Hydrogen isotopes in serial hair samples record season of death in a mummified child from 19th century San Francisco, CA.

BACKGROUND: The mummified body of a small child was found in a sealed Barstow cast iron casket during construction activity in San Francisco in 2016. Using historical records and ancient DNA the child was determined to be Edith H. Cook. She was born 28 November, 1873 in the city of San Francisco, and died of "marasmus" on 13 October, 1876 also in San Francisco. AIMS: Currently, there are few techniques for estimating human season of death in archaeological cases. Hydrogen isotope ratios (δ2H) in hair keratin is known to strongly correlate with drinking water. We explore δ2H in serial hair samples as a potential technique to estimate season of death by comparing the δ2H record from hair to the known date of death. MATERIALS & METHODS: Approximately 50 hairs were removed from the scalp, aligned from the root, and cut into 5cm serial sections, each representing approximately 2 weeks of growth, and the total sequence a total of 1 year of growth. δ2H was measured on each 5cm segment and compared to previously-reported δ13C, δ15N, and δ34S values. RESULTS: δ2H in the serial hair samples ranged between -56‰ and -48‰, consistent with her water values recorded in surface waters from San Francisco, and follow a sinusoidal pattern. Decreasing δ2H in terminal samples before death suggest Fall as the season of death, consistent with the known date of death. DISCUSSION: This test case shows that archaeological hair preserves a seasonal signature in the form of changing keratin δ2H values that correlate to changing δ2H in surface drinking water. Terminal values in root record water ingested during the final week(s) before death. CONCLUSIONS: We argue that this technique can be used to estimate season of death in future archaeological or forensic cases where hair has been preserved but date of death is unknown.

Transfusion Medicine Equations Made Internet Accessible.

Multiple mathematical equations inform the practice of transfusion medicine. These equations apply to a wide range of topics: dosage of blood products, calculation of fluid volumes, and even specific treatment decisions (e.g. corrected count increment for determination of platelet refractoriness). The calculation of these equations can be complicated, prone to error, and time-consuming. A trusted source is needed to accurately perform these calculations 24 hours a day without error and without monetary cost. We sought to build internet-enabled calculators relevant to the practice of transfusion medicine. We partnered with MDCalc, an online host of medical calculators with 1 million monthly users in 196 countries, to design and host the calculators. The calculators guide users in the application of transfusion medicine equations by providing indications for use, inputs for the equations variables, error-checking, warnings for bad inputs, and interpretive guidance of the result. The following calculators were built: blood volume, corrected count increment (CCI), plasma dosage, cryoprecipitated antihemophilic factor dosage, approximate number of units for compatibility testing, maternal-fetal hemorrhage Rh(D) immune globulin dosage, intrauterine RBC transfusion dosage, neonatal polycythemia partial exchange, theoretical removal of a substance by plasmapheresis, sickle cell RBC exchange volume, peripheral blood stem cell collection, and a calculator relevant to donor lymphocyte infusion. Clinicians can now utilize this reputable and highly visible online source to access these common transfusion medicine equations at any time with an internet-enabled device (https://www.mdcalc.com/search?filter=transfusion+medicine).

Platelet Transfusions in the PICU: Does Disease Severity Matter?

