Chemotherapy negatively impacts the tumor immune microenvironment in NSCLC: an analysis of pre- and post-treatment biopsies in the multi-center SAKK19/09 study.

BACKGROUND: Over the past few years, immune checkpoint inhibitors have changed the therapeutic landscape of non-small-cell lung cancer (NSCLC). Response to immune checkpoint inhibitors correlates with a pre-existing anti-tumoral immune response. Checkpoint inhibitors have been introduced as second-line therapy and are only very recently used as monotherapy or in combination with chemotherapy as first-line treatment of NSCLC. However, the effect of conventional first-line platinum-based chemotherapy on the immune infiltrate in the tumor is largely unknown. METHODS: We measured the gene expression of a custom set of 201 cancer- and immune-related genes in 100 NSCLC tumor biopsies collected before chemotherapy and 33 re-biopsies after platinum-based chemotherapy at the time point of progression. For 29 patients matched pre- and post-chemotherapy samples could be evaluated. RESULTS: We identified a cluster of 47 co-expressed immune genes, including PDCD1 (PD1) and CD274 (PD-L1), along with three other co-expression clusters. Chemotherapy decreased the average gene expression of the immune cluster while no effect was observed on the other three cluster. Within this immune cluster, CTLA4, LAG3, TNFRSF18, CD80 and FOXP3 were found to be significantly decreased in patient-matched samples after chemotherapy. CONCLUSION: Our results suggest that conventional platinum-based chemotherapy negatively impacts the immune microenvironment at the time point of secondary progression.

Comprehensive validation of published immunohistochemical prognostic biomarkers of prostate cancer -what has gone wrong? A blueprint for the way forward in biomarker studies.

BACKGROUND: Treatment planning of localised prostate cancer remains challenging. Besides conventional parameters, a wealth of prognostic biomarkers has been proposed so far. None of which, however, have successfully been implemented in a routine setting so far. The aim of our study was to systematically verify a set of published prognostic markers for prostate cancer. METHODS: Following an in-depth PubMed search, 28 markers were selected that have been proposed as multivariate prognostic markers for primary prostate cancer. Their prognostic validity was examined in a radical prostatectomy cohort of 238 patients with a median follow-up of 60 months and biochemical progression as endpoint of the analysis. Immunohistochemical evaluation was performed using previously published cut-off values, but allowing for optimisation if necessary. Univariate and multivariate Cox regression were used to determine the prognostic value of biomarkers included in this study. RESULTS: Despite the application of various cut-offs in the analysis, only four (14%) markers were verified as independently prognostic (AKT1, stromal AR, EZH2, and PSMA) for PSA relapse following radical prostatectomy. CONCLUSIONS: Apparently, many immunohistochemistry-based studies on prognostic markers seem to be over-optimistic. Codes of best practice, such as the REMARK guidelines, may facilitate the performance of conclusive and transparent future studies.

MicroRNA-29b is involved in the Src-ID1 signaling pathway and is dysregulated in human lung adenocarcinoma.

The c-Src kinase regulates cancer cell invasion through inhibitor of DNA binding/differentiation 1 (ID1). Src and ID1 are frequently overexpressed in human lung adenocarcinoma. The current study aimed at identifying microRNAs (miRNAs) involved in the Src-ID1 signaling in lung cancer. Incubation of lung cancer cells with the Src inhibitor saracatinib led to the upregulation of several miRNAs including miR-29b, which was the most highly upregulated miRNA with predicted binding to the ID1 3'-untranslated region (UTR). Luciferase reporter assays confirmed direct binding of miR-29b to the ID1 3'-UTR. Expression of miR-29b suppressed ID1 levels and significantly reduced migration and invasion. Expression of antisense-miR-29b (anti-miR-29b), on the other hand, enhanced ID1 mRNA and protein levels, and significantly increased lung cancer cell migration and invasion, a hallmark of the Src-ID1 pathway. The ectopic expression of ID1 in miR-29b-overexpressing cells was able to rescue the migratory potential of these cells. Both, anti-miR-29b and ID1 overexpression diminished the effects of the Src inhibitors saracatinib and dasatinib on migration and invasion. Saracatinib and dasatinib decreased c-Myc transcriptional repression on miR-29b and led to increased ID1 protein levels, whereas forced expression of c-Myc repressed miR-29b and induced ID1. In agreement, we showed direct recruitment of c-Myc to the miR-29b promoter. miR-29b was significantly downregulated in primary lung adenocarcinoma samples compared with matched alveolar lung tissue, and miR-29b expression was a significant prognostic factor for patient outcome. These results suggest that miR-29b is involved in the Src-ID1 signaling pathway, is dysregulated in lung adenocarcinoma and is a potential predictive marker for Src kinase inhibitors.

