Development and Validation of Three Automated High-Throughput Molecular Tests to Detect Monkeypox Virus Infections.

BACKGROUND: The 2022 outbreak of the clade IIb monkeypox virus and subsequent global spread lead to an urgent need for the development of high-throughput, sensitive, and reproducible diagnostic tests. METHODS: We developed 3 assays to detect monkeypox virus, 2 (MPXV+ and MPXV) for m2000 RealTime and 1 (MPXV) for Alinity m platforms. Dual targets in E9L and B6R (MPXV+) and J2L and B7R (MPXV) increased mutation resistance. In silico prediction indicates MPXV+ cross-reactivity with orthopox viruses and specific monkeypox virus detection with MPXV. RESULTS: m2000 RealTime MPXV+ and MPXV assay sensitivity was determined to be 3.2 plaque-forming units/mL using a reference virus culture diluted into universal transport medium (UTM). Alinity m MPXV lower limit of detection was 200 copies/mL using monkeypox virus plasmids in pooled UTM matrix. m2000 RealTime MPXV+ and MPXV assays were validated with lesion swabs in UTM and 1:1 saliva to UTM mixtures. Commercially available and remnant clinical lesion specimens in UTM were tested with RealTime MPXV+, RealTime MPXV and Alinity m MPXV assays and demonstrated high agreement to known mpox (MPX)-positive specimens. CONCLUSIONS: RealTime MPXV+, RealTime MPXV, and Alinity MPXV are high throughput and sensitive assays used for the detection of monkeypox virus. These assays maybe useful during MPX outbreaks.

Purifying selection decreases the potential for Bangui orthobunyavirus outbreaks in humans.

Pathogens carried by insects, such as bunyaviruses, are frequently transmitted into human populations and cause diseases. Knowing which spillover events represent a public health threat remains a challenge. Metagenomic next-generation sequencing (mNGS) can support infectious disease diagnostics by enabling the detection of any pathogen from clinical specimens. mNGS was performed on blood samples to identify potential viral coinfections in human immunodeficiency virus (HIV)-positive individuals from Kinshasa, the Democratic Republic of the Congo (DRC), participating in an HIV diversity cohort study. Time-resolved phylogenetics and molecular assay development assisted in viral characterization. The nearly complete genome of a novel orthobunyavirus related to Nyangole virus, a virus previously identified in neighboring Uganda, was assembled from a hepatitis B virus-positive patient. A quantitative polymerase chain reaction assay was designed and used to screen >2,500 plasma samples from Cameroon, the DRC, and Uganda, failing to identify any additional cases. The recent sequencing of a US Center for Disease Control Arbovirus Reference Collection revealed that this same virus, now named Bangui virus, was first isolated in 1970 from an individual in the Central African Republic. Time-scaled phylogenetic analyses of Bangui with the related Anopheles and Tanga serogroup complexes indicate that this virus emerged nearly 10,000 years ago. Pervasive and episodic models further suggest that this virus is under purifying selection and that only distant common ancestors were subject to positive selection events. This study represents only the second identification of a Bangui virus infection in over 50 years. The presumed rarity of Bangui virus infections in humans can be explained by its constraint to an avian host and insect vector, precluding efficient transmission into the human population. Our results demonstrate that molecular phylogenetic analyses can provide insights into the threat posed by novel or re-emergent viruses identified by mNGS.

The practical role of the respiratory therapist in the standard apnea test procedure for declaration of death via neurologic criteria in adults.

Introduction: Respiratory therapists (RTs) in the intensive care unit can at times find themselves involved in and assisting during the performance of the apnea test (ApT). The ApT is a clinically complex procedure and is the last part of the clinical declaration of death by neurologic criteria (DNC) protocol and requires close collaboration between the physicians and the RTs. As such, the ApT should be performed with the upmost attention to detail. Context and Aims: The RTs need to be versed on the intricacies of the ApT. Except in very large medical centers, the ApT is not a procedure performed with high enough frequency as to maintain high level of proficiency. For a successful ApT, structured knowledge and preparation are paramount. This publication attempts to fill that gap, for adult hospitalized patients not on ECMO (extracorporeal membrane oxygenation). To generate this report, we make use of the published guidelines, and our personal experience on performing ApTs in large medical centers. Conclusion: We provide a structure by means of a checklist, from the RTs' perspective, to guide and help them lead on the efficient performance of the ApT.

