A non-volitional skeletal muscle endurance test measures functional changes associated with impaired blood flow.

Introduction: An electrically stimulated intermittent fatigue test using mechanomyography was recently proposed as a possible tool for detecting clinically relevant changes in muscle function. This study was designed to determine whether the proposed test can detect additional fatigue when it should be present. Methods: Subjects (n = 10) underwent two trials each (occluded and normal blood flow) with a standardized fatigue protocol on the Ankle Dorsiflexors (AD) and Wrist Extensors (WE) using a clinical electrical stimulator. Results: Mean normalized twitch acceleration was strongly predictive of mean normalized torque (R 2 = 0.828). The WE experienced lower twitch magnitudes throughout the tourniquet trial (10.81 ± 1.25 m/s2) compared to normal blood flow (18.05 ± 1.06 m/s2). The AD twitches were overall reduced in the tourniquet trial (3.87 ± 0.48 m/s2) compared with the control trial (8.57 ± 0.91 m/s2). Conclusion: Occluding blood flow to a muscle should cause greater muscle fatigue. The ability to detect reduced contraction magnitudes during an electrically stimulated fatigue protocol resulting from low blood flow suggests the proposed test may be capable of detecting clinically relevant muscle deficits.

Development of a Kinetic ELISA and Reactive B Cell Frequency Assay to Detect Respiratory Syncytial Virus Pre-Fusion F Protein-Specific Immune Responses in Infants.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of respiratory disease in infants, making vaccination an attractive preventive strategy. Due to earlier reports of vaccine-enhanced disease in RSV-naive children, assessing prior RSV infection is critical for determining eligibility for future infant vaccine trials. However, this is complicated by the presence of maternally transferred maternal antibodies. We sought to develop assays that measure immune responses to RSV pre-fusion (F) protein that discriminates between maternal and infant responses. METHODS: We measured RSV-specific responses in two groups of children <3 years of age; those with laboratory-confirmed RSV (RSV-infected) and those enrolled prior to their first RSV season (RSV-uninfected). Serial blood samples were obtained and recent infections with RSV and other respiratory viruses were assessed during follow-up. An RSV pre-F-specific kinetic enzyme-linked immunosorbent assay (kELISA) and an F-specific reactive B cell frequency (RBF) assay were developed. RESULTS: One hundred two young children were enrolled between July 2015 and April 2017; 74 were in the RSV-uninfected group and 28 were in the RSV-infected group. Participants were asked to provide sequential blood samples over time, but only 53 participants in the RSV-uninfected group and 22 participants in the RSV-infected groups provided multiple samples. In the RSV-infected group, most had positive kELISA and RBF during the study. In the RSV-uninfected group, two patterns emerged: declining kELISA values without reactive B cells, due to maternal transplacental antibody transfer, and persistently positive kELISA with reactive B cells, due to asymptomatic undiagnosed RSV infection. CONCLUSIONS: A kELISA targeting RSV pre-F epitopes and an RBF assay targeting RSV F-specific B cells generally allow discrimination between maternally and infant-derived antibodies.

Potently neutralizing human mAbs against the zoonotic pararubulavirus Sosuga virus.

Sosuga virus (SOSV) is a recently discovered paramyxovirus with a single known human case of disease. There has been little laboratory research on SOSV pathogenesis or immunity, and no approved therapeutics or vaccines are available. Here, we report the discovery of human mAbs from the circulating memory B cells of the only known human case and survivor of SOSV infection. We isolated 6 mAbs recognizing the functional attachment protein hemagglutinin-neuraminidase (HN) and 18 mAbs against the fusion (F) protein. The anti-HN mAbs all targeted the globular head of the HN protein and could be organized into 4 competition-binding groups that exhibited epitope diversity. The anti-F mAbs can be divided into pre- or postfusion conformation-specific categories and further into 8 competition-binding groups. The only Ab in the panel that did not display neutralization activity was the single postfusion-specific anti-F mAb. Most of the anti-HN mAbs were more potently neutralizing than the anti-F mAbs, with mAbs in 1 of the HN competition-binding groups possessing ultrapotent (<1 ng/mL) half-maximal inhibitory virus neutralization values. These findings provide insight into the molecular basis for human Ab recognition of paramyxovirus surface proteins and the mechanisms of SOSV neutralization.

