Mechanism of Action and Epitopes of Clostridium difficile Toxin B-neutralizing Antibody Bezlotoxumab Revealed by X-ray Crystallography

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.     

Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH₄Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.     

Understanding bias in geographic range size estimates

Aim Estimates of geographic range size derived from natural history museum specimens are probably biased for many species. We aim to determine how bias in these estimates relates to range size. Location We conducted computer simulations based on herbarium specimen records from localities ranging from the southern United States to northern Argentina. Methods We used theory on the sampling distribution of the mean and variance to develop working hypotheses about how range size, defined as area of occupancy (AOO), was related to the inter‐specific distribution of: (1) mean collection effort per area across the range of a species (MC); (2) variance in collection effort per area across the range of a species (VC); and (3) proportional bias in AOO estimates (PBias: the difference between the expected value of the estimate of AOO and true AOO, divided by true AOO). We tested predictions from these hypotheses using computer simulations based on a dataset of more than 29,000 herbarium specimen records documenting occurrences of 377 plant species in the tribe Bignonieae (Bignoniaceae). Results The working hypotheses predicted that the mean of the inter‐specific distribution of MC, VC and PBias were independent of AOO, but that the respective variance and skewness decreased with increasing AOO. Computer simulations supported all but one prediction: the variance of the inter‐specific distribution of VC did not decrease with increasing AOO. Main conclusions Our results suggest that, despite an invariant mean, the dispersion and symmetry of the inter‐specific distribution of PBias decreases as AOO increases. As AOO increased, range size was less severely underestimated for a large proportion of simulated species. However, as AOO increased, range size estimates having extremely low bias were less common.     

Targeting of aberrant mRNAs to cytoplasmic processing bodies

In eukaryotes, a specialized pathway of mRNA degradation termed nonsense-mediated decay (NMD) functions in mRNA quality control by recognizing and degrading mRNAs with aberrant termination codons. We demonstrate that NMD in yeast targets premature termination codon (PTC)-containing mRNA to P-bodies. Upf1p is sufficient for targeting mRNAs to P-bodies, whereas Upf2p and Upf3p act, at least in part, downstream of P-body targeting to trigger decapping. The ATPase activity of Upf1p is required for NMD after the targeting of mRNAs to P-bodies. Moreover, Upf1p can target normal mRNAs to P-bodies but not promote their degradation. These observations lead us to propose a new model for NMD wherein two successive steps are used to distinguish normal and aberrant mRNAs.     

HIV-specific T-cells accumulate in the liver in HCV/HIV co-infection

Background and Aims

Hepatitis C Virus (HCV)-related liver disease progresses more rapidly in individuals co-infected with Human Immunodeficiency Virus-1 (HIV), although the underlying immunologic mechanisms are unknown. We examined whether HIV-specific T-cells are identified in the liver of HCV/HIV co-infected individuals and promote liver inflammation through bystander immune responses.

Methods

Ex-vivo intra-hepatic lymphocytes from HCV mono-infected and HCV/HIV co-infected individuals were assessed for immune responses to HIV and HCV antigens by polychromatic flow cytometry.

Results

HCV/HIV liver biopsies had similar frequencies of lymphocytes but lower percentages of CD4⁺ T-cells compared to HCV biopsies. In co-infection, intra-hepatic HIV-specific CD8⁺ and CD4⁺ T-cells producing IFN-γ and TNF-α were detected and were comparable in frequency to those that were HCV-specific. In co-infected individuals, viral-specific CD8⁺ T-cells produced more of the fibrogenic cytokine, TNF-α. In both mono- and co-infected individuals, intra-hepatic HCV-specific T-cells were poorly functional compared to HIV-specific T-cells. In co-infection, HAART was not associated with a reconstitution of intra-hepatic CD4⁺ T-cells and was associated with reduction in both HIV and HCV-specific intra-hepatic cytokine responses.

Conclusion

The accumulation of functional HIV-specific T-cells in the liver during HCV/HIV co-infection may represent a bystander role for HIV in inducing faster progression of liver disease.     

The epithelial cell response to rotavirus infection.