Use of a Viral Filter to Reduce Exposure to Exhaled Aerosol Does Not Affect Methacholine Dose Delivery During Bronchoprovocation Testing.

BACKGROUND: Methacholine challenge testing (MCT) is a common bronchoprovocation technique used to assess airway hyper-responsiveness. We previously demonstrated that the addition of a viral filter to the nebulizer exhalation limb substantially reduced expelled particles during MCT. Our aim was to evaluate whether this modification affects the delivered dose of methacholine. METHODS: A mechanical ventilator was connected to a lung simulator with breathing frequency 15 breaths/min, tidal volume 500 mL, inspiratory-expiratory ratio 1:1, with a sinusoidal waveform. We compared methacholine dose delivery using the Hudson Micro Mist or AeroEclipse II BAN nebulizers powered by either a dry gas source or a compressor system. A filter placed in line between the nebulizer and test lung was weighed before and after 1 min of nebulized methacholine delivery. Mean inhaled mass was measured with and without a viral filter on the exhalation limb. Dose delivery was calculated by multiplying the mean inhaled mass by the respirable fraction (particles < 5 µm) and inhalation time. Unpaired t test was used to compare methacholine dose delivery with and without viral filter placement. RESULTS: The addition of a viral filter did not significantly affect methacholine dose delivery across all devices tested. Using a 50-psi dry gas source, dose delivered with or without a viral filter did not differ with the Hudson (422.3 µg vs 282.0 µg, P = .11) or the AeroEclipse nebulizer (563.0 µg vs 657.6 µg, P = .59). Using the compressor, dose delivered with and without a viral filter did not differ with the Hudson (974.0 µg vs 868.0 µg, P = .03) or the AeroEclipse nebulizer (818.0 µg vs 628.5 µg, P = .42). CONCLUSIONS: The addition of a viral filter to the nebulizer exhalation limb did not affect methacholine dose during bronchoprovocation testing. Routine use of a viral filter should be considered to improve pulmonary function technician safety and infection control measures during the ongoing COVID-19 pandemic.

The early SARS-CoV-2 epidemic in Senegal was driven by the local emergence of B.1.416 and the introduction of B.1.1.420 from Europe.

Molecular surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is growing in west Africa, especially in the Republic of Senegal. Here, we present a molecular epidemiology study of the early waves of SARS-CoV-2 infections in this country based on Bayesian phylogeographic approaches. Whereas the first wave in mid-2020 was characterized by a significant diversification of lineages and predominance of B.1.416, the second wave in late 2020 was composed primarily of B.1.1.420. Our results indicate that B.1.416 originated in Senegal and was exported mainly to Europe. In contrast, B.1.1.420 was introduced from Italy, gained fitness in Senegal, and then spread worldwide. Since both B.1.416 and B.1.1.420 lineages carry several positive selected mutations in the spike and nucleocapsid genes, each of which may explain their local dominance, their mutation profiles should be carefully monitored. As the pandemic continues to evolve, molecular surveillance in all regions of Africa will play a key role in stemming its spread.

The Abbott Pandemic Defense Coalition: a unique multisector approach adds to global pandemic preparedness efforts.

Detection and epidemiologic characterization of infectious disease outbreaks are key for early identification and response to potential pandemic threats. The rapid global spread of severe SARS-CoV-2 in 2020 highlighted the critical role of diagnostics in understanding the epidemiology of the virus early in the pandemic. As a natural extension of Abbott's work in diagnostics, virus discovery, and virus surveillance, the Abbott Pandemic Defense Coalition (APDC) was launched in early 2021. The APDC is a global multisector scientific and public health partnership whose primary objective is the early detection and mitigation of infectious disease threats of pandemic potential. As of January 2022, the APDC network has partners on 5 continents including academic institutions, governmental, and nongovernmental organizations. A novel element of the APDC is the capacity for early development and rapid deployment of scalable, quality diagnostics targeting newly identified pathogens of pandemic potential.

