Pharmacogenetic angiogenesis profiling for first-line Bevacizumab plus oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer

PURPOSE: There is substantial germline genetic variability within angiogenesis pathway genes, thereby causing interindividual differences in angiogenic capacity and resistance to antiangiogenesis therapy. We investigated germline polymorphisms in genes involved in VEGF-dependent and -independent angiogenesis pathways to predict clinical outcome and tumor response in metastatic colorectal cancer (mCRC) patients treated with bevacizumab and oxaliplatin-based chemotherapy.

EXPERIMENTAL DESIGN: A total of 132 patients treated with first-line bevacizumab and FOLFOX or XELOX were included in this study. Genomic DNA was isolated from whole-blood samples by PCR-RFLP or direct DNA sequencing. The endpoints of the study were progression-free survival (PFS), overall survival (OS), and response rate (RR).

RESULTS: The minor alleles of EGF rs444903 A>G and IGF-1 rs6220 A>G were associated with increased OS and remained significant in multivariate Cox regression analysis (HR: 0.52; 95% CI: 0.31-0.87; adjusted P = 0.012 and HR: 0.60; 95% CI: 0.36-0.99; adjusted P = 0.046, respectively). The minor allele of HIF1α rs11549465 C>T was significantly associated with increased PFS but lost its significance in multivariate analysis. CXCR1 rs2234671 G>C, CXCR2 rs2230054 T>C, EGFR rs2227983 G>A, and VEGFR-2 rs2305948 C>T predicted tumor response, with CXCR1 rs2234671 G>C remaining significant in multiple testing (P(act) = 0.003).

CONCLUSION: In this study, we identified common germline variants in VEGF-dependent and -independent angiogenesis genes predicting clinical outcome and tumor response in patients with mCRC receiving first-line bevacizumab and oxaliplatin-based chemotherapy.

The role of antimicrobial peptides in periodontal disease.

Objectives Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

Phylloseptin-PBa—A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity

Antimicrobial peptides from amphibian skin secretion display remarkable broad-spectrum antimicrobial activity and are thus promising for the discovery of new antibiotics. In this study, we report a novel peptide belonging to the phylloseptin family of antimicrobial peptides, from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea, which was named Phylloseptin-PBa. Degenerate primers complementary to putative signal peptide sites of frog skin peptide precursor-encoding cDNAs were designed to interrogate a skin secretion-derived cDNA library from this frog. Subsequently, the peptide was isolated and identified using reverse phase HPLC and MS/MS fragmentation. The synthetic replicate was demonstrated to have activity against S. aureus, E. coli and C. albicans at concentrations of 8, 128 and 8 mg/L, respectively. In addition, it exhibited anti-proliferative activity against the human cancer cell lines, H460, PC3 and U251MG, but was less active against a normal human cell line (HMEC). Furthermore, a haemolysis assay was performed to assess mammalian cell cytotoxicity of Phylloseptin-PBa. This peptide contained a large proportion of α-helical domain, which may explain its antimicrobial and anticancer activities.

Achieving optimal dose intensity with adjuvant chemotherapy in elderly breast cancer patients:A 10-year retrospective study in a UK institution

The study was to determine if breast cancer patients aged 65 and above could be given adjuvant chemotherapy safely while achieving an acceptable relative dose intensity of at least 85%. We identified all patients aged 65 and over who received adjuvant chemotherapy over the 10 year period, November 1999 to October 2009, and determined the proportion that achieved a relative dose intensity of at least 85% as well as the tolerability of their treatment. A total of 101 patients were identified, with a median age of 69 years (range 65-78).Of these, 25.7% of patients had at least one major comorbidity, 84.2% had a tumor size of 5 cm or less, 73.3% were node positive and 58.4% were hormone receptor positive. The chemotherapy regimens used were AC (Doxorubicin and Cyclophosphamide), FEC (Fluorouracil, Epirubicin, and Cyclophosphamide), CMF (Cyclophosphamide, Methotrexate, and Fluorouracil) and ECMF (Epirubicin followed by CMF). Seventy-nine patients (78.2%) achieved the relative dose intensity of at least 85%. With respect to toxicity, 11.9% of patients developed febrile neutropenia and 23.8% of patients required hospital admission during the treatment period, but there were no treatment-related deaths in the group. A significant proportion of patients aged 65 and above achieved the intended dose intensity of at least 85% over this 10-year period, with manageable toxicity levels. This supports the use of these regimens as adjuvant chemotherapy for breast cancer in this age group. © 2011 Wiley Periodicals, Inc.

