Biology subjects

Rocha, S. M.

Publications and source records attributed to Rocha, S. M..

6 recordsLinked to original sources

bioRxiv2024

Discrimination of Jamaican fruit bat lymphocytes by flow cytometry

Bats are natural reservoir hosts of many important zoonotic viruses but because there are few immunological reagents and breeding colonies available for infectious disease research, little is known about their immune responses to infection. We established a breeding colony Jamaican fruit bats (Artibeus jamaicensis) to study bat virology and immunology. The species is used as a natural reservoir model for H18N11 influenza A virus, and as a surrogate model for SARS-CoV-2, MERS-CoV and Tacaribe virus. As part of our ongoing efforts to develop this model organism, we sought to identify commercially available monoclonal antibodies (mAb) for profiling Jamaican fruit bat lymphocytes. We identified several cross-reactive mAb that can be used to identify T and B cells; however, we were unable to identify mAb for three informative T cell markers, CD3{gamma}, CD4 and CD8. We targeted these markers for the generation of hybridomas, and identified several clones to each that can be used with flow cytometry and fluorescence microscopy. Specificity of the monoclonal antibodies was validated by sorting lymphocytes, followed by PCR identification of confirmatory transcripts. Spleens of Jamaican fruit bats possess about half the number of T cells than do human or mouse spleens, and we identified an unusual population of cells that expressed the B cell marker CD19 and the T cell marker CD3. The availability of these monoclonal antibodies will permit a more thorough examination of adaptive immune responses in Jamaican fruit bats that should help clarify how the bats control viral infections and without disease. ImportanceBats naturally host a number of viruses without disease, but which can cause significant disease in humans. Virtually nothing is known about adaptive immune responses in bats because of a lack of immunological tools to examine such responses. We have begun to address this deficiency by identifying several commercially available monoclonal antibodies to human and mouse antigens that are cross-reactive to Jamaican fruit bat lymphocyte orthologs. We also generated monoclonal antibodies to Jamaican fruit bat CD3{gamma}, CD4 and CD8 that are suitable for identifying T cell subsets by flow cytometry and immunofluorescent staining of fixed tissues. Together, these reagents will allow a more detailed examination of lymphocyte populations in Jamaican fruit bats.

immunology↗

bioRxiv2024

Neuroprotective efficacy of the glucocorticoid receptor modulator PT150 in the rotenone mouse model of Parkinsons disease

Parkinsons disease (PD) is the most common neurodegenerative movement disorder worldwide. Current treatments for PD largely center around dopamine replacement therapies and fail to prevent the progression of pathology, underscoring the need for neuroprotective interventions. Approaches that target neuroinflammation, which occurs prior to dopaminergic neuron (DAn) loss in the substantia nigra (SN), represent a promising therapeutic strategy. The glucocorticoid receptor (GR) has been implicated in the neuropathology of PD and modulates numerous neuroinflammatory signaling pathways in the brain. Therefore, we investigated the neuroprotective effects of the novel GR modulator, PT150, in the rotenone mouse model of PD, postulating that inhibition of glial inflammation would protect DAn and reduce accumulation of neurotoxic misfolded -synuclein protein. C57Bl/6 mice were exposed to 2.5 mg/kg/day rotenone by intraperitoneal injection for 14 days, immediately followed by oral treatment with 30 mg/kg/day or 100 mg/kg/day PT150 in the 14-day post-lesioning incubation period, during which the majority of DAn loss and -synuclein (-syn) accumulation occurs. Our results indicate that treatment with PT150 reduced both loss of DAn and microgliosis in the nigrostriatal pathway. Although morphologic features of astrogliosis were not attenuated, PT150 treatment promoted potentially neuroprotective activity in these cells, including increased phagocytosis of hyperphosphorylated -syn. Ultimately, PT150 treatment reduced the loss of DAn cell bodies in the SN, but not the striatum, and prohibited intra-neuronal accumulation of -syn. Together, these data indicate that PT150 effectively reduced SN pathology in the rotenone mouse model of PD.

neuroscience↗

bioRxiv2021

A novel glucocorticoid and androgen receptor modulator reduces viral entry and innate immune inflammatory responses in the Syrian Hamster model of SARS-CoV-2