Epitope-focused immunogen design based on the ebolavirus glycoprotein HR2-MPER region.

The three human pathogenic ebolaviruses: Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) virus, cause severe disease with high fatality rates. Epitopes of ebolavirus glycoprotein (GP) recognized by antibodies with binding breadth for all three ebolaviruses are of major interest for rational vaccine design. In particular, the heptad repeat 2 -membrane-proximal external region (HR2-MPER) epitope is relatively conserved between EBOV, BDBV, and SUDV GP and targeted by human broadly-neutralizing antibodies. To study whether this epitope can serve as an immunogen for the elicitation of broadly-reactive antibody responses, protein design in Rosetta was employed to transplant the HR2-MPER epitope identified from a co-crystal structure with the known broadly-reactive monoclonal antibody (mAb) BDBV223 onto smaller scaffold proteins. From computational analysis, selected immunogen designs were produced as recombinant proteins and functionally validated, leading to the identification of a sterile alpha motif (SAM) domain displaying the BDBV-HR2-MPER epitope near its C terminus as a promising candidate. The immunogen was fused to one component of a self-assembling, two-component nanoparticle and tested for immunogenicity in rabbits. Robust titers of cross-reactive serum antibodies to BDBV and EBOV GPs and moderate titers to SUDV GP were induced following immunization. To confirm the structural composition of the immunogens, solution NMR studies were conducted and revealed structural flexibility in the C-terminal residues of the epitope. Overall, our study represents the first report on an epitope-focused immunogen design based on the structurally challenging BDBV-HR2-MPER epitope.

An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer.

The protective human antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) focuses on the spike (S) protein, which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope ("supersite") on the N-terminal domain (NTD). Using the single B cell technology called linking B cell receptor to antigen specificity through sequencing (LIBRA-Seq), we isolated a large panel of NTD-reactive and SARS-CoV-2-neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies against the NTD supersite were commonly encoded by the IGHV1-24 gene, forming a genetic cluster representing a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface (TI) and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited the cell-to-cell spread of the virus in culture, and conferred protection in human angiotensin-converting enzyme 2-transgenic (ACE2-transgenic) mice against the SARS-CoV-2 challenge. This study provides insight into antibody targeting of the S protein TI region, suggesting this region may be a site of virus vulnerability.

Genetic and structural basis for SARS-CoV-2 variant neutralization by a two-antibody cocktail.

Understanding the molecular basis for immune recognition of SARS-CoV-2 spike glycoprotein antigenic sites will inform the development of improved therapeutics. We determined the structures of two human monoclonal antibodies-AZD8895 and AZD1061-which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor-binding domain (RBD) of SARS-CoV-2 to define the genetic and structural basis of neutralization. AZD8895 forms an 'aromatic cage' at the heavy/light chain interface using germ line-encoded residues in complementarity-determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals. AZD1061 has an unusually long LCDR1; the HCDR3 makes interactions with the opposite face of the RBD from that of AZD8895. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the crucial binding residues of both antibodies and identified positions of concern with regards to virus escape from antibody-mediated neutralization. Both AZD8895 and AZD1061 have strong neutralizing activity against SARS-CoV-2 and variants of concern with antigenic substitutions in the RBD. We conclude that germ line-encoded antibody features enable recognition of the SARS-CoV-2 spike RBD and demonstrate the utility of the cocktail AZD7442 in neutralizing emerging variant viruses.

Cooperativity mediated by rationally selected combinations of human monoclonal antibodies targeting the henipavirus receptor binding protein.

Hendra virus and Nipah virus (NiV), members of the Henipavirus (HNV) genus, are zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans. We isolated a panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior exposure to equine Hendra virus (HeV) vaccine, targeting distinct antigenic sites. The most potent class of cross-reactive antibodies achieves neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3, with a second class being enhanced by receptor binding. mAbs from both classes display synergistic activity in vitro. In a stringent hamster model of NiV Bangladesh (NiVB) infection, antibodies from both classes reduce morbidity and mortality and achieve synergistic protection in combination. These candidate mAbs might be suitable for use in a cocktail therapeutic approach to achieve synergistic potency and reduce the risk of virus escape.