OBJECTIVES: Pediatric intensivists frequently prescribe platelet transfusions to critically ill children, but there are limited data on platelet transfusion practice and platelet transfusion-related outcomes in the PICU. In this study, we evaluated the current platelet transfusion practice and platelet transfusion-related outcomes in the PICU. DESIGN: Institutional review board-approved, retrospective cohort study from January 2010 to March 2016. SETTING: Tertiary-level PICU. PATIENTS: Children less than 19 years old who received platelet transfusions in the PICU. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Thirty-six percent (1,547/4,339) of platelet transfusions in the institution were given to 2.4% of PICU patients (232/9,659). The patients who received a platelet transfusion (platelet transfusions, n = 232) compared with those who did not receive platelets (no platelet transfusions, n = 9,427) were younger, had similar gender distribution, had a higher median Pediatric Risk of Mortality-3 score, and stayed longer in the PICU. Fifty percent of platelet transfusions were prescribed prophylactically for thrombocytopenia to patients without extracorporeal membrane oxygenation support. The mortality was higher for platelet transfusions group (30% vs 2.3%) with an 18 times increased unadjusted odds of mortality when compared with no platelet transfusion group (odds ratio, 18.2; 95% CI, 13.3-24.8; p < 0.0001). In a multiple logistic regression analysis, the predicted probability of dying for platelet transfusion group compared with no platelet transfusion group depended on the median Pediatric Risk of Mortality-3 score. Patients who received platelet transfusion versus no platelet transfusion have increased odds of dying at lower median Pediatric Risk of Mortality-3 scores, but decreased odds of dying at higher median Pediatric Risk of Mortality-3 scores. CONCLUSIONS: This PICU cohort demonstrates that the odds or predicted probability of dying change in patients who received platelet transfusions based on underlying disease severity measured by Pediatric Risk of Mortality-3 score compared with patients who did not receive platelet transfusions. A large, prospective trial is required to confirm this association and determine whether to consider underlying disease severity in estimating risks and benefits of prophylactic platelet transfusions in critically ill children.

LabRS: A Rosetta stone for retrospective standardization of clinical laboratory test results.

Objective: Clinical laboratories in the United States do not have an explicit result standard to report the 7 billion laboratory tests results they produce each year. The absence of standardized test results creates inefficiencies and ambiguities for secondary data users. We developed and tested a tool to standardize the results of laboratory tests in a large, multicenter clinical data warehouse. Methods: Laboratory records, each of which consisted of a laboratory result and a test identifier, from 27 diverse facilities were captured from 2000 through 2015. Each record underwent a standardization process to convert the original result into a format amenable to secondary data analysis. The standardization process included the correction of typos, normalization of categorical results, separation of inequalities from numbers, and conversion of numbers represented by words (eg, "million") to numerals. Quality control included expert review. Results: We obtained 1.266 × 109 laboratory records and standardized 1.252 × 109 records (98.9%). Of the unique unstandardized records (78.887 × 103), most appeared <5 times (96%, eg, typos), did not have a test identifier (47%), or belonged to an esoteric test with <100 results (2%). Overall, these 3 reasons accounted for nearly all unstandardized results (98%). Conclusion: Current results suggest that the tool is both scalable and generalizable among diverse clinical laboratories. Based on observed trends, the tool will require ongoing maintenance to stay current with new tests and result formats. Future work to develop and implement an explicit standard for test results would reduce the need to retrospectively standardize test results.

Unit conversions between LOINC codes.

Logical Observation Identifiers Names and Codes (LOINC) is the most widely used controlled vocabulary to identify laboratory tests. A given laboratory test can often be reported in more than 1 unit of measure (eg, grams or moles), and LOINC defines unique codes for each unit. Consequently, an identical laboratory test performed by 2 different clinical laboratories may have different LOINC codes. The absence of unit conversions between compatible LOINC codes impedes data aggregation and analysis of laboratory results. To develop such conversions, a computational process was developed to review the LOINC standard for potential conversions, and multiple expert reviewers oversaw and finalized the conversion list. In all, 285 bidirectional conversions were identified, including conversions for routine clinical tests such as sodium, magnesium, and human immunodeficiency virus (HIV). Unit conversions were applied to the aggregation of laboratory test results to demonstrate their usefulness. Diverse informatics projects may benefit from the ability to interconvert compatible results.

Vaccination with Vesicular Stomatitis Virus-Vectored Chimeric Hemagglutinins Protects Mice against Divergent Influenza Virus Challenge Strains.

