BackgroundLeptospirosis is a zoonosis of worldwide distribution caused by infection with pathogenic serovars of Leptospira spp. The most common species, L. interrogans, can survive in the environment for lengthy periods of time in between infection of mammalian hosts. Transmission of pathogenic Leptospira to humans mostly occurs through abraded skin or mucosal surfaces after direct or indirect contact with infected animals or contaminated soil or water. The spirochete then spreads hematogenously, resulting in multi-organ failure and death in severe cases. Previous DNA microarray studies have identified differentially expressed genes required for adaptation to temperature and osmolarity conditions inside the host compared to those of the environment.ResultsIn order to identify genes involved in survival in the early spirochetemic phase of infection, we performed a transcriptional analysis of L. interrogans serovar Copenhageni upon exposure to serum in comparison with EMJH medium. One hundred and sixty-eight genes were found to be differentially expressed, of which 55 were up-regulated and 113 were down-regulated. Genes of known or predicted function accounted for 54.5 and 45.1% of up- and down-regulated genes, respectively. Most of the differentially expressed genes were predicted to be involved in transcriptional regulation, translational process, two-component signal transduction systems, cell or membrane biogenesis, and metabolic pathways.ConclusionsOur study showed global transcriptional changes of pathogenic Leptospira upon exposure to serum, representing a specific host environmental cue present in the bloodstream. The presence of serum led to a distinct pattern of gene expression in comparison to those of previous single-stimulus microarray studies on the effect of temperature and osmolarity upshift. The results provide insights into the pathogenesis of leptospirosis during the early bacteremic phase of infection.

The impact of gut fungi and (1→3)-β-d-glucan (BG), a major fungal cell wall component, on uremia was explored by Candida albicans oral administration in bilateral nephrectomy (BiNx) mice because of the prominence of C. albicans in the human intestine but not in mice. As such, BiNx with Candida administration (BiNx-Candida) enhanced intestinal injury (colon cytokines and apoptosis), gut leakage (fluorescein isothiocyanate [FITC]-dextran assay, endotoxemia, serum BG, and bacteremia), systemic inflammation, and liver injury at 48 h postsurgery compared with non-Candida BiNx mice. Interestingly, uremia-induced enterocyte apoptosis was severe enough for gut translocation of viable bacteria, as indicated by culture positivity for bacteria in blood, mesenteric lymph nodes (MLNs), and other organs, which was more severe in BiNx-Candida than in non-Candida BiNx mice. Candida induced alterations in the gut microbiota of BiNx mice as indicated by (i) the higher fungal burdens in the feces of BiNx-Candida mice than in sham-Candida mice by culture methods and (ii) increased Bacteroides with decreased Firmicutes and reduced bacterial diversity in the feces of BiNx-Candida mice compared with non-Candida BiNx mice by fecal microbiome analysis. In addition, lipopolysaccharide plus BG (LPS+BG), compared with each molecule alone, induced high supernatant cytokine levels, which were enhanced by uremic mouse serum in both hepatocytes (HepG2 cells) and macrophages (RAW264.7 cells). Moreover, LPS+BG, but not each molecule alone, reduced the glycolysis capacity and mitochondrial function in HepG2 cells as determined by extracellular flux analysis. Additionally, a probiotic, Lactobacillus rhamnosus L34 (L34), attenuated disease severity only in BiNx-Candida mice but not in non-Candida BiNx mice, as indicated by liver injury and serum cytokines through the attenuation of gut leakage, the fecal abundance of fungi, and fecal bacterial diversity but not fecal Gram-negative bacteria. In conclusion, Candida enhanced BiNx severity through the worsening of gut leakage and microbiota alterations that resulted in bacteremia, endotoxemia, and glucanemia. IMPORTANCE The impact of fungi in the intestine on acute uremia was demonstrated by the oral administration of Candida albicans in mice with the removal of both kidneys. Because fungi in the mouse intestine are less abundant than in humans, a Candida-administered mouse model has more resemblance to patient conditions. Accordingly, acute uremia, without Candida, induced intestinal mucosal injury, which resulted in the translocation of endotoxin, a major molecule of gut bacteria, from the intestine into blood circulation. In acute uremia with Candida, intestinal injury was more severe due to fungi and the alteration in intestinal bacteria (increased Bacteroides with decreased Firmicutes), leading to the gut translocation of both endotoxin from gut bacteria and (1→3)-β-d-glucan from Candida, which synergistically enhanced systemic inflammation in acute uremia. Both pathogen-associated molecules were delivered to the liver and induced hepatocyte inflammatory responses with a reduced energy production capacity, resulting in acute uremia-induced liver injury. In addition, Lactobacillus rhamnosus attenuated intestinal injury through reduced gut Candida and improved intestinal bacterial conditions.

