Cost and Use Trends of Endomyocardial Biopsy in Heart Transplant Patients: A 4-Year Claims Data Analysis.

BACKGROUND: This study evaluated patterns of utilization, complications, and costs of endomyocardial biopsies (EMB) in heart transplant patients. METHODS: The IBM Treatment Pathways tool was used to analyze claims data selected from IBM's MarketScan de-identified Health Insurance Portability and Accountability Act (HIPAA)-compliant dataset. Differences in EMB paid amounts and utilization patterns were assessed for commercial payers and Medicare (2016-2019). The type, frequency, and overall cost of complications of the EMB procedure in these patients were also evaluated. RESULTS: A total of 8,170 records (6,385 commercial payers and 1,785 Medicare) of heart transplant patients with evidence of EMB procedures performed between 2016 and 2019 were identified in the database. In 2019, the median paid amount for an outpatient EMB in a heart transplant patient was US $7,918 (commercial) and US $2,980 (Medicare). Heart transplant patients received between 4.6 and 6.8 (median; Medicare, commercial) EMBs the first year after the transplant. Approximately 25% of EMB procedures were associated with complications. In 2019, the total median cost of EMB complications per patient was US $9,049. CONCLUSIONS: Analysis showed that the paid amount for the EMB procedure increased by almost 25% from 2016 to 2019 for commercial payers. Given the high frequency of complications after the EMB procedure and the associated cost of the complications, it is estimated that the median paid amounts are closer to US $10,000 per patient per EMB. Given the number of EMBs provided, the associated risks, and the paid amount trends, non-invasive alternatives to EMB should be considered for the surveillance of heart transplant patients.

Centenarian clocks: epigenetic clocks for validating claims of exceptional longevity.

Claims surrounding exceptional longevity are sometimes disputed or dismissed for lack of credible evidence. Here, we present three DNA methylation-based age estimators (epigenetic clocks) for verifying age claims of centenarians. The three centenarian clocks were developed based on n = 7039 blood and saliva samples from individuals older than 40, including n = 184 samples from centenarians, 122 samples from semi-supercentenarians (aged 105 +), and 25 samples from supercentenarians (aged 110 +). The oldest individual was 115 years old. Our most accurate centenarian clock resulted from applying a neural network model to a training set composed of individuals older than 40. An epigenome-wide association study of age in different age groups revealed that age effects in young individuals (age < 40) are correlated (r = 0.55) with age effects in old individuals (age > 90). We present a chromatin state analysis of age effects in centenarians. The centenarian clocks are expected to be useful for validating claims surrounding exceptional old age.

Adipose tissue measurement in clinical research for obesity, type 2 diabetes and NAFLD/NASH.

INTRODUCTION: Excess body fat is linked to higher risks for metabolic syndrome, type 2 diabetes mellitus (T2DM), and cardiovascular disease (CV), among other health conditions. However, it is not only the level but also the distribution of body fat that contributes to increased disease risks. For example, an increased level of abdominal fat, or visceral adipose tissue (VAT), is associated with a higher risk of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). METHODS: A review of the most relevant primary and secondary sources on body composition from the last 25 years was conducted. Relevant articles were identified using PUBMED and Google Scholar. Narrative synthesis was performed as statistical pooling was not possible due to the heterogeneous nature of the studies. RESULTS: The body mass index (BMI) is commonly used as a proxy measure of body fatness. However, BMI does not reflect the level and distribution of body fat. Other anthropometric methods such as waist circumference measurement and waist-hip ratio, as well as methodologies like hydro densitometry, bioelectrical impedance, and isotope dilution are also limited in their ability to determine body fat distribution. Imaging techniques to define body composition have greatly improved performance over traditional approaches. Ultrasound (US), computed tomography (CT), dual-energy X-ray absorptiometry (DXA), magnetic resonance imaging (MRI), are now commonly used in clinical research. Of these, MRI can provide the most accurate and high-resolution measure of body composition. In addition, MRI techniques are considered the best for the determination of fat at the organ level. On the other hand, imaging modalities require specialized, often expensive equipment and expert operation. CONCLUSIONS: Anthropometric methods are suitable for rapid, high-volume screening of subjects but do not provide information on body fat distribution. Imaging techniques are more accurate but are expensive and do not lend themselves for high throughput. Therefore, successful trial strategies require a tiered approach in which subjects are first screened using anthropometric methods followed by more sophisticated modalities during the execution of the trial. This article provides a brief description of the most clinically relevant adipose tissue measurement techniques and discusses their value in obesity, diabetes, and NAFLD/NASH clinical research.

Disparities in Emergency Transport of Women with a Preterm Labor Diagnosis in Urban vs Rural Areas in the US.