PRO_10--a new tissue-based prognostic multigene marker in patients with early estrogen receptor-positive breast cancer.

BACKGROUND/AIMS: Clinicopathological and molecular factors determine the prognosis of breast cancer. PRO_10 is a prognostic score based on quantitative RT-PCR of 10 proliferation-associated genes obtained from formalin-fixed, paraffin-embedded breast cancer tissues. We revalidated PRO_10 in patients treated in a non-trial setting. METHODS: The charts of 315 patients with postmenopausal estrogen receptor (ER)-positive breast cancer between 1996 and 2004 were reviewed. Forty-eight cases relapsed within 5 years of diagnosis; they were paired with controls by matching the N and T stage, histological grade, percent ER-positive cells, human epidermal growth factor receptor 2, age, adjuvant chemo- and endocrine therapy. The score was tested by conditional logistic regression. RESULTS: Despite strict matching, PRO_10 remained prognostic for recurrence in the whole group (odds ratio, OR = 4.7, p = 0.005) and in subgroups of grade 2 (OR = 5.5, p = 0.009) and N0 cancers (OR = 15, p = 0.002). Five-year recurrence-free survival was 29% in patients with high and 67% in patients with low scores (p = 0.002). PRO_10 was prognostic for overall survival (5-year overall survival 71 vs. 91%). CONCLUSION: PRO_10 is an independent prognostic marker in postmenopausal ER-positive breast cancer. It is based on formalin-fixed, paraffin-embedded tissue and could be integrated easily into the routine diagnostic workflow.

Sulphur management in onion (Allium cepa) cultivation in hills of Himachal Pradesh.

Field experiment were conducted at CSK HPKV Research Farm, Palampur during Rabi seasons of 2000-01 and 2001-02, to study the response of onion (Allium cepa var Patna red) at four sulphur levels (0, 15, 30 and 60 kg ha(-1)) applied through Gypsum and S95. The analysis was done to allocate the limited availability of sulphur for maximizing net profit over fertilizer cost. The results show that the dose of sulphur under its full availability is 43.02 kg ha(-1). But under its scarce availability the maximum benefit would occur when it is applied up to 32.11 kg ha(-1) followed by even distribution of fertilizer i.e. 20 kg ha(-1). The returns following sulphur application at these rates, would be Rs 69340, 73092 and 68700 ha(-1) respectively.

The low-dose combination preparation Vertigoheel activates cyclic nucleotide pathways and stimulates vasorelaxation.

Vertigo of various and often unknown aetiologies has been associated with and attributed to impaired microvascular perfusion in the inner ear or the vertebrobasilar system. Vertigoheel is a low-dose combination preparation of proven value in the symptomatic treatment of vertigo. In the present study we tested the hypothesis that Vertigoheel's anti-vertiginous properties may in part be due to a vasodilatory effect exerted via stimulation of the adenylate and/or guanylate cyclase pathways. Thus, the influence of Vertigoheel or its single constituents on synthesis and degradation of cyclic nucleotides was measured. Furthermore, vessel myography was used to observe the effect of Vertigoheel on the vasoreactivity of rat carotid arteries. Vertigoheel and one of its constituents, Anamirta cocculus, stimulated adenylate cyclase activity, while another constituent, Conium maculatum, inhibited phosphodiesterase 5, suggesting that the individual constituents of Vertigoheel contribute differentially to a synergistic stimulation of cyclic nucleotide signalling pathways. In rat carotid artery rings, Vertigoheel counteracted phenylephrine-induced tonic vasoconstriction. The present data demonstrate a vasorelaxant effect of Vertigoheel that goes along with a synergistic stimulation of cyclic nucleotide pathways and may provide a mechanistic basis for the documented anti-vertiginous effects of this combination preparation.

