Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: A multicenter study

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1β and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1β and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal−Wallis analysis of variance with Dunn’s multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences (P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1β determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface

Determinations of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma: Reassessment of Parameters Affecting Assay Outcome

Appropriate interpretation of HIV-1 RNA levels requires an understanding of differences in test results due to multiple factors, which include assay and biological variation as well as specimen-handling conditions. Multiple investigations with diverse patient populations and assays have suggested that the contributions of technical and biological variations to RNA levels were quite consistent and predictable and in the range of 0.3 to 0.6 log10 RNA copies/ml. To date, all of the studies that have assessed variations in the levels of HIV-1 RNA measured have been limited primarily to isolates of the B clade; thus, what is lacking is knowledge of the degree to which the clade subtype influences assay variation and whether the biological variation observed with the clade B subtype is consistent for other clades. The major finding from the workshop was the unexpected stability of the HIV-1 RNA collected and stored under a variety of specimen handling conditions. HIV-1 RNA was shown to be relatively stable in whole blood, plasma, and serum, with the greatest stability being in plasma. Separated plasma was found to have stable titers even after storage at room temperature for 24 to 48 h and repeated freeze-thaw cycles. Within the constraints of the studies described here, the potential differences in RNA levels due to various specimen- handling conditions were not large (10 to 20% due to the anticoagulant type used in the collection tube [30 to 80% if serum rather than plasma is used], 10 to 30% due to time at RT prior to processing within 24 h, 30 to 80% due to the use of a storage temperature of -20 or -80°C). Thus, the anticipated RNA levels for nonideally collected and processed plasma specimens may be only about 130% (0.11 log10) less than those for plasma specimens collected and processed ideally (assuming that these differences are additive). This 130% difference is relatively small compared to the potential total average standard deviation of up to about 400% or 0.6 log10 RNA copies/ml due to intra- and interassay (both 0.1 to 0.2 log10) and biological (0.1 to 0.2 log10) RNA copies/ml factors. On the basis of these findings, workshop participants concluded that retrospective studies, including those which have used sera or heparinized samples, should show biological comparability to studies performed under ideal conditions, and thus both retrospective and prospective studies are useful in providing an understanding of the role of HIV-1 RNA levels in blood in transmission and disease progression. However, for prospectively designed studies, workshop participants recommended that blood for quantitative HIV-1 RNA testing ideally be collected in tubes containing EDTA, processed within 6 h of collection (but up to 24 h is still acceptable), and then stored at -80°C until assayed. Novel methodological approaches which could be useful in diagnosing and quantitating viral load in developing countries were also described, i.e., the use of DPSs, or in other body fluids such as cervical-vaginal secretions, i.e., sno-strip wicks. Finally, workshop participants determined what laboratory evaluations, including assays of HIV-1 RNA levels, with blood samples should be a priority in pediatric cohort studies while acknowledging that this ultimately depends on the study question being asked. Recommendations concerning specimen handling were then developed for international and domestic studies that use assays for detection of HIV-1 RNA. The findings reported herein underscore the continued need for the exchange of information among investigation and industry with the aim of elucidating the technological parameters that influence the assays used to evaluate HIV-1 disease and therapeutic interventions. Only by understanding the factors that affect assay outcome can we appropriately discern their value and use in clinical studies and for patient management

Illumination of Parainfluenza Virus Infection and Transmission in Living Animals Reveals a Tissue-Specific Dichotomy