AKI is one of the most serious complications of leptospirosis, an important zoonosis in the tropics. Recently, NGAL, one of the novel AKI biomarkers, is extensively studied in various specific settings such as sepsis, cardiac surgery, and radiocontrast nephropathy. In this multicenter study, we aimed to study the role of NGAL as an early marker and an outcome predictor of leptospirosis associated AKI. Patients who presented with clinical suspiciousness of leptospirosis were prospectively enrolled in 9 centers from August 2012 to November 2014. The first day of enrollment was the first day of clinical suspicious leptospirosis. Blood and urine samples were serially collected on the first three days and day 7 after enrollment. We used three standard techniques (microscopic agglutination test, direct culture, and PCR technique) to confirm the diagnosis of leptospirosis. KDIGO criteria were used for AKI diagnosis. Recovery was defined as alive and not requiring dialysis during hospitalization or maintaining maximum KDIGO stage at hospital discharge. Of the 221 recruited cases, 113 cases were leptospirosis confirmed cases. Thirty seven percent developed AKI. Median uNGAL and pNGAL levels in those developing AKI were significantly higher than in patients not developing AKI [253.8 (631.4) vs 24.1 (49.6) ng/ml, p < 0.001] and [1,030 (802.5) vs 192.0 (209.0) ng/ml, p < 0.001], respectively. uNGAL and pNGAL levels associated with AKI had AUC-ROC of 0.91, and 0.92, respectively. Both of urine NGAL and pNGAL level between AKI-recovery group and AKI-non recovery were comparable. From this multicenter study, uNGAL and pNGAL provided the promising result to be a marker for leptospirosis associated AKI. However, both of them did not show the potential role to be the predictor of renal recovery in this specific setting.

More than 65 million people have been confirmed infection with SARS-CoV-2 and more than 1 million have died from COVID-19 and this pandemic remains critical worldwide. Effective vaccines are one of the most important strategies to limit the pandemic. Here, we report a construction strategy of DNA vaccine candidates expressing full length wild type SARS-CoV-2 spike (S) protein, S1 or S2 region and their immunogenicity in mice. All DNA vaccine constructs of pCMVkan-S, -S1 and -S2 induced high levels of specific binding IgG that showed a balance of IgG1/IgG2a response. However, only the sera from mice vaccinated with pCMKkan-S or -S1 DNA vaccines could inhibit viral RBD and ACE2 interaction. The highest neutralizing antibody (NAb) titer was found in pCMVkan-S group, followed by -S1, while -S2 showed the lowest PRNT50 titers. The geometric mean titers (GMTs) were 2,551, 1,005 and 291 for pCMVkan-S, -S1 and -S2, respectively. pCMVkan-S construct vaccine also induced the highest magnitude and breadth of T cells response. Analysis of IFN-γ positive cells after stimulation with SARS-CoV-2 spike peptide pools were 2,991, 1,376 and 1,885 SFC/106 splenocytes for pCMVkan-S, -S1 and -S2, respectively. Our findings highlighted that full-length S antigen is more potent than the truncated spike (S1 or S2) in inducing of neutralizing antibody and robust T cell responses.

Chitosan conjugated with maleimide (CS-Mal) as a potential precursor for bioconjugation and the example of the application in in situ injectable adhesive gel is proposed. The homogeneous reaction in water/dimethyl sulfoxide (DMSO) (1:1) followed by dialysis in HCl (10 × 10 m) is a good condition to obtain CS-Mal. When SH-PEG-SH is applied as the crosslinker, the gel can be obtained in a few seconds without any by-products at room temperature. The gel formation and properties are controllable by simply varying the concentration and the molecular weight of CS, the Mal substitution degree, and the temperature. The gel is injectable and shows adhesive property for soft tissue. Moreover, the gel shows not only biocompatibility to SAOS2 cells but also antimicrobial activity against both gram-negative and gram-positive bacteria. CS-Mal is useful not only for bioconjugation of CS but also for development of biomaterials.