Immune and behavioral correlates of protection against symptomatic post-vaccination SARS-CoV-2 infection.

Introduction: We sought to determine pre-infection correlates of protection against SARS-CoV-2 post-vaccine inzfections (PVI) acquired during the first Omicron wave in the United States. Methods: Serum and saliva samples from 176 vaccinated adults were collected from October to December of 2021, immediately before the Omicron wave, and assessed for SARS-CoV-2 Spike-specific IgG and IgA binding antibodies (bAb). Sera were also assessed for bAb using commercial assays, and for neutralization activity against several SARS-CoV-2 variants. PVI duration and severity, as well as risk and precautionary behaviors, were assessed by questionnaires. Results: Serum anti-Spike IgG levels assessed by research assay, neutralization titers against Omicron subvariants, and low home risk scores correlated with protection against PVIs after multivariable regression analysis. Commercial assays did not perform as well as research assay, likely due to their lower dynamic range. Discussion: In the 32 participants that developed PVI, anti-Spike IgG bAbs correlated with lower disease severity and shorter duration of illness.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens, including fungal infections. Herein, we show that the simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from that of the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and neglected tropical diseases (NTDs) targeted for elimination by 2030.

Natural killer cells and BNT162b2 mRNA vaccine reactogenicity and durability.

Introduction: Natural killer (NK) cells can both amplify and regulate immune responses to vaccination. Studies in humans and animals have observed NK cell activation within days after mRNA vaccination. In this study, we sought to determine if baseline NK cell frequencies, phenotype, or function correlate with antibody responses or inflammatory side effects induced by the Pfizer-BioNTech COVID-19 vaccine (BNT162b2). Methods: We analyzed serum and peripheral blood mononuclear cells (PBMCs) from 188 participants in the Prospective Assessment of SARS-CoV-2 Seroconversion study, an observational study evaluating immune responses in healthcare workers. Baseline serum samples and PBMCs were collected from all participants prior to any SARS-CoV-2 infection or vaccination. Spike-specific IgG antibodies were quantified at one and six months post-vaccination by microsphere-based multiplex immunoassay. NK cell frequencies and phenotypes were assessed on pre-vaccination PBMCs from all participants by multi-color flow cytometry, and on a subset of participants at time points after the 1st and 2nd doses of BNT162b2. Inflammatory side effects were assessed by structured symptom questionnaires, and baseline NK cell functionality was quantified by an in vitro killing assay on participants that reported high or low post-vaccination symptom scores. Results: Key observations include: 1) circulating NK cells exhibit evidence of activation in the week following vaccination, 2) individuals with high symptom scores after 1st vaccination had higher pre-vaccination NK cytotoxicity indices, 3) high pre-vaccination NK cell numbers were associated with lower spike-specific IgG levels six months after two BNT162b2 doses, and 4) expression of the inhibitory marker NKG2A on immature NK cells was associated with higher antibody responses 1 and 6 months post-vaccination. Discussion: These results suggest that NK cell activation by BNT162b2 vaccination may contribute to vaccine-induced inflammatory symptoms and reduce durability of vaccine-induced antibody responses.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens including fungal infections. Since these diseases target the most vulnerable communities who are disadvantaged by health and socio-economic factors, new agents should be, if possible, easy-to-prepare to allow for commercialization based on their low cost. In this study, we show that simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and NTDs targeted for elimination by 2030. Overall, the discovery of these new compounds with broad-spectrum activity opens new avenues for the development of treatments for several current human infections, either caused by fungi or by parasites, including other NTDs, as well as newly emerging diseases. ONE-SENTENCE SUMMARY: Simple derivatives of the well-known antifungal drug fluconazole were found to be highly effective in vivo against fungal infections, and also potent against the parasitic nematode Brugia, which causes lymphatic filariasis and against Trichuris, one of the soil-transmitted helminths that infects millions of people globally.

Use of a Pericranial Flap Technique for Deep Brain Stimulation Hardware Protection and Improved Cosmesis.