UNLABELLED: Seasonal influenza virus infections continue to cause significant disease each year, and there is a constant threat of the emergence of reassortant influenza strains causing a new pandemic. Available influenza vaccines are variably effective each season, are of limited scope at protecting against viruses that have undergone significant antigenic drift, and offer low protection against newly emergent pandemic strains. "Universal" influenza vaccine strategies that focus on the development of humoral immunity directed against the stalk domains of the viral hemagglutinin (HA) show promise for protecting against diverse influenza viruses. Here, we describe such a strategy that utilizes vesicular stomatitis virus (VSV) as a vector for chimeric hemagglutinin (cHA) antigens. This vaccination strategy is effective at generating HA stalk-specific, broadly cross-reactive serum antibodies by both intramuscular and intranasal routes of vaccination. We show that prime-boost vaccination strategies provide protection against both lethal homologous and heterosubtypic influenza challenge and that protection is significantly improved with intranasal vaccine administration. Additionally, we show that vaccination with VSV-cHAs generates greater stalk-specific and cross-reactive serum antibodies than does vaccination with VSV-vectored full-length HAs, confirming that cHA-based vaccination strategies are superior at generating stalk-specific humoral immunity. VSV-vectored influenza vaccines that express chimeric hemagglutinin antigens offer a novel means for protecting against widely diverging influenza viruses. IMPORTANCE: Universal influenza vaccination strategies should be capable of protecting against a wide array of influenza viruses, and we have developed such an approach utilizing a single viral vector system. The potent antibody responses that these vaccines generate are shown to protect mice against lethal influenza challenges with highly divergent viruses. Notably, intranasal vaccination offers significantly better protection than intramuscular vaccination in a lethal virus challenge model. The results described in this study offer insights into the mechanisms by which chimeric hemagglutinin (HA)-based vaccines confer immunity, namely, that the invariant stalk of cHA antigens is superior to full-length HA antigens at inducing cross-reactive humoral immune responses and that VSV-cHA vaccine-induced protection varies by site of inoculation, and contribute to the further development of universal influenza virus vaccines.

Hemagglutinin Stalk Immunity Reduces Influenza Virus Replication and Transmission in Ferrets.

We assessed whether influenza virus hemagglutinin stalk-based immunity protects ferrets against aerosol-transmitted H1N1 influenza virus infection. Immunization of ferrets by a universal influenza virus vaccine strategy based on viral vectors expressing chimeric hemagglutinin constructs induced stalk-specific antibody responses. Stalk-immunized ferrets were cohoused with H1N1-infected ferrets under conditions that permitted virus transmission. Hemagglutinin stalk-immunized ferrets had lower viral titers and delayed or no virus replication at all following natural exposure to influenza virus.

A viable recombinant rhabdovirus lacking its glycoprotein gene and expressing influenza virus hemagglutinin and neuraminidase is a potent influenza vaccine.

UNLABELLED: The emergence of novel influenza viruses that cause devastating human disease is an ongoing threat and serves as an impetus for the continued development of novel approaches to influenza vaccines. Influenza vaccine development has traditionally focused on producing humoral and/or cell-mediated immunity, often against the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Here, we describe a new vaccine candidate that utilizes a replication-defective vesicular stomatitis virus (VSV) vector backbone that lacks the native G surface glycoprotein gene (VSVΔG). The expression of the H5 HA of an H5N1 highly pathogenic avian influenza virus (HPAIV), A/Vietnam/1203/04 (VN1203), and the NA of the mouse-adapted H1N1 influenza virus A/Puerto Rico/8/34 (PR8) in the VSVΔG vector restored the ability of the recombinant virus to replicate in cell culture, without the requirement for the addition of trypsin. We show here that this recombinant virus vaccine candidate was nonpathogenic in mice when given by either the intramuscular or intranasal route of immunization and that the in vivo replication of VSVΔG-H5N1 is profoundly attenuated. This recombinant virus also provided protection against lethal H5N1 infection after a single dose. This novel approach to vaccination against HPAIVs may be widely applicable to other emerging strains of influenza virus. IMPORTANCE: Preparation for a potentially catastrophic influenza pandemic requires novel influenza vaccines that are safe, can be produced and administered quickly, and are effective, both soon after administration and for a long duration. We have created a new influenza vaccine that utilizes an attenuated vesicular stomatitis virus (VSV) vector, to deliver and express influenza virus proteins against which vaccinated animals develop potent antibody responses. The influenza virus hemagglutinin and neuraminidase proteins, expressed on the surface of VSV particles, allowed this vaccine to grow in cell culture and induced a potent antibody response in mice that was effective against infection with a lethal influenza virus. The mice showed no adverse reactions to the vaccine, and they were protected against an otherwise lethal influenza infection after only 14 days postvaccination and after as many as 140 days postvaccination. The ability to rapidly produce this safe and effective vaccine in cell culture is additionally advantageous.

