYP2J9 is a developmentally regulated P450 that is abundant in brain, localized to cerebellar Purkinje cells, and active in the biosynthesis of 19-HETE, an eicosanoid that inhibits activity of P/Q-type Ca(2+) channels. We postulate that CYP2J9 arachidonic acid products play important functional roles in the brain.

Rheumatoid synovial CD4+ T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells.

CD4+ memory T cells (Tm) from rheumatoid arthritis peripheral blood (RAPB) or peripheral blood from normal donors produced IL-2, whereas fewer cells secreted IFN-gamma or IL-4 after a brief stimulation. RAPB Tm contained significantly more IFN-gamma producers than normal cells. Many rheumatoid arthritis (RA) synovial Tm produced IFN-gamma alone (40%) and fewer cells produced IL-2 or IL-4. An in vitro model was employed to generate polarized T-helper (Th) effectors. Normal and RAPB Tm differentiated into both IFN-gamma- and IL-4-producing effectors. RA synovial fluid (RASF) Tm demonstrated defective responsiveness, exhibiting diminished differentiation of IL-4 effectors, whereas RA synovial tissue (RAST) Tm exhibited defective generation of IFN-gamma and IL-4 producers.

Measurement of human and murine interleukin 2 and interleukin 4.

This unit describes protocols employing cell lines or bioassays that can be used for the quantitation of murine total T cell growth factor (TCGF) activity, interleukin 2 (IL-2), and interleukin 4 (IL-4), and of human IL-2 and IL-4. The ability to distinguish between different growth factors is crucial to understanding the regulation of the immune response. The Basic Protocol describes the use of the CTLL-2 line to detect murine IL-2 and IL-4 in supernatants. One alternate protocol describes the detection of IL-2 in samples of human serum or supernatants using CTLL-2 cells, while other alternate procedures describe the detection and quantitation of murine IL-4 using a mutagenized subline of CTLL-2, CT.4S, and the detection of human IL-4 using a derivative of the CT.4S mouse cell line, CT.h4S. Support protocols are provided for the quantitation of CTLL-2, CT4.S, or CT.h4S proliferation using a standard [3H]thymidine incorporation method or by using the 3-(4,5-dimenthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Support Protocol 1). Support protocols also describe the calculation of cytokine units from samples based on DNA synthesis data and procedures for the maintenance of the CTLL-2, CT.4S, and CT.h4S cell lines.

A comparison of teaching strategies for integrating information technology into clinical nursing education.

As health care becomes more information-intensive and diverse, there is a need to integrate information technology (IT) into clinical education. Little is known, however, about how to design instructional strategies for integrating information technology into clinical nursing education. This article outlines the instructional strategies used by faculty in five nursing programs who taught students to use a point-of-care information technology system. The article also reports students' computer acceptance and summarizes IT clinical teaching recommendations.

Knuckle pads: does knuckle cracking play an etiologic role?

Very little has been published on knuckle pads and the cosmetic and psychological effects they can have. In children, most knuckle pads are considered to be idiopathic; however, familial cases as well as those caused by trauma have been described. We report a 14-year-old African American girl with a 3-year history of slowly enlarging hyperkeratotic nodules over multiple finger joints and on the lateral aspects of several fingers. These lesions were initially confined to the patient's left hand but subsequently involved the right hand. The patient and her mother denied any unusual hobbies or sports in which the patient's fingers might be traumatized. The patient, however, was noted to crack her knuckles during her clinic visit. To the mother's surprise, the patient admitted to doing this quite frequently on a daily basis. A diagnosis of knuckle pads was made and confirmed histologically. To our knowledge, this is the first reported case of knuckle pads with cracking of the knuckles as the possible etiology.

Dehydration activates an NF-kappaB-driven, COX2-dependent survival mechanism in renal medullary interstitial cells.