OBJECTIVES: Deep brain stimulation (DBS) has become an established neuromodulation therapy; however, surgical site complications such as hardware skin erosion remain an important risk and can predispose to infection, requiring explantation of the system. Nuances of surgical technique can affect wound healing, cosmetic outcome, comfort, and risk of infection. In this study, we describe our experience with a layered closure technique using a vascularized pericranial flap for improving cosmesis and protection of the implanted hardware against skin erosion and infection. MATERIALS AND METHODS: We retrospectively reviewed 636 individuals (746 lead implantations) who underwent DBS surgery by a single academic neurosurgeon between 2001 and 2020. A layered pericranial flap closure technique for the burr-hole and connector sites was instituted in 2015. We assessed the effects of a multimodal infection prevention approach that included the pericranial flap on hardware complication rates compared with the premultimodality cohort, and we report the nuances of the technique. RESULTS: In our institutional experience, we found that implementation of a pericranial flap closure technique can enhance the subjective cosmetic result at the burr-hole cover site and increase patient comfort and satisfaction. In addition, we found a decrease in hardware infection rates in the current cohort with a multimodal infection prevention regimen that includes the pericranial-flap technique (n = 256, 2015-2020 period) to 1.2% (p = 0.006), from 6.9% in the earlier cohort (n = 490, 2001-2015 period). CONCLUSIONS: The report highlights the potential of a pericranial-flap closure technique as a surgical adjunct to improve DBS surgical site healing and cosmesis and may, as part of a multimodal strategy, contribute to decreased risk of skin breakdown and hardware infection.

Prospective Assessment of Symptoms to Evaluate Asymptomatic SARS-CoV-2 Infections in a Cohort of Health Care Workers.

BACKGROUND: The frequency of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is unclear and may be influenced by how symptoms are evaluated. In this study, we sought to determine the frequency of asymptomatic SARS-CoV-2 infections in a prospective cohort of health care workers (HCWs). METHODS: A prospective cohort of HCWs, confirmed negative for SARS-CoV-2 exposure upon enrollment, were evaluated for SARS-CoV-2 infection by monthly analysis of SARS-CoV-2 antibodies as well as referral for polymerase chain reaction testing whenever they exhibited symptoms of coronavirus disease 2019 (COVID-19). Participants completed the standardized and validated FLU-PRO Plus symptom questionnaire scoring viral respiratory disease symptom intensity and frequency at least twice monthly during baseline periods of health and each day they had any symptoms that were different from their baseline. RESULTS: Two hundred sixty-three participants were enrolled between August 25 and December 31, 2020. Through February 28, 2021, 12 participants were diagnosed with SARS-CoV-2 infection. Symptom analysis demonstrated that all 12 had at least mild symptoms of COVID-19, compared with baseline health, near or at time of infection. CONCLUSIONS: These results suggest that asymptomatic SARS-CoV-2 infection in unvaccinated, immunocompetent adults is less common than previously reported. While infectious inoculum doses and patient factors may have played a role in the clinical manifestations of SARS-CoV-2 infections in this cohort, we suspect that the high rate of symptomatic disease was due primarily to participant attentiveness to symptoms and collection of symptoms in a standardized, prospective fashion. These results have implications for studies that estimate SARS-CoV-2 infection prevalence and for public health measures to control the spread of this virus.

Durability of Antibody Response and Frequency of SARS-CoV-2 Infection 6 Months after COVID-19 Vaccination in Healthcare Workers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies decay but persist 6 months postvaccination; lower levels of neutralizing titers persist against Delta than wild-type virus. Of 227 vaccinated healthcare workers tested, only 2 experienced outpatient symptomatic breakthrough infections, despite 59/227 exhibiting serologic evidence of SARS-CoV-2 infection, defined as presence of nucleocapsid protein antibodies.

Adverse Effects and Antibody Titers in Response to the BNT162b2 mRNA COVID-19 Vaccine in a Prospective Study of Healthcare Workers.

