A Clinical and Biological Guide for Understanding Chemotherapy-Induced Alopecia and its Prevention

Chemotherapy-induced alopecia (CIA) is the most visibly distressing side effect of commonly administered chemotherapeutic agents. As psychological health has huge relevance on lifestyle, diet and self-esteem, it is important for clinicians to fully appreciate the psychological burden that CIA can place on patients. Here, for the first time, we provide a comprehensive review encompassing the molecular characteristics of the human hair follicle (HF), how different anticancer agents damage the HF to cause CIA, subsequent HF pathophysiology and we assess known and emerging prevention modalities that have aimed to reduce or prevent CIA. We argue that, at present, scalp cooling is the only safe and FDA-cleared modality available, and we highlight the extensive available clinical and experimental (biological) evidence for its efficacy. The likelihood of a patient that uses scalp cooling during chemotherapy maintaining enough hair to not require a wig is approximately 50%. This is despite different types of chemotherapy regimens, patient-specific differences and possible lack of staff experience in effectively delivering scalp cooling. The increased use of scalp cooling and an understanding of how to deliver it most effectively to patients has enormous potential to ease the psychological burden of CIA, until other, more efficacious, equally safe treatments become available

The role of the bronchial microvasculature in the airway remodelling in asthma and COPD

In recent years, there has been increased interest in the vascular component of airway remodelling in chronic bronchial inflammation, such as asthma and COPD, and in its role in the progression of disease. In particular, the bronchial mucosa in asthmatics is more vascularised, showing a higher number and dimension of vessels and vascular area. Recently, insight has been obtained regarding the pivotal role of vascular endothelial growth factor (VEGF) in promoting vascular remodelling and angiogenesis. Many studies, conducted on biopsies, induced sputum or BAL, have shown the involvement of VEGF and its receptors in the vascular remodelling processes. Presumably, the vascular component of airway remodelling is a complex multi-step phenomenon involving several mediators. Among the common asthma and COPD medications, only inhaled corticosteroids have demonstrated a real ability to reverse all aspects of vascular remodelling. The aim of this review was to analyze the morphological aspects of the vascular component of airway remodelling and the possible mechanisms involved in asthma and COPD. We also focused on the functional and therapeutic implications of the bronchial microvascular changes in asthma and COPD

A genome-wide meta-analysis of palmoplantar pustulosis implicates T_H2 responses and cigarette smoking in disease pathogenesis

Background Palmoplantar pustulosis (PPP) is an inflammatory skin disorder that mostly affects smokers and manifests with painful pustular eruptions on the palms and soles. Although the disease can present with concurrent plaque psoriasis, TNF and IL-17/IL-23 inhibitors show limited efficacy. There is therefore a pressing need to uncover PPP disease drivers and therapeutic targets. Objectives We sought to identify genetic determinants of PPP and investigate whether cigarette smoking contributes to disease pathogenesis. Methods We performed a genome-wide association meta-analysis of 3 North-European cohorts (n = 1,456 PPP cases and 402,050 controls). We then used the scGWAS program to investigate the cell-type specificity of the association signals. We also undertook genetic correlation analyses to examine the similarities between PPP and other immune-mediated diseases. Finally, we applied Mendelian randomization to analyze the causal relationship between cigarette smoking and PPP. Results We found that PPP is not associated with the main genetic determinants of plaque psoriasis. Conversely, we identified genome-wide significant associations with the FCGR3A/FCGR3B and CCHCR1 loci. We also observed 13 suggestive (P &lt; 5 × 10−6) susceptibility regions, including the IL4/IL13 interval. Accordingly, we demonstrated a significant genetic correlation between PPP and TH2-mediated diseases such as atopic dermatitis and ulcerative colitis. We also found that genes mapping to PPP-associated intervals were preferentially expressed in dendritic cells and often implicated in T-cell activation pathways. Finally, we undertook a Mendelian randomization analysis, which supported a causal role of cigarette smoking in PPP. ConclusionsThe first genome-wide association study of PPP points to a pathogenic role for deregulated TH2 responses and cigarette smoking.</p

