Markers Of Biological Activity Research Articles

P0218 Dietary Inflammatory Index and disease activity in patients with IBD: is there an association? A Preliminary Analysis

Abstract Background Inflammatory bowel disease (IBD) is increasing in incidence in recently industrialized and developing countries, and this increment is paralleled by Westernization of lifestyle.1 In fact, specific nutrients and additives instigate gut inflammation and increasing evidence demonstrates its impact on developing IBD and disease behavior.2,3 Dietary Inflammatory Index (DII) was developed to assess the inflammatory properties of a diet.4 The aim of this study is to assess the association between higher DII scores and disease activity in patients with ulcerative colitis (UC) and Crohn’s disease (CD). Methods A cross-sectional study was conducted from June to October 2024. Patients undergoing ambulatory treatment at a day care IBD unit were consecutively recruited. A self-applied semiquantitative food frequency questionnaire, validated for the adult Portuguese population, was applied and medical charts reviewed. Blood and feces were collected, and anthropometric parameters measured by a body composition analyzer (Tanita DC-430MA). DII score was calculated according to the Shivappa et al. method, using 30 parameteres.4 The primary outcome was to assess the association between DII scores and 4 disease activity levels defined by clinical and biological activity markers (table 1). Clinical activity was classified according to Mayo Partial score for UC and Harvey-Bradshaw index (HBI) for CD. Biological activity was evaluated through fecal calprotectin. Results A total of 100 IBD patients (37 UC, 61 CD and 2 non-classified) were included; mean age 43.47 ±16.03 years, 41% female. Mean disease duration was 9.72±7.62 years. Baseline characteristics are described at table 2. Mean DII score was 1.49±2.43. The calculated DII score was categorized into quartiles. Comparison of calprotectin, CRP and Mayo Partial score by DII quartile was performed using Kruskal-Wallis test, and HBS using one-way ANOVA. No statistically significant correlations were found. Chi-square test for the association between activity levels and DII quartiles showed no statistically significant differences (p 0.496 DC; p 0.492 UC). Conclusion An association between DII scores and disease activity was not observed. Further studies with larger and more homogeneous samples are needed in the future to clarify the utility of this score in clinical practice. Given the undeniable and growing importance of these tools, other scales should also be studied and validated in IBD population.

Raman spectroscopy assisted by other analytical techniques to identify the most deteriorated carbonate‐stones to be consolidated in two monuments of Vitoria‐Gasteiz (Spain)

AbstractThis work describes the diagnostic study on the building materials, mostly carbonated, belonging to Santa Maria Cathedral and the Medieval Wall of Vitoria‐Gasteiz (Spain) with the aim to design the best conservation procedure. Both the studies of the lithology and the secondary compounds originated by environmental impacts on the Cathedral and on the Medieval Wall were carried out using laboratory instruments (μ‐Raman and micro‐energy‐dispersive X‐ray spectroscopy, X‐ray diffraction and ion chromatography) on selected samples provided by the restorers. The systematic presence of black crusts in the stones of the Cathedral was related to the growth of microcrystalline structures of secondary compounds and biological patinas and the deposition of atmospheric particles from traffic and house heating systems. In fact, the main components identified were carbon, and iron compounds such as hematite, goethite, magnetite and lepidocrocite. In addition, the detection of lead compounds (lead‐rich hydroxyapatite) suggested in the same way the impact of the urban environment on the degradation and blackening of stone materials. The presence of sulfates, mainly gypsum, and, to a lesser extent, epsomite, anhydrite and bloedite could be caused by the sulfation of carbonated compounds as a result of an acid attack of atmospheric pollutants. The results on the secondary products of the Medieval Wall showed a greater presence of degradation by microorganisms compared to the Cathedral. This is probably related to the large garden surrounding the fortification, where the grass is in direct contact to the lower part of the structure. Markers of biological activity, such as carotenoid pigments and calcium oxalate weddellite, together with other soluble oxalates were identified. The presence of ammonium nitrate, characterised by means ion chromatography, causes a chemical degradation of carbonate stone materials over time, due to the acidic nature of the ammonium ion. In both cases considered in this study, the presence of nitrate compounds, nitratine and potassium nitrate, was attributed to both natural factors (ammonium nitrate is coming from the decomposition of plant and animal excretions), and anthropogenic contamination.