OBJECTIVE: This study evaluated patterns of utilization and costs of emergency transport among women with a diagnosis of preterm labor in the US. METHODS: The IBM® Treatment Pathways® tool was used to interrogate a cohort randomly selected from the IBM's MarketScan ® dataset. Differences in costs and utilization patterns were assessed by the type of emergency transport service and geography. RESULTS: A cohort of 12,995 women between the ages of 16 and 45 met the inclusion criteria. About 1,029 (7.9%) of these women had evidence of emergency transport within a day of the preterm labor diagnosis. In this cohort, the median cost of emergency ground transportation was US$834; air transport had a median cost of US$22,922. Additionally, 3.1% (284) women out of a cohort of 8,728 women ages of 16 and 45 with a diagnosis of false labor required emergency transport within 7 days suggesting that they were discharged too soon. DISCUSSION: The prevalence of emergency transport for preterm labor in rural areas is significantly higher compared to non-rural areas. In addition, the disproportionate use of air transport in rural areas increases the costs of the preterm labor event. Moreover, disparities in both utilization rates and costs were identified for different parts of the country.

Vaxfectin adjuvant improves antibody responses of juvenile rhesus macaques to a DNA vaccine encoding the measles virus hemagglutinin and fusion proteins.

DNA vaccines formulated with the cationic lipid-based adjuvant Vaxfectin induce protective immunity in macaques after intradermal (i.d.) or intramuscular (i.m.) delivery of 0.5 to 1 mg of codon-optimized DNA encoding the hemagglutinin (H) and fusion (F) proteins of measles virus (MeV). To characterize the effect of Vaxfectin at lower doses of H+F DNA, rhesus macaques were vaccinated twice with 20 µg of DNA plus Vaxfectin i.d., 100 µg of DNA plus Vaxfectin i.d., 100 µg of DNA plus Vaxfectin i.m. or 100 µg of DNA plus phosphate-buffered saline (PBS) i.m. using a needleless Biojector device. The levels of neutralizing (P = 0.036) and binding (P = 0.0001) antibodies were higher after 20 or 100 µg of DNA plus Vaxfectin than after 100 µg of DNA plus PBS. Gamma interferon (IFN-γ)-producing T cells were induced more rapidly than antibody, but were not improved with Vaxfectin. At 18 months after vaccination, monkeys were challenged with wild-type MeV. None developed rash or viremia, but all showed evidence of infection. Antibody levels increased, and IFN-γ- and interleukin-17-producing T cells, including cells specific for the nucleoprotein absent from the vaccine, were induced. At 3 months after challenge, MeV RNA was detected in the leukocytes of two monkeys. The levels of antibody peaked 2 to 4 weeks after challenge and then declined in vaccinated animals reflecting low numbers of bone marrow-resident plasma cells. Therefore, Vaxfectin was dose sparing and substantially improved the antibody response to the H+F DNA vaccine. This immune response led to protection from disease (rash/viremia) but not from infection. Antibody responses after challenge were more transient in vaccinated animals than in an unvaccinated animal.

Analysis of biomarkers after intramuscular injection of Vaxfectin-formulated hCMV gB plasmid DNA.

Cationic lipids have been used as delivery systems to enhance the performance of vaccines and immunotherapeutics. However, little is known about the effect of administration of cationic lipid-formulated vaccines on gene expression. This study used DNA microarrays (39,000 transcripts) to characterize early changes in gene expression patterns in mouse muscle 1 and 2 days after intramuscular (i.m.) injection of a hCMV gB plasmid DNA (pDNA) vaccine formulated with the cationic lipid system Vaxfectin; gene expression profiles were compared to those obtained after i.m. injection of pDNA in PBS. Analysis of the DNA microarray data indicated that approximately 1% of the represented transcripts were modulated at least 2-fold compared to the PBS samples at both time points. Functional analysis of the modulated genes revealed that transcripts involved in antigen processing and presentation, apoptosis and the Toll-like receptor pathway were significantly enriched. In addition, confirmation of local and systemic modulation of subsets of biomarkers was achieved using Real-Time PCR and Cytometric Bead Assays. Time course and magnitude of cellular infiltration (F4/80+ and CD11b+ cells) to the injection site was changed in response to formulation of hCMVgB pDNA with Vaxfectin. Since the expression level of the pDNA-encoded transgene in the muscle was not affected by formulation Vaxfectin mechanism of action is expected to rely primarily on modulation of immune pathways and not on an increase in transfection of the antigen-encoding pDNA. Taken together, these data help explain the Vaxfectin-dependent robust enhancement of immune responses.

Vaxfectin-adjuvanted seasonal influenza protein vaccine: correlation of systemic and local immunological markers with formulation parameters.

