Clinical and pathological correlation between urinary exosome miR-223 and IgAN patients.

OBJECTIVE: To investigate the association between urine exosome miR-223 and clinical markers with pathological severity of IgA nephropathy (IgAN) in order to offer a new perspective for the evaluation of IgAN patients. MATERIALS AND METHODS: Western blotting and transmission electron microscopy were used to identify the exosomes collected and isolated from subjects' urine. qRT-PCR was then performed to determine the expression level of miR-223. Following that, the relationship between miR-223 expression, clinical markers, and the severity of pathology in IgAN patients was examined. RESULTS: (1) Urine can be used to isolate exosomes since its marker protein was visible by Western blotting, and its size and structure were observable using transmission electron microscopy. (2) Expression levels of miR-223 in urinary exosomes were much higher in IgAN patients than in healthy subjects, and these were also positively correlated with creatinine (Cr) (rho = 0.396; p = 0.006), blood urea nitrogen (BUN) (rho = 0.371; p = 0.011), 24-hour urinary microalbumin (24hU-mALB) (rho = 0.341; p = 0.036), mesangial cell proliferation (rho = 0.359; p = 0.014), glomerular segmental sclerosis (rho = 0.417; p = 0.004), cell/fibroblast crescents (rho = 0.612; p = 0.000), glomerulosclerosis, and renal interstitial fibrosis (rho = 0.331; p = 0.025). CONCLUSION: In urine exosomes, miR-223 might be considered a non-invasive biomarker for the assessment of IgAN disease progression.

Synthesis of structure-defined ß-1,4-GlcNAc-modified wall teichoic acids as potential vaccine against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a high priority pathogen due to its life-threating infections to human health. Development of prophylactic or therapeutic anti-MRSA vaccine is a potential approach to treat S. aureus infections and overcome the resistance crisis. ß-1,4-GlcNAc glycosylated wall teichoic acids (WTAs) derived from S. aureus are a new type of antigen that is closely associated with ß-lactam resistance. In this study, structure-defined ß-1,4-GlcNAc-modified WTAs varied in chain length and numbers of GlcNAc modification were synthesized by an ionic liquid-supported oligosaccharide synthesis (ILSOS) strategy in high efficiency and chromatography-free approach. Then the obtained WTAs were conjugated with tetanus toxin (TT) as vaccine candidates and were further evaluated in a mouse model to determine the structure-immunogenicity relationship. In vivo immunological studies revealed that the WTAs-TT conjugates provoked robust T cell-dependent responses and elicited high levels of specific anti-WTAs IgG antibodies production associated with the WTAs structure including chain length as well as the ß-1,4-GlcNAc modification pattern. Heptamer WTAs conjugate T6, carrying three copy of ß-1,4-GlcNAc modified RboP, was identified to elicit the highest titers of specific antibody production. The T6 antisera exhibited the highest recognition and binding affinity and the most potent OP-killing activities to MSSA and MRSA cells. This study demonstrated that ß-1,4-GlcNAc glycosylated WTAs are promising antigens for further development against MRSA.

Repurposing the Pentameric B-Subunit of Shiga Toxin for Gb3-Targeted Immunotherapy of Colorectal Cancer by Rhamnose Conjugation.

Globotriaosylceramide (Gb3 or CD77) is a tumor-associated carbohydrate antigen implicated in several types of cancer that serves as a potential cancer marker for developing target-specific diagnosis and therapy. However, the development of Gb3-targeted therapeutics has been challenging due to its carbohydrate nature. In the present work, taking advantage of its natural pentamer architecture and Gb3-specific targeting of shiga toxin B subunit (StxB), we constructed a pentameric antibody recruiting chimera by site-specifically conjugating StxB with the rhamnose hapten for immunotherapy of colorectal cancer. The Sortase A-catalyzed enzymatic tethering of rhamnose moieties to the C terminus of Stx1B and Stx2B had very moderate effect on their pentamer architectures and thus the resultant conjugates maintained the potent ability to bind to Gb3 antigen both immobilized on an assay plate and expressed on colorectal cancer cells. All StxB-rhamnose constructs were capable of efficiently mediating the binding of rhamnose antibodies onto HT29 colorectal cancer cells, which was further shown to be able to induce cancer cell lysis by eliciting potent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in vitro. Finally, the best StxB-rhamnose conjugate, i.e. 1B-3R, was confirmed to be able to inhibit the colorectal tumor growth using a HT29-derived xenograft murine model. Taken together, our data demonstrated the potential of repurposing StxB as an excellent multivalent scaffold for developing Gb3-targeted biotherapeutics and StxB-rhamnose conjugates might be promising candidates for targeted immunotherapy of Gb3-related colorectal cancer.

Nanobody-Based Bispecific Neutralizer for Shiga Toxin-Producing E. coli.

Currently, no specific therapeutics are available for foodborne Shiga toxin-producing Escherichia coli (STEC) infections that cause severe gastroenteritis and life-threatening complications of hemolytic uremic syndrome (HUS). As STEC attachment to intestinal epithelium might increase the host absorption of Shiga toxins and severity of the disease, we were inspired to develop a bispecific neutralizer capable of blocking its Shiga toxin and adhesin intimin simultaneously. Two nanobodies against the B subunit of Shiga toxin 2 (Stx2B) and the C terminus of Intimin (IntC280) were genetically fused together as the bispecific neutralizer, and it can be efficiently produced in a conventional E. coli expression system. We demonstrated that each of the nanobody modules in the bispecific format showed increased antigen binding capability and was able to functionally neutralize the binding of Stx2B or IntC280 to the respective host receptors even in the presence of the two virulence factors together. Moreover, the bispecific neutralizer was relatively stable to harsh storage conditions and gastrointestinal pH extremes. Taking into account its easy and economical production and superior pharmaceutical properties, we believe that a nanobody-based bispecific neutralizer would be more favorable and practical to be developed as a therapeutic to fight STEC in the developing world.