Biomineralization of primary carbonate cements: a new biosignature in the fossil record from the Anisian of Southern Italy

Biomineralization is a generic term used to indicate biological-mediated mineral formation. In carbonate mineralization, nucleation of crystals can be: (1) controlled directly by the organisms, like in the skeletal formation of most metazoans; (2) induced by microbial communities, by indirect precipitation mediated by their metabolic activities; or (3) influenced by organic matter decay, with mineral precipitation on specific non-living organic cell surfaces. Recognition of these products is a direct marker of biological activity in time and space and is a key element in the study of the biological evolution and of its interactions with the geological processes. In this paper, primary carbonate cements from the Anisian microbial build-up of the ‘Monte Facito’ Formation (Basilicata region, Southern Italy) have been studied from a geobiological point of view. Optical microscopy, UV-epifluorescence and micro-Raman spectroscopy have been applied to investigate the organic mediation on their precipitation. The cements formed in microcavities or on grain substrates, and often show a microstromatolite-like pattern of growth. They are composed of alternations of cloudy organic and whitish inorganic bands that point to a double phase of mineralization. In the first phase, a biologically induced/influenced biomineralization is confirmed by the presence of organic matter strictly connected with the cloudy bands. This phase is followed by a pure abiotic mineralization that leads to the formation of whitish bands. This process repeated cyclically, ending at the complete filling of the microcavities or because of changes in the chemical conditions of the microsystem, for example, due to burial processes. This model of mineralization is similar to that proposed for primary cements forming in recent beach rocks. The Monte Facito Formation cements could be considered as the product of unconventional biomineralization, and the understanding of their growth process could provide an innovative tool in the research of biological signatures in the fossil record. The term unconventional is here utilized to discriminate this type of biomineralizations from those related to well-known biotic mineralization processes, like those involved in skeletons and microbialites growth, which can be considered as conventional biomineralizations.

Echo Intima-Media Thickness (IMT) in the Evaluation of Early Atherosclerosis in Inflammatory Bowel Disease (IBD) Patients – a Prospective Study

Introduction: Inflammatory bowel diseases (IBD) are accompanied by an early and accelerated atherosclerotic process. Previous studies showed that Doppler ultrasonography and intima media thickness (IMT) is a reliable marker for early atherosclerosis diagnosis. The aim of our study was to evaluate the presence of early atherosclerosis in a group of subjects with inflammatory bowel diseases, using Doppler ultrasonography and intima-media thickness (IMT) as a marker of subclinical atherosclerosis. Methods: A case-control study was conducted on 61 patients with inflammatory bowel disease and 19 healthy individuals. The included patients had an established IBD diagnosis, based on clinical, endoscopic, and histological criteria for at least 12 months. Results: In our study echo IMT was not significantly higher in IBD patients than in matched healthy controls. We found no difference between Crohn’s disease and ulcerative colitis patients or between conventional or biologic treatment regarding the echo IMT. Severity of the disease and its duration were predictors of an increased IMT. We found that disease activity (measured by the endoscopic scores and CRP) correlates with higher values of echo IMT. Also the presence of anemia reached statistical significance and was correlated with increased echo IMT in the biologic treatment group. Conclusions: In our study we found an increased echo IMT in IBD patients versus control, but the values did not reach statistical significance. We found no difference between Crohn’s disease and UC, conventional or biologic treatment. Age and duration of disease were corelated with increased IMT. Disease that was not controlled (high endoscopic, clinical and biological markers of activity) was correlated with increased echo IMT. The limitations of our study are that in our patients therapy was started early in the course of the disease and that the majority of the patients were in remission when echography was made. Further studies are necessary to evaluate factors correlated with early atherosclerosis in IBD and how echo IMT can be used as a tool in the proactive evaluation of these patients.

Phenolic Fractions from Vaccinium vitis-idaea L. and Their Antioxidant and Anticancer Activities Assessment.