Detection of SARS-CoV-2 variants by Abbott molecular, antigen, and serological tests.

BACKGROUND: Viral diversity presents an ongoing challenge for diagnostic tests, which need to accurately detect all circulating variants. The Abbott Global Surveillance program monitors severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants and their impact on diagnostic test performance. OBJECTIVES: To evaluate the capacity of Abbott molecular, antigen, and serologic assays to detect circulating SARS-CoV-2 variants, including all current variants of concern (VOC): B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.2 (delta). STUDY DESIGN: Dilutions of variant virus cultures (B.1.1.7, B.1.351, B.1.429, B.1.526.1, B.1.526.2, B.1.617.1, B.1.617.2, P.1, R.1 and control isolate WA1) and a panel of N = 248 clinical samples from patients with sequence confirmed variant infections (B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, B.1.526.1, B.1.526.2, P.1, P.2, R.1) were evaluated on at least one assay: Abbott ID NOW COVID-19, m2000 RealTime SARS-CoV-2, Alinity m SARS-CoV-2, and Alinity m Resp-4-Plex molecular assays; the BinaxNOW COVID-19 Ag Card and Panbio COVID-19 Ag Rapid Test Device; and the ARCHITECT/Alinity i SARS-CoV-2 IgG and AdviseDx IgM assays, Panbio COVID-19 IgG assay, and ARCHITECT/Alinity i AdviseDx SARS-CoV-2 IgG II assay. RESULTS: Consistent with in silico predictions, each molecular and antigen assay detected VOC virus cultures with equivalent sensitivity to the WA1 control strain. Notably, 100% of all tested variant patient specimens were detected by molecular assays (N = 197 m2000, N = 88 Alinity m, N = 99 ID NOW), and lateral flow assays had a sensitivity of >94% for specimens with genome equivalents (GE) per device above 4 log (85/88, Panbio; 54/57 Binax). Furthermore, Abbott antibody assays detected IgG and IgM in 94-100% of sera from immune competent B.1.1.7 patients 15-26 days after symptom onset. CONCLUSIONS: These data confirm variant detection for 11 SARS-CoV-2 assays, which is consistent with each assay target region being highly conserved. Importantly, alpha, beta, gamma, and delta VOCs were detected by molecular and antigen assays, indicating that these tests may be suitable for widescale use where VOCs predominate.

Emergence of novel combinations of SARS-CoV-2 spike receptor binding domain variants in Senegal.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that carry mutations in the spike gene are of concern for potential impact to treatment and prevention efforts. To monitor for new SARS-CoV-2 mutations, a panel of specimens were sequenced from both wave one (N = 96), and wave two (N = 117) of the pandemic in Senegal by whole genome next generation sequencing. Amongst these genomes, new combinations of SARS-CoV-2 spike mutations were identified, with E484K + N501T, L452R + N501Y, and L452M + S477N exclusively found in second wave specimens. These sequences are evidence of local diversification over the course of the pandemic and parallel evolution of escape mutations in different lineages.

Emergence of a Distinct Picobirnavirus Genotype Circulating in Patients Hospitalized with Acute Respiratory Illness.

Picobirnaviruses (PBV) are found in a wide range of hosts and typically associated with gastrointestinal infections in immunocompromised individuals. Here, a divergent PBV genome was assembled from a patient hospitalized for acute respiratory illness (ARI) in Colombia. The RdRp protein branched with sequences previously reported in patients with ARI from Cambodia and China. Sputa from hospitalized individuals (n = 130) were screened by RT-qPCR which enabled detection and subsequent metagenomic characterization of 25 additional PBV infections circulating in Colombia and the US. Phylogenetic analysis of RdRp highlighted the emergence of two dominant lineages linked to the index case and Asian strains, which together clustered as a distinct genotype. Bayesian inference further established capsid and RdRp sequences as both significantly associated with ARI. Various respiratory-tropic pathogens were detected in PBV+ patients, yet no specific bacteria was common among them and four individuals lacked co-infections, suggesting PBV may not be a prokaryotic virus nor exclusively opportunistic, respectively. Competing models for the origin and transmission of this PBV genotype are presented that attempt to reconcile vectoring by a bacterial host with human pathogenicity. A high prevalence in patients with ARI, an ability to reassort, and demonstrated global spread indicate PBV warrant greater public health concern.