BACKGROUND: The relationship between postvaccination symptoms and strength of antibody responses is unclear. The goal of this study was to determine whether adverse effects caused by vaccination with the Pfizer/BioNTech BNT162b2 vaccine are associated with the magnitude of vaccine-induced antibody levels. METHODS: We conducted a single-center, observational cohort study consisting of generally healthy adult participants that were not severely immunocompromised, had no history of coronavirus disease 2019, and were seronegative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein before vaccination. Severity of vaccine-associated symptoms was obtained through participant-completed questionnaires. Testing for immunoglobulin G antibodies against SARS-CoV-2 spike protein and receptor-binding domain was conducted using microsphere-based multiplex immunoassays performed on serum samples collected at monthly visits. Neutralizing antibody titers were determined by microneutralization assays. RESULTS: Two hundred six participants were evaluated (69.4% female, median age 41.5 years old). We found no correlation between vaccine-associated symptom severity scores and vaccine-induced antibody titers 1 month after vaccination. We also observed that (1) postvaccination symptoms were inversely correlated with age and weight and more common in women, (2) systemic symptoms were more frequent after the second vaccination, (3) high symptom scores after first vaccination were predictive of high symptom scores after second vaccination, and (4) older age was associated with lower titers. CONCLUSIONS: Lack of postvaccination symptoms after receipt of the BNT162b2 vaccine does not equate to lack of vaccine-induced antibodies 1 month after vaccination.

Recrudescent infection after deep brain stimulator reimplantation.

OBJECTIVE: The efficacy of deep brain stimulation (DBS) in treating the symptoms of movement disorders can be life changing for patients. Thus, the 5%-15% incidence of stimulator-related infection requiring removal of the device can be particularly disheartening. Although DBS system reimplantation is generally successful, this is not always the case. The literature is replete with publications describing the incidence of infection and the associated features. However, the literature is sparse in terms of information on the incidence of recurrent or recrudescent infection after system reimplantation. The goal of this paper was to evaluate factors leading to unsuccessful reimplantation of a DBS system following initial infection. METHODS: Data were reviewed for all DBS procedures performed by one surgeon (K.L.H.) over 19 years including the infectious agent, location of infection, treatment regimen, and subsequent reimplantation of a DBS system and long-term outcome. RESULTS: In this series of 558 patients who had undergone DBS surgery, 37 (6.6%) subsequently developed an infection. Infections with methicillin-sensitive Staphylococcus aureus, Enterobacter species, or coagulase-negative staphylococci were predominant. Four patients had cerebritis, one had meningitis, and the rest had soft tissue infections of the pocket or scalp. All had their entire DBS system explanted, followed by 4-6 weeks of intravenous antibiotics and surveillance for recrudescence for an additional period of at least 30 days. Twenty-five patients subsequently underwent DBS system reimplantation, and the procedure was successful in 22. Three of the 4 patients with cerebritis developed a subsequent wound infection after system reimplantation. None of the other 22 patients developed a recurrence. The odds ratio for developing a recurrent infection after cerebritis was 28.5 (95% CI 1.931-420.5, p = 0.007). CONCLUSIONS: This study, the largest series of DBS system reimplantations following infection, demonstrated that most patients can have successful reimplantations without recurrent infection. However, patients who have had DBS-related cerebritis have a nearly 30-fold increased risk of developing reinfection after reimplantation. Alternative strategies for these patients are discussed.

Adverse effects and antibody titers in response to the BNT162b2 mRNA COVID-19 vaccine in a prospective study of healthcare workers.

BACKGROUND: mRNA COVID-19 vaccines are playing a key role in controlling the COVID-19 pandemic. The relationship between post-vaccination symptoms and strength of antibody responses is unclear. OBJECTIVE: To determine whether adverse effects caused by vaccination with the Pfizer/BioNTech BNT162b2 vaccine are associated with the magnitude of vaccine-induced antibody levels. DESIGN: Single center, prospective, observational cohort study. SETTING: Participants worked at Walter Reed National Military Medical Center and were seen monthly at the Naval Medical Research Center Clinical Trials Center. PARTICIPANTS: Generally healthy adults that were not severely immunocompromised, had no history of COVID-19, and were seronegative for SARS-CoV-2 spike protein prior to vaccination. MEASURES: Severity of vaccine-associated symptoms was obtained through participant completed questionnaires. Testing for IgG antibodies against SARS-CoV-2 spike protein and receptor binding domain was conducted using microsphere-based multiplex immunoassays. RESULTS: 206 participants were evaluated (69.4% female, median age 41.5 years old). We found no correlation between vaccine-associated symptom severity scores and vaccine-induced antibody titers one month after vaccination. We also observed that 1) post-vaccination symptoms were inversely correlated with age and weight and more common in women, 2) systemic symptoms were more frequent after the second vaccination, 3) high symptom scores after first vaccination were predictive of high symptom scores after second vaccination, and 4) older age was associated with lower titers. LIMITATIONS: Study only observes antibody responses and consists of healthy participants. CONCLUSIONS: Lack of post-vaccination symptoms following receipt of the BNT162b2 vaccine does not equate to lack of vaccine-induced antibodies one month after vaccination. This study also suggests that it may be possible to design future mRNA vaccines that confer robust antibody responses with lower frequencies of vaccine-associated symptoms. FUNDING: This study was executed by the Infectious Disease Clinical Research Program (IDCRP), a Department of Defense (DoD) program executed by the Uniformed Services University of the Health Sciences (USUHS) through a cooperative agreement by the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF). This project has been funded by the Defense Health Program, U.S. DoD, under award HU00012120067. Project funding for JHP was in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The funding bodies have had no role in the study design or the decision to submit the manuscript for publication.