Clinical outcomes and response to treatment of patients receiving topical treatments for pyoderma gangrenosum: a prospective cohort study

Background: pyoderma gangrenosum (PG) is an uncommon dermatosis with a limited evidence base for treatment. Objective: to estimate the effectiveness of topical therapies in the treatment of PG. Methods: prospective cohort study of UK secondary care patients with a clinical diagnosis of PG suitable for topical treatment (recruited July 2009 to June 2012). Participants received topical therapy following normal clinical practice (mainly Class I-III topical corticosteroids, tacrolimus 0.03% or 0.1%). Primary outcome: speed of healing at 6 weeks. Secondary outcomes: proportion healed by 6 months; time to healing; global assessment; inflammation; pain; quality-of-life; treatment failure and recurrence. Results: Sixty-six patients (22 to 85 years) were enrolled. Clobetasol propionate 0.05% was the most commonly prescribed therapy. Overall, 28/66 (43.8%) of ulcers healed by 6 months. Median time-to-healing was 145 days (95% CI: 96 days, ∞). Initial ulcer size was a significant predictor of time-to-healing (hazard ratio 0.94 (0.88;80 1.00); p = 0.043). Four patients (15%) had a recurrence. Limitations: No randomised comparator Conclusion: Topical therapy is potentially an effective first-line treatment for PG that avoids possible side effects associated with systemic therapy. It remains unclear whether more severe disease will respond adequately to topical therapy alone

Risk of chronic kidney disease after cancer nephrectomy.

The incidence of early stage renal cell carcinoma (RCC) is increasing and observational studies have shown equivalent oncological outcomes of partial versus radical nephrectomy for stage I tumours. Population studies suggest that compared with radical nephrectomy, partial nephrectomy is associated with decreased mortality and a lower rate of postoperative decline in kidney function. However, rates of chronic kidney disease (CKD) in patients who have undergone nephrectomy might be higher than in the general population. The risks of new-onset or accelerated CKD and worsened survival after nephrectomy might be linked, as kidney insufficiency is a risk factor for cardiovascular disease and mortality. Nephron-sparing approaches have, therefore, been proposed as the standard of care for patients with type 1a tumours and as a viable option for those with type 1b tumours. However, prospective data on the incidence of de novo and accelerated CKD after cancer nephrectomy is lacking, and the only randomized trial to date was closed prematurely. Intrinsic abnormalities in non-neoplastic kidney parenchyma and comorbid conditions (including diabetes mellitus and hypertension) might increase the risks of CKD and RCC. More research is needed to better understand the risk of CKD post-nephrectomy, to develop and validate predictive scores for risk-stratification, and to optimize patient management