Therapeutic alphavirus cross-reactive E1 human antibodies inhibit viral egress.

Alphaviruses cause severe arthritogenic or encephalitic disease. The E1 structural glycoprotein is highly conserved in these viruses and mediates viral fusion with host cells. However, the role of antibody responses to the E1 protein in immunity is poorly understood. We isolated E1-specific human monoclonal antibodies (mAbs) with diverse patterns of recognition for alphaviruses (ranging from Eastern equine encephalitis virus [EEEV]-specific to alphavirus cross-reactive) from survivors of natural EEEV infection. Antibody binding patterns and epitope mapping experiments identified differences in E1 reactivity based on exposure of epitopes on the glycoprotein through pH-dependent mechanisms or presentation on the cell surface prior to virus egress. Therapeutic efficacy in vivo of these mAbs corresponded with potency of virus egress inhibition in vitro and did not require Fc-mediated effector functions for treatment against subcutaneous EEEV challenge. These studies reveal the molecular basis for broad and protective antibody responses to alphavirus E1 proteins.

Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail.

The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-21301, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an 'aromatic cage' at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals1-4. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.

Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection.

Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.

Investigation of Drowning Deaths: A Practical Review.

Drowning, which typically involves a watery environment, remains a serious public health concern claiming an estimated 362 000 lives per year worldwide across all socioeconomic classifications and has remained under close observation by the World Health Organization and its signatories. A significant number of water-related deaths are attributed to accidental drowning, while a smaller but still significant number represent suicidal or homicidal drowning. Others involve a combination of drowning precipitated by injury, intoxication, or environmental extremes. Still others involve victims that die from injury, intoxication, or a natural disease entity of such significance as to preclude the drowning process, while near or in water. While there may be an initial presumption that all water-related deaths are accidental drownings, other possibilities must be considered in the investigation of these types of deaths, as drowning as a cause of death is a diagnosis based on the exclusion of other potential causes. The coordinated investigative efforts of multiple agencies and disciplines are required not only for the designation as drowning as the cause of death but also for death certification. The ongoing analysis and dissemination of data generated from all levels of investigation augment our understanding of the impact on public health and safety, guiding allocation of monetary and educational resources in an effort to prevent further mortality and disability.

Hiding in Plain Sight: Clinically Unrecognized Fatal Tooth Asphyxia Revealed by the Forensic Autopsy.

Fatal asphyxia by choking whether by food or foreign material remains an uncommon occurrence affecting mainly those at the extremes of age and with variable and sometimes misleading clinical presentations. Prompt clinical recognition of impending airway obstruction afforded by complete physical examination and assessment is paramount for prevention of morbidity and mortality in these cases. In the elderly, a death initially presenting with sudden cardiorespiratory collapse may be erroneously certified as due to natural disease without performance of an autopsy. Fortunately, deaths subsequent to cardiorespiratory collapse, where results of the clinical work-up fail to identify an etiology and medical history is insufficient, are reportable, falling under the jurisdiction of the medical examiner/coroner. The performance of an autopsy in the evaluation of a sudden death arising after hospitalization in which the etiology remains unclear can provide valuable information to our clinical colleagues that they can apply to more timely diagnosis and treatment. Furthermore, the forensic autopsy offers clarification and answers to questions of medicolegal importance. This is particularly true for choking deaths. Presented is a choking death after tooth aspiration whereby the forensic autopsy provided specific anatomic correlation to the clinical clues not recognized before death and provided the true cause of death.

Allergy in extremis: a case of sudden unexpected death due to an allergy-associated disease.

