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Anti-ceramide single-chain variable fragment mitigates radiation GI syndrome mortality independent of DNA repair.

ABSTRACT: After 9/11, threat of nuclear attack on American urban centers prompted government agencies to develop medical radiation countermeasures to mitigate hematopoietic acute radiation syndrome (H-ARS) and higher-dose gastrointestinal acute radiation syndrome (GI-ARS) lethality. While repurposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS in preclinical models, no mitigator potentially deliverable under mass casualty conditions preserves GI tract. Here, we report generation of an anti-ceramide 6B5 single-chain variable fragment (scFv) and show that s.c. 6B5 scFv delivery at 24 hours after a 90% lethal GI-ARS dose of 15 Gy mitigated mouse lethality, despite administration after DNA repair was complete. We defined an alternate target to DNA repair, an evolving pattern of ceramide-mediated endothelial apoptosis after radiation, which when disrupted by 6B5 scFv, initiates a durable program of tissue repair, permitting crypt, organ, and mouse survival. We posit that successful preclinical development will render anti-ceramide 6B5 scFv a candidate for inclusion in the Strategic National Stockpile for distribution after a radiation catastrophe.

SUBMITTER: Rotolo JA

PROVIDER: S-EPMC8119204 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Anti-ceramide single-chain variable fragment mitigates radiation GI syndrome mortality independent of DNA repair.

Rotolo Jimmy A JA Fong Chii Shyang CS Bodo Sahra S Nagesh Prashanth Kb PK Fuller John J Sharma Thivashnee T Piersigilli Alessandra A Zhang Zhigang Z Fuks Zvi Z Singh Vijay K VK Kolesnick Richard R

JCI insight 20210422 8

After 9/11, threat of nuclear attack on American urban centers prompted government agencies to develop medical radiation countermeasures to mitigate hematopoietic acute radiation syndrome (H-ARS) and higher-dose gastrointestinal acute radiation syndrome (GI-ARS) lethality. While repurposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS in preclinical models, no mitigator potentially deliverable under mass casualty conditions preserves GI tract. Here, we report ge ...[more]

PMID: 33724956

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Project description:PurposeAfter September 11, 2001, nuclear threat prompted government agencies to develop medical countermeasures to mitigate two syndromes, the hematopoietic-acute radiation syndrome (H-ARS) and the higher-dose gastrointestinal-acute radiation syndrome (GI-ARS), both lethal within weeks. While repurposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS, no mitigator potentially deliverable under mass casualty conditions preserves the GI tract. We recently reported that anti-ceramide single-chain variable fragment (scFv) mitigates GI-ARS lethality, abrogating ongoing small intestinal endothelial apoptosis to rescue Lgr5+ stem cells. Here, we examine long-term consequences of prevention of acute GI-ARS lethality.Methods and materialsFor these studies, C57BL/6J male mice were treated with 15 Gy whole body irradiation, the 90% GI-ARS lethal dose for this mouse strain.ResultsMice irradiated with 15 Gy alone or with 15 Gy + bone marrow transplantation (BMT) or anti-ceramide scFv, succumb to an ARS within 8 to 10 days. Autopsies reveal only mice receiving anti-ceramide scFv at 24 hours post-whole body irradiation display small intestinal rescue. No marrow reconstitution occurs in any group with attendant undetectable circulating blood elements. Mice receiving 15 Gy + BMT + scFv, however, normalize blood counts by day 12, suggesting that scFv also improves marrow reconstitution, a concept for which we provide experimental support. We show that at 14 Gy, the upper limit dose for H-ARS lethality before transition to GI-ARS lethality, anti-ceramide scFv markedly improves marrow take, reducing the quantity of marrow-conferring survival by more than 3-fold. Consistent with these findings, mice receiving 15 Gy + BMT + scFv exhibit prolonged survival. At day 90, before sacrifice, they display normal appearance, behavior, and serum biochemistries, and surprisingly, at full autopsy, near-normal physiology in all 42 tissues examined.ConclusionsAnti-ceramide scFv mitigates GI-ARS lethality and improves marrow reconstitution rendering prolonged survival with near normal autopsies.

Project description:BackgroundIntimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). Both pathogens are still important causes of diarrhea in children and adults in many developing and industrialized countries. Considering the fact that antibodies are important tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667). In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv).FindingsAnti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3)pLys strain was transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv, expressed as inclusion bodies (insoluble fraction), was denatured, purified and submitted to refolding. The protein yield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin, resulting in an absorbance of 0.75 at 492 nm. The immunofluorescence assay showed a strong reactivity with EPEC E2348/69.ConclusionThis study demonstrated that the recombinant anti-intimin antibody obtained is able to recognize the conserved region of intimin (Int388-667) in purified form and the EPEC isolate.

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Anti-ceramide single-chain variable fragment mitigates radiation GI syndrome mortality independent of DNA repair.

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Anti-ceramide single-chain variable fragment mitigates radiation GI syndrome mortality independent of DNA repair.

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