Antinociceptive activity of chronic administration of different extracts of Terminalia bellerica Roxb. and Terminalia chebula Retz. fruits.

The petroleum ether (PE), chloroform (CH), ethanol (ETH) and water extracts of Terminalia bellerica and T. chebula fruits were evaluated for their analgesic activity using the tail immersion model in mice. The ethanolic extracts of both the plants exhibited analgesic response at 200,400 and 800mg/kg. The studies were further carried for 15 days to evaluate the effect of these extracts in chronic pain and maximum analgesic response was observed on 14th day in both the plants. Phytochemical investigation of ethanolic extract of the fruits of Terminalia bellerica and T. chebula revealed the presence of saponins, triterpenoids, carbohydrates, tannins and proteins. The results indicate that fruits of T. bellerica and T. chebula could be considered as potential candidate for bioactivity-guided isolation of natural analgesic agents used in the management of chronic pain.

Non-invasive gene-expression-based detection of well-developed collateral function in individuals with and without coronary artery disease.

BACKGROUND: In patients with coronary artery disease (CAD), a well grown collateral circulation has been shown to be important. The aim of this prospective study using peripheral blood monocytes was to identify marker genes for an extensively grown coronary collateral circulation. METHODS: Collateral flow index (CFI) was obtained invasively by angioplasty pressure sensor guidewire in 160 individuals (110 patients with CAD, and 50 individuals without CAD). RNA was extracted from monocytes followed by microarray-based gene-expression analysis. 76 selected genes were analysed by real-time polymerase chain reaction (PCR). A receiver operating characteristics analysis based on differential gene expression was then performed to separate individuals with poor (CFI<0.21) and well-developed collaterals (CFI>or=0.21) Thereafter, the influence of the chemokine MCP-1 on the expression of six selected genes was tested by PCR. RESULTS: The expression of 203 genes significantly correlated with CFI (p = 0.000002-0.00267) in patients with CAD and 56 genes in individuals without CAD (p = 00079-0.0430). Biological pathway analysis revealed 76 of those genes belonging to four different pathways: angiogenesis, integrin-, platelet-derived growth factor-, and transforming growth factor beta-signalling. Three genes in each subgroup differentiated with high specificity among individuals with low and high CFI (>or=0.21). Two out of these genes showed pronounced differential expression between the two groups after cell stimulation with MCP-1. CONCLUSIONS: Genetic factors play a role in the formation and the preformation of the coronary collateral circulation. Gene expression analysis in peripheral blood monocytes can be used for non-invasive differentiation between individuals with poorly and with well grown collaterals. MCP-1 can influence the arteriogenic potential of monocytes.

Direct, residual and direct + residual effects of sulphur in garlic (Allium sativum)-maize (Zea mays) cropping sequence.

Significant positive effects of 30 kg/ha of sulphur as manifested on yield and yield parameters of garlic were further carried over to following maize crop. Garlic bulb and foliage yield (6.3 and 0.8 t/ha respectively) obtained at 30 kg/ha of sulphur dose was significantly higher over without sulphur (3.7 and 0.5 t/ha respectively) as revealed from two years' pooled data. Similarly number of leaves/plant, weight of cloves/5bulbs and weight/100 cloves at the said sulphur dose significantly increased over without sulphur from 10.5 to 11.9, 98.3 to 141.2 g and from 159 to 217 g in respective manner Increase in grain yield of maize (residual effect) and in the economic yield of the whole cropping sequence (Bulb yield of garlic and grain yield of maize) i.e. direct plus residual effect at 30 kg/ha of sulphur dose over without sulphur was from 28.3 to 47.2 and from 71 to 116 q/ha in respective manner i.e. with significant differences. Sulphur use efficiencies (kg yield/kg sulphur) of these crops at 15, 30 and 45 kg/ha over no sulphur were 57, 43 and 32; 53, 63 and 6 and 160, 150 and 67, all in respective order An optimum sulphur dose of 44.3 kg/ha produced increased bulb yield (over no S) worth Rs 34892 over fertilizer cost giving B:C ratio of 31.5:1. Utilization of sulphur added at 15, 30 and 45 kg/ha rates was 24.1, 19.3 and 15.7% by the garlic crop; and 29.6. 24.5 and 9.02% by the following maize crop, thus, adding up to 54.1, 43.8 and 24.9% by the cropping sequence, all in respective order.

