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)-1beta 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-1beta 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-1beta 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.

PRO 2000 elicits a decline in genital tract immune mediators without compromising intrinsic antimicrobial activity.

OBJECTIVE: Vaginal microbicides should protect against infection without disrupting the mucosal environment or its mediators of host defense. The objective of this study was to examine the effect of 14 daily applications of 0.5% PRO 2000 or placebo gel on mediators of mucosal immunity and intrinsic antimicrobial activity. DESIGN AND METHODS: A randomized, prospective, double-blind, placebo-controlled study was conducted among 24 healthy, abstinent women. Levels of cytokines, chemokines, defensins, and other protective factors and intrinsic antimicrobial activity were determined in cervicovaginal lavage samples collected on study days 0, 7, 14, and 21. RESULTS: No increase in pro-inflammatory cytokines was observed. Rather cytokines and protective factors including interleukin (IL)-1 receptor antagonist, immunoglobulins and human beta-defensin 2 were lower in the drug compared with the placebo group. All of the mediators returned towards baseline on day 21. Women who were cycling had lower levels of most proteins on study days 7 and/or 14 compared with women on oral contraceptives; however, the magnitude of decline was greater in women who received PRO 2000 compared with placebo gel. The reduction in protective factors was not associated with a loss in the intrinsic anti-viral (HIV or herpes simplex virus) activity or anti-bacterial activity (Escherichia coli or Staphylococcus aureus). CONCLUSION: In contrast to experience with nonoxynol-9, PRO 2000 did not trigger an inflammatory response in cervicovaginal secretions. There was a modest reduction in mucosal immune mediators, but this loss was not associated with a reduction in intrinsic antimicrobial activity.

Amphipathic DNA polymers are candidate vaginal microbicides and block herpes simplex virus binding, entry and viral gene expression.

BACKGROUND: Amphipathic DNA polymers are promising therapies for the prevention of HIV and genital herpes infections. Recent studies on a panel of such compounds indicated potent activity against HIV binding and entry. This current study was conducted to explore the anti-herpes simplex virus (HSV) activity of the same panel of compounds and to determine their mechanism of activity. METHODS: The anti-HSV activity of a 40-nucleotide degenerate polymer (REP 9), a 40-nucleotide polycytidine amphipathic DNA polymer (REP 9C) and an analogue lacking amphipathic activity (Randomer 3) were compared in plaque reduction assays in the absence or presence of human genital tract secretions; the mechanisms of anti-HSV activity were explored. RESULTS: REP 9 inhibited HSV infection 10,000-fold, whereas Randomer 3 displayed no anti-HSV activity. The antiviral activity was independent of sequence but was dependent on size: the most potent activity was observed for analogues of 40 nucleotides in length. Mechanistic studies indicated that REP 9 and REP 9C blocked HSV-2 binding and entry, were active when added post-entry, inhibited viral gene expression and blocked HSV-induced apoptosis. Confocal microscopy studies showed rapid delivery of fluorescently tagged REP 9 and REP 9C into human epithelial cells, and delivery was significantly greater in infected cells as compared with uninfected cells. REP 9 exhibited no cytotoxicity and retained anti-HSV activity in the presence of cervicovaginal secretions and when virus was introduced in seminal plasma. CONCLUSIONS: REP 9 and REP 9C represent a novel class of antiviral agents that act by multiple mechanisms. These compounds warrant further development for systemic or topical delivery for the prevention and treatment of HIV and HSV.

Ley matemática exponencial aplicada a la evaluación de la dinámica cardíaca en 18 horas / Mathematical exponential law applied to the evaluation of the cardiac dynamics in 18 hours