Renal prostaglandin (PG) synthesis is mediated by cyclooxygenase-1 and -2 (COX1 and COX2). After dehydration, the maintenance of normal renal function becomes particularly dependent upon PG synthesis. The present studies were designed to examine the potential link between medullary COX1 and COX2 expression in hypertonic stress. In response to water deprivation, COX2, but not COX1, mRNA levels increase significantly in the renal medulla, specifically in renal medullary interstitial cells (RMICs). Water deprivation also increases renal NF-kappaB-driven reporter expression in transgenic mice. NF-kappaB activity and COX2 expression could be induced in cultured RMICs with hypertonic sodium chloride and mannitol, but not urea. RMIC COX2 expression was also induced by driving NF-kappaB activation with a constitutively active IkappaB kinase alpha (IKKalpha). Conversely, introduction of a dominant-negative IkappaB mutant reduced COX2 expression after hypertonicity or IKKalpha induction. RMICs failed to survive hypertonicity when COX2 was downregulated using a COX2-selective antisense or blocked with the selective nonsteroidal anti-inflammatory drug (NSAID) SC58236, reagents that did not affect cell survival in isotonic media. In rabbits treated with SC58236, water deprivation induced apoptosis of medullary interstitial cells in the renal papilla. These results demonstrate that water deprivation and hypertonicity activate NF-kappaB. The consequent increase in COX2 expression favors RMIC survival in hypertonic conditions. Inhibition of RMIC COX2 could contribute to NSAID-induced papillary injury.

Strategic allocation of ejaculates by male Adélie penguins.

Sperm competition theory suggests that males should strategically allocate sperm to those females that will bring them the best possible genetic returns. Although males of a number of species of insects and fishes have been shown to allocate sperm strategically, we provide, to our knowledge, the first evidence that an avian species is also capable of allocating ejaculates. Male Adélie penguins (Pygoscelis adeliae) are more likely to transfer sperm during extra-pair copulations (EPCs) than during pair copulations. We investigated the question of how males allocate ejaculates within the constraints of limited sperm availability and found (i) that males that engaged in EPC attempts ejaculated less often when copulating with their social partner than males that made no EPC attempts, and (ii) that there was no difference between males that were involved in failed EPC attempts and those that were involved in successful EPCs in the proportion of copulations that resulted in sperm transfer. These results indicate that males achieve strategic allocation of sperm within the constraints of limited sperm availability by withholding ejaculates from their social partners.

Granuloma annulare of the eyelid: a case report and review of the literature.

Granuloma annulare is a benign, relatively common dermatosis of childhood. Lesions typically occur on the extremities and resolve spontaneously over a period of several months to years. Localized facial involvement is rare. We report a case of granuloma annulare confined to the left eyelid. The literature on periocular granuloma annulare is reviewed. This diagnosis should be considered for any acquired papules of the periorbital area, especially if there is a history of antecedent trauma. Unnecessary surgical excision can then be avoided.

Lupus tumidus.

Lupus tumidus is a rare subtype of chronic cutaneous lupus erythematosus that was first described by Gougerot and Bournier in 1930. Clinically, lupus tumidus presents as smooth, shiny, red-violet plaques of the head and neck that may be pruritic and have a fine scale. These lesions characteristically clear without scarring and recur in their original distribution. Histologic features include superficial and deep lymphohistiocytic infiltrates and abundant dermal deposits of mucin. We describe lupus tumidus as a distinct form of cutaneous lupus erythematosus and report 4 cases.

Human CD4+ T cell differentiation and effector function: implications for autoimmunity.

Human CD4+ memory T cells progress through stages of postthymic differentiation that have been characterized by distinct phenotypes. We have investigated the factors regulating cytokine production, and the correlation between phenotype and effector function in normal and autoimmune individuals. These studies suggest that antigen-induced proliferation in the periphery drives CD4+ T cells through successive stages of differentiation that culminate in optimal effector function and resistance to external modulatory influences. Moreover, these studies support the concept that in autoimmune individuals, the chronic accumulation of differentiated proinflammatory T cells perpetuate the inflammatory response resulting in aggressive disease.

Molecular cloning, enzymatic characterization, developmental expression, and cellular localization of a mouse cytochrome P450 highly expressed in kidney.

