New insights for vaccine development against Clostridium difficile infections.

Increased antibiotic usage is the main risk factor for gut microbiota dysbiosis. In dysbiosis, there is an increased susceptibility to intestinal pathogens, such as Clostridium difficile infection, the leading cause of hospital-acquired infection worldwide. High-spectrum antibiotics, such as vancomycin or metronidazole, also increases the risk of developing CDI symptoms after the treatment. An impaired immune response could also be responsible for the high incidence of recurrence of CDI (R-CDI), suggesting that immune system stimulation could help eradicate the infection in patients suffering multiple episodes in CDI or prevent the infective course. Here, we discuss novel immunotherapeutic approaches that aid the immune system to target C. difficile and how these can be improved.

Immunogenicity and protective efficacy of Clostridium difficile spore proteins.

Clostridium difficile is a spore-forming, anaerobic, Gram-positive organism that is the leading cause of antibiotic-associated infectious diarrhea, commonly known as C. difficile infection (CDI). C. difficile spores play an important role in the pathogenesis of CDI. Spore proteins, especially those that are surface-bound may play an essential role in the germination, colonization and persistence of C. difficile in the human gut. In our current study, we report the identification of two surface-bound spore proteins, CdeC and CdeM that may be utilized as immunization candidates against C. difficile. These spore proteins are immunogenic in mice and are able to protect mice against challenge with C. difficile UK1, a clinically-relevant 027/B1/NAP1 strain. These spore proteins are also able to afford high levels of protection against challenge with C. difficile 630Δerm in golden Syrian hamsters. This unprecedented study shows the vaccination potential of C. difficile spore exosporium proteins.

Use of improved memory type control charts for monitoring cancer patients recovery time censored data.

Control charts are a statistical approach for monitoring cancer data that can assist discover patterns, trends, and unusual deviations in cancer-related data across time. To detect deviations from predicted patterns, control charts are extensively used in quality control and process management. Control charts may be used to track numerous parameters in cancer data, such as incidence rates, death rates, survival time, recovery time, and other related indicators. In this study, CDEC chart is proposed to monitor the cancer patients recovery time censored data. This paper presents a composite dual exponentially weighted moving average Cumulative sum (CDEC) control chart for monitoring cancer patients recovery time censored data. This approach seeks to detect changes in the mean recovery time of cancer patients which usually follows Weibull lifetimes. The results are calculated using type I censored data under known and estimated parameter conditions. We combine the conditional expected value (CEV) and conditional median (CM) approaches, which are extensively used in statistical analysis to determine the central tendency of a dataset, to create an efficient control chart. The suggested chart's performance is assessed using the average run length (ARL), which evaluates how efficiently the chart can detect a change in the process mean. The CDEC chart is compared to existing control charts. A simulation study and a real-world data set related to cancer patients recovery time censored data is used for results illustration. The proposed CDEC control chart is developed for the data monitoring when complete information about the patients are not available. So, instead of doping the patients information we can used the proposed chart to monitor the patients information even if it is censored. The authors conclude that the suggested CDEC chart is more efficient than competitor control charts for monitoring cancer patients recovery time censored data. Overall, this study introduces an efficient new approach for cancer patients recovery time censored data, which might have significant effect on quality control and process improvement across a wide range of healthcare and medical studies.

Oral therapy for myelodysplastic syndromes/neoplasms and acute myeloid leukemia: a revolution in progress.

INTRODUCTION: Patients with myeloid neoplasms such as myelodysplastic syndromes/neoplasms (MDS) and acute myeloid leukemia (AML) are generally older, and many are not eligible for curative intent intensive therapies and/or allogeneic hematopoietic stem cell transplantation. While lower intensity, hypomethylating agent (HMA)-based therapies such as azacitidine+venetoclax have improved patient outcomes significantly, responses are not durable, and most patients die from disease-related complications. The approvals of oral HMAs such as cedazuridine-decitabine (C-DEC) and oral azacitidine (CC-486) have kindled the hope that myeloid malignancies may soon be treated with total oral therapy. AREAS COVERED: We review all-oral therapies including the approvals of C-DEC and CC-486 in MDS and AML, respectively, in addition to emerging all-oral therapies, both monotherapy and combination, in higher-risk (HR) MDS and AML. EXPERT OPINION: Oral HMAs have the potential to be a convenient and efficacy-equivalent treatment option for patients with HR-MDS or AML and improve their quality of life by reducing clinic visits for medication administration. Total-oral therapy combinations, largely including an oral HMA 'backbone,' are in the early phases of clinical development, and it is our hope that well-designed trials employing these agents may soon allow the identification of optimal regimens that deliver effective disease-directed therapy with good tolerability.

Role of the Spore Coat Proteins CotA and CotB, and the Spore Surface Protein CDIF630_02480, on the Surface Distribution of Exosporium Proteins in Clostridioides difficile 630 Spores.