Lingonberry leaves and fruits are associated with a range of potential bioactivities related to their phenolic content and composition, but the identification of major biological activity markers remains limited. The present study aimed at the isolation of lingonberry phenolic fractions and biological activity evaluation of them. Crude dry extracts of lingonberry leaves and fruits were fractionated by chromatography using Sephadex LH-20 and analyzed by validated HPLC-PDA method. For each fraction, the anticancer activity against human clear cell renal cell carcinoma (CaKi-1), human colon adenocarcinoma (HT-29), and human malignant melanoma (IGR39) cell lines was determined using MTT assay, and the radical scavenging, reducing, and chelating activities were investigated using ABTS, FRAP, and FIC assays, respectively. Further, 28 phenolics were identified and quantified in the crude extract of lingonberry leaves and 37 in the extract of fruits. These compounds, during fractionation steps, were selectively eluted into active fractions, enriched with different groups of phenolics—monophenols, anthocyanins, phenolic acids, catechins, flavonols, or proanthocyanidins. Fractions of lingonberry leaves and fruits, obtained by the last fractionation step, proved to be the most active against tested cancer cell lines and possessed the greatest antioxidant activity. In this perspective, the predominant compounds of these fractions—polymeric and mainly A-type dimeric proanthocyanidins—also quercetin can be considered to be anticancer and antioxidant activity markers of lingonberries.

Biological Activity Markers Useful in the Diagnosis of Chronic Diarrhea

Biological Activity Markers Useful in the Diagnosis of Chronic Diarrhea

Transcriptional profiling of Salmonella enterica serovar Enteritidis exposed to ethanolic extract of organic cranberry pomace.

Non-typhoidal Salmonella enterica serovars continue to be an important food safety issue worldwide. Cranberry (Vaccinium macrocarpon Ait) fruits possess antimicrobial properties due to their various acids and phenolic compounds; however, the underlying mechanism of actions is poorly understood. We evaluated the effects of cranberry extracts on the growth rate of Salmonella enterica serovars Typhimurium, Enteritidis and Heidelberg and on the transcriptomic profile of Salmonella Enteritidis to gain insight into phenotypic and transcriptional changes induced by cranberry extracts on this pathogen. An ethanolic extract from cranberry pomaces (KCOH) and two of its sub-fractions, anthocyanins (CRFa20) and non-anthocyanin polyphenols (CRFp85), were used. The minimum inhibitory (MICs) and bactericidal (MBCs) concentrations of these fractions against tested pathogens were obtained using the broth micro-dilution method according to the Clinical Laboratory Standard Institute’s guidelines. Transcriptional profiles of S. Enteritidis grown in cation-adjusted Mueller-Hinton broth supplemented with or without 2 or 4 mg/ml of KCOH were compared by RNASeq to reveal gene modulations serving as markers for biological activity. The MIC and MBC values of KCOH were 8 and 16 mg/mL, respectively, against all tested S. enterica isolates. The MIC value was 4 mg/mL for both CRFa20 and CRFp85 sub-fractions, and a reduced MBC value was obtained for CRFp85 (4 mg/ml). Treatment of S. Enteritidis with KCOH revealed a concentration-dependent transcriptional signature. Compared to the control, 2 mg/ml of KCOH exposure resulted in 89 differentially expressed genes (DEGs), of which 53 and 36 were downregulated and upregulated, respectively. The upregulated genes included those involved in citrate metabolism, enterobactin synthesis and transport, and virulence. Exposure to 4 mg/ml KCOH led to the modulated expression of 376 genes, of which 233 were downregulated and 143 upregulated, which is 4.2 times more DEGs than from exposure to 2 mg/ml KCOH. The downregulated genes were related to flagellar motility, Salmonella Pathogenicity Island-1 (SPI-1), cell wall/membrane biogenesis, and transcription. Moreover, genes involved in energy production and conversion, carbohydrate transport and metabolism, and coenzyme transport and metabolism were upregulated during exposure to 4 mg/ml KCOH. Overall, 57 genes were differentially expressed (48 downregulated and 9 upregulated) in response to both concentrations. Both concentrations of KCOH downregulated expression of hilA, which is a major SPI-1 transcriptional regulator. This study provides information on the response of Salmonella exposed to cranberry extracts, which could be used in the control of this important foodborne pathogen.

EVALUATION OF SERUM PROCALCITONIN, SERUM IRON AND RE.D CELL DISTRIBUTION WIDTH (RDW) AS NOVEL BIOLOGICAL MARKERS IN INFLAMMATORY BOWEL DISEASES.