Etiology, incidence, and outcomes of patient-ventilator asynchrony in critically-ill patients undergoing invasive mechanical ventilation.

Patient-ventilator asynchrony (PVA) is commonly encountered during mechanical ventilation of critically ill patients. Estimates of PVA incidence vary widely. Type, risk factors, and consequences of PVA remain unclear. We aimed to measure the incidence and identify types of PVA, characterize risk factors for development, and explore the relationship between PVA and outcome among critically ill, mechanically ventilated adult patients admitted to medical, surgical, and medical-surgical intensive care units in a large academic institution staffed with varying provider training background. A single center, retrospective cohort study of all adult critically ill patients undergoing invasive mechanical ventilation for ≥ 12 h. A total of 676 patients who underwent 696 episodes of mechanical ventilation were included. Overall PVA occurred in 170 (24%) episodes. Double triggering 92(13%) was most common, followed by flow starvation 73(10%). A history of smoking, and pneumonia, sepsis, or ARDS were risk factors for overall PVA and double triggering (all P < 0.05). Compared with volume targeted ventilation, pressure targeted ventilation decreased the occurrence of events (all P < 0.01). During volume controlled synchronized intermittent mandatory ventilation and pressure targeted ventilation, ventilator settings were associated with the incidence of overall PVA. The number of overall PVA, as well as double triggering and flow starvation specifically, were associated with worse outcomes and fewer hospital-free days (all P < 0.01). Double triggering and flow starvation are the most common PVA among critically ill, mechanically ventilated patients. Overall incidence as well as double triggering and flow starvation PVA specifically, portend worse outcome.

The Impact of a Standardized Refractory Hypoxemia Protocol on Outcome of Subjects Receiving Venovenous Extracorporeal Membrane Oxygenation.

BACKGROUND: Current mechanical ventilation practice and the use of treatment adjuncts in patients requiring extracorporeal membrane oxygenation (ECMO) for refractory hypoxemia (RH) vary widely and their impact on outcomes remains unclear. In 2015, we implemented a standardized approach to protocolized ventilator settings and guide the escalation of adjunct therapies in patients with RH. This study aimed to investigate ICU mortality, its associated risk factors, and mechanical ventilation practice before and after the implementation of a standardized RH guideline in patients requiring venovenous ECMO (VV-ECMO). METHODS: This was a single-center, retrospective cohort study of patients undergoing VV-ECMO due to RH respiratory failure between January 2008 and March 2015 (before RH protocol implementation) and between April 2015 and October 2019 (after RH protocol implementation). RESULTS: A total of 103 subjects receiving VV-ECMO for RH were analyzed. After implementation of the RH protocol, more subjects received prone positioning (6.7% vs 23.3%, P = .02), and fewer received high-frequency oscillatory ventilation than before launching the RH protocol (0% vs 13.3%, P = .01). Plateau pressure was also lower before initiation of ECMO (P = .04) and at day 1 during ECMO (P = .045). Driving pressure was consistently lower at days 1, 2, and 3 after ECMO initiation: median 13.0 (interquartile range [IQR] 10.6-18.0) vs 16.0 (IQR 14.0-20.0) cm H2O at day 1 (P = .003); 13.0 (IQR 11.0-15.9) vs 15.5 (IQR 12.0-20.0) cm H2O at day 2 (P = .03); and 12.0 (IQR 10.0-14.5) vs 15.0 (IQR 12.0-19.0) cm H2O at day 3 (P = .005). CONCLUSIONS: The implementation of a standardized RH guideline improved compliance with a lung-protective ventilation strategy and utilization of the prone position and was associated with lower driving pressure during the first 3 days after ECMO initiation in subjects with refractory hypoxemia.