Assessment of influenza virus hemagglutinin stalk-based immunity in ferrets.

UNLABELLED: Therapeutic monoclonal antibodies that target the conserved stalk domain of the influenza virus hemagglutinin and stalk-based universal influenza virus vaccine strategies are being developed as promising countermeasures for influenza virus infections. The pan-H1-reactive monoclonal antibody 6F12 has been extensively characterized and shows broad efficacy against divergent H1N1 strains in the mouse model. Here we demonstrate its efficacy against a pandemic H1N1 challenge virus in the ferret model of influenza disease. Furthermore, we recently developed a universal influenza virus vaccine strategy based on chimeric hemagglutinin constructs that focuses the immune response on the conserved stalk domain of the hemagglutinin. Here we set out to test this vaccination strategy in the ferret model. Both strategies, pretreatment of animals with a stalk-reactive monoclonal antibody and vaccination with chimeric hemagglutinin-based constructs, were able to significantly reduce viral titers in nasal turbinates, lungs, and olfactory bulbs. In addition, vaccinated animals also showed reduced nasal wash viral titers. In summary, both strategies showed efficacy in reducing viral loads after an influenza virus challenge in the ferret model. IMPORTANCE: Influenza virus hemagglutinin stalk-reactive antibodies tend to be less potent yet are more broadly reactive and can neutralize seasonal and pandemic influenza virus strains. The ferret model was used to assess the potential of hemagglutinin stalk-based immunity to provide protection against influenza virus infection. The novelty and significance of the findings described in this report support the development of vaccines stimulating stalk-specific antibody responses.

Factors Influencing RBC Alloimmunization: Lessons Learned from Murine Models.

Red blood cell (RBC) alloimmunization may occur following transfusion or pregnancy/delivery. Although observational human studies have described the immunogenicity of RBC antigens and the clinical significance of RBC alloantibodies, studies of factors influencing RBC alloimmunization in humans are inherently limited by the large number of independent variables involved. This manuscript reviews data generated in murine models that utilize transgenic donor mice, which express RBC-specific model or authentic human blood group antigens. Transfusion of RBCs from such donors into nontransgenic but otherwise genetically identical recipient mice allows for the investigation of individual donor or recipient-specific variables that may impact RBC alloimmunization. Potential donor-related variables include methods of blood product collection, processing and storage, donor-specific characteristics, RBC antigen-specific factors, and others. Potential recipient-related variables include genetic factors (MHC/HLA type and polymorphisms of immunoregulatory genes), immune activation status, phenotype of regulatory immune cell subsets, immune cell functional characteristics, prior antigen exposures, and others. Although murine models are not perfect surrogates for human biology, these models generate phenomenological and mechanistic hypotheses of RBC alloimmunization and lay the groundwork for follow-up human studies. Long-term goals include improving transfusion safety and minimizing the morbidity/mortality associated with RBC alloimmunization.

Seroepidemiologies of human metapneumovirus and respiratory syncytial virus in young children, determined with a new recombinant fusion protein enzyme-linked immunosorbent assay.

We compared antibodies against human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) in children. The antibody nadirs for both viruses were at 3 to 5 months, and the majority of children were seropositive for both by 2 years. There was no significant difference in the kinetics of maternal antibody decline or seroconversion relative to the two viruses.