Background: Ulcerative colitis and Crohn’s disease are idiopathic inflammatory bowel diseases (IBD). Although some clinical activity indexes are commonly used in IBD, specific and sensitive laboratory markers that correlate with disease activity and associated complication are still lacking. Traditional markers, such as C-reactive protein (CRP), the erythrocyte sedimentation rate (ESR), and the white blood cell (WBC) count, are still the most common markers usedin clinical practice. Procalcitonin (PCT) plays a major role in systemic inflammation and induces a dose-dependent increase in TNFα secretion. Plasma level of PCT increases during bacterial infections and sepsis. There are some data showing that serum PCT level is a useful marker in many inflammatory disorders. Previous findings suggested that inflammatory and infectious disease of the bowel might increase serum PCT levels. Anemia is the most prevalent extra intestinal complication of IBD. Serum iron was previously included in several studies as inflammatory marker of IBD.RDW is a quantitative measurement of anisocytosis. Pro-inflammatory cytokines have been reported to inhibit the maturation of erythrocytes, which is caused by erythropoietin. Thus, inflammation causes immature red blood cells to be released into the peripheral circulation, which may result in anisocytosis. Aim of the work: This study aims to evaluate sensitivity andspecificity of serum procalcitonin, serum iron and red cell distribution width (RDW) as novel biological markers of activity in IBD patients. Patients and methods:60 Patients with confirmed IBD diagnosis (Crohn's and ulcerative colitis) were divided into 2groups (30 remission and 30 exacerbation) classified according to Truelove and Witts index in ulcerative colitis and Harvey Bradshaw index in Crohn’s disease. Routine diagnostic investigations, serum Procalcitonin and serum iron level were withdrawn. Serum procalcitonin was done by commercially available ELISA kits. Results: The study showed significant difference between the 2 groups as regards serum iron where median in remission was 52 mcg/dl while in activity was 26 mcg/dl (P value:0.033),also for RDW (P value:0.014) and serum procalcitonin where mean in remissionwas 0.62 ng/ml while in activity was 0.98 ng/ml ( P value:0.029). Conclusion: RDW, serum procalcitonin and serum iron in IBD patients need to be included in further studies with larger sample size for the possibility to be used as future laboratory markers for disease activity.

Prognostic value of thymidine kinase activity in patients with chronic lymphocytic leukemia.

Thymidine kinase (TK) activity is a marker of biological activity that allows the indolent and aggressive forms of chronic lymphocytic leukemia (CLL) to be distinguished. The aims of the study were to determine the relationship between TK activity and clinical status and prognosis, as well as to compare its activity with that of other prognostic and predictive factors. TK activity was measured in patient sera at the time of diagnosis using the DiviTum method, with the mean value being 439 Du/L. A correlation was found between TK activity and risk of disease progression (p=0.045). The optimal discriminative value of TK activity in the prediction of CLL progression was found to be 600 Du/L. TK activity significantly differed between the patients who achieved complete remission and those who only partially responded to therapy. In 93% of patients without any response to treatment and 18 out of 20 patients with progressive disease, TK activity over 600 Du/L was noted. In addition, all of the 10 patients with 17p13 deletion displayed TK activity of over 600 Du/L (p=0.0004). High TK activity also correlated with elevated levels of LDH (p=0.001) and β2‑microglobulin (p=0.03) in the study group. The results of the study indicated the importance of TK activity as a prognostic factor in patients with CLL.

