Serum protein profile in systemic-onset juvenile idiopathic arthritis differentiates response versus nonresponse to therapy

Systemic-onset juvenile idiopathic arthritis (SJIA) is a disease of unknown etiology with an unpredictable response to treatment. We examined two groups of patients to determine whether there are serum protein profiles reflective of active disease and predictive of response to therapy. The first group (n = 8) responded to conventional therapy. The second group (n = 15) responded to an experimental antibody to the IL-6 receptor (MRA). Paired sera from each patient were analyzed before and after treatment, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Despite the small number of patients, highly significant and consistent differences were observed before and after response to therapy in all patients. Of 282 spectral peaks identified, 23 had mean signal intensities significantly different (P < 0.001) before treatment and after response to treatment. The majority of these differences were observed regardless of whether patients responded to conventional therapy or to MRA. These peaks represent potential biomarkers of active disease. One such peak was identified as serum amyloid A, a known acute-phase reactant in SJIA, validating the SELDI-TOF MS platform as a useful technology in this context. Finally, profiles from serum samples obtained at the time of active disease were compared between the two patient groups. Nine peaks had mean signal intensities significantly different (P < 0.001) between active disease in patients who responded to conventional therapy and in patients who failed to respond, suggesting a possible profile predictive of response. Collectively, these data demonstrate the presence of serum proteomic profiles in SJIA that are reflective of active disease and suggest the feasibility of using the SELDI-TOF MS platform used as a tool for proteomic profiling and discovery of novel biomarkers in autoimmune diseases

Expression analysis onto microarrays of randomly selected cDNA clones highlights HOXB13 as a marker of human prostate cancer

In a strategy aimed at identifying novel markers of human prostate cancer, we performed expression analysis using microarrays of clones randomly selected from a cDNA library prepared from the LNCaP prostate cancer cell line. Comparisons of expression profiles in primary human prostate cancer, adjacent normal prostate tissue, and a selection of other (nonprostate) normal human tissues, led to the identification of a set of clones that were judged as the best candidate markers of normal and/or malignant prostate tissue. DNA sequencing of the selected clones revealed that they included 10 genes that had previously been established as prostate markers: NKX3.1, KLK2, KLK3 (PSA), FOLH1 (PSMA), STEAP2, PSGR, PRAC, RDH11, Prostein and FASN. Following analysis of the expression patterns of all selected and sequenced genes through interrogation of SAGE databases, a further three genes from our clone set, HOXB13, SPON2 and NCAM2, emerged as additional candidate markers of human prostate cancer. Quantitative RT–PCR demonstrated the specificity of expression of HOXB13 in prostate tissue and revealed its ubiquitous expression in a series of 37 primary prostate cancers and 20 normal prostates. These results demonstrate the utility of this expression-microarray approach in hunting for new markers of individual human cancer types

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Z-1,1-Dichloro-2,3-diphenylcyclopropanes Block Human Prostate Carcinoma Cell Proliferation, Inhibit Prostate-specific Antigen Expression, and Initiate Apoptosis

BACKGROUND: Z-1,1-Dichloro-2,3-diphenylcyclopropane (A(II)) has long been known to be active against models of breast carcinoma. Microtubule perturbation and interaction at type II estrogen binding sites mediate its actions. METHODS: Since these targets are potentially useful for treatment of prostate tumors, we studied the drug\u27s effects on androgen-sensitive (LNCaP) and -independent (PC-3) human prostatic carcinoma lines. Effects on cell growth and morphology, prostate-specific antigen (PSA) expression, and cell cycle kinetics were determined by microscopy, antibody-based methods, flow cytometry, and electrophoresis. RESULTS: At 100 microM, A(II) reduced survival of both lines by 50% in 12-24 hr, whereas 10 microM A(II) caused a prolonged block of proliferation in both lines, and parallel and complete block of PSA in LNCaP cells. At 10 microM, A(II) caused no major changes in chromatin, morphology or cell cycle distributions, whereas 100 microM drug caused rapid, large-scale cell detachment, nuclear and internucleosomal DNA fragmentation, and hypodiploidy. These effects were also accompanied by dissolution of cellular microtubule arrays. A more potent tubulin assembly-inhibiting congener of A(II), Z-1, 1-dichloro-2-(4-methoxy-phenyl)-3-phenylcyclopropane, slightly more effectively inhibited cell growth, caused little hypodiploidy, but potently and dose-dependently caused G(2)/M accumulation. CONCLUSIONS: These and previous data suggest that the Z-1, 1-dichloro-2,3-diarylcyclo-propanes may be useful in the treatment of human prostate disease