Antecedentes: la teoría de los sistemas dinámicos estudia la evolución de los sistemas. Mediante esta teoría y la geometría fractal se desarrolló una ley matemática de ayuda diagnóstica a los sistemas dinámicos cardiacos, que permite diferenciar entre normalidad y enfermedad, y la evolución entre los dos estados. Objetivo: confirmar la capacidad diagnóstica de la ley matemática exponencial desarrollada inicialmente para dinámicas cardiacas en 21 horas, para dinámicas evaluadas en 18 horas. Método: se tomaron 400 registros electrocardiográficos, 80 de dinámicas normales y 320 de dinámicas anormales. Se generó una sucesión pseudoaleatoria con el número de latidos/hora y las frecuencias máximas y mínimas cada hora; luego, se construyó el atractor de cada dinámica, para así calcular los espacios de ocupación y la dimensión fractal. Finalmente, se estableció el diagnóstico físico-matemático en 18 y 21 horas y se comparó con el diagnóstico clínico tomado como Gold Standard, obteniendo valores de sensibilidad, especificidad y coeficiente Kappa. Resultados: se encontraron valores de ocupación espacial en la rejilla Kp para normalidad entre 236 y 368 y para estados patológicos entre 22 y 189, lo que permitió diferenciar entre normalidad, enfermedad, y estados de evolución hacia la enfermedad en 18 horas. Se obtuvieron valores de sensibilidad y especificidad del 100 por ciento y coeficiente Kappa igual a 1. Conclusión: la ley matemática permitió dictaminar diagnósticos reduciendo el tiempo de evaluación a 18 horas confirmando así su aplicabilidad clínica(AU)

Dynamical systems theory aims to study the evolution of systems. With this theory and fractal geometry, it was developed a mathematical law of diagnostic utility in cardiac dynamical systems that may differentiate normality from disease and evolution between these two states. Objective: To confirm the diagnostic capacity of the exponential mathematical law initially developed for cardiac dynamics in 21 hours, for dynamics evaluated in 18 hours. Method: There were taken 400 electrocardiographic records, 80 from normal dynamics and 320 from abnormal dynamics. A pseudorandom sequence was generated with the number of beats per hour and the maximum and minimum frequencies each hour; then, the attractors were built for each dynamic, in order to calculate the space occupation and the fractal dimension. Finally the physical and mathematical diagnosis in 18 and 21 hours was established, and compared to clinical diagnosis taken as Gold Standard, obtaining values of sensitivity, specificity and Kappa coefficient. Results: There were found values for spatial occupation in the Kp grid between 236 and 368 for normal cases, and between 22 and 189 for pathological states, which allowed distinguish normality from disease and states of progression to disease in 18 hours. There were obtained values for sensitivity and specificity of 100 percent and a Kappa coefficient equal to 1. Conclusion: The mathematical law allowed to stablish diagnostics by reducing the evaluation time to 18 hours confirming its clinical applicability(AU)

Sistemas dinámicos aplicados a la dinámica cardiaca en 18 horas mediante una ley matemática exponencial / Dynamical systems applied to cardiac dynamics in 18 hours by an exponential mathematical law

Objetivo: confirmar la capacidad diagnostica de una ley exponencial de ayuda diagnostica, desarrollada para 21 horas con base en la teoría de sistemas dinámicos junto con la geometría fractal, en evaluaciones realizadas en 18 horas, mediante un estudio de concordancia diagnóstica con respecto al Gold estándar. Materiales y métodos:se realizó un estudio de 60 dinámicas cardiacas evaluadas en Holter y registros electrocardiográficos continuos, de los cuales 15 provienen de sujetos normales y 45 de pacientes con diferentes tipos de patologías cardiacas. Se desarrollaron simulaciones teóricas de la secuencia de las frecuencias cardiacas durante 18 horas, y se construyeron atractores. Se calculó la dimensión fractal de cada atractor y su ocupación espacial en el espacio generalizado de Box-Counting. Se determinó el diagnóstico matemático a partir de la ley y se calculó sensibilidad, especificidad y coeficiente Kappa. Resultados: se encontraron valores para normalidad entre 219 y 373 en la rejilla Kp y entre 49 y 70 para enfermedad aguda, evidenciando que el método permite diferenciar normalidad de enfermedad aguda mediante la ocupación espacial de los atractores valorados desde la ley matemática en 18 horas. Se encontraron valores de sensibilidad y especificidad del 100% y un coeficiente Kappa de 1 al comparar el diagnóstico físico-matemático con el Gold estándar. Conclusión: la ley exponencial de los sistemas dinámicos cardiacos aplicada en 18 horas es útil como herramientade ayuda diagnóstica, permitiendo cuantificar casos normales, en evolución hacia la enfermedad y en estados agudos...(AU)

