Coagulation factor replacement therapy for the X-linked bleeding disorder Haemophilia, characterized by a deficiency of coagulation protein factor VIII (FVIII), is severely complicated by antibody (inhibitors) formation. The development of FVIII inhibitors drastically alters the quality of life of the patients and is associated with a tremendous increase in morbidity as well as treatment costs. The ultimate goal of inhibitor control is antibody elimination. Immune tolerance induction (ITI) is the only clinically established approach for developing antigen-specific tolerance to FVIII. This work aims to establish a novel cost-effective strategy to produce FVIII molecules in fusion with cholera toxin B (CTB) subunit at the N terminus using the Bacillus subtilis expression system for oral tolerance, as the current clinical immune tolerance protocols are expensive. Regions of B-Domain Deleted (BDD)-FVIII that have potential epitopes were identified by employing Bepipred linear epitope prediction; 2 or more epitopes in each domain were combined and cDNA encoding these regions were fused with CTB and cloned in the Bacillus subtilis expression vector pHT43 and expression analysis was carried out. The expressed CTB-fused FVIII epitope domains showed strong binding affinity towards the CTB-receptor GM1 ganglioside. To conclude, Bacillus subtilis expressing FVIII molecules might be a promising candidate for exploring for the induction of oral immune tolerance.

Dengue virus is an enveloped virus with an icosahedral assembly of envelope proteins (E). The E proteins are arranged as a head-to-tail homodimer, and domain III (EDIII) is placed at the edge of the dimer, converging to a pentamer interface. For a structure-based approach, cholera toxin B (CTB) was harnessed as a structural scaffold for the five-fold symmetry of EDIII. Pivoted by an RNA-mediated chaperone for the protein folding and assembly, CTB-EDIII of dengue serotype 1 (DV1) was successfully produced as soluble pentamers in an E. coli host with a high yield of about 28 mg/L. Immunization of mice with CTB-DV1EDIII elicited increased levels of neutralizing antibodies against infectious viruses compared to the control group immunized with DV1EDIII without CTB fusion. IgG isotype switching into a balanced Th1/Th2 response was also observed, probably triggered by the intrinsic adjuvant activity of CTB. Confirming the immune-enhancing potential of CTB in stabilizing the pentamer assembly of EDIII, this study introduces a low-cost bacterial production platform designed to augment the soluble production of subunit vaccine candidates, particularly those targeting flaviviruses.

BACKGROUND: Early diagnosis is essential in the field of lysosomal storage disorders for the proper management of patients and for starting therapies before irreversible damage occurs, particularly in neurodegenerative conditions. Currently, specific biomarkers for the diagnosis of lysosomal storage disorders are lacking in routine laboratory practice, except for enzymatic tests, which are available only in specialized metabolic centers. Recently, we established a method for measuring and verifying changes in GM1 ganglioside levels in peripheral blood lymphocytes in patients with GM1 gangliosidosis. However, fresh blood is not always available, and using frozen/thawed lymphocytes can lead to inaccurate results.
METHODS: We used frozen/thawed fibroblasts obtained from stored biopsies to explore the feasibility of fluorescent imaging and flow-cytometric methods to track changes in storage materials in fibroblasts from patients with three lysosomal neurodegenerative conditions: GM1 gangliosidosis, Sialidosis, and Niemann-Pick type C. We used specific markers for each pathology.
CONCLUSIONS: We demonstrated that with our methods, it is possible to clearly distinguish the levels of accumulated metabolites in fibroblasts from affected and unaffected patients for all the three pathologies considered. Our methods proved to be rapid, sensitive, unbiased, and potentially applicable to other LSDs.

