Clostridium difficile infection is the leading cause of antibiotic- and healthcare-associated diarrhea, and its containment and treatment imposes a significant financial burden, estimated to be over $3 billion in the USA alone. Since the year 2000, CDI epidemics/outbreaks have occurred in North America, Europe and Asia. These outbreaks have been variously associated with, or attributed to, the emergence of Clostridium difficile strains with increased virulence, an increase in resistance to commonly used antimicrobials such as the fluoroquinolones, or host susceptibilities, including the use of gastric acid suppressants, to name a few. Efforts to elucidate C. difficile pathogenic mechanisms have been hampered by a lack of molecular tools, manipulatable animal models, and genetic intractability of clinical C. difficile isolates. However, in the past 5 y, painstaking efforts have resulted in the unraveling of multiple C. difficile virulence-associated pathways and mechanisms. We have recently reviewed the disease, its associated risk factors, transmission and interventions (Viswanathan, Gut Microbes 2010). This article summarizes genetics, non-toxin virulence factors, and host-cell biology associated with C. difficile pathogenesis as of 2011, and highlights those findings/factors that may be of interest as future intervention targets.

Clostridium difficile is responsible for 15-20% of antibiotic-associated diarrheas, and nearly all cases of pseudomembranous colitis. Among the cell wall proteins involved in the colonization process, Cwp84 is a protease that cleaves the S-layer protein SlpA into two subunits. A cwp84 mutant was previously shown to be affected for in vitro growth but not in its virulence in a hamster model. In this study, the cwp84 mutant elaborated biofilms with increased biomass compared with the parental strain, allowing the mutant to grow more robustly in the biofilm state. Proteomic analyses of the 630Δerm bacteria growing within the biofilm revealed the distribution of abundant proteins either in cell surface, matrix or supernatant fractions. Of note, the toxin TcdA was found in the biofilm matrix. Although the overall proteome differences between the cwp84 mutant and the parental strains were modest, there was still a significant impact on bacterial surface properties such as altered hydrophobicity. In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo. Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins.

Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA - and lcpB - mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB - mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA - and lcpB - strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence.

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Background: Chemotherapy induced peripheral neuropathy (CIPN) can affect up to a third of patients undergoing chemotherapy. CIPN is thought to be caused by drug induced damage to the peripheral motor or sensory nervous system. Symptoms range from tingling and numbness to balance issues and falls. Based on severity of symptoms, chemotherapy (CTX) is delayed, reduced, or discontinued which can adversely affect outcomes. Currently there are no predictive biomarkers to identify those at risk of developing CIPN. Diffuse tensor imaging (DTI), a subtype of diffusion-weighted imaging (DWI), measured by routine magnetic resonance imaging (MRI) is being increasingly evaluated to assess nerve fiber trajectory. DTI allows for quantitative measurements such as fractional anisotropy (FA) and apparent diffusion coefficient (ADC) which have shown some promising but mixed results in evaluating peripheral neuropathy and CIPN in some studies. We conducted a pilot study to evaluate if quantitative DTI measurements, FA and ADC, at mid-calf and ankle can evaluate CTX induced nerve damage and predict development of CIPN. Methods: We conducted a prospective study in patients with breast cancer who were treated with a taxane-based CTX regimen. Study was approved by our institutional review board and registered on clinicaltrials.gov (NCT03365895). Patients who were eligible to get CTX with a taxane based regimen (paclitaxel or docetaxel) were included. Patients with prior exposure to neurotoxic CTX, significant peripheral neuropathy at baseline, or history of diabetes were excluded. All patients completed pre and post CTX MRI of bilateral leg and ankles and a self-reported Functional Assessment of Cancer Therapy/Gynecological Oncology Group neurotoxic questionnaire (FACT-NTX). Patients were diagnosed with developing symptomatic neuropathy if the absolute increase in their FACT-NTX score was ≥3 at post treatment evaluation. Results: Twenty patients consented however 6 were ineligible. Fourteen patients completed all the study procedures. Median age of the evaluable patients was 53 years (33-72 years); 11/14 had paclitaxel CTX. For all patients, median baseline FACT-NTX score was significantly lower (2.5) than post treatment score (5.5) (p=0.009). Based on FACT-NTX score changes, 64% of patients developed symptomatic neuropathy. For all patients, the minimum FA values post-chemotherapy at mid-calf and ankle (0.42 and 0.41 respectively) are significantly lower than baseline (0.54 and 0.49 respectively, p=<0.0001 at mid-calf, p=0.03 at ankle) indicating change in nerve structure with CTX. There was no significant change in mean ADC measurements at mid-calf and ankle pre CTX (1.21 and 1.17 respectively) versus post CTX (1.29 and 1.9 respectively, p=0.14 for mid-calf, p=0.9 at ankle). In patients who developed neuropathy, the median minimum FA value at mid-calf at baseline was lower compared to those who did not (p=0.016). Conclusions: Our study highlights the use of measuring minimum FA value at the mid-calf and ankle to evaluate for CIPN. Our results indicate that minimum FA value decreased with CTX induced nerve damage and a lower baseline measurement is likely predictive of developing CIPN. This suggests that minimum FA value can be used as a non-invasive imaging biomarker to help predict those at risk for CIPN and potentially be used to implement prevention strategies. Additional clinical trials are warranted to further evaluate this promising predictive biomarker. Citation Format: Hninyee Win, Lana Gimber, Jennifer Segar, Michele Chu, Sima Ehsani, Pavani Chalasani. Imaging predictors for development of chemotherapy induced peripheral neuropathy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-27.