Improving a regional chemotherapy service by learning from deaths occurring within 30 days of treatment with systemic anti-cancer therapy.

Background: Outwith clinical trials, patient outcomes specifically related to SACT (systemic anti-cancer therapy) are not well reported despite a significant proportion of patients receiving active treatment at the end of life. The NCEPOD reviewing deaths within 30 days of SACT found SACT caused or hastened death in 27% of cases.

Method: Across the Northern Ireland cancer network, 95 patients who died within 30 days of SACT for solid tumours were discussed at the Morbidity and Mortality monthly meeting during 2013. Using a structured template, each case was independently reviewed, with particular focus on whether SACT caused or hastened death.

Results: Lung, GI and breast cancers were the most common sites. Performance status was recorded in 92% at time of final SACT cycle (ECOG PS 0-2 89%).

In 57% the cause of death was progressive disease. Other causes included thromboembolism (13%) and infection (5% neutropenic sepsis, 6% non-neutropenic sepsis). In 26% with death from progressive disease, the patient was first cycle of first line treatment for metastatic disease. In the majority discussion regarding treatment aims and risks was documented. Only one patient was receiving SACT with curative intent, who died from appropriately managed neutropenic sepsis.

A definitive decision regarding SACT's role in death was made in 60%: in 49% SACT was deemed non-contributory and in 11% SACT was deemed the cause of death. In 40% SACT did not play a major role, but a definitive negative association could not be made.

Conclusion: Development of a robust review process of 30-day mortality after SACT established a benchmark for SACT delivery for future comparisons and identified areas for SACT service organisation improvement. Moreover it encourages individual practice reflection and highlights the importance of balancing patients' needs and concerns with realistic outcomes and risks, particularly in heavily pre-treated patients or those of poor performance status.

Host Defence Peptide LL-37; Effects of truncation on antimicrobial activity

Background: The oral cavity is an ideal environment for colonisation by micro-organisms. A first line of defence against microbial infection is the secretion of broad spectrum host defence peptides (HDPs). In the current climate of antibiotic resistance, exploiting naturally occurring HDPs or synthetic derivatives (mimetics) to combat infection is particularly appealing. The human cathelicidin, LL-37 is one such HDP expressed ubiquitously by epithelial cells and neutrophils. LL-37 exhibits the ability to bind lipopolysaccharide (LPS) and displays broad spectrum activity against a wide range of bacteria. The current study focuses on truncation of LL-37 and defining the antimicrobial and LPS binding activity of the resultant mimetics. Objectives: To assess the antimicrobial and LPS binding activity of LL-37 and three truncated mimetics (KE-18, EF-14 and KR-12). Methods: Peptides were synthesised in-house by Fmoc solid phase peptide synthesis or obtained commercially. Antimicrobial activity was determined using a radial diffusion assay and ability to bind LPS was determined by indirect ELISA. Results: LL-37 and mimetics displayed antimicrobial activity against Streptococcus mutans and Enterococcus Faecalis. KE-18 and KR-12 were shown to possess antimicrobial activity against both pathogens whereas EF-14 was the least antimicrobial. In terms of LPS binding, KE-18 and KR-12 were both effective whereas EF-14 showed the least activity of the three mimetics. Conclusion: Truncation of LL-37 can yield peptides which retain antimicrobial activities and have the ability to bind LPS. Interestingly in some cases the truncation of LL-37 produced mimetics with greater potency than the parent molecule in terms of antimicrobial activity and LPS binding. This work was funded by DEL and the Diabetes Wellness Foundation.

