Using the systematic review methodology to evaluate factors that influence the persistence of influenza virus in environmental matrices.

Understanding factors that influence persistence of influenza virus in an environment without host animals is critical to appropriate decision-making for issues such as quarantine downtimes, setback distances, and eradication programs in livestock production systems. This systematic review identifies literature describing persistence of influenza virus in environmental samples, i.e., air, water, soil, feces, and fomites. An electronic search of PubMed, CAB, AGRICOLA, Biosis, and Compendex was performed, and citation relevance was determined according to the aim of the review. Quality assessment of relevant studies was performed using criteria from experts in virology, disease ecology, and environmental science. A total of 9,760 abstracts were evaluated, and 40 appeared to report the persistence of influenza virus in environmental samples. Evaluation of full texts revealed that 19 of the 40 studies were suitable for review, as they described virus concentration measured at multiple sampling times, with viruses detectable at least twice. Seven studies reported persistence in air (six published before 1970), seven in water (five published after 1990), two in feces, and three on surfaces. All three fomite and five air studies addressed human influenza virus, and all water and feces studies pertained to avian influenza virus. Outcome measurements were transformed to half-lives, and resultant multivariate mixed linear regression models identified influenza virus surviving longer in water than in air. Temperature was a significant predictor of persistence over all matrices. Salinity and pH were significant predictors of persistence in water conditions. An assessment of the methodological quality review of the included studies revealed significant gaps in reporting critical aspects of study design.

Finding PRRSV in sow herds: Family oral fluids vs. serum samples from due-to-wean pigs.

The aim of this study was to compare the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in due-to-wean litters in commercial swine breeding herds using family oral fluids (FOF) vs. individual piglet serum samples. FOF and piglet serum samples were collected in 199 due-to-wean litters on six farms containing 2177 piglets. All samples were individually tested for PRRSV RNA by RT-rtPCR. A litter was considered PRRSV-positive when PRRSV RNA was detected in ≥ 1 piglet serum sample or the FOF sample. Mixed effect logistic regression with farm as a random effect was used 1) to evaluate the probability of obtaining a PRRSV RNA positive FOF as a function of the proportion of viremic piglets in a litter and 2) the effect of litter size and parity on the probability that a litter would test PRRSV RNA positive in FOF. A Bayesian prevalence estimation under misclassification (BayesPEM) analysis was used to calculate the PRRSV prevalence and 95 % credible interval given the condition that all samples (FOF and serum) tested negative. In total, 34 of 199 litters (17.1 %) contained ≥ 1 viremic piglet(s), and 28 of 199 litters (14.1 %) were FOF positive. When all piglet serum samples within a litter tested negative, 1 of 165 FOF (0.6 %) tested PRRSV RNA positive. The probability of a PCR-positive FOF sample from litters with 10 %, 20 %, 30 %, 40 %, and 50 % within-litter PRRSV prevalence was 3.5 %, 35.1 %, 88.8 %, 99.2 %, and >99.9 %, respectively. The odds of a PCR-positive FOF in a first parity litter were 3.36 times (95 % CI: 2.10-5.38) that of a parity ≥ 2 litter. The odds of a positive FOF result in a litter with ≤ 11 piglets were 9.90 times (95 % CI: 4.62-21.22) that of a litter with > 11 piglets. FOF was shown to be an efficacious sample type for PRRSV detection in farrowing rooms. A risk-based approach for litter selection combined with FOF collection can be used to improve on-farm PRRSV detection with a limited sample size, compared to sampling multiple individual pigs. Finally, the BayesPEM analysis showed that PRRSV may still be present in breeding herds when all samples (serum and FOF) test PRRSV RNA negative, i.e., negative surveillance results should be interpreted with caution.

A comparison of three sampling approaches for detecting PRRSV in suckling piglets.