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Breast cancers (BC) expressing estrogen and/or progesterone receptors, referred to as hormone receptor (HR)-positive represent the largest molecular subset of breast tumors. Fortunately, estrogen receptor expression on the cell surface serves as a drug target for endocrine therapy. In HR+, human epidermal growth factor receptor-2 (HER2) negative BC, neoadjuvant endocrine therapy (NET) is being increasingly used. While NET has been shown to reduce tumor cell proliferation causing apoptosis, up to 20% of tumors do not respond. Currently there are no validated predictive gene signatures distinguishing responders from non-responders to NET. Furthermore, there are limited data on the effects of different NET regimens in vivo and how they vary between responders and non-responders. Methods: We conducted a retrospective translational study in patients who were treated with NET at our institution. This study was approved by our institutional review board. Paired pre-treatment and surgical samples were collected from 24 patients along with clinico-pathological information. Responders were defined as those with a preoperative endocrine prognostic index (PEPI) score of 0. RNA isolation was performed using the Roche HighPure FFPET RNA Isolation spin-column kit. Purified RNA was hybridized with the PanCancer Code Set and finally the cartridge was scanned on the nCounter Digital Analyzer. Raw counts from each gene were imported into the nSolver Analysis. Log2 normalized data obtained from Nanostring nCounter analysis was analyzed for differentially expressed genes between pre- and post-treatment samples. The bioinformatics analysis was carried out using nCounter analysis software. Results: The median age at diagnosis was 64 years and NET used was anastrozole alone or in combination with fulvestrant. At baseline the majority of tumors were invasive ductal carcinomas (n=18), grade II (n=18), with a Ki67% >15% (n=16). Median duration of treatment with NET was 5 months (range 2-10 months). Nine tumors were defined as responders to NET and 15 were non-responders. In all of the 24 paired samples, we found 123 genes with a statistically significant change in expression (defined by greater than two-fold change and a false discovery rate adjusted p-value <0.05) in the surgical specimen compared to pre-treatment biopsy. Genes in the Janus kinase-signal transducers and activators of transcription (Jak-STAT) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, RAS signaling, cytosine-cytosine receptor interaction and the Phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K-Akt) signaling pathway were found to be upregulated whereas genes in cell cycle pathway were downregulated in the surgical specimens after NET. Comparing pre-treatment samples of responders to non-responders 25 genes involving PI3K-Akt, Notch signaling and Fanconi anemia pathway were differentially expressed. Conclusions: In our retrospective translational study we found that NET upregulates genes in the Jak-STAT, MAPK, RAS, cytosine-cytosine receptor interaction and PI3K-Akt signaling pathways and downregulates cell cycle pathway genes independent of type or duration of NET. We also found multiple genes involving the PI3K-Akt and Notch pathway were differentially expressed in pre-treatment specimens among responders compared to non-responders. While the sample size comparing responders versus non-responders was inadequate, our results suggest some early trends which warrant further investigation. Citation Format: Darien E Reed, Ritu Pandey, Jennifer M Segar, Michele Chu, Lauren LeBeau, Pavani Chalasani. Gene expression changes with neoadjuvant hormonal treatment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-13-05.