Since its initial discovery in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID19, has spread worldwide and despite significant research efforts, treatment options remain limited. Replication of SARS-CoV-2 in lung is associated with marked infiltration of macrophages and activation of innate immune inflammatory responses triggered, in part, by heightened production of interleukin-6 (IL-6) that recruits lymphocytes to the site of infection that amplify tissue injury. Antagonists of the glucocorticoid and androgen receptors have shown promise in experimental models of COVID19 and in clinical studies, because cell surface proteins required for viral entry, angiotensin converting enzyme 2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2), are transcriptionally regulated by these receptors. We therefore postulated that the glucocorticoid (GR) and androgen receptor (AR) antagonist, PT150, would reduce infectivity of SARS-CoV-2 and prevent inflammatory lung injury in the Syrian golden hamster model of COVID19. Animals were infected intranasally with 2.5 x 104 TCID50/ml equivalents of SARS-CoV-2 (strain 2019-nCoV/USA-WA1/ 2020) and PT150 was administered by oral gavage at 30 and 100 mg/Kg/day for a total of 7 days. Animals were then examined at days 3, 5 and 7 post-infection (DPI) for lung histopathology, viral load and production of proteins regulating the initiation and progression of SARS-CoV-2 infection. Results of these studies indicated that oral administration of PT150 decreased replication of SARS-CoV-2 in lung, as well as expression of ACE2 and TMPRSS2 protein. Hypercellularity and inflammatory cell infiltration driven by macrophage responses were dramatically decreased in PT150-treated animals, as was tissue damage and expression of IL-6. Molecular modeling suggested that PT150 binds to the co-activator interface of the ligand binding domain of both AR and GR and thereby acts as an allosteric modulator and transcriptional repressor of these receptors. Phylogenetic analysis of AR and GR across multiple species permissive to SARS-CoV-2 infection revealed a high degree of sequence identity maintained across species, including human, suggesting that the mechanism of action and therapeutic efficacy observed in Syrian hamsters would likely be predictive of positive outcomes in patients. PT150 is therefore a strong candidate for further clinical development for the treatment of COVID19 across variants of SARS-CoV-2.

immunology↗

bioRxiv2020

A potent SARS-CoV-2 neutralizing human monoclonal antibody that reduces viral burden and disease severity in Syrian hamsters

The emergence of COVID-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. Currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of COVID-19 are needed. We identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the SARS-CoV-2 spike protein receptor binding domain that neutralized the virus in vitro. Administration of the lead antibody clone to Syrian hamsters challenged with SARS-CoV-2 significantly reduced viral load and histopathology score in the lungs. Moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. The use of this antibody could provide an important therapy for treatment of COVID-19 patients.

microbiology↗

bioRxiv2020

SARS-CoV-2 infection, neuropathogenesis and transmission among deer mice: Implications for reverse zoonosis to New World rodents

Coronavirus disease-19 (COVID-19) emerged in November, 2019 in China and rapidly became pandemic. As with other coronaviruses, a preponderance of evidence suggests the virus originated in horseshoe bats (Rhinolophus spp.) and likely underwent a recombination event in an intermediate host prior to entry into human populations. A significant concern is that SARS-CoV-2 could become established in secondary reservoir hosts outside of Asia. To assess this potential, we challenged deer mice (Peromyscus maniculatus) with SARS-CoV-2 and found robust virus replication in the upper respiratory tract, lungs and intestines, with detectable viral RNA for up to 21 days in oral swabs and 14 days in lungs. Virus entry into the brain also occurred, likely via gustatory-olfactory-trigeminal pathway with eventual compromise to the blood brain barrier. Despite this, no conspicuous signs of disease were observed and no deer mice succumbed to infection. Expression of several innate immune response genes were elevated in the lungs, notably IFN, Cxcl10, Oas2, Tbk1 and Pycard. Elevated CD4 and CD8{beta} expression in the lungs was concomitant with Tbx21, IFN{gamma} and IL-21 expression, suggesting a type I inflammatory immune response. Contact transmission occurred from infected to naive deer mice through two passages, showing sustained natural transmission. In the second deer mouse passage, an insertion of 4 amino acids occurred to fixation in the N-terminal domain of the spike protein that is predicted to form a solvent-accessible loop. Subsequent examination of the source virus from BEI Resources indicated the mutation was present at very low levels, demonstrating potent purifying selection for the insert during in vivo passage. Collectively, this work has determined that deer mice are a suitable animal model for the study of SARS-CoV-2 pathogenesis, and that they have the potential to serve as secondary reservoir hosts that could lead to periodic outbreaks of COVID-19 in North America.

microbiology↗

bioRxiv2016

Surveillance to Establish Elimination of Transmission and Freedom from Dog-mediated Rabies

BackgroundWith a global target set for zero human deaths from dog-mediated rabies by 2030 and some regional programmes close to eliminating canine rabies, there is an urgent need for enhanced surveillance strategies suitable for declaring freedom from disease and elimination of transmission with known confidence.\n\nMethodsUsing exhaustive contact tracing across settings in Tanzania we generated detailed data on rabies incidence, rabid dog biting behaviour and health-seeking behaviour of bite victims. Using these data we compared case detection of sampling-based and enhanced surveillance methodologies and investigated elimination verification procedures.\n\nFindingsWe demonstrate that patients presenting to clinics with bite injuries are sensitive sentinels for identifying dog rabies cases. Triage of patients based on bite history criteria and investigation of suspicious incidents can confirm >10% of dog rabies cases and is an affordable approach that will enable validation of disease freedom following two years without case detection. Approaches based on sampling the dog population without using bite-injury follow-up were found to be neither sensitive nor cost-effective.\n\nInterpretationThe low prevalence of rabies, and short window in which disease can be detected, preclude sampling-based surveillance. Instead, active case finding guided by bite-patient triage is needed as elimination is approached. Our proposed methodology is affordable, practical and supports the goal of eliminating human rabies deaths by improving administration of lifesaving post-exposure prophylaxis for genuinely exposed but untreated contacts. Moreover, joint investigations by public health and veterinary workers will strengthen intersectoral partnerships and capacity for control of emerging zoonoses.

epidemiology↗