Determining whether porcine reproductive and respiratory syndrome virus (PRRSV) is circulating within a breeding herd is a longstanding surveillance challenge. Most commonly, piglets in farrowing rooms are sampled to infer the PRRSV status of the sow herd, with sample size based on the expectation of hypergeometric distribution and piglet selection based on simple random sampling (SRS), i.e., randomly selecting individuals from a population in a manner that all individuals have equal chance of being selected. Conceptually straightforward, the assumptions upon which it is based (homogeneous population and independence of individuals) rarely hold in modern swine facilities. Alternative approaches for sample selection include two-stage stratified sampling (2SS), i.e., randomly selecting litters (first stratum) and randomly selecting piglets (second stratum) within selected litters, and risk-based sampling (RBS), i.e., selecting litters with a higher risk of having viremic piglets, and randomly selecting pigs within those litters. The objectives of this study were to 1) characterize the pattern of distribution of PRRSV-viremic piglets in farrowing rooms and 2) compare the efficiency of SRS, 2SS, and RBS for the detection of PRRSV-viremic piglets. In 12 sow farms, serum samples were collected from all 4510 piglets in 422 litters housed in 23 farrowing rooms and tested for PRRSV RNA. At the population level, the distribution of PRRSV-viremic pigs was analyzed for population homogeneity and spatial clustering. At the litter level, litter size and sow parity were evaluated as risk factors. A non-homogeneous distribution of PRRSV-viremic piglets was observed in nearly all farrowing rooms (15/16), and spatial clustering detected on 11 occasions (11/16). Simulated sampling based on farrowing room data determined that 2SS required 1-to-25 fewer samples than SRS to detect ≥ 1 viremic piglet in 13 of 16 rooms and the same number of samples in 3 rooms. RBS required 1-to-7 fewer samples than 2SS to detect ≥ 1 viremic piglet in 7 of 16 rooms, the same number of samples in 6 rooms, and 1 more sample in 3 rooms. Notably, SRS was less efficient than either 2SS or RBS in detecting PRRSV-viremic piglets in farrowing rooms, regardless of the confidence level. It may be concluded that the core assumptions upon which most current surveillance methods are based do not hold in modern farrowing room facilities. Simulation-based sample size tables for SRS and 2SS are provided.

Collecting oral fluid samples from due-to-wean litters.

Oral fluids are a common diagnostic sample in group-housed nursery, grow-finish, and adult swine. Although oral fluids from due-to-wean litters could be a valuable tool in monitoring pathogens and predicting the health status of pig populations post-weaning, it is generally not done because of inconsistent success in sample collection. The objective of this study was to determine the optimum procedure for collecting oral fluid samples from due-to-wean litters. Successful collection of oral fluids from due-to-wean litters using "Litter Oral Fluid" (LOF) or "Family Oral Fluid" (FOF) sampling techniques were compared in 4 phases involving 920 attempts to collect oral fluids. Phase 1 testing showed that prior exposure to a rope improved the success rates of both LOF (33.4%) and FOF (16.4%) techniques. Phase 2 determined that longer access to the rope (4 h vs 30 min) did not improve the success rate for either LOF or FOF. Phase 3 evaluated the effect of attractants and found that one (Baby Pig Restart®) improved the success rate when used with the FOF technique. Phase 4 compared the success rates of "optimized LOF" (litters previously trained) vs "optimized FOF" (litter previously trained and rope treated with Baby Pig Restart®) vs standard FOF. No difference was found between the FOF-based techniques, but both were superior to the "optimized LOF" technique. Thus, FOF-based procedures provided a significantly higher probability of collecting oral fluids from due-to-wean litters (mean success rate 84.9%, range 70% to 92%) when compared to LOF-based methods (mean success rate 24.1%, range 16.5% to 32.2%).

A method to provide improved dose-response estimates for airborne pathogens in animals: an example using porcine reproductive and respiratory syndrome virus.