Implementation of a Goal-Directed Mechanical Ventilation Order Set Driven by Respiratory Therapists Improves Compliance With Best Practices for Mechanical Ventilation.

PURPOSE: Data regarding best practices for ventilator management strategies that improve outcomes in acute respiratory distress syndrome (ARDS) are readily available. However, little is known regarding processes to ensure compliance with these strategies. We developed a goal-directed mechanical ventilation order set that included physician-specified lung-protective ventilation and oxygenation goals to be implemented by respiratory therapists (RTs). We sought as a primary outcome to determine whether an RT-driven order set with predefined oxygenation and ventilation goals could be implemented and associated with improved adherence with best practice. METHODS: We evaluated 1302 patients undergoing invasive mechanical ventilation (1693 separate episodes of invasive mechanical ventilation) prior to and after institution of a standardized, goal-directed mechanical ventilation order set using a controlled before-and-after study design. Patient-specific goals for oxygenation partial pressure of oxygen in arterial blood (Pao 2), ARDS Network [Net] positive end-expiratory pressure [PEEP]/fraction of inspired oxygen [Fio 2] table use) and ventilation (pH, partial pressure of carbon dioxide) were selected by prescribers and implemented by RTs. RESULTS: Compliance with the new mechanical ventilation order set was high: 88.2% compliance versus 3.8% before implementation of the order set ( P < .001). Adherence to the PEEP/Fio 2 table after implementation of the order set was significantly greater (86.0% after vs 82.9% before, P = .02). There was no difference in duration of mechanical ventilation, intensive care unit (ICU) length of stay, and in-hospital or ICU mortality. CONCLUSIONS: A standardized best practice mechanical ventilation order set can be implemented by a multidisciplinary team and is associated with improved compliance to written orders and adherence to the ARDSNet PEEP/Fio 2 table.

The effect of temperature exposure during shipment on a commercially available demineralized bone matrix putty.

During August and September of 2013, temperature data loggers were shipped to and from an AATB accredited and FDA registered allograft tissue processing facility in Belgrade, MT (Bacterin International, Inc.) to five warm climate cities (Dallas, TX, El Paso, TX, New Orleans, LA, Phoenix, AZ, and Tampa, FL). Shipping data acquired from 72 independent shipments were analyzed to generate an assessment of temperature exposure, shipment times, and shipping event durations experienced during routine distribution. Overall the packages experienced an average temperature of 26.2 ± 2.3 °C which mirrored the average external ambient temperature of 25.8 ± 3.0 °C. However, temperature spikes above 40 °C were frequently observed. The data from the model shipments were extrapolated to provide a worst-case high temperature spike of 52.9 °C for 12 h and 14 min. Multiple lots of a commercially available demineralized bone matrix (DBM) putty (OsteoSelect® DBM Putty) were subjected to continuous heating at 50 °C, to multiple worst-case temperature spikes, and to multiple freeze-thaw cycles to assess the effects of these temperature extremes on the handling and osteoinductivity of the allograft tissue. Five weeks of continuous exposure to 50 °C and 12 simulated worst-case one-way shipments did not adversely affect the handling characteristics or the in vivo osteoinductivity of the product.

Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes.

Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.

Functional characterization of a VEGF-A-targeting Anticalin, prototype of a novel therapeutic human protein class.

