Johid Reza Malik

Coronavirus disease 2019 (COVID‐19) survivors frequently experience a wide range of symptoms known as post‐acute sequelae of SARS‐CoV‐2 (PASC) or long COVID. Importantly, complications arising from microvascular dysfunction, blood‐brain barrier (BBB) disruption, and chronic neuroinflammation have been implicated in driving PASC within the central nervous system (CNS), known as neuro‐PASC. Notably, people with HIV (PWH), who suffer from chronic neuroinflammation, BBB impairment, and glial cell dysfunction, collectively known as neuro‐HIV, are generally at higher risk of neuro‐PASC. The overlap between neuro‐PASC and neuro‐HIV raises concerns that HIV and SARS‐CoV‐2 co‐infection may exacerbate neurological dysfunctions among PWH. In this study, using an in‐vitro cell culture model, we examine the effects of HIV and SARS‐CoV‐2 mono‐ and co‐infection in microglia, astrocytes, and pericytes. Our results demonstrated that majority of brain cell types support SARS‐CoV‐2 replication, in the presence and absence of HIV infection. Furthermore, in both mono‐ and co‐infected cells, there were varying degree of up‐ and downregulation of SARS‐CoV‐2 host cell entry factors, such as ACE2, TMPRSS2, NRP1, and TRIM28, and inflammatory cytokines including IL‐6, TNF‐α, and IL‐1β. Moreover, conditioned media collected from HIV, SARS‐CoV‐2, and HIV/SARS‐CoV‐2 co‐infected astrocytes and pericytes were shown to be neurotoxic. Additionally, proteomic analysis has revealed a unique set of proteins significantly up/down regulated in HIV/SARS‐CoV‐2 co‐infected astrocytes and pericytes. The gene set enrichment analysis of these proteins indicates dysregulation of lipid, energy, and immune metabolism pathways linked to neurodegenerative disorders like Alzheimer's, Parkinson's, Huntington's disease, and amyotrophic lateral sclerosis. These in‐vitro findings indicate that astrocytes and pericytes from HIV/SARS‐CoV‐2 co‐infection exhibit altered protein expression profiles, implicating dysregulated signaling pathways associated with neurodegenerative dysfunction.

Antigen-binding fragments (Fabs) are emerging biochemical tools for treating and managing several diseases and conditions. Though full-length monoclonal antibodies (mAbs) have great potential, their higher molecular size, time required for generation, and cost remain major limiting factors. Fabs, being smaller and having similar affinity to mAbs for their target, can reach the affected site easily, where penetration of mAb might be hindered. Additionally, with fewer amino acids than mAbs, Fabs can be genetically engineered for the required function. In the present work, we have used a combination of state-of-the-art technologies and classical methods for Fab generation from a single B cell of rhesus macaque previously immunized with factor H binding protein (FHbp) of Neisseria meningitidis (Nm). FHbp was selected for its potency as an immunogen and importance in vaccine development against Nm. Using conjugated FHbp as a bait to sort single B cells, expressing antibodies against FHbp increases the probability of finding antigen-specific B cells. We have not only developed a protocol for Fab generation but also provided proof of concept for increasing the yield of toxic or poorly expressed proteins in bacteria by the inclusion of ethanol in bacterial culture.

Three years after SARS-CoV-2 emerged as a global infectious threat, the virus has become endemic. The neurological complications such as depression, anxiety, and other CNS complications after COVID-19 disease are increasing. The brain, and CSF have been shown as viral reservoirs for SARS-CoV-2, yielding a potential hypothesis for CNS effects. Thus, we investigated the CNS pharmacology of orally dosed nirmatrelvir/ritonavir (NMR/RTV). Using both an in vitro and an in vivo rodent model, we investigated CNS penetration and potential pharmacodynamic activity of NMR. Through pharmacokinetic modeling, we estimated the median CSF penetration of NMR to be low at 18.11% of plasma with very low accumulation in rodent brain tissue. Based on the multiples of the 90% maximal effective concentration (EC₉₀) for SARS-CoV-2, NMR concentrations in the CSF and brain do not achieve an exposure level similar to that of plasma. A median of only 16% of all the predicted CSF concentrations in rats were > 3xEC₉₀ (unadjusted for protein binding). This may have implications for viral persistence and neurologic post-acute sequelae of COVID-19 if increased NMR penetration in the CNS leads to decreased CNS viral loads and decreased CNS inflammation.