This paper describes a method to provide improved probability estimates that exposure to a specific dose of an airborne infectious pathogen will result in animal infection. Individual animals were exposed to a specific dose of airborne pathogen. Following exposure, animals were individually housed and monitored for evidence of infection. The detection of specific antibodies and/or the pathogen in diagnostic specimens was evidence that the exposure dose resulted in infection. If replicated over a range of doses, the results can be used to derive a dose-response curve for a variety of animal species and infectious pathogens. This information is useful in estimating the likelihood of infection associated with exposure to airborne infectious microorganisms. Applications include predicting the risk of transmission associated with exposure to airborne pathogens, modeling the transmission of airborne pathogens, and determining requirements for effective exposure doses for vaccines delivered in aerosols.

Immune response against porcine reproductive and respiratory syndrome virus during acute and chronic infection.

A significant obstacle to the prevention and control of porcine reproductive and respiratory syndrome virus (PRRSV) is the inability of current diagnostic tests to provide information concerning the stage of PRRSV infection. To explore possible prognostic combinations of cell-mediated and humoral immune responses, 3-week-old pigs (n=10) were intramuscularly (IM) inoculated with PRRSV isolate VR-2332 and followed for 193 days post-inoculation (DPI). Negative control pigs (n=10) were IM inoculated with minimum essential medium (MEM). At approximately 2-week intervals, blood samples were collected from all animals and tested for the number of interferon (IFN)-gamma-secreting peripheral blood mononuclear cells (enzyme-linked immunosorbent spot, Elispot), PRRSV viremia (quantitative reverse-transcriptase polymerase chain reaction, qRT-PCR), and serum antibodies using PRRSV protein ELISAs (N, GP5 3', GP5 5', M 5', M 3', GP5-M, and nsp2p) and a commercial PRRSV ELISA (IDEXX Laboratories Inc.). All pigs were viremic by 7 days post-inoculation, with 50% of the pigs resolving viremia by 56 DPI. A PRRSV-specific IFN-gamma response was detected at DPI 28, reached a plateau at 42 DPI, declined slightly, and remained relatively stable from 56 to 193 DPI. On the basis of ROC area under the curve (AUC) analysis, the ELISAs that most reliably differentiated PRRSV-inoculated pigs from negative control pigs were the commercial ELISA (AUC=0.97), the N ELISA (AUC=0.96), and the M 3' ELISA (AUC=0.93). Multivariate analyses were performed to evaluate the relationship between the immune response and the duration and level of viremia. With all antibody assays and Elispot included in the models, the analysis determined that the serum-virus neutralizing antibody response was the best predictor of both level and duration of viremia. It was concluded that humoral antibody responses, particularly the commercial ELISA, N ELISA, and M 3' ELISA were good predictors of prior exposure to PRRSV, but provided little information regarding the ontogeny of the protective immune response. Likewise, cell-mediated immunity based on the number of IFN-gamma-secreting lymphocytes was a poor prognosticator of PRRSV infection status.

Assessment of abattoir based monitoring of PRRSV using oral fluids.

Various porcine reproductive and respiratory syndrome virus (PRRSV) regional elimination projects have been implemented in the U.S., but none have yet succeeded. In part, this reflects the need for efficient methods to monitor over time the progress of PRRSV status of participating herds. This study assessed the feasibility of monitoring PRRSV using oral fluids collected at the abattoir. A total of 36 pig lots were included in the study. On-farm oral fluid (n = 10) and serum (n = 10) collected within two days of shipment to the abattoir were used to establish the reference PRRSV status of the population. Oral fluids (n = 3 per lot) were successfully collected from 32 lots (89%) at the lairage. Three veterinary diagnostic laboratories (VDLs) tested the sera (VDL1 and VDL3: n = 316, VDL2: n = 315) and oral fluids (VDL1 and VDL3: n = 319, VDL2: n = 320) for PRRSV antibodies (ELISA) and RNA (rRT-PCR). Environmental samples (n = 64, 32 before and 32 after pigs were placed in lairage) were tested for PRRSV RNA at one VDL. All oral fluids (farm and abattoir) tested positive for PRRSV antibody at all VDLs. PRRSV positivity frequency on serum ranged from 92.4% to 94.6% among VDLs, with an overall agreement of 97.6%. RNA was detected on 1.3% to 1.9%, 8.1% to 17.7%, and 8.3% to 17.7% of sera, on-farm and abattoir oral fluids, respectively. Between-VDLs rRT-PCR agreement on sera and oral fluids (farm and abattoir) ranged from 97.8% to 99.0%, and 79.0% to 81.2%, respectively. Between-locations agreement of oral fluids varied from 31.3% to 50% depending on the VDL. This study reported the application of swine oral fluids collected at the abattoir for monitoring PRRSV, and describes the between-VDL agreement for PRRS testing of serum and oral fluid field samples.