Approximately 2.9 million people worldwide suffer from cholera each year, many of whom are destitute. However, understanding of immunity against cholera is still limited. Several studies have reported the duration of antibodies following cholera; however, systematic reviews including a quantitative synthesis are lacking.
To meta-analyze cohort studies that have evaluated vibriocidal, cholera toxin B subunit (CTB), and lipopolysaccharide (LPS) antibody levels following a clinical cholera case.
Design: Systematic review and meta-analysis. We searched PubMed and Web of science for studies assessing antibodies against Vibrio cholerae in cohorts of patients with clinical cholera. Two authors independently extracted data and assessed the quality of included studies. Random effects models were used to pool antibody titers in adults and older children (aged ≥ 6 years). In sensitivity analysis, studies reporting data on young children (2-5 years) were included.
Nine studies met our inclusion criteria for systematic review and seven for meta-analysis. The pooled mean of vibriocidal antibody titers in adults and older children (aged ≥ 6 years) was 123 on day 2 post-symptom onset, which sharply increased on day 7 (pooled mean = 6956) and gradually waned to 2247 on day 30, 578 on day 90, and 177 on day 360. Anti-CTB IgA antibodies also peaked on day 7 (pooled mean = 49), followed by a rapid decrease on day 30 (pooled mean = 21), and further declined on day 90 (pooled mean = 10), after which it plateaued from day 180 (pooled mean = 8) to 360 (pooled mean = 6). Similarly, anti-CTB IgG antibodies peaked in early convalescence between days 7 (pooled mean = 65) and 30 (pooled mean = 69), then gradually waned on days 90 (pooled mean = 42) and 180 (pooled mean = 30) and returned to baseline on day 360 (pooled mean = 24). Anti-LPS IgA antibodies peaked on day 7 (pooled mean = 124), gradually declined on day 30 (pooled mean = 44), which persisted until day 360 (pooled mean = 10). Anti LPS IgG antibodies peaked on day 7 (pooled mean = 94). Thereafter, they decreased on day 30 (pooled mean = 85), and dropped further on days 90 (pooled mean = 51) and 180 (pooled mean = 47), and returned to baseline on day 360 (pooled mean = 32). Sensitivity analysis including data from young children (aged 2-5 years) showed very similar findings as in the primary analysis.
This study confirms that serological antibody (vibriocidal, CTB, and LPS) titers return to baseline levels within 1 year following clinical cholera, i.e., before the protective immunity against subsequent cholera wanes. However, this decay should not be interpreted as waning immunity because immunity conferred by cholera against subsequent disease lasts 3-10 years. Our study provides evidence for surveillance strategies and future research on vaccines and also demonstrates the need for further studies to improve our understanding of immunity against cholera.

Sympathetic neurons that innervate the heart are located primarily in the stellate ganglia (SG), which also contains neurons that project to brown adipose tissue (BAT). These studies were designed to examine the morphology of these two populations (cardiac- and BAT-projecting) and their target connectivity. We examined SG neurons in C57BL/6J mice following injections of the retrograde tracer cholera toxin B (CTb) conjugated to Alexa Fluor 488 and Alexa Fluor 555, into cardiac tissue and intrascapular BAT. BAT-projecting SG neurons were widely dispersed in SG, while cardiac-projecting SG neurons were localized primarily near the inferior cardiac nerve base. SG neurons were not dual-labeled, suggesting that sympathetic innervation is specific to the heart and BAT, supporting the idea of \"labeled lines\" of efferents. Morphologically, cardiac-projecting SG somata had more volume and were less abundant than BAT-projecting neurons using our tracer-labeling paradigm. We found a positive correlation between the number of primary dendrites per neuron and soma volume in cardiac-projecting SG neurons, though not in BAT-projecting neurons. In both SG subpopulations, the number of cholinergic inputs marked with vesicular acetylcholine transporter (VAChT) puncta contacting the soma was positively correlated to soma volume, suggesting scaling of inputs across a range of neuronal sizes. In separate studies using dual tracing from left and right BAT, we found that BAT-projecting SG neurons were located predominately ipsilateral to the injection, but a small subset of SG neurons project bilaterally to BAT. This tracing approach will allow the assessment of cell-specific mechanisms of plasticity within subpopulations of SG neurons.

The fact that Parkinson\'s disease (PD) pathologies are well advanced in most PD patients by the time of clinical elucidation attests to the importance of early diagnosis. Our attempt to achieve this has capitalized on our previous finding that GM1 ganglioside is expressed at subnormal levels in virtually all tissues of sporadic PD (sPD) patients including blood cells. GM1 is present in most vertebrate cells, is especially abundant in neurons where it was shown essential for their effective functioning and long term viability. We have utilized peripheral blood mononuclear cells (PBMCs) which, despite their low GM1, we found to be significantly lower in sPD patients compared to age-matched healthy controls. To quantify GM1 (and GD1a) we used high performance thin-layer chromatography combined with cholera toxin B linked to horseradish peroxidase, followed by densitometric quantification. GM1 was also deficient in PBMCs from PD patients with mutations in the glucocerebrosidase gene (PD-GBA), apparently even lower than in sPD. Reasons are given why we believe these results obtained with patients manifesting fully developed PD will apply as well to PD patients in preclinical stages-a topic for future study. We also suggest that these findings point to a potential disease altering therapy for PD once the early diagnosis is established.