The role of CD40 in epithelial cell fate

CD40 belongs to the tumour necrosis factor receptor (TNFR) family and its ligation by membrane- presented CD40 ligand (mCD40L) causes extensive apoptosis in malignant cells, while sparing their normal epithelial counterparts which, can respond to CD40 ligation by proliferation, thus representing an intriguing paradox in TNFR biology. Although the apoptotic potential of mCD40L is well-documented, the precise intracellular signalling pathways of cell death remain largely unknown. Moreover, due to the practical constraints imposed by the necessity to deliver surface-CD40L for therapeutic purposes, deciphering the molecular nature of the CD40 signalling ‘black-box’ may permit the design of novel therapeutic approaches. This thesis aimed to unravel for the first time the precise cell signalling pathways responsible for mCD40L-mediated apoptosis and attempted to determine the mechanisms underpinning the tumour-specificity of CD40 ligation. The first part of this thesis focused on optimisation of cell models for the delivery of the mCD40L signal to epithelial cells (via co-culture with third party cells and based on the urothelial cell culture system). A number of assays were optimised for the detection of several apoptosis hallmarks (membrane integrity loss, caspase activation, DNA fragmentation and mitochondrial cytochrome c release). Immunoblotting techniques were improved for the sensitive detection of intracellular CD40-signalling mediators, followed by the optimisation of methodologies for retrovirus transduction-mediated delivery of short hairpin RNA (shRNA) molecules for functional (knockdown) experiments. Expression studies and knockdown experiments reinforced the role of TRAF3 as a key mediator of apoptosis and cemented its essential role in both JNK/AP-1 activation and subsequent induction of pro-apoptotic Bak and Bax, with Bak and particularly Bax loss alone demonstrating as essential for apoptosis. The project also revealed for the first time the proximal events in the CD40 pathway that provided a link between TRAF3 induction and JNK/AP-1 activation followed by Bak/Bax induction. A series of functional experiments involving RNAi-mediated knockdown and/or pharmacological inhibition of critical target proteins suggested that mitogen activated protein kinases (MAPKs) ASK1 and MKK4 (but not MKK7) drive the activation of JNK/AP-1 and CD40-mediated death. Because of the well-documented sensitivity of ASK1 towards oxidative stress and its direct regulation by reactive oxygen species (ROS), a CD40-ROS paradigm was explored. It was found that oxidative stress is essential in CD40-mediated apoptosis as pharmacological inhibitors of ROS attenuated cytotoxicity. Additionally, the work provided novel evidence for a functional role for the NADPH oxidase (Nox) enzyme in apoptosis, as pharmacological inactivation of Nox attenuated ROS induction and blocked apoptosis, signifying that ROS-mediated oxidative stress is Nox-dependant. More specifically, it was found that CD40 ligation caused phosphorylation of p40phox, a Nox-2 subunit previously reported to interact with TRAF3, but not in the context of apoptosis induction. These observations implied that TRAF3-mediated CD40-Nox interactions may be crucial for CD40-induced apoptosis. Importantly, the observation that adequate ROS elevation, to permit efficient thioredoxin (Trx) down-regulation and ASK1 activation, was only observed following receptor ligation by pro-apoptotic mCD40L, but not by non-apoptotic soluble agonist, led to the hypothesis that pharmacological interference with Trx may compensate for the lack of signal strength and sensitise cells to apoptosis. Strikingly, combinatorial treatment of carcinoma cells with a specific Trx-1 inhibitor and agonistic anti-CD40 antibody showed efficient synergy and resulted in extensive apoptosis. The combinatorial treatment appeared functionally equivalent to mCD40L and employed ASK-1/JNK signalling to induce apoptosis. Finally, to enhance our understanding of the role of malignant transformation in CD40 susceptibility, the effect of over-expression of the telomerase catalytic sub-unit (hTERT) in normal epithelial cells was assessed. It was found that, unlike normal human urothelial (NHU) cells where CD40 ligation did not cause apoptosis (but appeared to be cyto-protective), para-malignant hTERT NHU expressers were highly susceptible to CD40-killing and to an extent equivalent to that observed in carcinoma-derived cells. By comparing normal, para-malignant and tumour-derived cells, the study added to increasing evidence that during malignant transformation cells exhibit higher basal ROS levels, which functions as ‘double-edged sword’ that renders them more susceptible to signals that elevate ROS past a lethal pro-apoptotic threshold, such as that triggered by CD40. Collectively, this thesis has unravelled for the first time the molecular nature of pro-apoptotic CD40 ligation revealing a key signalling axis triggered CD40 ligation that involves TRAF3, Nox-2 (p40phox), ROS, ASK1, MKK4, JNK, Bak/Bax and intrinsic apoptosis, whilst the new knowledge of the signalling pathway has now provided novel avenues for exploiting CD40 as a target for anti-cancer therapy

Expression and purification of a recombinant metal-binding T4 lysozyme fusion protein