A Four-Biomarker Blood Signature Discriminates Systemic Inflammation Due to Viral Infection Versus Other Etiologies.

The innate immune system of humans and other mammals responds to pathogen-associated molecular patterns (PAMPs) that are conserved across broad classes of infectious agents such as bacteria and viruses. We hypothesized that a blood-based transcriptional signature could be discovered indicating a host systemic response to viral infection. Previous work identified host transcriptional signatures to individual viruses including influenza, respiratory syncytial virus and dengue, but the generality of these signatures across all viral infection types has not been established. Based on 44 publicly available datasets and two clinical studies of our own design, we discovered and validated a four-gene expression signature in whole blood, indicative of a general host systemic response to many types of viral infection. The signature's genes are: Interferon Stimulated Gene 15 (ISG15), Interleukin 16 (IL16), 2',5'-Oligoadenylate Synthetase Like (OASL), and Adhesion G Protein Coupled Receptor E5 (ADGRE5). In each of 13 validation datasets encompassing human, macaque, chimpanzee, pig, mouse, rat and all seven Baltimore virus classification groups, the signature provides statistically significant (p < 0.05) discrimination between viral and non-viral conditions. The signature may have clinical utility for differentiating host systemic inflammation (SI) due to viral versus bacterial or non-infectious causes.

Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine.

Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti-IAV antibodies using homologous and heterologous haemagglutination-inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)-blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut-off of S/N ≤ 0.60, the sensitivity and specificity of the NP-blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post-inoculation and the interactions between those factors (P < 0.0001). Serum antibodies against NP provide an ideal universal diagnostic screening target and could provide a cost-effective approach for the detection and surveillance of IAV infections in swine populations.

Probability of porcine reproductive and respiratory syndrome (PRRS) virus infection as a function of exposure route and dose.

At the most elemental level, the design of effective strategies to control and/or eliminate porcine reproductive and respiratory syndrome (PRRS) virus depend on an accurate and comprehensive understanding of virus transmission. As a general rule, transmission is highly dependent on the route of exposure and the dose of virus. The objective of this study was to derive PRRS virus isolate VR-2332 dose-response curves for oral and intranasal routes of exposure, i.e., determine the probability that a specific virus dose would result in infection. Individually housed pigs approximately 21 days of age were exposed to specific doses of PRRS virus isolate VR-2332 by either oral or intranasal routes. Positive controls were intramuscularly inoculated with 10(2.2) 50% tissue culture infective dose (TCID50) of PRRS virus and negative controls were orally administered 100ml of diluent with no virus. Pigs were monitored for evidence of infection for 21 days following exposure, i.e., serum samples were collected on days 0, 7, 14, 21, and tested for virus and PRRS virus-specific antibodies. Dose-response curves and 95% confidence intervals for oral and intranasal routes of exposure were derived using logistic models (logit and probit). The infectious dose50 (ID50) for oral exposure was estimated to be 10(5.3) TCID50 (95% CI, 10(4.6) and 10(5.9)); the ID50 for intranasal exposure was estimated to be 10(4.0) TCID50 (95% CI, 10(3.0) and 10(5.0)). Given these estimates, it is worth noting that intramuscular exposure of animals to 10(2.2) TCID50 (positive controls) resulted in infection in all animals. Thus pigs were the most susceptible to infection via parenteral exposure.

Pharmacokinetic interaction between single oral doses of diltiazem and sirolimus in healthy volunteers.