Rotavirus is the most important worldwide cause of severe gastroenteritis in infants and young children. Intestinal epithelial cells are the principal targets of rotavirus infection, but the response of enterocytes to rotavirus infection is largely unknown. We determined that rotavirus infection of HT-29 intestinal epithelial cells results in prompt activation of NF-kappaB (<2 h), STAT1, and ISG F3 (3 h). Genetically inactivated rotavirus and virus-like particles assembled from baculovirus-expressed viral proteins also activated NF-kappaB. Rotavirus infection of HT-29 cells induced mRNA for several C-C and C-X-C chemokines as well as IFNs and GM-CSF. Mice infected with simian rotavirus or murine rotavirus responded similarly with the enhanced expression of a profile of C-C and C-X-C chemokines. The rotavirus-stimulated increase in chemokine mRNA was undiminished in mice lacking mast cells or lymphocytes. Rotavirus induced chemokines only in mice <15 days of age despite documented infection in older mice. Macrophage inflammatory protein-1beta and IFN-stimulated protein 10 mRNA responses occurred, but were reduced in p50-/- mice. Macrophage inflammatory protein-1beta expression during rotavirus infection localized to the intestinal epithelial cell in murine intestine. These results show that the intestinal epithelial cell is an active component of the host response to rotavirus infection.     

Dysregulated B Cell Expression of RANKL and OPG Correlates with Loss of Bone Mineral Density in HIV Infection

HIV infection is associated with high rates of osteopenia and osteoporosis, but the mechanisms involved are unclear. We recently reported that bone loss in the HIV transgenic rat model was associated with upregulation of B cell expression of the key osteoclastogenic cytokine receptor-activator of NF-κB ligand (RANKL), compounded by a simultaneous decline in expression of its physiological moderator, osteoprotegerin (OPG). To clinically translate these findings we performed cross-sectional immuno-skeletal profiling of HIV-uninfected and antiretroviral therapy-naïve HIV-infected individuals. Bone resorption and osteopenia were significantly higher in HIV-infected individuals. B cell expression of RANKL was significantly increased, while B cell expression of OPG was significantly diminished, conditions favoring osteoclastic bone resorption. The B cell RANKL/OPG ratio correlated significantly with total hip and femoral neck bone mineral density (BMD), T- and/or Z-scores in HIV infected subjects, but revealed no association at the lumbar spine. B cell subset analyses revealed significant HIV-related increases in RANKL-expressing naïve, resting memory and exhausted tissue-like memory B cells. By contrast, the net B cell OPG decrease in HIV-infected individuals resulted from a significant decline in resting memory B cells, a population containing a high frequency of OPG-expressing cells, concurrent with a significant increase in exhausted tissue-like memory B cells, a population with a lower frequency of OPG-expressing cells. These data validate our pre-clinical findings of an immuno-centric mechanism for accelerated HIV-induced bone loss, aligned with B cell dysfunction.     

Effects of CdCl2 on the secretory functions of sex accessory glands of rats

The effects of cadmium chloride (Cd) alone (1 mg given as a single injection) or in combination with ascorbic acid (AA; 100 mg/day for 10 days) on the secretory functions of sex accessory glands of rats were studied in healthy male albino rats. Animals were sacrificed after 10 days treatment and the seminal vesicles (SVs), dorsolateral prostate (DLP), ventral prostate (VP), bulbourethral glands (BU), and coagulating glands (CO) were excised and weighed. Weight of all accessory glands were decreased by 10 days treatment with Cd. Cd + AA gave similar results. AA concentration increased in all glands and was significantly increases in CO (p less than .01). Levels of ascorbogin increased in all glands except CO and BU and in the latter a significant (p less than .001) increase was found. The rate of AA utilization increased significantly (p less that .001) in the accessories. A significant (p less than .001) reduction in the activity of succinate dehydrogenase was observed in Cd-treated rats with a further reduction with combined treatment. Alkaline phosphatase decreased (p less than .001) after Cd treatment but AA in combination restored it to control levels. Cd increased acid phosphatase (p less than .001) and was further activated by Cd + AA. Phosphorylase activity was elevated with Cd (p less than .001) but recovery occurred in SV and BU with Cd + AA. Glycogen increased (p less than .001; .01) with both treatments as did citric acid. Protein results were inconsistent with Cd but activation was found in most glands under combined treatment. The results reveal that most androgen-dependent biochemical constituents and organ weights were affected significantly by a single injection of Cd. AA had a protective and beneficial influence on the restoration of structural integrity and metabolism in sex accessory glands.     

Immunoreactive-FSH in human normal gastric mucosa

Using indirect immuno-peroxidase staining technique, localization of immunoreactive follicle-stimulating hormone (IR-FSH) is demonstrated in the cytoplasm of the epithelial cells of normal human stomach. In view of their triangular shape and central nucleus and their predominance in the intermediate glands of the gastric mucosa, these cells are identified as parietal cells. The stromal tissue is devoid of staining reaction.