Increased Sensitivity of the Antiestrogen-resistant MCF7/LY2 Human Breast Carcinoma Cell Line to Apoptosis Induced by the Novel Microtubule-stabilizing Agent (+)-Discodermolide

(+)-Discodermolide is a sponge-derived natural product with the most potent microtubule stabilizing activity yet discovered. Its actions parallel that of the promising antibreast cancer agent paclitaxel despite the lack of any apparent similarities in the drugs\u27 structures. To complement our previous studies on human breast cancer cells, we compared the effects of the two drugs against the estrogen receptor positive but tamoxifen-resistant MCF-7/LY2 line. Growth inhibition, cell, and nuclear morphological, electrophoretic, and flow cytometric analyses were performed. (+)-Discodermolide potently inhibited the growth of the cells (e.g., 48-hours IC50 of 1.5 nM) at concentrations similar to those observed with paclitaxel, and somewhat lower than the values observed previously with estrogen responsive MCF-7 cells and estrogen nonresponsive MDA-MB231 cells. (+)-Discodermolide-treated MCF-7/LY2 cells had condensed and highly fragmented nuclei, as well as micronuclei, suggesting mitotic block and the induction of apoptosis. Flow cytometric comparison of cells treated with either drug at 10 nM showed both caused accumulation into the G2/M portion of the cell cycle as well as induction of a pronounced hypodiploid cell population, with (+)-discodermolide yielding a greater effect. The timing and type of high molecular weight DNA fragmentation induced by the two agents was fully consistent with induction of apoptosis, again with (+)-discodermolide showing an advantage over paclitaxel in this regard. More extensive DNA fragmentation was noted in MCF-7/LY2 than has been observed in MCF-7 and MDA-MB231 cells. These in vitro results, coupled with those obtained previously, suggest that (+)-discodermolide might have promise as a new chemotherpeutic agent against breast cancers. In addition, its novel and synthetically approachable structure make (+)-discodermolide a promising lead compound for design and discovery of new microtubule stabilizing agents as alternatives to taxoids

The Potent Microtubule-stabilizing Agent (+)-Discodermolide Induces Apoptosis in Human Breast Carcinoma Cells—Preliminary Comparisons to Paclitaxel

(+)-Discodermolide, a sponge-derived natural product, stabilizes microtubules more potently than paclitaxel despite the lack of any obvious structural similarities between the drugs. It competitively inhibits the binding of paclitaxel to tubulin polymers, hypernucleates microtubule assembly more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant ovarian and colon carcinoma cells. Because paclitaxel shows clinical promise for breast cancer treatment, its effects in a series of human breast cancer cells were compared to those of (+)-discodermolide. Growth inhibition, cell and nuclear morphological, and electrophoretic and flow cytometric analyses were performed on (+)-discodermolide-treated MCF-7 and MDA-MB231 cells. (+)-Discodermolide potently inhibited the growth of both cell types (IC50 \u3c 2.5 nM) at concentrations similar to those observed with paclitaxel. Complete inhibition of growth occurred with 10 nM or greater of each drug and was not reversed by removal. (+)-Discodermolide-treated cells exhibited condensed and highly fragmented nuclei. Flow cytometric comparison of cells treated with either drug at 10 nM, a concentration well below that achieved clinically with paclitaxel, showed both caused cell cycle perturbation and induction of a hypodiploid cell population. (+)-Discodermolide caused these effects more extensively and at earlier time points. The timing and type of high molecular weight DNA fragmentation induced by the two agents was consistent with induction of apoptosis. The results suggest that (+)-discodermolide has promise as a new chemotherapeutic agent against breast and other cancers

Induction of Human Breast Cancer Cell Apoptosis from G2/M Preceded by Stimulation into the Cell Cycle by Z-1,1-dichloro-2,3-diphenylcyclopropane