Human tear lipocalin (Tlc) was utilized as a protein scaffold to engineer an Anticalin that specifically binds and functionally blocks vascular endothelial growth factor A (VEGF-A), a pivotal inducer of physiological angiogenesis that also plays a crucial role in several neovascular diseases. Starting from a naive combinatorial library where residues that form the natural ligand-binding site of Tlc were randomized, followed by affinity maturation, the final Anticalin PRS-050 was selected to bind all major splice forms of VEGF-A with picomolar affinity. Moreover, this Anticalin cross-reacts with the murine ortholog. PRS-050 efficiently antagonizes the interaction between VEGF-A and its cellular receptors, and it inhibits VEGF-induced mitogenic signaling as well as proliferation of primary human endothelial cells with subnanomolar IC50 values. Intravitreal administration of the Anticalin suppressed VEGF-induced blood-retinal barrier breakdown in a rabbit model. To allow lasting systemic neutralization of VEGF-A in vivo, the plasma half-life of the Anticalin was extended by site-directed PEGylation. The modified Anticalin efficiently blocked VEGF-mediated vascular permeability as well as growth of tumor xenografts in nude mice, concomitantly with reduction in microvessel density. In contrast to bevacizumab, the Anticalin did not trigger platelet aggregation and thrombosis in human FcγRIIa transgenic mice, thus suggesting an improved safety profile. Since neutralization of VEGF-A activity is well known to exert beneficial effects in cancer and other neovascular diseases, including wet age-related macular degeneration, this Anticalin offers a novel potent small protein antagonist for differentiated therapeutic intervention in oncology and ophthalmology.

CD32a antibodies induce thrombocytopenia and type II hypersensitivity reactions in FCGR2A mice.

The CD32a immunoglobulin G (IgG) receptor (Fcγ receptor IIa) is a potential therapeutic target for diseases in which IgG immune complexes (ICs) mediate inflammation, such as heparin-induced thrombocytopenia, rheumatoid arthritis, and systemic lupus erythematosus. Monoclonal antibodies (mAbs) are a promising strategy for treating such diseases. However, IV.3, perhaps the best characterized CD32a-blocking mAb, was recently shown to induce anaphylaxis in immunocompromised "3KO" mice. This anaphylactic reaction required a human CD32a transgene because mice lack an equivalent of this gene. The finding that IV.3 induces anaphylaxis in CD32a-transgenic mice was surprising because IV.3 had long been thought to lack the intrinsic capacity to trigger cellular activation via CD32a. Such an anaphylactic reaction would also limit potential therapeutic applications of IV.3. In the present study, we examine the molecular mechanisms by which IV.3 induces anaphylaxis. We now report that IV.3 induces anaphylaxis in immunocompetent CD32a-transgenic "FCGR2A" mice, along with the novel finding that IV.3 and 2 other well-characterized CD32a-blocking mAbs, AT-10 and MDE-8, also induce severe thrombocytopenia in FCGR2A mice. Using recombinant variants of these same mAbs, we show that IgG "Fc" effector function is necessary for the induction of anaphylaxis and thrombocytopenia in FCGR2A mice. Variants of these mAbs lacking the capacity to activate mouse IgG receptors not only failed to induce anaphylaxis or thrombocytopenia, but also very potently protected FCGR2A mice from near lethal doses of IgG ICs. Our findings show that effector-deficient IV.3, AT-10, and MDE-8 are promising candidates for developing therapeutic mAbs to treat CD32a-mediated diseases.

CDP7657, an anti-CD40L antibody lacking an Fc domain, inhibits CD40L-dependent immune responses without thrombotic complications: an in vivo study.