The auditory cortex sends massive projections to the inferior colliculus, but the organization of this pathway is not yet well understood. Previous work has shown that the corticocollicular projection emanates from both layers 5 and 6 of the auditory cortex and that neurons in these layers have different morphological and physiological properties. It is not yet known in the mouse if both layer 5 and layer 6 project bilaterally, nor is it known if the projection patterns differ based on projection location. Using targeted injections of Fluorogold into either the lateral cortex or dorsal cortex of the inferior colliculus, we quantified retrogradely labeled neurons in both the left and right lemniscal regions of the auditory cortex, as delineated using parvalbumin immunostaining. After dorsal cortex injections, we observed that approximately 18-20% of labeled cells were in layer 6 and that this proportion was similar bilaterally. After lateral cortex injections, only ipsilateral cells were observed in the auditory cortex, and they were found in both layer 5 and layer 6. The ratio of layer 5:layer 6 cells after lateral cortex injection was similar to that seen after dorsal cortex injection. Finally, injections of different tracers were made into the two inferior colliculi, and an average of 15-17% of cells in the auditory cortex were double-labeled, and these proportions were similar in layers 5 and 6. These data suggest that (1) only the dorsal cortex of the inferior colliculus receives bilateral projections from the auditory cortex, (2) both the dorsal and lateral cortex of the inferior colliculus receive similar layer 5 and layer 6 auditory cortical input, and (3) a subpopulation of individual neurons in both layers 5 and 6 branch to innervate both dorsal cortices of the inferior colliculus.

Cervical spinal cord injury (cSCI) severs bulbospinal projections to respiratory motor neurons, paralyzing respiratory muscles below the injury. C2 spinal hemisection (C2Hx) is a model of cSCI often used to study spontaneous and induced plasticity and breathing recovery post-injury. One key assumption is that C2Hx dennervates motor neurons below the injury, but does not affect their survival. However, a recent study reported substantial bilateral motor neuron death caudal to C2Hx. Since phrenic motor neuron (PMN) death following C2Hx would have profound implications for therapeutic strategies designed to target spared neural circuits, we tested the hypothesis that C2Hx minimally impacts PMN survival. Using improved retrograde tracing methods, we observed no loss of PMNs at 2- or 8-weeks post-C2Hx. We also observed no injury-related differences in ChAT or NeuN immunolabeling within labelled PMNs. Although we found no evidence of PMN loss following C2Hx, we cannot rule out neuronal loss in other motor pools. These findings address an essential prerequisite for studies that utilize C2Hx as a model to explore strategies for inducing plasticity and/or regeneration within the phrenic motor system, as they provide important insights into the viability of phrenic motor neurons as therapeutic targets after high cervical injury.

Exploiting plants as biological bioreactors for production and delivery of edible oral subunit vaccines is a promising application of biotechnology. Efforts to enhance expression levels of transgenes coding for antigenic proteins by exploiting promoters, targeting sequences, and enhancer elements have produced rather low quantities of the antigen in plant tissues, but enough to induce immune responses in feeding studies. This review will cover components of various gene constructs used in developing plant-based vaccines against a myriad of viral and bacterial diseases. Specifically, it will focus on sequences that are involved in targeting the antigen to mucosal tissues of the intestinal tract, thus enhancing the immunogenicity of the plant-based vaccine as well as those components that result in higher accumulation of the protein within the plant.

Cholera toxin B (CTB) is a subunit of cholera toxin, a bacterial enterotoxin secreted by Vibrio cholerae and also functions as an immune adjuvant. However, it remains unclear how CTB activates immune cells. We here evaluated whether or how CTB induces production of a pro-inflammatory cytokine, interleukin-1β (IL-1β). CTB induced IL-1β production not only from bone marrow-derived macrophages (BMMs) but also from resident peritoneal macrophages in synergy with O111:B4-derived lipopolysaccharide (LPS O111:B4) that can bind to CTB. Meanwhile, when prestimulated with O55:B5-derived LPS (LPS O55:B5) that fails to bind to CTB, resident peritoneal macrophages, but not BMMs, produced IL-1β in response to CTB. The CTB-induced IL-1β production in synergy with LPS in both peritoneal macrophages and BMMs was dependent on ganglioside GM1, which is required for internalization of CTB. Notably, not only the NLRP3 inflammasome but also the pyrin inflammasome were involved in CTB-induced IL-1β production from resident peritoneal macrophages, while only the NLRP3 inflammasome was involved in that from BMMs. In response to CTB, a Rho family small GTPase, RhoA, which activates pyrin inflammasome upon various kinds of biochemical modification, increased its phosphorylation at serine-188 in a GM1-dependent manner. This phosphorylation as well as CTB-induced IL-1β productions were dependent on protein kinase A (PKA), indicating critical involvement of PKA-dependent RhoA phosphorylation in CTB-induced IL-1β production. Taken together, these results suggest that CTB, incorporated through GM1, can activate resident peritoneal macrophages to produce IL-1β in synergy with LPS through novel mechanisms in which pyrin as well as NLRP3 inflammasomes are involved.