Hepatic effects of Cimicifuga racemosa extract in vivo and in vitro.

Extracts of Cimicifuga racemosa are used frequently for menopausal complaints. Cimicifuga is well tolerated but can occasionally cause liver injury. To assess hepatotoxicity of cimicifuga in more detail, ethanolic C. racemosa extract was administered orally to rats, and liver sections were analyzed by electron microscopy. Tests for cytotoxicity, mitochondrial toxicity and apoptosis/necrosis were performed using HepG2 cells. Mitochondrial toxicity was studied using isolated rat liver mitochondria. Microvesicular steatosis was found in rats treated with > 1,000 mg/kg [DOSAGE ERROR CORRECTED] body weight cimicifuga extract. In vitro, cytotoxicity was apparent at concentrations > or =75 microg/mL, and mitochondrial beta-oxidation was impaired at concentrations > or =10 microg/mL. The mitochondrial membrane potential was decreased at concentrations > or =100 microg/mL, and oxidative phosphorylation was impaired at concenq trations > or =300 microg/mL. The mechanism of cell death was predominantly apoptosis. C. racemosa exerts toxicity in vivo and in vitro, eventually resulting in apoptotic cell death. The results are compatible with idiosyncratic hepatotoxicity as observed in patients treated with cimicifuga extracts.

HPLC based activity profiling for 5-lipoxygenase inhibitory activity in Isatis tinctoria leaf extracts.

In the pursuit of the anti-inflammatory constituents in lipophilic woad extracts, the 5-lipoxygenase (5-LOX) inhibitory activity was investigated by HPLC-based activity profiling. In a low-resolution profiling, two time windows with peaks of activity were found. The first coincided with tryptanthrin, a known dual inhibitor of cyclooxygenase-2 (COX-2) and 5-LOX, whereas the major inhibitory fraction was towards the end of the HPLC run. The active fractions were profiled in a peak-resolved manner, and the compounds analyzed by LC-MS, GC and TLC. The activity in the lipophilic fractions of the Isatis extract could be linked to an unsaturated fatty acid, alpha-linolenic acid.

Apoptotic cell death in the lactating mammary gland is enhanced by a folding variant of alpha-lactalbumin.

Apoptosis is essential to eliminate secretory epithelial cells during the involution of the mammary gland. The environmental regulation of this process is however, poorly understood. This study tested the effect of HAMLET (human alpha-lactalbumin made lethal to tumor cells) on mammary cells. Plastic pellets containing HAMLET were implanted into the fourth inguinal mammary gland of lactating mice for 3 days. Exposure of mammary tissue to HAMLET resulted in morphological changes typical for apoptosis and in a stimulation of caspase-3 activity in alveolar epithelial cells near the HAMLET pellets but not more distant to the pellet or in contralateral glands. The effect was specific for HAMLET and no effects were observed when mammary glands were exposed to native a-lactalbumin or fatty acid alone. HAMLET also induced cell death in vitro in a mouse mammary epithelial cell line. The results suggest that HAMLET can mediate apoptotic cell death in mammary gland tissue.

Dual inhibition of 5-lipoxygenase/cyclooxygenase by a reconstituted homeopathic remedy; possible explanation for clinical efficacy and favourable gastrointestinal tolerability.