INTRODUCTION: CD40 ligand (CD40L) blockade has demonstrated efficacy in experimental autoimmune models. However, clinical trials of hu5c8, an anti-human CD40L IgG1 antibody, in systemic lupus erythematosus (SLE) were halted due to an increased incidence of thrombotic events. This study evaluated CDP7657, a high affinity PEGylated monovalent Fab' anti-CD40L antibody fragment, to assess whether an Fc-deficient molecule retains efficacy while avoiding the increased risk of thrombotic events observed with hu5c8. METHODS: The potency and cross-reactivity of CDP7657 was assessed in in vitro assays employing human and non-human primate leukocytes, and the capacity of different antibody formats to activate platelets in vitro was assessed using aggregometry and dense granule release assays. Given the important role CD40L plays in regulating humoral immunity, in vivo efficacy was assessed by investigating the capacity of Cynomolgus monkeys to generate immune responses to the tetanus toxoid antigen while the potential to induce thrombotic events in vivo was evaluated after repeat dosing of antibodies to Rhesus monkeys. A PEGylated anti-mouse CD40L was generated to assess efficacy in the New Zealand Black/White (NZB/W) mouse model of SLE. RESULTS: CDP7657 dose-dependently inhibited antigen-specific immune responses to tetanus toxoid in Cynomolgus monkeys, and in contrast to hu5c8, there was no evidence of pulmonary thrombovasculopathy in Rhesus monkeys. Aglycosyl hu5c8, which lacks Fc receptor binding function, also failed to induce thrombotic events in Rhesus monkeys. In vitro experiments confirmed that antibody constructs lacking an Fc, including CDP7657, did not induce human or monkey platelet activation. A PEGylated monovalent Fab' anti-mouse CD40L antibody also inhibited disease activity in the NZB/W mouse model of SLE after administration using a therapeutic dosing regimen where mice received antibodies only after they had displayed severe proteinuria. CONCLUSIONS: These findings demonstrate for the first time that anti-CD40L antibodies lacking a functional Fc region do not induce thrombotic events in Rhesus monkeys and fail to activate platelets in vitro but, nevertheless retain pharmacological activity and support the investigation of CDP7657 as a potential therapy for systemic lupus erythematosus and other autoimmune diseases.

Ultrasonic delivery of silica-gold nanoshells for photothermolysis of sebaceous glands in humans: Nanotechnology from the bench to clinic.

Recent advances in nanotechnology have provided numerous opportunities to transform medical therapies for the treatment of diseases including cancer, atherosclerosis, and thrombosis. Here, we report, through in vitro studies and in vivo human pilot clinical studies, the use of inert, inorganic silica-gold nanoshells for the treatment of a widely prevalent and researched, yet poorly treated disease of acne. We use ~150nm silica-gold nanoshells, tuned to absorb near-IR light and near-IR laser irradiation to thermally disrupt overactive sebaceous glands in the skin which define the etiology of acne-related problems. Low-frequency ultrasound was used to facilitate deep glandular penetration of the nanoshells. Upon delivery of the nanoshells into the follicles and glands, followed by wiping of superficial nanoshells from skin surface and exposure of skin to near-infrared laser, nanoshells localized in the follicles absorb light, get heated, and induce focal thermolysis of sebaceous glands. Pilot human clinical studies confirmed the efficacy of ultrasonically-delivered silica-gold nanoshells in inducing photothermal disruption of sebaceous glands without damaging collateral skin.

Acne Treatment Based on Selective Photothermolysis of Sebaceous Follicles with Topically Delivered Light-Absorbing Gold Microparticles.

The pathophysiology of acne vulgaris depends on active sebaceous glands, implying that selective destruction of sebaceous glands could be an effective treatment. We hypothesized that light-absorbing microparticles could be delivered into sebaceous glands, enabling local injury by optical pulses. A suspension of topically applied gold-coated silica microparticles exhibiting plasmon resonance with strong absorption at 800 nm was delivered into human pre-auricular and swine sebaceous glands in vivo, using mechanical vibration. After exposure to 10-50 J cm(-2), 30 milliseconds, 800 nm diode laser pulses, microscopy revealed preferential thermal injury to sebaceous follicles and glands, consistent with predictions from a computational model. Inflammation was mild; gold particles were not retained in swine skin 1 month after treatment, and uptake in other organs was negligible. Two independent prospective randomized controlled clinical trials were performed for treatment of moderate-to-severe facial acne, using unblinded and blinded assessments of disease severity. Each trial showed clinically and statistically significant improvement of inflammatory acne following three treatments given 1-2 weeks apart. In Trial 2, inflammatory lesions were significantly reduced at 12 weeks (P=0.015) and 16 weeks (P=0.04) compared with sham treatments. Optical microparticles enable selective photothermolysis of sebaceous glands. This appears to be a well-tolerated, effective treatment for acne vulgaris.