AIM AND BACKGROUND: The pharmacokinetic interaction between sirolimus, a macrolide immunosuppressant metabolized by CYP3A4, and the calcium channel blocker diltiazem was studied in 18 healthy subjects. Several clinically important interactions have previously been reported for other immunosuppressive drugs that are metabolized by the same enzyme and for calcium antagonists. METHODS: Healthy subjects who were 20 to 43 years old participated in an open, three-period, randomized, crossover study of the pharmacokinetics of a single 10-mg oral dose of sirolimus, a single oral 120-mg dose of diltiazem, and the two drugs given together. The three study periods were separated by a 21-day washout phase. RESULTS: The geometric mean (90% confidence interval) whole blood sirolimus area under the plasma concentration time-curve increased 60% (35%-90%), from 736 to 1178 ng x h/mL, and maximum concentration increased 43% (14%-81%), from 67 to 96 ng/mL, with diltiazem coadministration, whereas the mean elimination half-life of sirolimus decreased slightly, from 79 to 67 hours. Apparent oral clearance and volume of distribution of sirolimus decreased with 38% and 45%, respectively, when sirolimus was given with diltiazem. The plasma maximum concentration and area under the plasma concentration-time curve of diltiazem, desacetyldiltiazem, and desmethyldiltiazem were unchanged after coadministration of sirolimus, and no potentiation of the effects of diltiazem on diastolic or systolic blood pressure or on the electrocardiographic parameters was seen. CONCLUSIONS: Single-dose diltiazem coadministration leads to higher sirolimus exposure, presumably by inhibition of the first-pass metabolism of sirolimus. Because of the pronounced intersubject variability in the extent of the sirolimus-diltiazem interaction, whole blood sirolimus concentrations should be monitored closely in patients treated with the two drugs.

Population pharmacokinetics of sirolimus in kidney transplant patients.

OBJECTIVE: To characterize the dose-related pharmacokinetics of the immunosuppressant agent sirolimus (formerly rapamycin) in kidney transplant patients by use of two-stage and nonlinear mixed-effect model population methods. METHODS: Patients (n = 36) from three centers (Germany, the United Kingdom, and Sweden) who received steady-state oral doses of cyclosporine (ciclosporin) were assessed after single oral administration of sirolimus at doses of 3, 5, 10, and 15 mg/m2. Plasma and whole blood sirolimus samples were analyzed by a high-performance liquid chromatographic/mass spectrophotometric method. Simultaneous fitting used biexponential functions with intercept/slope or clearance/volume terms, as well as first-order absorption (ka) and a lag-time. RESULTS: The nonlinear mixed-effect model method (P-Pharm) provided a better characterization of sirolimus kinetics, especially for the absorption and distribution phases where fewer data were available per patient. Sirolimus distribution between whole blood and plasma was concentration-independent, with a mean blood/plasma ratio (coefficient of variation) of 30.9 (48.5%). Elimination was not influenced by dose, as shown by estimates of the terminal half-life of 63 hours (27.5%) and apparent oral blood clearance of 8.9 L/hr (38.2%). Sirolimus distribution parameters were influenced by body weight and surface area. Sirolimus was rapidly absorbed, as shown by the absorption lag-time of 0.27 hour (35.1%), and ka of 2.77 hr-1 (48.4%). The concomitant administration of sirolimus and cyclosporine did not reveal any pharmacokinetic interactions. CONCLUSION: This report provides an initial population pharmacokinetics of sirolimus in kidney transplant recipients receiving cyclosporine concurrently. Sirolimus blood and plasma pharmacokinetics were biexponential and linear for doses from 3 to 15 mg/m2. No pharmacokinetic interaction was found between sirolimus and cyclosporine.

Central venous catheter insertion by femoral vein: safety and effectiveness for the pediatric patient.