Abstract 4994: SEA-CD40: from bench to bedside

Abstract SEA-CD40 is a non-fucosylated, humanized IgG1 monoclonal antibody directed against human CD40, a co-stimulatory receptor of the TNF receptor superfamily. The consequence of enhanced SEA-CD40/FcγRIIIa binding is potent immune stimulatory activity. CD40 receptor ligation induces multiple pathways; to pave the road for identification of a specific activity signature in the clinical setting, in vitro preclinical assays were developed to monitor the immune modulatory activity of SEA-CD40. Human PBMCs stimulated with increasing concentrations of SEA-CD40 were assessed for immune changes including cytokine production, cellular activation, and modulation of cellular subsets. SEA-CD40 PBMC stimulation elicited a unique set of cytokines including MIP-1β, MCP-1, and IL-8. In addition to inducing cytokines, specific immune cell changes were also observed including up-regulation of stimulatory molecules on monocyte/ macrophages, activation of NK cells, and changes in cellular subsets such as deletion of B-cells. While some of these changes were common across the other CD40 therapeutic antibodies being tested in the clinic, SEA-CD40-specific changes were identified. These changes included a reduction in the immune dampening cytokine IL-10, induction of Th1 CXCR3 positive cells, and reduction of T-regulatory cells, all potentially contributing to an antitumor immune response. The specific in vitro SEA-CD40 signature was also observed in vivo in cynomolgus monkeys. SEA-CD40 treatment induced the same signature cytokines observed in vitro and elicited the same cellular changes including depletion of B-cells, and activation of CD8+ T-cells. A CD40 receptor occupancy assay was also created to correlate receptor engagement with activity. Interestingly, while SEA-CD40 is rapidly cleared from plasma, it is detectable on the surface of antigen-presenting cells for up to 3 weeks. The initial starting dose for SEA-CD40 clinical trials was calculated using the minimal anticipated biological effect level (MABEL). SEA-CD40 cytokine induction was the most sensitive preclinical marker of biologic activity and was, therefore, used for MABEL dose calculation. In the ongoing phase 1 First-In-Human clinical trial, this preclinical SEA-CD40 activity signature is being monitored in adult patients with advanced solid tumors (study NCT02376699). Establishing a clear immune biomarker strategy from pre-clinical research to clinical trials is vital for tracking the activity of our immuno-oncology drugs in patients and for identifying a safe and efficacious regimen. Citation Format: Shyra J. Gardai, Haley Neff-LaFord, Angela Epp, Jing Yang, Thomas Manley, Che-Leung Law. SEA-CD40: from bench to bedside. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4994.

Pharmacokinetic and Pharmacodynamic Characteristics of Subcutaneously Applied PTH-1-37

Background/Aims: Parathyroid hormone (PTH) derivatives exert pronounced renal and osteoanabolic properties when given intermittently. The current study was performed to assess the pharmacokinetic and pharmacodynamic properties as well as safety of subcutaneously applied PTH-1-37 after repeated dosing in healthy subjects. Methods: This randomized, double-blind, dose-escalating, placebo and active comparator controlled study was conducted in 33 healthy postmenopausal women. Subjects were allocated to one of five treatment options: 10, 20, or 40 µg PTH-1-37, 20 µg PTH-1-34 or placebo, administered as once daily subcutaneous doses for three days. Plasma drug concentrations and serum levels of endogenous PTH-1-84, and calcium as markers of biological activity were monitored during the treatment. Results: PTH was absorbed rapidly from the subcutaneous tissue with a median t_max of 30 minutes for 20 and 40 µg of PTH-1-37. t_max was 45 minutes for 20 µg PTH-1-34. Elimination half-lives were estimated as 76 ± 34 min and 70 ± 13 min for 20 µg and 40 µg PTH-1-37 (mean ± SD), and 78 ± 34 for 20 µg PTH-1-34. Both PTH fragments (PTH-1-37 and PTH-1-34) increased serum calcium. For PTH-1-37 the effect on serum calcium was dose-dependent. Suppression of endogenous PTH-1-84 was seen after the application of both PTH-1-37 and PTH-1-34. During the study period, the subjects experienced no unexpected or serious adverse events. Conclusions: PTH-1-37 is rapidly absorbed after s.c. injection, has a short plasma elimination half-life, and does not accumulate during multiple dosing. Biological activity was demonstrated by rising serum calcium and decreasing endogenous PTH-1-84 in blood plasma. The study drugs were well tolerated and safe. Our investigation presents data that PTH-1-37 is an excellent drug candidate for intervening with syndromes of dysregulation of calcium metabolism.

Role of Organics in Regulating ClNO₂ Production at the Air-Sea Interface.

We report measurements of the product yield for nitryl chloride (ClNO2) production following the reactive uptake of dinitrogen pentoxide (N2O5) to a wide variety of ambient seawater samples as well as seawater mimics. The ClNO2 yield, as measured for ambient seawater collected from both coastal and open ocean waters, was found to be both insensitive to chlorophyll-a, a marker for biological activity, and significantly lower (0.16-0.30) than that expected for equivalent salt-containing solutions (0.82 ± 0.05). Suppression in the ClNO2 yield can be induced by the addition of aromatic organic compounds (e.g., phenol and humic acid) to synthetic seawater matrices. In the case of phenol, surface tension measurements reveal that the surface phenol:chloride ratio can be enhanced by more than a factor of 100 as compared to bulk ratios for subtle changes in surface tension (<1.5 mN m(-1)), providing a mechanism to suppress ClNO2 production at low bulk phenol concentrations. We interpret measurements of the dependence of the ClNO2 yield on phenol using a kinetic model, where we confine the surface enhancement in phenol to the top 1 nm of the interface. Our results are most consistent with a model where N2O5 is ionized within the first three water monolayers (<1 nm), where the product nitronium ions react rapidly with interfacial phenol molecules. These results suggest that ClNO2 may not be formed at the air-sea interface at the yield expected for NaCl, and that the reactive uptake of N2O5 and the subsequent product yield of ClNO2 may serve as a unique probe for the composition of the interfacial region of the sea surface microlayer.