Vaxfectin, a cationic lipid-based adjuvant, when combined with a seasonal influenza protein vaccine has been reported to enhance predominantly either antibody or cellular responses depending upon the ratio of adjuvant to antigen. Preliminary physical characterization showed that particle size was dependent on the antigen to Vaxfectin ratio. In an effort to identify potential predictive markers helpful in formulation development, a panel of biomarkers was assayed both at the site of administration and in the serum. Local upregulation of IFN-gamma, IL-6, Cxcl9, CCL2, TNF-alpha, CD274 as well as Toll-like receptor pathway transcripts MyD88, TLR2, TLR3 and TLR9 was observed. Also, systemic levels of IL-6, TNF-alpha and CCL2 were elevated in response to Vaxfectin formulation in a ratio-dependent manner. These results have identified biomarkers that may be useful in testing Vaxfectin-protein formulations to produce balanced humoral and cell-mediated immune responses.

Vaxfectin, a cationic lipid-based adjuvant for protein-based influenza vaccines.

Mice were immunized either with unadjuvanted seasonal trivalent influenza vaccine (TIV) or TIV formulated with Vaxfectin, a cationic lipid-based adjuvant. Increasing doses of Vaxfectin resulted in increased hemagglutination-inhibition or anti-TIV ELISA antibody titers, with up to a 200-fold increase obtained with 900 microg of Vaxfectin. A >or=10-fold dose-sparing effect was demonstrated with Vaxfectin formulations. Vaxfectin preferentially increased IgG2 titers compared to IgG1 titers, resulting in a balanced IgG isotype distribution. Lower doses of Vaxfectin (30 microg) did not enhance antibody responses, but increased the number of IFN-gamma secreting T-cells by up to 18-fold. The data demonstrate that Vaxfectin enhances Th1 responses with protein-based seasonal influenza vaccine, and suggest that cellular or humoral immune responses may be preferentially induced by modifying the Vaxfectin:antigen ratio in the vaccine formulation.

Herpes simplex virus type 2 tegument proteins contain subdominant T-cell epitopes detectable in BALB/c mice after DNA immunization and infection.

Cytotoxic T cells are important in controlling herpes simplex virus type 2 (HSV-2) reactivation and peripheral lesion resolution. Humans latently infected with HSV-2 have cytotoxic T cells directed against epitopes present in tegument proteins. Studies in mice of immunity to HSV have commonly focused on immunodominant responses in HSV envelope glycoproteins. These antigens have not proved to be an effective prophylactic vaccine target for most of the human population. The murine immune response against HSV tegument proteins has not been explored. We analysed cellular responses in BALB/c mice directed against the tegument proteins encoded by UL46, UL47 and UL49 and against the envelope glycoprotein gD after DNA vaccination or HSV-2 infection. After DNA vaccination, the splenocyte T-cell response to overlapping peptides from UL46 and UL47 was more than 500 gamma interferon spot-forming units per 10(6) responder cells. Peptide truncation studies, responder cell fractionation and major histocompatibility complex binding studies identified several CD8(+) and CD4(+) epitopes. Cellular responses to tegument protein epitopes were also detected after HSV-2 infection. Tegument proteins are rational candidates for further HSV-2 vaccine research.

Use of Vaxfectin adjuvant with DNA vaccine encoding the measles virus hemagglutinin and fusion proteins protects juvenile and infant rhesus macaques against measles virus.

A measles virus vaccine for infants under 6 months of age would help control measles. DNA vaccines hold promise, but none has provided full protection from challenge. Codon-optimized plasmid DNAs encoding the measles virus hemagglutinin and fusion glycoproteins were formulated with the cationic lipid-based adjuvant Vaxfectin. In mice, antibody and gamma interferon (IFN-gamma) production were increased by two- to threefold. In macaques, juveniles vaccinated at 0 and 28 days with 500 microg of DNA intradermally or with 1 mg intramuscularly developed sustained neutralizing antibody and H- and F-specific IFN-gamma responses. Infant monkeys developed sustained neutralizing antibody and T cells secreting IFN-gamma and interleukin-4. Twelve to 15 months after vaccination, vaccinated monkeys were protected from an intratracheal challenge: viremia was undetectable by cocultivation and rashes did not appear, while two naïve monkeys developed viremia and rashes. The use of Vaxfectin-formulated DNA is a promising approach to the development of a measles vaccine for young infants.

Vaccination with polymerase chain reaction-generated linear expression cassettes protects mice against lethal influenza A challenge.

The feasibility of a linear expression cassette (LEC)-based influenza A DNA vaccine was demonstrated in mice, using a lethal dose (LD90) of a mouse-adapted A/Hong Kong/8/68 (H3N2) influenza strain. LECs expressing hemagglutinin (HA) from either the homotypic H3N2 or the heterotypic H1N1 (A/Puerto Rico/8/34) influenza virus were produced by polymerase chain reaction and either phosphodiester- or phosphorothioate-modified oligonucleotide primers. Survival subsequent to lethal viral challenge was used as a primary end point; weight loss was the secondary end point. Survival and weight loss data showed that protection can be achieved in mice with 50 microg of phosphate-buffered saline-formulated LEC DNA or 2 microg of Vaxfectin-formulated LEC DNA. Survival correlated with neutralizing antibody titers (hemagglutination inhibition, HAI); titers obtained after vaccination with LEC were equivalent to those obtained with HA (H3N2) plasmid DNA control. Vaccination with heterotypic H1 HA-LEC DNA provided no protection against viral challenge.