Objective: to confirm the diagnostic capacity of an exponential diagnostic aid law, developed for 21 hours, based on the theory of dynamic systems along with the fractal geometry, in evaluations carried out in 18 hours, through a diagnostic concordance study with respect to the gold standard. Materials and methods: a study of 60 cardiac dynamics evaluated in Holter and continuous electrocardiographic recordings was performed, of which 15 come from normal subjects and 45 from patients with different types of heart diseases. Theoretical simulations of the sequence of heart rates were developed for 18 hours, and were built attractors. The fractal dimension of each attractor and its spatial occupation in the generalized Box-Counting space was calculated.Mathematical diagnosis is determined from the law and sensitivity, specificity and Kappa coefficient was calculated. Results: values normally found between 219 and 373 in the Kp grid and between 49 and 70 for acute disease, showing that the method can differentiate normal acute disease by spatial occupation of attractors assessed from the mathematical law in 18 hours. Sensitivity and specificity of 100% and a Kappa coefficient of 1 were found by comparing the physical-mathematical diagnosis with the Gold standard. Conclusion: the exponential mathematical law of cardiac dynamic systems applied in 18 hours is useful as a diagnostic aid tool, allowing quantifying normal cases, evolving towards disease and acute conditions...(AU)

Female genital tract secretions inhibit herpes simplex virus infection: correlation with soluble mucosal immune mediators and impact of hormonal contraception.

PROBLEM: Female genital tract secretions inhibit herpes simplex virus (HSV) infection, however, the intra- and inter-subject variability, contribution of specific mediators, and impact of reproductive hormones have not been defined. METHOD: of study Cervicovaginal lavage (CVL) (n = 89) obtained from nine cyclers and seven women on hormonal contraception (HC), who completed between three and eight weekly visits, were examined for anti-herpes simplex virus activity and concentrations of mediators. RESULTS: The CVL inhibited HSV infection by a mean value of approximately 57% during the follicular or luteal phase, but only by 36% in hormonal contraceptive users. Human neutrophil peptides 1-3 (HNP1-3) (P = 0.03), IL-8 (P = 0.003), lactoferrin (P = 0.005), lysozyme (P = 0.003), IgA (P = 0.002), and IgG (P = 0.02) correlated with antiviral activity. Intra-subject and inter-subject variability was observed, suggesting that factors other than hormones contribute to innate defense. CONCLUSION: Endogenous antimicrobial activity may provide a biomarker of healthy mucosal immunity and may be reduced in the setting of HC. However, larger prospective studies are needed.

Disruption of tight junctions by cellulose sulfate facilitates HIV infection: model of microbicide safety.

BACKGROUND: The lack of biomarkers that are predictive of safety is a critical gap in the development of microbicides. The present experiments were designed to evaluate the predictive value of in vitro models of microbicide safety. METHODS: Changes in the epithelial barrier were evaluated by measuring transepithelial electrical resistance (TER) after exposure of human epithelial cells to candidate microbicides in a dual-chamber system. The significance of observed changes was addressed by challenging cultures with human immunodeficiency virus (HIV) and measuring the ability of virus to cross the epithelium and infect target T cells cultured in the lower chamber. RESULTS: Exposure to nonoxynol-9 (N-9) or cellulose sulfate (CS), but not 9-[2-(phosphonomethoxy)propyl]adenine (also referred to as tenofovir) or PRO2000, resulted in a rapid and sustained reduction in TER and a marked increase in HIV infection of T cells cultured in the lower chamber. Moreover, CS triggered nuclear factor kappaB activation in peripheral blood mononuclear cells and increased HIV replication in chronically infected U1 cells. CONCLUSIONS: Epithelial barrier disruption and enhanced viral replication may have contributed to the increased risk of HIV acquisition observed in phase 3 trials of N-9 and CS. Expansion of in vitro safety testing to include these models would provide a more stringent preclinical assessment of microbicide safety and may prove to be more predictive of clinical outcomes.