T Cell receptor clonotype influences epitope hierarchy in the CD8+ T cell response to respiratory syncytial virus infection.

CD8+ T cell responses are important for recognizing and resolving viral infections. To better understand the selection and hierarchy of virus-specific T cell responses, we compared the T cell receptor (TCR) clonotype in parent and hybrid strains of respiratory syncytial virus-infected mice. K(d)M2(82-90) (SYIGSINNI) in BALB/c and D(b)M(187-195) (NAITNAKII) in C57Bl/6 are both dominant epitopes in parent strains but assume a distinct hierarchy, with K(d)M2(82-90) dominant to D(b)M(187-195) in hybrid CB6F1/J mice. The dominant K(d)M2(82-90) response is relatively public and is restricted primarily to the highly prevalent Vß13.2 in BALB/c and hybrid mice, whereas D(b)M(187-195) responses in C57BL/6 mice are relatively private and involve multiple Vß subtypes, some of which are lost in hybrids. A significant frequency of TCR CDR3 sequences in the D(b)M(187-195) response have a distinct "(D/E)WG" motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but D(b)M(187-195) showed a distinctive structure formed by Lys(7). The data suggest that common recombination events in prevalent Vß genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines.

Assessment of Clostridium difficile infections by quantitative detection of tcdB toxin by use of a real-time cell analysis system.

We explored the use of a real-time cell analysis (RTCA) system for the assessment of Clostridium difficile toxins in human stool specimens by monitoring the dynamic responses of the HS27 cells to tcdB toxins. The C. difficile toxin caused cytotoxic effects on the cells, which resulted in a dose-dependent and time-dependent decrease in cell impedance. The RTCA assay possessed an analytical sensitivity of 0.2 ng/ml for C. difficile toxin B with no cross-reactions with other enterotoxins, nontoxigenic C. difficile, or other Clostridum species. Clinical validation was performed on 300 consecutively collected stool specimens from patients with suspected C. difficile infection (CDI). Each stool specimen was tested in parallel by a real-time PCR assay (PCR), a dual glutamate dehydrogenase and toxin A/B enzyme immunoassay (EIA), and the RTCA assay. In comparison to a reference standard in a combination of the three assays, the RTCA had a specificity of 99.6% and a sensitivity of 87.5% (28 of 32), which was higher than the EIA result (P = 0.005) but lower than the PCR result (P = 0.057). In addition, the RTCA assay allowed for quantification of toxin protein concentration in a given specimen. Among RTCA-positive specimens collected prior to treatment with metronidazole and/or vancomycin, a significant correlation between toxin protein concentrations and clinical CDI severities was observed (R(2) = 0.732, P = 0.0004). Toxin concentrations after treatment (0.89 ng/ml) were significantly lower than those prior to the treatment (15.68 ng/ml, Wilcoxon P = 0.01). The study demonstrates that the RTCA assay provides a functional tool for the potential assessment of C. difficile infections.

Soluble recombinant human metapneumovirus G protein is immunogenic but not protective.

Human metapneumovirus (HMPV) expresses the major surface glycoproteins F and G. We evaluated the protective efficacy of immunization with G. We generated a recombinant form of G ectodomain (GDeltaTM) that was secreted from mammalian cells and purified by affinity chromatography. We tested the immunogenicity of GDeltaTM in cotton rats. Animals were immunized with PBS, GDeltaTM alone or adjuvanted, or were infected once with HMPV, and challenged with live HMPV at 28 days. Animals vaccinated with adjuvanted and non-adjuvanted GDeltaTM developed high levels of serum antibodies to both recombinant and native G protein; however, vaccinated animals did not develop neutralizing antibodies and were not protected against virus challenge. Unlike the analogous non-fusion glycoproteins of other human paramyxoviruses, HMPV G does not appear to be a protective antigen. This represents an unusual feature of HMPV.