Comparison of rabbit and mouse models for persistence analysis of plasmid-based vaccines.

Experiments were conducted with a cationic lipid-formulated pDNA vaccine (VCL-AB01) to evaluate the models used to determine biodistribution, persistence and the potential for integration (into genomic DNA) of plasmid DNA-based vaccines. Mice were injected with a high-dose volume of 50 microL unilaterally containing approximately 1.33 x 10(13) plasmid copy numbers (PCN) or a low-dose volume of 20 microL bilaterally ( approximately 5.3 x 10(12) PCN). Rabbits were injected bilaterally with a 0.5 mL ( approximately 1.33 x 10(14) PCN) volume. Injection site muscle tissue was harvested two days, one month, and two months postinjection for the low-dose murine and rabbit models and two days and two months postinjection for the high-dose murine model. Total DNA was extracted and analyzed by real-time quantitative PCR for sequences specific to the injected pDNA. The geometric mean PCN/microg of total DNA from the high and low dose models were compared to determine if injection volume impacts clearance and/or persistence. Results from these studies showed that PCN clearance over two months was similar in mice injected with 20 microL and rabbits injected with 0.5 mL, but PCN clearance was slower in mice injected with similar PCN in 50 microL (1.33 x 10(13) PCN) compared to 20 microL (5.3 x 10(12) PCN). Persistence at two months in the rabbit and low-dose murine models was comparable, with geometric mean of 5.22 x 10(3) PCN/microg of total DNA for the low-dose volume murine model and 2.81 x 10(3)/microg DNA for the rabbit model. Interanimal variability in persistence was not impacted by dose volume.

Sustained protective rabies neutralizing antibody titers after administration of cationic lipid-formulated pDNA vaccine.

Published data indicate that formulation of pDNA with cationic lipids could greatly enhance the response to a pDNA vaccine in larger mammals. The present work tested the influence of several pDNA:cationic lipid formulations on rabies neutralizing titers. Plasmid expressing Rabies G protein (CVS strain) was evaluated in vivo for ability to elicit neutralizing titers. pDNA:DMRIE-DOPE formulated at two DNA:cationic lipid molar ratios was compared in mice to a Vaxfectin-pDNA formulation. Mouse data indicate that Vaxfectin is more effective than DMRIE-DOPE in eliciting neutralizing titers. In addition, the ratio of pDNA to DMRIE-DOPE can also affect neutralizing titers. Our data show that sustained neutralizing titers (120 days) can be obtained after a single administration of DMRIE-DOPE-formulated pDNA in rabbits.

I. Poloxamer-formulated plasmid DNA-based human cytomegalovirus vaccine: evaluation of plasmid DNA biodistribution/persistence and integration.

Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.

II. Cationic lipid-formulated plasmid DNA-based Bacillus anthracis vaccine: evaluation of plasmid DNA persistence and integration potential.

Several formulated plasmid DNA (pDNA)-based vaccines are being evaluated for safety and efficacy in healthy human subjects. A safety concern for any vaccine that contains genetic material, be it whole organism, live-attenuated, or gene-based, is the potential for integration into genomic DNA (gDNA). To address this concern, a preclinical pDNA persistence/integration study was conducted in rabbits to determine the level of pDNA in muscle 2, 28, and 64 days after intramuscular injection of DMRIE:DOPE-formulated pDNAs encoding Bacillus anthracis detoxified LF and PA proteins (VCL-AB01 vaccine). Total DNA was extracted from day 64 muscle tissue and fractionated by column agarose gel electrophoresis (CAGE). Plasmid copy number (PCN) in muscle 64 days after injection (geometric mean, 2808 PCN/microg of total DNA or 150,000 diploid genomes) was determined by quantitative polymerase chain reaction. Analysis of total DNA from five VCLAB01- injected rabbits revealed that two of five samples had no detectable PCN in the high molecular weight fraction after one round of CAGE, two samples had PCN under the lower limit of quantitation, and the remaining sample had 123 PCN/microg. All PCN in the latter sample cleared after an additional round of CAGE. It appears, therefore, that persisting PCN fractionate as low molecular weight material and are most likely not integrated into gDNA. Even if the worst-case assumption is made that the highest PCN found associated with gDNA represented covalently integrated pDNA inserts, the frequency of mutation would still be 500-fold lower than the autosomal spontaneous mutation rate.