OBJECTIVE: In order to elucidate potential anti-inflammatory activities of Zeel comp. N and its constituents, the inhibition of the synthesis of Leukotriene B4 (LTB4) and Prostaglandin (PGE2) by 5-lipoxygenase (5-LOX) and cyclo-oxygenase 1 and 2 (COX 1 and 2) respectively were examined in vitro. MATERIALS: Human HL-60 cells, differentiated for 6-8 days with DMSO (1.2% v/v) were used for the 5-LOX assay. The COX activity assays were carried out with purified enzymes, COX 1 (ram seminal vesicles), COX 2 (sheep placenta) and with human THP-1 cells, differentiated for 24 h with PMA (50 nM). METHODS: LTB4 and PGE2 production in the 5-LOX and COX assays respectively were determined by enzyme linked immunoassays. RESULTS: A reconstituted Zeel comp. N combination as well as its constituent mother tinctures of Arnica montana, Sanguinaria canadensis and Rhus toxicodendron (Toxicodendron quercifolium) showed distinct inhibitory effects on the production of LTB4 by 5-LOX (IC50 values of 10, 20, 2 and 5 microg/ml respectively) and on the synthesis of PGE2 by COX 1 (IC50 values of 50, 80, 40 and 20 microg/ml respectively) and COX 2 enzymes (IC50 values of 60, 110, 50 and 20 microg/ml respectively). The mother tincture of Solanum dulcamara inhibited the production of PGE2 by COX 1 (IC50 40 microg/ml) and COX 2 (IC50 150 microg/ml) but not production of leukotriene LTB4 by 5-LOX. CONCLUSIONS: The observed dual inhibition of both LOX- and COX-metabolic pathways may offer an explanation for the reported clinical efficacy and the favorable gastrointestinal tolerability of the original remedy Zeel comp. N.

Higher succinate than acetate levels differentiate cerebral degenerating cysticerci from anaerobic abscesses on in-vivo proton MR spectroscopy.

We present three patients with large intraparenchymal isolated degenerating cysticerci in whom the diagnosis was primarily based on in-vivo proton MR spectroscopy, and subsequently confirmed histologically. We suggest that the presence of succinate alone or more succinate acetate indicates the presence of degenerating cysticerci and differentiates them from anaerobic brain abscesses, which show acetate alone or in higher concentration than succinate, even when the clinical and imaging features are similar.

Conjugation of desmethylnaproxen in the rat--a novel acyl glucuronide-sulfate diconjugate as a major biliary metabolite.

The nonsteroidal anti-inflammatory drug naproxen is primarily metabolized in humans by acyl glucuronidation to form naproxen acyl glucuronide and by O-dealkylation to form 6-O-desmethylnaproxen (DMN). DMN contains both carboxy and phenolic groups and has been shown to form acyl glucuronide and sulfate conjugates. This project aimed to investigate whether DMN formed a phenolic glucuronide and diglucuronide(s) (with both the carboxy and phenolic groups glucuronidated). Male Sprague-Dawley rats (300-350 g) with exteriorized bile flow were dosed i.v. with DMN at 50 mg/kg. Four major DMN-related peaks were detected in bile by high-performance liquid chromatography (HPLC) analysis at 225 nm, including the known acyl glucuronide and sulfate conjugates. Selective hydrolyses using acidic and alkaline conditions and digestion with beta-glucuronidase allowed tentative identification of the two unknown peaks as the phenolic glucuronide of DMN and a novel acyl glucuronide-sulfate diconjugate of DMN (i.e., formed by sulfonation of the phenolic group and glucuronidation of the carboxy group). The identities were confirmed by liquid chromatography-tandem mass spectrometry analysis of individual HPLC fractions. Total recovery of the DMN dose was approximately 80%, with the sulfate conjugate (50%) and unchanged DMN (10%) being excreted predominantly in urine and the acyl glucuronide (10%), phenolic glucuronide (6%), and acyl glucuronide-sulfate diconjugate (4%) being excreted predominantly or exclusively in bile. No evidence for a diglucuronide metabolite of DMN was found in either bile or urine of the DMN-dosed rats.

Mouse mammary gland involution is associated with cytochrome c release and caspase activation.