The Circulating Level of Soluble Receptor for Advanced Glycation End Products Displays Different Patterns in Ulcerative Colitis and Crohn's Disease: A Cross-Sectional Study.

RAGE is a transmembrane receptor expressed on immune and endothelial cells, whose binding with its ligands, the S100 calgranulins, leads to chronic inflammation. Conversely, its soluble form (sRAGE) plays a protective role by acting as a decoy. We carried out a cross-sectional analysis of the sRAGE and S100A12 serum levels in patients with Crohn's disease (CD) and ulcerative colitis (UC) and searched for a correlation with clinical and biological markers of activity. We enrolled 60 CD, 67 UC patients, and 66 controls (all adults). Disease activity was scored through the clinical, endoscopic, and histologic indexes of severity, whilst disease location and behaviour were assessed according to the Montreal classification. In all cases, the levels of serum sRAGE, S100A12, C-reactive protein, and faecal calprotectin were measured. sRAGE levels were significantly lower in UC, both active and inactive, than in controls and CD (817.35, range 437.3-1449; 1211, range 843.7-1618; 1207.5, range 743.15-1875.75; P<0.05 for both), and inversely correlated with clinical and endoscopic indexes of activity in both IBD groups (P<0.05 for all) and with the histologic score in the CD group. Moreover, those CD patients with a penetrating behaviour showed a significant reduction in both sRAGE (P=0.006) and S100A12 (P=0.034) as compared to those with an inflammatory/stricturing pattern. Although S100A12 levels were not found up-regulated, a negative correlation appeared evident with the clinical (r=-0.38) and endoscopic (r=-0.32) indexes of activity in UC and CD, respectively. These data suggest a different role for RAGE in CD and UC, and a potential use of sRAGE as a new biomarker.

Preclinical pharmacokinetics, pharmacodynamics, and efficacy of RG7116: a novel humanized, glycoengineered anti-HER3 antibody.

PurposeRG7116 is a novel anti-HER3 therapeutic antibody that inhibits HER3 signalling and induces antibody-dependent cellular cytotoxicity of tumor cells due to a glycoengineered antibody Fc moiety. We investigated the efficacy and pharmacokinetic/pharmacodynamic properties of HER3 signal inhibition by RG7116 in a murine xenograft model of human head and neck cancer.MethodsSCID-beige mice bearing FaDu cells were treated with RG7116 at a weekly dose of 0.3–10 mg/kg, and tumor growth control and modulation of selected proteins (HER3 and AKT) were examined.ResultsComplete tumor stasis up to Day 46 was observed at a dose >3 mg/kg, and this dose down-modulated membrane HER3 expression and inhibited HER3 and AKT phosphorylation. Systemic RG7116 exposure was greater than dose-proportional and total clearance declined with increasing dose, indicating that RG7116 elimination is target-mediated. This is consistent with the better efficacy, and the HER3 and pAKT inhibition, that was observed at doses >1 mg/kg. Tumor regrowth occurred from Day 46 onwards and was associated with HER1 and HER2 upregulation, indicating the activation of alternative HER escape pathways. Modulation of HER3 and phospho-HER3 was also demonstrated in the skin and mucosa of an RG7116-treated cynomolgus monkey, suggesting that these may be useful surrogate tissues for monitoring RG7116 activity.ConclusionsThese data confirm the promising efficacy of RG7116 and highlight the value of assessing the PK behavior of the antibody and measuring target protein modulation as a marker of biological activity. Clinical development of RG7116 has now begun, and phase I trials are ongoing.

Sonic hedgehog paracrine signaling activates stromal cells to promote perineural invasion in pancreatic cancer.