Clostridioides difficile is Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea. During disease, C. difficile forms metabolically dormant spores that persist in the host and contribute to recurrence of the disease. The outermost surface of C. difficile spores, termed the exosporium, plays an essential role in interactions with host surfaces and the immune system. The main exosporium proteins identified to date include three orthologues of the BclA family of collagen-like proteins, and three cysteine-rich proteins. However, how the underlying spore coat influences exosporium assembly remains unclear. In this work, we explore the contribution of spore coat proteins cotA and cotB, and the spore surface protein, CDIF630_02480, to the exosporium ultrastructure, formation of the polar appendage and the surface accessibility of exosporium proteins. Transmission electron micrographs of spores of insertional inactivation mutants demonstrate that while cotB contributes to the formation of thick-exosporium spores, cotA and CDIF630_02480 contribute to maintain proper thickness of the spore coat and exosporium layers, respectively. The effect of the absence of cotA, cotB and CDIF630_02480 on the surface accessibility of the exosporium proteins CdeA, CdeC, CdeM, BclA2 and BclA3 to antibodies was affected by the presence of the spore appendage, suggesting that different mechanisms of assembly of the exosporium layer might be implicated in each spore phenotype. Collectively, this work contributes to our understanding of the associations between spore coat and exosporium proteins, and how these associations affect the assembly of the spore outer layers. These results have implications for the development of anti-infecting agents targeting C. difficile spores.

The Clostridioides difficile Cysteine-Rich Exosporium Morphogenetic Protein, CdeC, Exhibits Self-Assembly Properties That Lead to Organized Inclusion Bodies in Escherichia coli.

Clostridioides difficile is an obligately anaerobic, spore-forming, Gram-positive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outermost layer of C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in Escherichia coli filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an E. coli strain with a more oxidative environment led to the loss of the lamella-like organization of CdeC IBs. Additionally, dithiothreitol (DTT) treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Deletions in different portions of CdeC did not affect CdeC's ability to aggregate and form oligomers stable under denaturation conditions but affected CdeC's self-assembly properties. Overall, these observations have important implications in further studies elucidating the role of CdeC in the exosporium assembly of C. difficile spores.IMPORTANCE The endospore of Clostridioides difficile is the vehicle for transmission and persistence of the pathogen, and, specifically, the exosporium is the first contact between the host and the spore. The underlying mechanisms that govern exosporium assembly in C. difficile remain understudied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. Understanding the exosporium assembly's molecular bases may be essential to developing new therapies against C. difficile infection.

Identification of Escherichia coli strains for the heterologous overexpression of soluble Clostridium difficile exosporium proteins.

Clostridium difficile infections are one of the leading causes of hospital-acquired infections. C. difficile spores are considered the morphotype of transmission and recurrent infection due to its natural spore resistance properties. The outermost spore layer, the exosporium, provides the first contact with the environment and the host. However, molecular biology studies on exosporium proteins are lacking primarily due to difficulties in over-expressing these proteins under soluble conditions. In this work, we have developed a protocol to express soluble exosporium proteins of C. difficile spores in the heterologous Escherichia coli host. We found that the optimum soluble expression conditions may vary between 21, 30 and 37 °C, depending on the protein, and at least CdeC, BclA1 and BclA3, required E. coli strains that provided an oxidative environment such as Shuffle T7. These results will allow further studies with recombinant proteins of the exosporium of C. difficile spores.

Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa.

Clostridium difficile is the causative agent of the most frequently reported nosocomial diarrhea worldwide. The high incidence of recurrent infection is the main clinical challenge of C. difficile infections (CDI). Formation of C. difficile spores of the epidemic strain R20291 has been shown to be essential for recurrent infection and transmission of the disease in a mouse model. However, the underlying mechanisms of how these spores persist in the colonic environment remains unclear. In this work, we characterized the adherence properties of epidemic R20291 spores to components of the intestinal mucosa, and we assessed the role of the exosporium integrity in the adherence properties by using cdeC mutant spores with a defective exosporium layer. Our results showed that spores and vegetative cells of the epidemic R20291 strain adhered at high levels to monolayers of Caco-2 cells and mucin. Transmission electron micrographs of Caco-2 cells demonstrated that the hair-like projections on the surface of R20291 spores are in close proximity with the plasma membrane and microvilli of undifferentiated and differentiated monolayers of Caco-2 cells. Competitive-binding assay in differentiated Caco-2 cells suggests that spore-adherence is mediated by specific binding sites. By using spores of a cdeC mutant we demonstrated that the integrity of the exosporium layer determines the affinity of adherence of C. difficile spores to Caco-2 cells and mucin. Binding of fibronectin and vitronectin to the spore surface was concentration-dependent, and depending on the concentration, spore-adherence to Caco-2 cells was enhanced. In the presence of an aberrantly-assembled exosporium (cdeC spores), binding of fibronectin, but not vitronectin, was increased. Notably, independent of the exosporium integrity, only a fraction of the spores had fibronectin and vitronectin molecules binding to their surface. Collectively, these results demonstrate that the integrity of the exosporium layer of strain R20291 contributes to selective spore adherence to components of the intestinal mucosa.