The parainfluenza viruses (PIVs) are highly contagious respiratory paramyxoviruses and a leading cause of lower respiratory tract (LRT) disease. Since no vaccines or antivirals exist, non-pharmaceutical interventions are the only means of control for these pathogens. Here we used bioluminescence imaging to visualize the spatial and temporal progression of murine PIV1 (Sendai virus) infection in living mice after intranasal inoculation or exposure by contact. A non-attenuated luciferase reporter virus (rSeV-luc(M-F*)) that expressed high levels of luciferase yet was phenotypically similar to wild-type Sendai virus in vitro and in vivo was generated to allow visualization. After direct intranasal inoculation, we unexpectedly observed that the upper respiratory tract (URT) and trachea supported robust infection under conditions that result in little infection or pathology in the lungs including a low inoculum of virus, an attenuated virus, and strains of mice genetically resistant to lung infection. The high permissivity of the URT and trachea to infection resulted in 100% transmission to naïve contact recipients, even after low-dose (70 PFU) inoculation of genetically resistant BALB/c donor mice. The timing of transmission was consistent with the timing of high viral titers in the URT and trachea of donor animals but was independent of the levels of infection in the lungs of donors. The data therefore reveals a disconnect between transmissibility, which is associated with infection in the URT, and pathogenesis, which arises from infection in the lungs and the immune response. Natural infection after transmission was universally robust in the URT and trachea yet limited in the lungs, inducing protective immunity without weight loss even in genetically susceptible 129/SvJ mice. Overall, these results reveal a dichotomy between PIV infection in the URT and trachea versus the lungs and define a new model for studies of pathogenesis, development of live virus vaccines, and testing of antiviral therapies

Ustekinumab as Induction and Maintenance Therapy for Crohn’s Disease

BACKGROUND Ustekinumab, a monoclonal antibody to the p40 subunit of interleukin-12 and inter-leukin-23, was evaluated as an intravenous induction therapy in two populations with moderately to severely active Crohn’s disease. Ustekinumab was also evaluated as subcutaneous maintenance therapy. METHODS We randomly assigned patients to receive a single intravenous dose of ustekinumab (either 130 mg or approximately 6 mg per kilogram of body weight) or placebo in two induction trials. The UNITI-1 trial included 741 patients who met the criteria for primary or secondary nonresponse to tumor necrosis factor (TNF) antagonists or had unacceptable side effects. The UNITI-2 trial included 628 patients in whom conventional therapy failed or unacceptable side effects occurred. Patients who completed these induction trials then participated in IM-UNITI, in which the 397 patients who had a response to ustekinumab were randomly assigned to receive subcutaneous maintenance injections of 90 mg of ustekinumab (either every 8 weeks or every 12 weeks) or placebo. The primary end point for the induction trials was a clinical response at week 6 (defined as a decrease from baseline in the Crohn’s Disease Activity Index [CDAI] score of ≥100 points or a CDAI score <150). The primary end point for the maintenance trial was remission at week 44 (CDAI score <150). RESULTS The rates of response at week 6 among patients receiving intravenous ustekinumab at a dose of either 130 mg or approximately 6 mg per kilogram were significantly higher than the rates among patients receiving placebo (in UNITI-1, 34.3%, 33.7%, and 21.5%, respectively, with P≤0.003 for both comparisons with placebo; in UNITI-2, 51.7%, 55.5%, and 28.7%, respectively, with P<0.001 for both doses). In the groups receiving maintenance doses of ustekinumab every 8 weeks or every 12 weeks, 53.1% and 48.8%, respectively, were in remission at week 44, as compared with 35.9% of those receiving placebo (P = 0.005 and P = 0.04, respectively). Within each trial, adverse-event rates were similar among treatment groups. CONCLUSIONS Among patients with moderately to severely active Crohn’s disease, those receiving intravenous ustekinumab had a significantly higher rate of response than did those receiving placebo. Subcutaneous ustekinumab maintained remission in patients who had a clinical response to induction therapy. (Funded by Janssen Research and Development; ClinicalTrials.gov numbers, NCT01369329, NCT01369342, and NCT01369355.

Standardized microtiter assay for determination of syncytium-inducing phenotypes of clinical human immunodeficiency virus type 1 isolates.

A standardized assay in 96-well microtiter plates for syncytium-inducing (SI) human immunodeficiency virus type 1 phenotype detection using MT-2 cells has been developed. SI variants were found in 67% of the patients with advanced human immunodeficiency virus disease. The occurrence of the SI phenotype increased with lower CD4+ counts. There was no association between p24 antigenemia and the SI phenotype

Stabilities of free and complexed human immunodeficiency virus p24 antigens during short- and long-term storage.

By the standard p24 assay there was a 25 to 27% decrease in free p24 antigen in serum after storage at 4 degrees C over 14 days but no loss at -70 degrees C. There was no loss at either temperature by the immune complex dissociation (ICD) procedure. Furthermore, there was no significant loss of detectable p24 in serum by either the ICD or the standard p24 assay after 700 days of storage at -70 degrees C