Pancreatic cancer is characterized by stromal desmoplasia and perineural invasion (PNI). We sought to explore the contribution of pancreatic stellate cells (PSC) activated by paracrine Sonic Hedgehog (SHH) in pancreatic cancer PNI and progression. In this study, the expression dynamics of SHH were examined via immunohistochemistry, real-time PCR, and Western blot analysis in a cohort of carcinomatous and nonneoplastic pancreatic tissues and cells. A series of in vivo and in vitro assays was performed to elucidate the contribution of PSCs activated by paracrine SHH signaling in pancreatic cancer PNI and progression. We show that SHH overexpression in tumor cells is involved in PNI in pancreatic cancer and is an important marker of biologic activity of pancreatic cancer. Moreover, the overexpression of SHH in tumor cells activates the hedgehog pathway in PSCs in the stroma instead of activating tumor cells. These activated PSCs are essential for the promotion of pancreatic cancer cell migration along nerve axons and nerve outgrowth to pancreatic cancer cell colonies in an in vitro three-dimensional model of nerve invasion in cancer. Furthermore, the coimplantation of PSCs activated by paracrine SHH induced tumor cell invasion of the trunk and nerve dysfunction along sciatic nerves and also promoted orthotropic xenograft tumor growth, metastasis, and PNI in in vivo models. These results establish that stromal PSCs activated by SHH paracrine signaling in pancreatic cancer cells secrete high levels of PNI-associated molecules to promote PNI in pancreatic cancer.

OP0224 A placebo-controlled phase II study of hyperimmune caprine serum as potential treatment for established diffuse cutaneous systemic sclerosis

ObjectivesHyperimmune caprine (goat) serum prepared under GMP conditions has been used to treat a number of inflammatory or autoimmune diseases including chronic inflammatory demyelinating polyneuropathy. We have performed a study...

In vitro and in vivo antibacterial activities of cranberry press cake extracts alone or in combination with β-lactams against Staphylococcus aureus

BackgroundCranberry fruits possess many biological activities partly due to their various phenolic compounds; however the underlying modes of action are poorly understood. We studied the effect of cranberry fruit extracts on the gene expression of Staphylococcus aureus to identify specific cellular processes involved in the antibacterial action.MethodsTranscriptional profiles of four S. aureus strains grown in broth supplemented or not with 2 mg/ml of a commercial cranberry preparation (Nutricran®90) were compared using DNA arrays to reveal gene modulations serving as markers for biological activity. Ethanol extracted pressed cakes from fresh fruits also produced various fractions and their effects on marker genes were demonstrated by qPCR. Minimal inhibitory concentrations (MICs) of the most effective cranberry fraction (FC111) were determined against multiple S. aureus strains and drug interactions with β-lactam antibiotics were also evaluated. Incorporation assays with [3H]-radiolabeled precursors were performed to evaluate the effect of FC111 on DNA, RNA, peptidoglycan (PG) and protein biosynthesis.ResultsTreatment of S. aureus with Nutricran®90 or FC111 revealed a transcriptional signature typical of PG-acting antibiotics (up-regulation of genes vraR/S, murZ, lytM, pbp2, sgtB, fmt). The effect of FC111 on PG was confirmed by the marked inhibition of incorporation of D-[3H]alanine. The combination of β-lactams and FC111 in checkerboard assays revealed a synergistic activity against S. aureus including strain MRSA COL, which showed a 512-fold drop of amoxicillin MIC in the presence of FC111 at MIC/8. Finally, a therapeutic proof of concept was established in a mouse mastitis model of infection. S. aureus-infected mammary glands were treated with amoxicillin, FC111 or a combination of both; only the combination significantly reduced bacterial counts from infected glands (P<0.05) compared to the untreated mice.ConclusionsThe cranberry fraction FC111 affects PG synthesis of S. aureus and acts in synergy with β-lactam antibiotics. Such a fraction easily obtained from poorly exploited press-cake residues, may find interesting applications in the agri-food sector and help reduce antibiotic usage in animal food production.