Prospective observations made during surveillance of routine central venous catheterizations for hemodynamic monitoring were evaluated to determine the safety and effectiveness of femoral insertion of central venous catheters and to demonstrate the feasibility of teaching pediatric residents to perform this procedure. During a 19-month period of observation, 29 pediatric patients requiring a central venous catheter underwent attempted percutaneous femoral vein catheterization. Femoral catheterization was successful in 86% of patients attempted, and insertions by pediatric residents were successful in 68% of patients attempted. Arterial puncture was the only significant complication of insertion, occurring in 14%, and was not associated with adverse sequelae. During 33 months of observations, complications of indwelling femoral central venous catheters did not significantly exceed the frequency for internal and external jugular, subclavian, and antecubital central venous catheters. During more than 4 years of observation, the significant complications associated with indwelling femoral central venous catheters were swelling of the leg or documented thrombosis in 11% of 74 critically ill patients. These observations indicate safety and effectiveness of femoral central venous catheters which compares favorably to central venous catheter insertion by other routes. In contrast to previous reports of central venous catheter insertion via subclavian and internal jugular veins, we observed no cardiorespiratory compromise as a result of femoral central venous catheter complications. Skill in this technique is a feasible educational goal for pediatric residents.

Immune components in porcine mammary secretions.

Immune components present in mammary secretions are reviewed. In swine, the histological structure of the placenta prevents in utero transfer of immunoglobulins and mammary secretions are the sole source of maternal antibody for the neonate. In addition to immunoglobulins, porcine mammary secretions contain significant numbers of maternal cells of various types that may contribute to neonatal immunity, including phagocytes (neutrophils and macrophages), lymphocytes (B and T cells), and epithelial cells. Immunomodulating and/or antimicrobial substances, including lactoferrin, lysozyme, lactoperoxidase, and cytokines, are also present in mammary secretions and may contribute to the protection of the neonate. While the role of immunoglobulins in mammary secretions is well understood, the contribution of cellular components and non-specific immune factors to neonatal immunity remains to be defined.

Antibody-dependent enhancement (ADE) of porcine reproductive and respiratory syndrome virus (PRRSV) infection in pigs.

Infection of porcine alveolar macrophages by the porcine reproductive and respiratory syndrome virus (PRRSV) was significantly enhanced in vitro by antibody raised against the PRRSV isolate ISU-P (p < 0.01). Increased yields and infection rates were highly correlated (r = 0.95) and the ratio of yield to infection rate was greater than 1.4, suggesting that more than one mechanism was responsible for enhanced infection. Antibody-dependent enhancement (ADE) of infection was also demonstrated in vivo using a completely randomized block design (n = 16). The mean level and duration of viremia were greater (p < 0.05) in pigs injected with subneutralizing amounts of PRRSV-specific IgG prior to virus challenge than in control pigs injected with normal IgG. In contrast, virus replication was significantly (p < 0.01) inhibited in pigs with neutralizing antibody titers of 4 log2. The period of time that subneutralizing levels of antibody can persist and contribute to ADE of PRRSV infection was estimated in 4 pigs injected with PRRSV-specific IgG to yield an initial neutralizing antibody titer of 3.8 log2. Neutralizing activity declined to undetectable levels by day 37 postinjection (PI). ADE activity was first detected in undiluted sera on day 20 PI and persisted through day 62 PI. Western immunoblot analysis of sera collected between days 37 and 62 PI detected antibodies specific for the 15-kDa nucleocapsid and 26-kDa glycosylated envelope proteins. These results strongly suggest that ADE has the potential to contribute to the pathogenesis of PRRSV infection and is mediated by antibody specific for the 26-kDa envelope protein.

Characterization of immune response of young pigs to porcine circovirus type 2 infection.