Prospective Assessment of SARS-CoV-2 Seroconversion (PASS) study: an observational cohort study of SARS-CoV-2 infection and vaccination in healthcare workers.

BACKGROUND: SARS-CoV-2 is a recently emerged pandemic coronavirus (CoV) capable of causing severe respiratory illness. However, a significant number of infected people present as asymptomatic or pauci-symptomatic. In this prospective assessment of at-risk healthcare workers (HCWs) we seek to determine whether pre-existing antibody or T cell responses to previous seasonal human coronavirus (HCoV) infections affect immunological or clinical responses to SARS-CoV-2 infection or vaccination. METHODS: A cohort of 300 healthcare workers, confirmed negative for SARS-CoV-2 exposure upon study entry, will be followed for up to 1 year with monthly serology analysis of IgM and IgG antibodies against the spike proteins of SARS-CoV-2 and the four major seasonal human coronavirus - HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63. Participants will complete monthly questionnaires that ask about Coronavirus Disease 2019 (COVID-19) exposure risks, and a standardized, validated symptom questionnaire (scoring viral respiratory disease symptoms, intensity and severity) at least twice monthly and any day when any symptoms manifest. SARS-CoV-2 PCR testing will be performed any time participants develop symptoms consistent with COVID-19. For those individuals that seroconvert and/or test positive by SARS-CoV-2 PCR, or receive the SARS-CoV-2 vaccine, additional studies of T cell activation and cytokine production in response to SARS-CoV-2 peptide pools and analysis of Natural Killer cell numbers and function will be conducted on that participant's cryopreserved baseline peripheral blood mononuclear cells (PBMCs). Following the first year of this study we will further analyze those participants having tested positive for COVID-19, and/or having received an authorized/licensed SARS-CoV-2 vaccine, quarterly (year 2) and semi-annually (years 3 and 4) to investigate immune response longevity. DISCUSSION: This study will determine the frequency of asymptomatic and pauci-symptomatic SARS-CoV-2 infection in a cohort of at-risk healthcare workers. Baseline and longitudinal assays will determine the frequency and magnitude of anti-spike glycoprotein antibodies to the seasonal HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63, and may inform whether pre-existing antibodies to these human coronaviruses are associated with altered COVID-19 disease course. Finally, this study will evaluate whether pre-existing immune responses to seasonal HCoVs affect the magnitude and duration of antibody and T cell responses to SARS-CoV-2 vaccination, adjusting for demographic covariates.

The Poised Cannula Technique Reduces the Stereotactic Error of the Fiducial-Less Frameless DBS Procedure.

BACKGROUND: In this study, we describe a technique of optimizing the accuracy of frameless deep brain stimulation (DBS) lead placement through the use of a cannula poised at the entry to predict the location of the fully inserted device. This allows real-time correction of error prior to violation of the deep gray matter. METHODS: We prospectively gathered data on radial error during the operative placements of 40 leads in 28 patients using frameless fiducial-less DBS surgery. Once the Nexframe had been aligned to target, a cannula was inserted through the center channel of the BenGun until it traversed the pial surface and a low-dose O-arm spin was obtained. Using 2 points along the length of the imaged cannula, a trajectory line was projected to target depth. If lead location could be improved, the cannula was inserted through an alternate track in the BenGun down to target depth. After intraoperative microelectrode recording and clinical assessment, another O-arm spin was obtained to compare the location of the inserted lead with the location predicted by the poised cannula. RESULTS: The poised cannula projection and the actual implant had a mean radial discrepancy of 0.75 ± 0.64 mm. The poised cannula projection identified potentially clinically significant errors (avg 2.07 ± 0.73 mm) in 33% of cases, which were reduced to a radial error of 1.33 ± 0.66 mm (p = 0.02) after correction using an alternative BenGun track. The final target to implant error for all 40 leads was 1.20 ± 0.52 mm with only 2.5% of errors being >2.5 mm. CONCLUSION: The poised cannula technique results in a reduction of large errors (>2.5 mm), resulting in a decline in these errors to 2.5% of implants as compared to 17% in our previous publication using the fiducial-less method and 4% using fiducial-based methods of DBS lead placement.