Serum Cytokine Profile by ELISA in Patients with Echinococcal Cysts of the Liver: A Stage-Specific Approach to Assess Their Biological Activity

To investigate the usefulness of serum cytokine dosage in the clinical management of cystic echinococcosis (CE), we analyzed serum levels of Th1 and Th2 cytokines in patients with hepatic CE in different cyst stages, CE1-2 (active), CE3a-3b (transitional), and CE4-5 (inactive). Ex vivo assessment of Th1 (IFN-γ) and Th2 (IL-4, IL-13, and IL-10) cytokines in sera was carried out using ELISA. IL-10 was undetectable in all serum samples of patients and controls, while a few sera contained measurable amounts of IFN-γ, IL-4, and IL-13. No statistically significant difference was found between the percentages of positive samples for each cytokine and the different groups analyzed (patients/controls, stage, number, location, and size of the cyst, serology, and sex of patients), with the exception of the association of IL-4 and IL-13 with the cyst stage. Overall, this investigation showed many limits of serum cytokine dosage as a marker of biological activity of echinococcal cysts. Because of low sensitivity and lack of specificity of this test, we believe that other ways to evaluate ex vivo biological activity of the cysts should be explored.

Association of rash with improved outcomes by the addition of cetuximab (C) to second-line mitoxantrone plus prednisone (MP) for progressive mCRPC after docetaxel-based chemotherapy.

e15040 Background: A previously reported phase II trial demonstrated that the addition of C, a chimeric monoclonal antibody that binds EGFR to MP for mCRPC following docetaxel did not extend overall time to progression (TTP) or overall survival (OS). In an unplanned analysis, we retrospectively analyzed the association of skin rash, a marker of biologic activity of C, with outcomes in CMP patients. Methods: The open-label phase II trial enrolled 115 patients with progressive mCRPC after docetaxel randomized 2:1 to CMP or MP. Therapy was M 12mg/m2 intravenously (IV) on Day 1, oral prednisone 10mg daily in both arms, and C 250mg/m2 IV (400mg/m2 Day 1, Cycle 1) on Days 1, 8, and 15 in the CMP arm. Cycles, a maximum of 10, were repeated every 21 days. Radiological assessments of disease and PSA occurred every 4 cycles. The primary endpoint was TTP defined as disease progression or death from any cause. 75 CMP patients were eligible for analyzing the association of rash with outcomes. Results: No MP arm patients developed rash. In the CMP arm, 51 patients (68%) had no rash, 24 (32%) had rash of any grade including 1 patient (1.3%) with grade 3 rash, and no grade 4 rash. Median TTP with and without rash was 10.3 months (95% CI: 5.4-11.8) vs 2.8 months (95% CI: 2.5-4.9), p(logrank)=0.004. Median survival with and without rash was 21.3 months (95% CI: 11.3-22.8) vs 9.3 months (95% CI: 5.3-12.3), p=0.01. On multivariable analysis including rash, baseline PSA (<median vs >/=median), performance status (0 vs 1-2) and visceral metastasis, rash was the only variable statistically significantly associated with TTP (HR=0.43, p=0.01), and demonstrating a trend for association with survival (HR=0.54, p=0.07). The overall CMP safety profile was consistent with profiles of components of study treatment. Conclusions: Patients with progressive mCRPC receiving CMP after docetaxel who developed rash demonstrated a significant extension of both TTP and OS. This finding is hypothesis-generating and may warrant the further study of higher doses of cetuximab coupled with pharmacodynamic and pharmacogenomic studies. Partially supported by ImClone Systems and Eli Lilly.

Thermodynamic and Kinetic Aspects of the Lipid Peroxidation of Arachidonic Acid

Lipid peroxidation is the oxidative modification of polyunsaturated fatty acids (PUFA) that leads to hydroperoxides. Hydroperoxides that are derived from the lipid arachidonic acid serve as regulators of the enzymes involved in prostaglandin synthesis. Further oxidation of these hydroperoxides produces short chain aldehydes, such as 4- hydroxynonenal. The mechanism of lipid peroxidation is well established, yet it is difficult to predict the ratio of aldehydes produced. Since these aldehydes are markers of biological activity, quantitative prediction of product ratios is desirable. Density functional calculations are used to study the thermodynamic and kinetic aspects of the first three steps in the mechanism of lipid peroxidation of arachidonic acid. It is found that the relative stability of peroxyl radical intermediates resulting from hydrogen abstraction and subsequent oxidation correlates with localization of the valence molecular orbitals on the peroxyl group. In addition, simple diene models are used to show that the most stable isomers of the peroxyl radicals are trans, trans, where the peroxyl group is located away from the bis-allylic carbon. Finally, the most stable peroxyl radicals originating from arachidonic acid are identified: these arise from hydrogen abstraction at the C13 position and exhibit trans, trans stereochemistry around the site of peroxidation.