Seminal plasma reduces the effectiveness of topical polyanionic microbicides.

The objective of this study was to test the activity of microbicides against herpes simplex virus type 2 (HSV-2) introduced in seminal plasma. We found that seminal plasma interfered with the activity of PRO 2000 and of cellulose sulfate, increasing by 100-fold the concentration of drug required to inhibit 90% of viral plaque formation. Seminal plasma competitively inhibited binding of the microbicides to the HSV-2 envelope. Most of the interference was found in a high molecular-weight fraction; tandem mass spectrometry identified the proteins as fibronectin-1 and lactoferrin. In a murine model, the interference translated in vivo into a loss in protection. We found that 2% PRO 2000 gel protected 100% of mice challenged intravaginally with HSV-2 introduced in PBS, whereas only 55% of mice were protected if virus was introduced in seminal plasma (P=.0007, log rank test). If these findings are reflective of what occurs in humans, modifications to microbicides to ensure that they retain activity in the presence of seminal plasma are indicated.

Molecular umbrellas: a novel class of candidate topical microbicides to prevent human immunodeficiency virus and herpes simplex virus infections.

Molecular umbrella compounds may function as novel topical microbicides to prevent human immunodeficiency virus (HIV) and herpes simplex virus (HSV) infections. In a preliminary structure-activity investigation, one umbrella compound, designated Spm8CHAS, was identified which inhibited both HIV and HSV infections with no cellular toxicity. The objectives of the current studies were to define its spectrum of antiviral activity, characterize its mechanism of action, and explore the possibility of combining Spm8CHAS with HIV-specific reverse transcriptase inhibitors. Spm8CHAS inhibited infections by laboratory and clinical R5 and X4 clade B and clade C HIV strains in cell culture. Ectocervical tissue explants exposed to HIV-1(BaL) in the presence of Spm8CHAS were completely protected (50% inhibitory concentration [IC(50)], 13.6 microg/ml), and transfer of virus to target T cells via migratory cells was abolished (IC(50), 3.8 microg/ml). Spm8CHAS inhibited HSV-2 infection of epithelial cells 10,000-fold if present throughout the infection. Notably, adding Spm8CHAS to cultures following HSV entry significantly reduced viral infection, indicating that the drug also acts postentry. Subsequent studies indicated that Spm8CHAS blocks cell-to-cell spread of HSV. Confocal microscopy using a fluorescently labeled analog of Spm8CHAS demonstrated that this conjugate crosses the plasma cell membrane and is transported to the nucleus. Combinations of Spm8CHAS with UC-781 or 9-[R-2-(phosphonylmethoxy)propyl] adenine monohydrate in vitro exhibited additive anti-HIV activity with preserved anti-HSV activity. The abilities of Spm8CHAS to inhibit primary isolates of HIV, block HSV infection postentry, and cross cell membranes support the development of a combination microbicide containing Spm8CHAS with an HIV-specific reverse transcriptase inhibitor to prevent both HIV and HSV infections by multiple mechanisms.

A comprehensive murine model to evaluate topical vaginal microbicides: mucosal inflammation and susceptibility to genital herpes as surrogate markers of safety.

A critical gap in microbicide development is the absence of surrogate safety markers. The objective of the present study was to develop a murine model to examine the mucosal response to microbicides and to assess the functional implication of observed changes. Mice received 14 daily intravaginal doses of nonoxynol-9, PRO 2000, or placebo gel. Nonoxynol-9 induced an inflammatory response characterized by increases in levels of cytokines and chemokines, recruitment of neutrophils and monocytes into the genital tract, and activation of the transcription factors NF- kappa B and activator protein-1. Minimal inflammation was observed in response to 2% PRO 2000. Nonoxynol-9-treated mice were significantly more susceptible to challenge with a low dose of herpes simplex virus type 2; the response of PRO 2000-treated mice was similar to the response to placebo. These findings suggest that PRO 2000 has little deleterious effect on mucosal immunity and, if validated by clinical experiences, support the inclusion of this model in the preclinical evaluation of future candidate microbicides.