At weaning, milk producing mammary epithelial cells undergo apoptosis and are removed by phagocytosis. Here, we show that mouse mammary gland involution is associated with mitochondrial cytochrome c release and processing of numerous caspases, including caspase-1, -3, -7, -8 and -9. Induction of caspase-3-like activity paralleled cleavage of poly-(ADP--ribose) polymerase. Dexamethasone inhibited processing of caspase-3, -7 and -8 and apoptosis, but had no effect on caspase-1 accumulation and cytochrome c release. In Bcl-2 transgenic animals, cytochrome c release, caspase activation and apoptosis were impaired. Thus, the pro-apoptotic signaling pathway in mammary epithelial cells during involution involves the release of cytochrome c and activation of caspases. It is inhibited by Bcl-2 at the mitochondrial level and by dexamethasone at a post-mitochondrial level.

Role of Fas ligand expression in promoting escape from immune rejection in a spontaneous tumor model.

Tumors escape immune-mediated rejection by a variety of mechanisms during tumor progression. The elucidation of these mechanisms in vivo suffers from a lack of suitable models of spontaneous tumor formation escaping active specific immunotherapy (ASI). In a rat neu transgenic (rNeu-TG) mouse model of spontaneous breast tumor formation, we showed that rNeu-TG mice developed late escape tumors despite the presence of a persistent rNeu-specific immune response after ASI. Cell suspensions derived from these escape tumors grew in vaccinated tumor-free mice, whereas injected spontaneous tumor cells were rejected. Escape tumors retained rNeu or MHC class I expression but significantly upregulated Fas (CD95, Apo-1) ligand. We further demonstrated that Fas-L on escape tumor cells correlated with apoptosis of infiltrating T lymphocytes. Thus, our results provide evidence that spontaneous breast tumors upregulate Fas-L expression after vaccination that may promote tumor escape in vivo after ASI.

Caspase-3 is essential for procaspase-9 processing and cisplatin-induced apoptosis of MCF-7 breast cancer cells.

In this study, we sought to investigate in more detail the role of caspase-3 in apoptotic processes in cultured cells and in cell-free extracts of breast cancer cells. We present evidence that apoptosis of caspase-3-deficient MCF-7 breast cancer cells is defective in response to cisplatin treatment, as determined by chromatin condensation, nuclear fragmentation, DNA fragmentation, and release of cytochrome c from the mitochondria. Reconstitution of MCF-7 cells by stable transfection of CASP-3 cDNA restores all these defects and results in an extensive apoptosis after cisplatin treatment. We further show that in extracts from caspase-3-deficient MCF-7 cells, procaspase-9 processing is strongly impaired after stimulation with either cytochrome c or recombinant caspase-8. Reconstitution of MCF-7 cell extracts with procaspase-3 corrects this defect, resulting in an efficient and complete processing of procaspase-9. Together, our data define caspase-3 as an important integrator of the apoptotic process in MCF-7 breast cancer cells and reveal an essential function of caspase-3 for procaspase-9 processing.

The tunel assay in the diagnosis of graft-versus-host disease: caveats for interpretation.

Acute graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following bone marrow transplantation, and early detection is important to allow effective therapy. Since the presence of apoptotic keratinocytes (dyskeratotic bodies) has been suggested as a useful diagnostic criterion for GVHD, attention has focused on the use of the TUNEL assay to detect apoptosis in clinical specimens. We reviewed clinical specimens upon which TUNEL had been performed for possible artifacts that might interfere with accurate evaluation for GVHD. Several distinct types of artifact were found and could be re-created in experimental systems. Artifacts in TUNEL staining generally resulted from the lack of specificity of this reaction for apoptotic cell death. Artifacts were found resulting from inadequate fixation, over-exposure of the TUNEL reaction, and proximity to the section edge. In addition, a novel artifact, apparently resulting from DNA shearing during the sectioning process, was noted and confirmed using confocal microscopy of experimental specimens. The TUNEL assay must therefore must be interpreted with caution in the clinical setting. In our laboratory, we consider TUNEL-positive cells as apoptotic only when accompanied by apoptotic morphology. Although these criteria clearly miss some cells in the early stages of apoptosis, they provide the highest specificity for apoptotic cell death.