A longitudinal study was conducted to characterize the immune response of young swine to infection with porcine circovirus type 2 (PCV-2). Five 8-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally and intramuscularly with a field isolate of PCV-2 at a concentration of 10(4) TCID50/mL. Along with monitoring for clinical signs and viremia, serum samples were collected from all pigs at day 0 and thereafter every 7 days postinoculation (PI) until the termination of the study on day 35 PI. No clinical signs were observed in any of the animals during the study period. In all pigs, PCV-2 was detected by polymerase chain reaction (PCR) in serum samples collected on days 7, 14, and 21 PI. Viral DNA and antigens were detected by in situ hybridization and immunohistochemistry in tonsil, spleen, medial iliac lymph nodes, and ileum collected from each pig at the end of the study. Collectively, naïve young swine were shown to be susceptible to PCV-2. Virus-specific antibody was detected by an indirect fluorescent antibody (IFA) assay on day 14 PI, but virus-neutralizing antibody was not detected until day 28 PI. As neutralizing antibodies developed, cross-reactivity with PCV type 1 (PCV-1) also developed on the IFA test. Western immunoblot analysis revealed three PCV-2 proteins with molecular masses of 28 kd, 28.5 kd, and 35 kd. The 35-kd protein was also demonstrated in PCV-1, suggesting that this protein induced the cross-reactivity between PCV types 1 and 2. Antibody to the 28-kd protein was detected on day 14 PI and later, indicating that this protein was the most immunogenic. Because of its immunogenicity and specificity to PCV-2, and 28-kd protein might provide the antigenic basis for the development of diagnostic tests for detection of PCV-2 antibody.

In vitro modulation of human neutrophil superoxide anion generation by various calcium channel antagonists used in ischemia-reperfusion resuscitation.

Generation of toxic oxygen species by activated polymorphonuclear leukocytes (PMNs) may represent an important mechanism of ischemia-reperfusion injury. Concentration-response data concerning inhibition of superoxide anion (O2-) generation by NADPH oxidoreductase (NADPH OR) from isolated human PMN were generated for five calcium channel antagonists commonly utilized in ischemia-reperfusion investigational therapeutics. Regression analysis derived IC50 values for verapamil, nimodipine, nicardipine and lidoflazine were 45, 20, 12 and 7 microM respectively. Inhibition of the extent of reaction at 5 min paralleled inhibition of initial velocity. No inhibition by flunarizine was noted at concentrations less than or equal to 25 microM (where it did not alter reaction mixture composition). Only nicardipine demonstrated a significant concentration-response effect relative to prolonging lag time preceding O2- synthesis. Inhibition appeared at least partially reversible for all five agents. Neither PMN activation/desensitization, free-radical scavenging, nor PMN cytotoxicity appeared to be involved in the inhibition of PMN O2- synthesis by these agents. Ca2+ antagonist inhibition of PMN NADPH OR appears to involve more than simple inhibition of Ca2+ flux across the PMN plasma membrane. Direct inhibition of the intracellular events involved in the activation and/or activity of NADPH OR may be operative.

Attenuation of endotoxin-induced pathophysiology by a new potent PAF receptor antagonist.

The role of platelet-activating factor (PAF) as a mediator of endotoxin-induced pathophysiology has been studied in several animal models with conflicting results. We evaluated the effect of a new, potent, and specific PAF receptor antagonist, ABT-299 (Abbott Laboratories) against endotoxin (lipopolysaccharide; LPS)-induced cardiopulmonary dysfunction in a porcine model. In initial experiments, the potency of ABT-299 was confirmed in vitro by its ability to inhibit PAF-induced porcine platelet aggregation at an IC50 of .047 +/- .01 microM, and in vivo by the ability of low doses (.12 mg/kg + .03 mg/kg/h) to block the cardiopulmonary pathologic response to exogenous PAF infusion. To evaluate the effect of ABT-299 administration during endotoxemia, pigs were randomly assigned to one of three groups: controls (n = 7), LPS (n = 9), or ABT-299 + LPS (n =7). ABT-299 was given at 1.0 mg/kg from -0.5 to 0 h plus .3 mg/kg/h from 0 to 6 h. LPS was given at .5 micrograms/kg/hr from 0 to 6 h. ABT-299 reduced the early LPS-induced fall in cardiac index and stroke volume, pulmonary hypertension and vasoconstriction, bronchoconstriction, and hypoxemia. Administration of LPS resulted in 44% mortality (before 6 h), which was blocked by ABT-299. Results with this antagonist indicate that PAF contributes to endotoxin-induced cardiopulmonary dysfunction in the pig, and is associated with mortality in this model.