Background: Pediatric brain tumors (PBT) stand as the leading cause of cancer‐related deaths in children. Chemoradiation protocols have improved survival rates, even for non‐resectable tumors. Nonetheless, radiation therapy carries the risk of numerous adverse effects that can have long‐lasting, detrimental effects on the quality of life for survivors. The pursuit of chemotherapeutics that could obviate the need for radiotherapy remains ongoing. Several anti‐tumor agents, including sunitinib, valproic acid, carboplatin, and panobinostat, have shown effectiveness in various malignancies but have not proven effective in treating PBT. The presence of the blood–brain barrier (BBB) plays a pivotal role in maintaining suboptimal concentrations of anti‐cancer drugs in the central nervous system (CNS). Ongoing research aims to modulate the integrity of the BBB to attain clinically effective drug concentrations in the CNS. However, current findings on the interaction of exogenous chemical agents with the BBB remain limited and do not provide a comprehensive explanation for the ineffectiveness of established anti‐cancer drugs in PBT.Methods: We conducted our search for chemotherapeutic agents associated with the blood–brain barrier (BBB) using the following keywords: Chemotherapy in Cancer, Chemotherapy in Brain Cancer, Chemotherapy in PBT, BBB Inhibition of Drugs into CNS, Suboptimal Concentration of CNS Drugs, PBT Drugs and BBB, and Potential PBT Drugs. We reviewed each relevant article before compiling the information in our manuscript. For the generation of figures, we utilized BioRender software.Focus: We focused our article search on chemical agents for PBT and subsequently investigated the role of the BBB in this context. Our search criteria included clinical trials, both randomized and non‐randomized studies, preclinical research, review articles, and research papers.Finding: Our research suggests that, despite the availability of potent chemotherapeutic agents for several types of cancer, the effectiveness of these chemical agents in treating PBT has not been comprehensively explored. Additionally, there is a scarcity of studies examining the role of the BBB in the suboptimal outcomes of PBT treatment, despite the effectiveness of these drugs for other types of tumors.

Angiotensin Converting Enzyme 2 (ACE-2), Transmembrane Serine Protease 2 (TMPRSS-2) and Neuropilin-1 cellular receptors support the entry of SARS-CoV-2 into susceptible human target cells and are characterized at the molecular level. Some evidence on the expression of entry receptors at mRNA and protein levels in brain cells is available, but co-expression of these receptors and confirmatory evidence on brain cells is lacking. SARS-CoV-2 infects some brain cell types, but infection susceptibility, multiple entry receptor density, and infection kinetics are rarely reported in specific brain cell types. Highly sensitive Taqman ddPCR, flow-cytometry and immunocytochemistry assays were used to quantitate the expression of ACE-2, TMPRSS-2 and Neuropilin-1 at mRNA and protein levels on human brain-extracted pericytes and astrocytes, which are an integral part of the Blood-Brain-Barrier (BBB). Astrocytes showed moderate ACE-2 (15.9 ± 1.3%, Mean ± SD, n = 2) and TMPRSS-2 (17.6%) positive cells, and in contrast show high Neuropilin-1 (56.4 ± 39.8%, n = 4) protein expression. Whereas pericytes showed variable ACE-2 (23.1 ± 20.7%, n = 2), Neuropilin-1 (30.3 ± 7.5%, n = 4) protein expression and higher TMPRSS-2 mRNA (667.2 ± 232.3, n = 3) expression. Co-expression of multiple entry receptors on astrocytes and pericytes allows entry of SARS-CoV-2 and progression of infection. Astrocytes showed roughly four-fold more virus in culture supernatants than pericytes. SARS-CoV-2 cellular entry receptor expression and “in vitro” viral kinetics in astrocytes and pericytes may improve our understanding of viral infection “in vivo”. In addition, this study may facilitate the development of novel strategies to counter the effects of SARS-CoV-2 and inhibit viral infection in brain tissues to prevent the spread and interference in neuronal functions.