Implementing Pool-Based Surveillance Testing for SARS-CoV-2 at the Army Public Health Center Laboratory and across the Army Public Health Laboratory Enterprise.

With limited clinical resources, burgeoning testing requests from Army and other Service units to clinical laboratories, and the continued spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) throughout the military population, the Army Public Health Laboratory (APHL) Enterprise was tasked to establish surveillance testing capabilities for active duty military populations in an expedient manner. Following a proof-of-concept study conducted by Public Health Command-Pacific, Public Health Command-Europe was the first public health laboratory to offer the capability to assess for SARS-CoV-2 in pooled samples, followed closely by the Army Public Health Center (APHC) at Aberdeen Proving Grounds, MD, paralleling the spread of the SARS-CoV-2 virus from China to Europe to the continental US. The APHLs have selected pool sizes of up to 10 samples per pool based on the best evidence available at the time of method development and validation. Real-Time quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) assays using RNA extracts from pooled nasopharyngeal swabs preserved in viral transport media were selected to assess the presence of SARS-CoV-2. The rapid development of initial surveillance testing capabilities depended on existing equipment in each laboratory, with a plan to implement full operational capability using additional staff and common high-throughput platforms. APHL Enterprise has successfully used existing resources to begin to address the changing and complex needs for COVID-19 testing within the Army population. Successful implementation of pooled surveillance testing at the APHC Laboratory has enabled more than 8,600 Soldiers to avoid clinical testing to date. The APHC Laboratory alone has tested over 10,000 samples and prevented approximately 8,600 soldiers from seeking testing with clinical diagnostic assays.

House dust mite sensitization drives cross-reactive immune responses to homologous helminth proteins.

The establishment of type 2 responses driven by allergic sensitization prior to exposure to helminth parasites has demonstrated how tissue-specific responses can protect against migrating larval stages, but, as a consequence, allow for immune-mediated, parasite/allergy-associated morbidity. In this way, whether helminth cross-reacting allergen-specific antibodies are produced and play a role during the helminth infection, or exacerbate the allergic outcome awaits elucidation. Thus, the main objective of the study was to investigate whether house dust mite (HDM) sensitization triggers allergen-specific antibodies that interact with Ascaris antigens and mediate antibody-dependent deleterious effects on these parasites as well as, to assess the capacity of cross-reactive helminth proteins to trigger allergic inflammation in house dust mite presensitized mice. Here, we show that the sensitization with HDM-extract drives marked IgE and IgG1 antibody responses that cross-react with Ascaris larval antigens. Proteomic analysis of Ascaris larval antigens recognized by these HDM-specific antibodies identified Ascaris tropomyosin and enolase as the 2 major HDM homologues based on high sequence and structural similarity. Moreover, the helminth tropomyosin could drive Type-2 associated pulmonary inflammation similar to HDM following HDM tropomyosin sensitization. The HDM-triggered IgE cross-reactive antibodies were found to be functional as they mediated immediate hypersensitivity responses in skin testing. Finally, we demonstrated that HDM sensitization in either B cells or FcγRIII alpha-chain deficient mice indicated that the allergen driven cell-mediated larval killing is not antibody-dependent. Taken together, our data suggest that aeroallergen sensitization drives helminth reactive antibodies through molecular and structural similarity between HDM and Ascaris antigens suggesting that cross-reactive immune responses help drive allergic inflammation.

Identification of anisomycin, prodigiosin and obatoclax as compounds with broad-spectrum anti-parasitic activity.