Neuropeptides display antimicrobial activity against cariogenic and endodontic bacteria

Introduction: Many neuropeptides are similar in size, amino acid composition and charge to antimicrobial peptides. It is therefore possible that the nervous system employs neuropeptides as antimicrobial agents by delivering them rapidly and precisely to innervated sites such as the dental pulp. Objectives: The aim of this study was to determine whether the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), which we have previously shown to be present in dental pulp, displayed antimicrobial activity against the cariogenic bacterium Streptococcus mutans and the endodontic bacterium Enterococcus faecalis. Methods: Neuropeptides were purchased from Bachem and utilised in antibacterial assays using a previously described ultra sensitive radial diffusion method. Results: Antimicrobial activity was identified as clear zones around neuropeptide-containing wells. NPY was found to exhibit antimicrobial against both Streptococcus mutans and Enterococcus faecalis. SP and VIP were shown to exhibit antimicrobial activity against Streptococcus mutans only. The neuropeptides NKA and CGRP did not show antimicrobial activity against either micro-organism. Conclusion: This study is the first to describe an antimicrobial role for neuropeptides in pulp biology. The antimicrobial actions of neuropeptides contribute a novel aspect to pulpal defence against cariogenic and endodontic bacteria worthy of further investigation.

Engineered alpha-defensin peptides display antimicrobial activity against oral pathogens

Introduction: Human alpha defensins are a family of neutrophil-derived antimicrobial peptides also known as human neutrophil peptides (HNPs). The defensin family of peptides are characterised by six invariant cysteine residues forming three disulphide bridges. The formation of the correct disulphide pairs complicates the synthesis of full length human alpha defensin and limits its therapeutic potential as an antimicrobial peptide. Objectives: The aim of this study was to determine whether truncated alpha defensins displayed antimicrobial activity against a range of micro-organisms including oral pathogens. Methods: Engineered peptides were synthesised by solid-phase methods using standard Fmoc chemistry. Antibacterial assays were performed using a previously described ultra sensitive radial diffusion method. A total of five engineered defensin peptides and full length alpha defensin were tested for their sensitivity against eight micro-organisms, including Gram negative bacteria, Gram positive bacteria and fungal pathogens Results: Antimicrobial activity was identified as clear zones around peptide-containing wells. Zone diameters were used to calculate minimum inhibitory concentrations (MICs) for each peptide. There was considerable variability in the susceptibility of the micro-organisms to the truncated analogues. Bacillus subtilis and Enterococcus faecalis were sensitive to the majority of the engineered peptides whereas Staphylococcus aureus, Escherichia coli and Candida albicans displayed resistance (defined as an MIC of greater than 250 ug/ml) to the truncated defensins. Of the five engineered peptides synthesised, the 2-aminobenzoic acid (Abz)-containing analogues based on the C-terminal sequence of alpha defensin displayed MIC values closest to that of the full length defensin in 5 out of 8 micro-organisms studied. Conclusion: This study demonstrates that truncated alpha defensins display variable antimicrobial activity against a range of micro-organisms, including oral pathogens. The generation of truncated defensins without disulphide bridges simplifies their synthesis and increases their therapeutic potential.

Eppin: A Novel Protein Possessing Antimicrobial Properties Against Oral Pathogens

Background: Epididymal protease inhibitor (eppin) is a dual motif protein belonging to the whey acidic protein (WAP) family. Although expressed in numerous different tissues, to date, its functional characterisation is limited. It has been shown to exhibit antibacterial activity against Gram-negative bacteria (Escherichia coli) and antiprotease activity against some proteases of the serine protease family. We are interested in determining the role of eppin in innate immune defence. Objectives: This study aims to determine eppin's potential function in the innate immune response in the oral cavity by investigating the antimicrobial activity of eppin against relevant oral pathogens. Methods: Eppin was recombinantly expressed in E. coli cells and purified by immobilised metal affinity chromatography (IMAC). The antimicrobial effects of the protein were then assessed against two oral pathogens, Fusobacterium nucleatum and Candida albicans, using a double layer radial diffusion assay. Results: Eppin displayed antimicrobial activities against both oral pathogens tested and these activities were shown to be comparable to the well characterised antimicrobial peptide, LL-37. The antifungal effects of eppin were shown to be more potent than those of the human cathelicidin, LL-37. Conclusions: Eppin has been shown to possess both antibacterial and antifungal properties against oral pathogens, suggesting an important role for this protein in the innate immune response in the oral cavity. This study furthers our knowledge of the physiological role exerted by eppin and its possible role in the modulation of chronic diseases such as periodontitis and oral candidiasis.

Two Novel Dermaseptin-Like Antimicrobial Peptides with Anticancer Activities from the Skin Secretion of Pachymedusa dacnicolor

The dermaseptin antimicrobial peptide family contains members of 27–34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 5′-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested.