We have shown previously that Z-1,1-dichloro-2,3-diphenylcyclopropane (a.k.a. Analog II, AII) inhibits human breast cancer cell proliferation regardless of estrogen receptor status or estrogen sensitivity, and that its cellular targets include microtubules. In the present study, we investigated the apoptosis-inducing effects of AII. MCF-7, MCF-7/LY2, and MDA-MB-231 cells all showed nuclear fragmentation in response to 100 μM AII when stained with Hoechst 33342 and examined by fluorescence microscopy. Pulsed field gel electrophoretic analysis showed that each of the cell lines also developed specific high molecular weight DNA fragments: a low level of 1–2 Mb fragments appeared after 6 hr, while 30–50 kb fragments accumulated subsequently. At 24 hr of drug exposure, the majority of cells became nonadherent, and the 30–50 kb fragments were restricted to detached MCF-7 and MDA-MB-231 cells. Both adherent and detached MCF-7/LY2 cells exhibited these fragments. A previous study by single-color (propidium) flow cytometry demonstrated that AII blocks MDA-MB-231 cells in G2/M of the cell cycle. More refined analyses in the present study showed this same result for MDA-MB-231 cells, but MCF-7 and MCF-7/LY2 cells did not reveal apparent drug-induced cell cycle block. AII demonstrated growth inhibitory, cell cycle-perturbing, and hypodiploidy-inducing activity against other human breast carcinoma lines, i.e. BT-20, CAMA-1, and SKBR-3, but no such actions in the non-tumorigenic, “normal” human breast epithelial line MCF-10A. Bromodeoxyuridine labeling and two-color flow cytometric analysis, however, suggested that AII caused stimulation into S phase, and that G2/M was the phase of the cell cycle from which cells apoptosed. AII caused cell rounding, detachment from the growth matrix, and nuclear shrinkage and fragmentation in parallel with biochemical changes. Cycloheximide inhibited AII-induced cell death, indicating that its toxicity requires de novo protein synthesis

Circadian Clock Proteins and Melatonin Receptors in Neurons and Glia of the Sapajus apella Cerebellum

Oscillations of brain proteins in circadian rhythms are important for determining several cellular and physiological processes in anticipation of daily and seasonal environmental rhythms. In addition to the suprachiasmatic nucleus, the primary central oscillator, the cerebellum shows oscillations in gene and protein expression. The variety of local circuit rhythms that the cerebellar cortex contains influences functions such as motivational processes, regulation of feeding, food anticipation, language, and working memory. The molecular basis of the cerebellar oscillator has been demonstrated by “clock gene” expression within cells of the cerebellar layers. Genetic and epidemiological evidence suggests that disruption of circadian rhythms in humans can lead to many pathological conditions. Despite this importance, data about clock gene and protein expression in the cerebellum of diurnal (day-active) species, specifically primates, is currently poorly explored, mainly in regard to cellular identity, as well as the relationship with other molecules also involved in cerebellar functions. These studies could contribute to clarification of the possible mechanisms behind cerebellar rhythmicity. Considering that calcium binding proteins (CaBPs) play crucial roles in preserving and modulating cerebellar functions and that clock gene expression can be controlled by afferent projections or paracrine circadian signals such as the hormone melatonin, the present study aimed to describe cellular identities, distribution patterns and day/night expression changes in PER1, PER2, CaBPs, and MT1 and MT2 melatonin receptors in the cerebellar cortex of a diurnal primate using conventional fluorescence and peroxidase-antiperoxidase immunocytochemical techniques. PER1 and PER2 immunoreactive (IR) cells were observed in the Purkinje cells of the cerebellum, and MT1 and MT2 receptors were localized around Purkinje cells in the Pj layer in Bergmann cells. This identity was confirmed by the S100β-IR of these cells. The highest expression of PER seen in the daytime analysis coincided with the highest expression of melatonin receptors. CaBPs showed day/night morphological and density changes in the cerebellar cortex. The presence of the same temporal variations in the expression of PER in the Pj neurons and in MT1 and MT2 receptors in Bergmann cells indicates a possible relation between these cells during the rhythmic processing of the cerebellum, in addition to the CaBP temporal morphological and density changes