Parasitic infections are a major source of human suffering, mortality, and economic loss, but drug development for these diseases has been stymied by the significant expense involved in bringing a drug though clinical trials and to market. Identification of single compounds active against multiple parasitic pathogens could improve the economic incentives for drug development as well as simplifying treatment regimens. We recently performed a screen of repurposed compounds against the protozoan parasite Entamoeba histolytica, causative agent of amebic dysentery, and identified four compounds (anisomycin, prodigiosin, obatoclax and nithiamide) with low micromolar potency and drug-like properties. Here, we extend our investigation of these drugs. We assayed the speed of killing of E. histolytica trophozoites and found that all four have more rapid action than the current drug of choice, metronidazole. We further established a multi-institute collaboration to determine whether these compounds may have efficacy against other parasites and opportunistic pathogens. We found that anisomycin, prodigiosin and obatoclax all have broad-spectrum antiparasitic activity in vitro, including activity against schistosomes, T. brucei, and apicomplexan parasites. In several cases, the drugs were found to have significant improvements over existing drugs. For instance, both obatoclax and prodigiosin were more efficacious at inhibiting the juvenile form of Schistosoma than the current standard of care, praziquantel. Additionally, low micromolar potencies were observed against pathogenic free-living amebae (Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba castellanii), which cause CNS infection and for which there are currently no reliable treatments. These results, combined with the previous human use of three of these drugs (obatoclax, anisomycin and nithiamide), support the idea that these compounds could serve as the basis for the development of broad-spectrum anti-parasitic drugs.

Side-to-side supercharging nerve allograft enhances neurotrophic potential.

INTRODUCTION: Critical limitations of processed acellular nerve allograft (PNA) are linked to Schwann cell function. Side-to-side bridge grafting may enhance PNA neurotrophic potential. METHODS: Sprague-Dawley rats underwent tibial nerve transection and immediate repair with 20-mm PNA (n = 33) or isograft (ISO; n = 9) or 40-mm PNA (n = 33) or ISO (n = 9). Processed acellular nerve allograft groups received zero, one, or three side-to-side bridge grafts between the peroneal nerve and graft. Muscle weight, force generation, and nerve histomorphology were tested 20 weeks after repair. Selected animals underwent neuron back labeling with fluorescent dyes. RESULTS: Inner axon diameters, g-ratios, and axon counts were smaller in the distal vs proximal aspect of each graft (P < .05). Schwann cell counts were greater, with a lower proportion of senescent cells for groups with bridges (P < .05). Retrograde labeling demonstrated that 6.6% to 17.7% of reinnervating neurons were from the peroneal pool. DISCUSSION: Bridge grafting positively influenced muscle recovery and Schwann cell counts and senescence after long PNA nerve reconstruction.

Functional Assessment of 2,177 U.S. and International Drugs Identifies the Quinoline Nitroxoline as a Potent Amoebicidal Agent against the Pathogen Balamuthia mandrillaris.

Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris-a proliferative trophozoite form and a nonproliferative cyst form, which is highly resistant to harsh physical and chemical conditions-have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multidrug regimens that often include the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low, and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more-effective treatments, we screened 2,177 clinically approved compounds for in vitro activity against B. mandrillaris The quinoline antibiotic nitroxoline (8-hydroxy-5-nitroquinoline), which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a pharmacologically relevant range. We compared the in vitro efficacy of nitroxoline to that of drugs currently used in the standard of care for GAE and found that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at pharmacologically relevant concentrations. Taken together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.IMPORTANCEBalamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This report addresses the need for new treatment options through a drug repurposing screen to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at pharmacologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings that we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life-threatening B. mandrillaris infections.

Cell Penetration Profiling Using the Chloroalkane Penetration Assay.

Biotherapeutics are a promising class of molecules in drug discovery, but they are often limited to extracellular targets due to their poor cell penetration. High-throughput cell penetration assays are required for the optimization of biotherapeutics for enhanced cell penetration. We developed a HaloTag-based assay called the chloroalkane penetration assay (CAPA), which is quantitative, high-throughput, and compartment-specific. We demonstrate the ability of CAPA to profile extent of cytosolic penetration with respect to concentration, presence of serum, temperature, and time. We also used CAPA to investigate structure-penetration relationships for bioactive stapled peptides and peptides fused to cell-penetrating sequences. CAPA is not only limited to measuring cytosolic penetration. Using a cell line where HaloTag is localized to the nucleus, we show quantitative measurement of nuclear penetration. Going forward, CAPA will be a valuable method for measuring and optimizing the cell penetration of biotherapeutics.