Immune-derived PD-L1 gene expression defines a subgroup of stage II/III colorectal cancer patients with favorable prognosis that may be harmed by adjuvant chemotherapy

A recent phase 2 study of metastatic colorectal carcinoma (CRC) patients showed that mismatch repair gene status was predictive of clinical response to PD-1-targeting immune checkpoint blockade. Further examination revealed strong correlation between PD-L1 protein expression and microsatellite instability (MSI) in stage IV CRC, suggesting that the amount of PD-L1 protein expression could identify late stage patients who may benefit from immunotherapy. To assess whether the clinical associations between PD-L1 gene expression and MSI identified in metastatic CRC are also present in stage II/III CRC, we used in silico analysis to elucidate the cell types expressing the PD-L1 gene. We found a significant association of PD-L1 gene expression with MSI in early stage CRC (P < 0.001) and show that unlike in non-CRC tumors, PD-L1 is derived predominantly from the immune infiltrate. We demonstrate that PD-L1 gene expression has positive prognostic value in the adjuvant disease setting (PD-L1low v PD-L1high HR = 9.09; CI, 2.11-39.10). PD-L1 gene expression had predictive value, as patients with high PD-L1 expression appear to be harmed by standard-of-care treatment (HR = 4.95; CI,1.10-22.35). Building on the promising results from the metastatic CRC PD-1-targeting trial, we provide compelling evidence that PD-L1high/MSI/immunehigh stage II/III CRC patients should not receive standard chemotherapy. This conclusion supports the rationale to clinically evaluate this patient subgroup for PD-1 blockade treatment.

CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS OF LAVANDULA SPP.

Lavenders belong to the family Labiatae and represent some of the most popular medicinal plants of great economic importance. Their essential oils are important for the perfume, cosmetic, flavouring and pharmaceutical industries. However, despite its popularity, and the long tradition of use, biological properties of the various Lavandula species are not yet been well sustained by scientific or clinical studies and some available data being inconclusive and controversial [1]. Although Lavandula spp. have similar ethnobotanical properties, however, chemical composition and therapeutic uses differ from different species and main composition of essential oils showed differences with species and with the region were they grow [1,2,3]. L. stoechas L. subsps. luisieri (Rozeira) Rozeira. L. pedunculata (Mill.) Cav. and L. viridis L’Hér are endemic to the Iberian Peninsula, widespread in the South of Portugal, namely in Alentejo and Algarve. In our work, essential oils from the stems or leaves from wild grown plants of L. luisieri (Alentejo), L. pedunculata (Alentejo) and L. viridis (Algarve), were extracted by hydrodistillation and analyzed by GC-FID. Antimicrobial activity was evaluated by solid diffusion disk assay and minimal inhibitory concentration (MIC) against pathogenic Gram-positive and Gram-negative bacteria and food spoilage fungi.

Antioxidant,antimicrobial and toxicological properties of Schinus molle L. essential oils

Ethnopharmacological relevance: Schinus molle L. has been used in folk medicine as antibacterial, antiviral, topical antiseptic, antifungal, antioxidant, anti-inflammatory, anti-tumoural as well as antispasmodic and analgesic; however, there are few studies of pharmacological and toxicological properties of S. molle essential oils. Aim of the study: The aim of this study was to evaluate the antioxidant and antimicrobial activities of S. molle leaf and fruit essential oils, correlated with their chemical composition and evaluate their acute toxicity. Materials and methods: The chemical composition of S. molle leaf and fruit essential oils were evaluated by GC-FID and GC-MS. Antioxidant properties were determined using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical and β-carotene/linoleic acid methods. Antimicrobial properties were evaluated by the agar disc diffusion method and minimal inhibitory concentration assay. Toxicity in Artemia salina and acute toxicity with behavioural screening in mice were evaluated. Results: The dominant compounds found in leaf and fruit essential oils (EOs) were monoterpene hydrocarbons, namely -phellandrene, β-phellandrene, β-myrcene, limonene and α-pinene. EOs showed low scavenging antioxidant activity by the DPPH free radical method and a higher activity by the β-carotene/linoleic acid method. Antimicrobial activity of EOs was observed for Gram+, Gram– pathogenic bacteria and food spoilage fungi. EOs showed cytotoxicity for Artemia salina and lower toxicity in Swiss mice. Conclusions: The result showed that EOs of leaves and fruits of S. molle demonstrated antioxidant and antimicrobial properties, suggesting their potential use in food or pharmaceutical industries.