Periplasmic expression of recombinant proteins presents many potential benefits that may aid recovery of the protein product. Muramidases are the preferred agents in effecting selective release of recombinant proteins from the periplasm of E. co/i and other Gram negative bacteria. Unfortunately cost restricts the use of pure lytic enzymes at large-scale and their removal as process contaminants adds to later purification demands. We constructed a reusable version of bacteriophage T4 lysozyme, by fusing a His-Gln-(His), peptide sequence to the C-terminus of a cysteine-free pseudo wild type bacteriophage T4 lysozyme. The peptide tail allowed rapid and high-level recovery on IDA Sepharose columns charged with Zn2+, Ni2+ and Cu2+ ions. The binding to metal-charged supports was specifically mediated by the histidine-rich tail as no binding was observed for the original cysteine-free pseudo wild type lysozyme. The strength of retention of polyhistidine recombinant T4 lysozyme on charged supports followed the expected Cu &gt; Ni &gt; Zn pattern, but there were few differences in the levels of purity and recovery of the modified enzyme, from columns charged with the different metal ions

The processing of a plasmid-based gene from <i>E. coli</i>. Primary recovery by filtration

We describe the primary recovery of plasmid DNA from alkaline lysis mixtures using a nutsche filter operated under pressure. Six different filter cloths constructed of polypropylene, polyester and stainless steel were tested, with pore sizes ranging from 5–160 micron. Both pore size and the material of the filter membranes employed in filtration experiments exerted considerable impact on the purity and yield of the plasmid DNA. The greatest degree of solids extrusion, shearing of chromosomal DNA and subsequent contamination of the filtrate was observed with the 160 lm polyester filter. The best compromise was obtained with a 5 micron polypropylene cloth. For an alkaline lysis mixture containing 101 g wetweight solids per litre, filtration through this cloth proceeded at an average rate of 22.5 cm h–1. Virtually complete removal of solids (99.4%) and protein (96.8%) was achieved, with a 8.2-fold purification of plasmid DNA at the expense of a 33% loss in yield. The filtration performance of this membrane was further modified by precoating with diatomaceous earths of different permeabilities (0.07–1.2 darcies). The finest filter aid resulted in very pure plasmid DNA (65%), complete suspended solids removal and &lt; 1% of the original protein remaining in the filtrate. However, the plasmid yield was only 30%, the processing rate was markedly reduced (8.2 cm h–1), and some losses of plasmidDNA,duetoadsorptionontothediatomaceousearth,were also observed (5.7%).<br/

The processing of a plasmid-based gene from <i>E. coli</i>. Primary recovery by filtration

We describe the primary recovery of plasmid DNA from alkaline lysis mixtures using a nutsche filter operated under pressure. Six different filter cloths constructed of polypropylene, polyester and stainless steel were tested, with pore sizes ranging from 5–160 micron. Both pore size and the material of the filter membranes employed in filtration experiments exerted considerable impact on the purity and yield of the plasmid DNA. The greatest degree of solids extrusion, shearing of chromosomal DNA and subsequent contamination of the filtrate was observed with the 160 lm polyester filter. The best compromise was obtained with a 5 micron polypropylene cloth. For an alkaline lysis mixture containing 101 g wetweight solids per litre, filtration through this cloth proceeded at an average rate of 22.5 cm h–1. Virtually complete removal of solids (99.4%) and protein (96.8%) was achieved, with a 8.2-fold purification of plasmid DNA at the expense of a 33% loss in yield. The filtration performance of this membrane was further modified by precoating with diatomaceous earths of different permeabilities (0.07–1.2 darcies). The finest filter aid resulted in very pure plasmid DNA (65%), complete suspended solids removal and &lt; 1% of the original protein remaining in the filtrate. However, the plasmid yield was only 30%, the processing rate was markedly reduced (8.2 cm h–1), and some losses of plasmidDNA,duetoadsorptionontothediatomaceousearth,were also observed (5.7%).<br/