Biology subjects

Chakravarty, S.

Publications and source records attributed to Chakravarty, S..

22 records · Page 2Linked to original sources

bioRxiv2020

Atypical B cells are a normal component of immune responses to vaccination and infection in humans

The full diversity of the circulating human B cell compartment is unknown. Flow cytometry analysis suggests that in addition to naive and memory B cells, there exists a population of CD11c+, CD27- CD21- "atypical" B cells, that are associated with chronic or recurrent infection and autoimmunity. We used single cell RNA-seq approaches to examine the diversity of both antigen-specific B cells and total B cells in healthy subjects and individuals naturally-exposed to recurrent malaria infections. This analysis revealed two B cell lineages: a classical lineage of activated and resting memory B cells, and an atypical-like lineage. Surprisingly, the atypical lineage was common in both malaria exposed individuals and non-exposed healthy controls. Using barcoded antibodies in conjunction with our transcriptomic data, we found that atypical lineage cells in healthy individuals lack many atypical B markers and thus represent an undercounted cryptic population. We further determined using antigen specific probes that atypical cells can be induced by primary vaccination in humans and can be recalled upon boosting. Collectively these data suggest that atypical cells are not necessarily pathogenic but can be a normal component of B responses to antigen.

immunology↗

bioRxiv2019

Antibody feedback limits the expansion of cognate memory B cells but drives the diversification of vaccine-induced antibody responses

Generating sufficient antibody to block infection is a key challenge for vaccines against malaria. Here we show that antibody titres to a key target, the repeat region of the Plasmodium falciparum circumsporozoite protein (PfCSP), plateaued after two immunizations in a clinical trial of the radiation-attenuated sporozoite vaccine. To understand the mechanisms limiting vaccine responsiveness, we developed Ig-knockin mice with elevated numbers of PfCSP-binding B cells. We determined that recall responses were inhibited by antibody feedback via epitope masking of the immunodominant PfCSP repeat region. Importantly, the amount of antibody that prevents boosting is below the amount of antibody required for protection. Finally, while antibody feedback limited responses to the PfCSP-repeat region in vaccinated volunteers, potentially protective subdominant responses to C-terminal regions did expand with subsequent boosts. These data suggest that antibody feedback drives the diversification of immune responses and that vaccination for malaria will require the targeting of multiple antigens.

immunology↗

bioRxiv2019

A simple and sensitive SYBR Gold-based assay to quantify DNA-protein interactions

A simple, accessible, and inexpensive assay to quantify the strength of DNA-protein interactions was developed. The assay relies on capturing DNA-protein complexes using an affinity resin that binds tagged, recombinant proteins. Sequential washes with filtration spin cups and centrifugation remove non-specific interactions in a gentle, uniform manner and a final elution isolates specific DNA-protein complexes. SYBR Gold nucleic acid stain is added to the eluted product and the fluorescence intensity accurately quantifies the amount of captured DNA, ultimately illustrating the relative strength of the DNA-protein interaction. The major utility of the assay resides in the versatility and quantitative nature of the SYBR Gold:nucleic acid interaction, eliminating the need for customized or labeled oligos and permitting relatively inexpensive quantification of binding capacity. The assay also employs DNA-protein complex capture by the very common purification tag, 6xHis, but other tags could likely be utilized. Further, SYBR Gold fluorescence is compatible with a wide variety of instruments, including UV transilluminators, a staple to any molecular biology laboratory. This assay was used to compare the binding capacities of different Auxin Response Factor (ARF) transcription factors to various dsDNA targets, including the classical AuxRE motif and several divergent sequences. Results from dose-response assays suggest that different ARF proteins might show distinct comparative affinities for AuxRE variants, emphasizing that specific ARF-AuxRE binding strengths likely contribute to the complex and fine-tuned cellular auxin response.

plant biology↗

bioRxiv2018

Age-dependent visualization of neural progenitor cells within the rostral migratory stream via MRI and endogenously labeled micron-sized iron oxide particles

The subventricular zone (SVZ) is one of the primary sources for rodent neural progenitor cells (NPC), however, aging greatly impacts the substructure of the region and rate of new cell birth. To determine if age impacts the rate of in vivo migration within animals, we examined the rostral migratory stream (RMS) of animals across 12 days using an established MRI technique. To visualize NPCs, we injected micron sized particles of iron oxide (MPIO) into the lateral ventricle to endogenously label cells within the SVZ, which then appeared as hypo-intensive spots within MR images. Our in vivo MRI data showed that the rate of migration was significantly different between all ages examined, with decreases in the distance traveled as age progressed. The total number of iron oxide labeled cells within the olfactory bulb on day 12, decrease significantly when compared across ages in ex vivo high-resolution scans. We also, for the first time, demonstrated the endogenous labeling of cells within the dentate gyrus (DG) of hippocampus. Here too, there was a significant decrease in the number of labeled cells within the structure across age. Histology of the NPCs verified the decrease in labeling of cells with doublecortin (DCX) as age progressed for both regions. The dramatic reduction of labeling in NPCs within the SVZ and DG observed with MRI, demonstrates the importance of understanding the impact of age on the relationship of NPC and disease.

neuroscience↗