The therapeutic potential of nitric oxide in lung transplantation.

Endogenously produced oxides of nitrogen appear to play important roles in tissue and organ homeostasis. Endogenous production of nitric oxide, which can be altered in response to various stimuli, can modulate vascular tone, oxyradical cascades, cell adhesion, and other aspects of inflammation. Because exogenously administered (inhaled) nitric oxide can mediate pulmonary vasodilatation and improve pulmonary function in some patients with lung injury, treatment of lung allograft recipients with inhaled nitric oxide may ameliorate ischemia-reperfusion injury, thereby improving perioperative pulmonary function and diminishing ventilatory support requirements. This review examines the biology of nitric oxide and present data that support its potential therapeutic effects for lung transplant recipients.

Function and composition of pulmonary surfactant and surfactant-derived fatty acid profiles are altered in young adults with cystic fibrosis.

STUDY OBJECTIVES: To determine whether chronic lung inflammation in young adult patients with cystic fibrosis (CF) alters the composition and function of surfactant and surfactant components in bronchoalveolar secretions. DESIGN: A prospective, descriptive study. SETTING: An adult CF center in a tertiary health-care center. PARTICIPANTS: Thirteen normal volunteer (NV) subjects recruited via local advertising and 15 CF patients recruited from the CF center. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: We performed BAL and measured surfactant-associated protein A (SP-A) via enzyme-linked immunosorbent assay in BAL fluid (BALF), and quantitated total phospholipid, phospholipid subclass, and fatty acid subclass content of extracted BALF. We also determined the protein and phospholipid content, SP-A content, and functional characteristics of surfactant isolated from BALF via high-speed centrifugation. The phospholipid-to-protein ratio (milligram/milligram) of surfactant isolated by centrifugation (mean +/- SEM) was 1.01 +/- 0.07 for NV subjects and 2.62 +/- 0.42 for CF patients (p = 0.0001). Minimal surface tension was < 1 dyne.s.cm(-5) in all samples from NV subjects, but 21.9 +/- 0.73 dyne.s.cm(-5) for surfactant from CF patients. Immunoblotting of isolated surfactant revealed a marked decrease in SP-A for CF patients, compared to NV subjects. However, mean concentrations of SP-A in BALF that had not been subjected to high-speed centrifugation to isolate surfactant were not significantly different for CF patients (4.7 +/- 0.8 microgram/mL) vs NV subjects (4.6 +/- 0.2 microgram/mL). Additionally, phospholipid-to-protein ratios (0.32 +/- 0.04 for NV subjects vs 0.10 +/- 0.02 for CF patients; p < 0.0001) in extracted uncentrifuged BALF, and SP-A-to-protein ratios (microgram/milligram) in BALF were significantly depressed (74 +/- 8 for NV subjects vs 16 +/- 3 for CF patients; p < 0.0001). The phospholipid and fatty acid subclass profiles of extracted CF BALF vs NV BALF revealed a decreased mean phosphatidylcholine-to-sphingomyelin ratio (20.7 +/- 10.0 vs 55.2 +/- 8.7; p = 0.002), increased oleic acid content (12.1 +/- 2.3 nmol/mL vs 3.2 +/- 0.9 nmol/mL; p < 0.01), and increased arachidonic acid content (2.2 +/- 0.5 nmol/mL vs 0.6 +/- 0.3 nmol/mL; p < 0.05) for CF patients. CONCLUSIONS: Altered phospholipid-to-protein ratios and phospholipid subclasses, altered surfactant-derived fatty acid profiles, high minimal surface tension, and decreased association of SP-A with lipid components of isolated surfactant indicate that surfactant components are considerably altered and dysfunctional in lower respiratory tract secretions of CF patients. Surfactant composition and function are altered in CF, and the pattern of phospholipid and surfactant-derived fatty acid subclass alterations in CF are characteristic of ongoing lung injury and may depress surfactant function.