Allergy-associated diseases have a multitude of confirmed or suspected etiologies and associations affecting organs and organ systems. The hyper-reactivity of the immune system in which the eosinophils are prominent plays a central role in organ-specific and systemic effects in these diseases. Patients may be plagued with nonspecific, episodic, or progressive signs and symptoms. Patients may also exhibit signs and symptoms that mimic more common conditions. Recognition of certain patterns of clinical signs and symptoms may lead to definitive clinical diagnosis and effective treatment, particularly of less common but potentially lethal disease entities. The lack of clinical recognition of these patterns, whether due to "red herring" historical information, lack of sign/symptom specificity, clinical presentation at early stage, or unfamiliarity with the constellation of disease presentation, may delay or preclude diagnosis and treatment. The forensic pathologist must also have an awareness of varied clinical presentations of diseases because these can provide us with direction toward the ultimate determination of cause and manner of death.The importance and contribution of the practice of forensic pathology are highlighted in the instance of sudden and unexpected death, whereby the previously unrecognized or unconfirmed disease entity that leads to the death is revealed via analysis of scene information, autopsy performance, and review of medical history and history of the most recent illness. Through this comprehensive approach, a greater understanding of the extent and potential lethality of disease with reiteration of the importance of early clinical diagnosis and treatment is reinforced. This benefits the medical community involved in diagnosis and treatment, clinicians and pathologists alike, with the ultimate goal of reduction of morbidity and mortality.Reported is a case of sudden unexpected death with historical, gross, and microscopic findings consistent with a multisystem, inflammatory, allergic disease entity.

Warzone stressor exposure, unit support, and emotional distress among U.S. Air force pararescuemen.

OBJECTIVES: Combat exposure is associated with increased mental health symptom severity among military personnel, whereas unit support is associated with decreased severity. However, to date no studies have examined these relationships among U.S. Air Force pararescuemen (PJs), who have a unique and specialized career field that serves in both medical and combatant capacities. DESIGN: Cross-sectional self-report survey. METHODS: Self-reported survey data regarding depression symptoms, posttraumatic stress disorder (PTSD) symptoms, perceived unit support, and exposure to traditional combat experiences (e.g., firefights) and medical consequences of combat (e.g., injuries and human remains) were collected from 194 PJs in seven rescue squadrons. RESULTS: Levels of combat exposure were compared with previously published findings from combat units, and levels of medical exposure were compared with previously published findings among military medical professionals. Medical exposure intensity showed a stronger relationship with PTSD severity (?=.365, p=.018) than with combat exposure intensity (?=.136, p=.373), but neither combat nor medical exposure was associated with depression severity (?s<.296, ps>.164). Unit support was associated with less severe PTSD (?=?.402, p<.001) and depression (?=?.259, p=.062) symptoms and did not moderate the effects of combat or medical exposure. CONCLUSIONS: Medical stressors contribute more to PTSD among PJs than do traditional combat stressors. Unit support is associated with reduced PTSD and depression severity regardless of intensity of warzone exposure among PJs.

Postmortem tissue distribution of MDPV following lethal intoxication by "bath salts".

3,4-Methylenedioxypyrovalerone (MDPV) is a psychoactive, synthetic analog of the central nervous system stimulant cathinone. Its recent popularity as a recreational drug in the United States has led to numerous reports to poison control centers across the country. As with other synthetic cathinones, the recreational use of MDPV has resulted in death. MDPV is thought to exert its pharmacologic effects by inhibiting the reuptake of dopamine and norepinephrine. This report describes the case of an exposure of a 39-year-old male to MDPV, which resulted in his death. Postmortem concentrations of MDPV in various tissues were measured. The detection of MDPV in tissues and fluids was accomplished using gas chromatography-mass spectrometry analysis after solid-phase extraction. Blood analysis also demonstrated therapeutic levels of lamotrigine, fluoxetine, risperidone, benztropine, pseudoephedrine and ibuprofen. The detection of cathinones in hair was conducted using high-performance liquid chromatography-tandem mass spectrometry after solid-phase extraction. MDPV was uniformly distributed among multiple tissues (blood, brain, muscle, cerebrospinal fluid and lung) at concentrations of approximately 0.4 to 0.6 µg/mL. Tissue and fluids responsible for detoxification/excretion had higher concentrations of MDPV (kidney, liver and bile > 0.8 µg/mL). A blood concentration ≥ 0.4 µg/mL was judged sufficient to cause death. The cause of death was ruled MDPV intoxication resulting in cardiac arrhythmia.