A cDNA encoding a new cytochrome P450 was isolated from a mouse liver library. Sequence analysis reveals that this 1,886-base pair cDNA encodes a 501-amino acid polypeptide that is 69-74% identical to CYP2J subfamily P450s and is designated CYP2J5. Recombinant CYP2J5 was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 cells using a baculovirus system. Microsomal fractions of CYP2J5/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolize arachidonic acid to 14,15-, 11,12-, and 8, 9-epoxyeicosatrienoic acids and 11- and 15-hydroxyeicosatetraenoic acids (catalytic turnover, 4.5 nmol of product/nmol of cytochrome P450/min at 37 degrees C); thus CYP2J5 is enzymologically distinct. Northern analysis reveals that CYP2J5 transcripts are most abundant in mouse kidney and present at lower levels in liver. Immunoblotting using a polyclonal antibody against a CYP2J5-specific peptide detects a protein with the same electrophoretic mobility as recombinant CYP2J5 most abundantly in mouse kidney microsomes. CYP2J5 is regulated during development in a tissue-specific fashion. In the kidney, CYP2J5 is present before birth and reaches maximal levels at 2-4 weeks of age. In the liver, CYP2J5 is absent prenatally and during the early postnatal period, first appears at 1 week, and then remains relatively constant. Immunohistochemical staining of kidney sections with anti-human CYP2J2 IgG reveals that CYP2J protein(s) are present primarily in the proximal tubules and collecting ducts, sites where the epoxyeicosatrienoic acids are known to modulate fluid/electrolyte transport and mediate hormonal action. In situ hybridization confirms abundant CYP2J5 mRNA within tubules of the renal cortex and outer medulla. Epoxyeicosatrienoic acids are endogenous constituents of mouse kidney thus providing direct evidence for the in vivo metabolism of arachidonic acid by the mouse renal epoxygenase(s). Based on these data, we conclude that CYP2J5 is an enzymologically distinct, developmentally regulated, protein that is localized to specific nephron segments and contributes to the oxidation of endogenous renal arachidonic acid pools. In light of the well documented effects of epoxyeicosatrienoic acids in modulating renal tubular transport processes, we postulate that CYP2J5 products play important functional roles in the kidney.

Sex steroid and corticosterone levels of Adélie penguins (Pygoscelis adeliae) during courtship and incubation.

Plasma levels of sex steroids (progesterone, 17alpha-hydroxyprogesterone, total androgens, and estradiol) were measured at six stages from courtship to late incubation in Adélie penguins. The pattern of change detected in the levels of plasma total androgens (males) and estradiol (females) was consistent with that found in many birds, with elevated levels during courtship (total androgens, 4 ng/ml; estradiol, 0.5 ng/ml) declining to low, stable levels during incubation. Progesterone levels declined moderately from 1.3 to 0.75-1.0 ng/ml in females following egg laying, but levels of 0.8-1.2 ng/ml persisted in males throughout the study period. 17alpha-Hydroxyprogesterone levels were consistently low (approximately 0.2 ng/ml) in females but progressively declined in males from 0.75 during courtship to <0.3 ng/ml at egg laying and during foraging. Plasma corticosterone levels were measured over the same period and were elevated in both males and females at courtship (16-18 ng/ml) and while fasting on the nest (11-15 ng/ml), but had declined in birds returning from foraging at sea, suggesting that elevated levels are related to the metabolic demands of fasting.

In vitro hepatic metabolism of a CYP3A-mediated drug, quinine, in Adélie penguins.

Very little is known about Antarctic animals' ability to metabolise or detoxify xenobiotics. The activity of cytochromes P450 subfamily 3A (CYP3A) in Adélie penguin liver was studied by incubating penguin liver microsomes with a human CYP3A substrate, quinine, and results were compared with those from human liver microsomes. The mean maximum rate of metabolism (Vmax) for quinine in penguin livers was approximately five times less (160+/-72 versus 574+/-416 pmol/mg/min; P<0.01), and the mean Km (substrate affinity) for the formation of quinine's major metabolite (3-hydroxyquinine) was significantly greater than that observed in human livers (160+/-73 versus 83+/-19 microM; P<0.01). The mean intrinsic clearance (Vmax/Km) was 1.1+/-0.4 microl/min (penguin), i.e. sevenfold less than in human livers (7.4+/-5.9 microl/min, P<0.005), suggesting that penguins have much less ability than humans to eliminate xenobiotics having a similar metabolic nature to quinine (i.e. CYP3A substrates). 3-Hydroxyquinine formation in penguin liver was inhibited by specific CYP3A inhibitors, midazolam and troleandomycin, but not by other CYP inhibitors, indicating that quinine metabolism to 3-hydroxyquinine in Adélie penguin liver is likely to be catalysed by a CYP isoform resembling human CYP3A. Adélie penguin liver CYP isoforms could serve as biomarkers for the impact of environmental pollution.