Multivisceral sarcoidosis: an unexpected finding in a water-related death.

Upon encountering a body submerged within or in close association with a watery environment, the temptation by the first responder may be to surmise that the death is probably an accidental drowning of some sort. The challenge, however, is to quickly move beyond such temptation, maintaining an open mind to other possibilities. Unearthing the circumstances surrounding a water-related death requires the collaborative efforts of groups of trained professionals including law enforcement officers, medicolegal death scene investigators, forensic scientists, and forensic pathologists. The forensic pathologist has the ultimate responsibility for the interpretation of all results arising from comprehensive autopsy and toxicological and other ancillary examinations within the context of all available investigative information, for the most accurate determination of cause and manner of death.A water-related death is presented in which investigation into the circumstances surrounding the death and ultimately comprehensive postmortem analysis lead to the discovery of multiorgan sarcoidosis and lack of supportive evidence of drowning. This in turn facilitated the proper classification of the manner of death as natural.

Homicidal ethylene glycol intoxication: a report of a case.

We report a case of a 75-year-old hypertensive, diabetic man who presented to the emergency room with symptoms and signs of nausea, acute intoxication, significant alteration in mental status with rapid neurologic deterioration, and blunt impact injuries sustained during a recent altercation with a 36-year-old female companion-caretaker. He denied a history of ethanol abuse or other recent toxic ingestion and had not been diagnosed with or treated for depression. Hospital laboratory tests revealed a metabolic acidosis and a negative urine toxicology screen. He was diagnosed with toxic encephalopathy with metabolic acidosis secondary to metformin. Despite treatments including hemodialysis, he expired after approximately 28 hours of hospitalization. A postmortem anatomic examination revealed recent blunt-impact injuries and cardiac and renal pathology. A subsequent histologic examination revealed the presence of calcium oxalate crystals in the kidneys and brain, in addition to cardiac and renal pathology. Comprehensive forensic toxicologic testing was performed on antemortem and postmortem samples and revealed lethal levels of ethylene glycol. The cause of death was as a result of acute intoxication by ethylene glycol with another condition of multiple blunt impacts to the head, trunk, and extremities. The manner of death was ruled as homicide. A trial by jury, involving the female companion-caretaker, resulted in her conviction, and she was sentenced to 23 years to life in prison. In this report, we present an unusual case of homicidal ethylene glycol intoxication in which legal proceedings have occurred.

Distinctive patterned injuries caused by an expandable baton.

Identification and documentation of patterned blunt-force injuries at autopsy is of utmost forensic importance, particularly when the object or surface producing the injury is unknown or uncertain. Documentation of patterned injuries produced by known objects contributes to the catalogue of forensic knowledge regarding those objects and the injuries they cause. This report presents a case in which a 27-year-old male sustained multiple nonlethal patterned blunt-force injuries produced by an expandable baton and subsequent multiple gunshot wounds during apprehension by police.

An unusual fatality in a child due to oxycodone.

An unusual fatality secondary to oxycodone in a child is reported. A 2-year-old female child was conveyed to a local hospital after exhibiting signs of rubbing of the mouth and staggering. A hospital toxicological immunoassay screen for drugs of abuse and tricyclic antidepressants was performed on a urine sample and reported as negative. She was discharged and found unresponsive the next morning. She was conveyed to a second hospital in full cardiopulmonary arrest and despite resuscitative efforts, was pronounced dead upon arrival. An autopsy was performed and postmortem specimens were submitted and screened for drugs using mainly chromatographic techniques. Quantitation was achieved by gas chromatography with nitrogen phosphorus detection. Confirmation was performed by gas chromatography/mass spectrometry. Oxycodone was the only drug detected in the following concentrations: heart blood, 1.36 mg/L; gastric contents, 7.33 mg in 33 mL (222.34 mg/L); liver, 0.2 mg/kg; and urine, 47.23 mg/L (47,230 ng/mL). In addition, immunoassay testing of the urine was positive for the opiate class of drugs. This case report demonstrates an unusual cause of death in a young child with emphasis on potential limitation in hospital urine screening tests and the importance of complete forensic toxicological testing in all child deaths.