Deriving Toxicity Values for Organophosphate Nerve Agents: A Position Paper in Support of the Procedures and Rationale for Deriving Oral RfDs for Chemical Warfare Nerve Agents

During the process of deriving oral Reference Dose (RfDs) values for chemical warfare agents, several issues arose regarding the identification of adverse effect levels and the application of uncertainty factors. For those agents that function as cholinesterase inhibitors (e.g., agents VX, GA, GB, and GD), these issues included the following: (1) Is the endpoint of blood cholinesterase inhibition an indicator of toxicity or a biomarker of exposure? (2) Can an experimental animal species be more sensitive than humans, thereby eliminating the need for an animal-to-human uncertainty factor? (3) Can the uncertainty factor that is used to extrapolate from a lowest-observed adverse-effect-level (LOAEL) to a no-observed-adverse-effect-level (NOAEL) be less than the default value of 10? (4) Can an oral RfD be derived from non-oral toxicity data? (5) Can an uncertainty factor of less than 10 be used to extrapolate from subchronic to chronic exposure (e.g., is the critical effect adequately described by the subchronic exposure data)? (6) What constitutes an adequate data base for organophosphate cholinesterase inhibitors, and what uncertainty factor should be used for an incomplete data base? Analysis of relevant data resulted in the following selection and justifications of uncertainty factors. For uncertainty associated with intraspecies extrapolation (UF_H), physiologic and pathologic conditions affecting cholinest-erase activity levels justified maintaining a UF_H of 10 for all of the nerve agents. Because available data indicated that humans tended to be more sensitive than rats regarding anticholinesterase effects, an interspecies variability (UF_A) factor of 10 was retained for agents GA, GB, and GD. For agent VX, however, the available data revealed that the domestic sheep test species exhibited sensitivity equivalent to or greater than that of humans thereby justifying a UF_A of 1. For uncertainties regarding extrapolation from subchronic-to- chronic exposure data, consideration of information on the physiology of cholinergic systems and the available toxicity data for the nerve agents and other cholinest-erase inhibitors indicated that a UF_S of 3 was justified for all four of the nerve agents. For uncertainties regarding LOAEL-to- NOAEL extrapolation (UF_L), the selection of agent GB, GD, and VX doses resulting in cholinesterase inhibition in the absence of clinical signs of toxicity (biomarker of exposure) justified this endpoint as a minimal LOAEL and a UF_L of 3. For agent GA, a NOAEL was used, and therefore no UF_L was required. The uncertainty factor for data base completeness (UF_D), was based upon several considerations. Of primary concern was the fact that chronic toxicity studies are not considered an essential component of the data base requirements for cholinesterase inhibitors because of the unlikelihood that the endpoint will change with an increase in exposure time beyond that defined as a subchronic exposure. Additionally, limited data regarding reproductive and developmental toxicity were not considered to represent critical toxicity endpoints for the nerve agents or cholinesterase inhibitors in general. Although the data base for agents GA, GB, and GD were lacking reproductive and developmental toxicity data to some extent, a UF_D of 3 was justified for the aforementioned reasons. The data base for agent VX was considered complete and a UF_D of 1 was selected for development of the RfD for this agent. A modifying factor (MF) to reflect qualitative assessment of additional uncertainties in the critical study or data base that are not addressed by uncertainty factors was limited to agent GA due to the route-to-route (i.e., intraperitoneal to oral) extrapolation and to insure the equivalent oral NOAEL was not overestimated. This article provides a brief overview of the nerve agents, information on cholinergic systems that is pertinent to deriving toxicity values for nerve agents and other organophosphate cholinesterase inhibitors, and a discussion of key issues regarding the use of uncertainty factors in RfD derivations.     

Differential Sensitivity of Children and Adults to Chemical Toxicity

Children, particularly neonates, can be biologically more sensitive to the same toxicant on a body weight basis than adults. Current understanding of the rates of maturation of metabolism and evidence from case studies indicate that human infants up to 6 months of age typically lack the capacity to detoxify and eliminate substances as readily as adults. For most chemicals, the infant physiologic systems usually produce higher blood levels for longer periods. The newborn's metabolic capacity rapidly matures and, by 6 months of age, children are usually not more sensitive than adults based on their pharmacokinetic competence. Whether children are at greater risk from chemical exposures is another question. Drawing conclusions about the ability of the U.S. Environmental Protection Agency's intraspecies (UF_H) and database (UF_D) uncertainty factors to protect children on the basis of the modest data available is challenging. However, virtually all studies available suggest that a high percentage of the population, including children, is protected by using a 10-fold UF_H or by using a 3.16-fold factor each for toxicokinetic and toxicodynamic variability. Based on specific comparisons for newborns, infants, children, adults and those with severe disease, the population protected is between 60% and 100%, with the studies in larger populations that include sensitive individuals suggesting that the value is closer to 100%. UF_D is likewise protective when used with databases that are missing substantive studies.     

Inter- and Intra-Species Extrapolation in Risk Assessment of Different Classes of Pesticides

Risk assessors routinely use the reference dose (RfD) approach for non-cancer risk assessment. In this approach, No-Observed-Adverse-Effect-Level (NOAEL) is divided by the product of uncertainty factors (UFs) and, occasionally, an additional modifying factor (MF), each usually employed by default as factors of 10. In the present investigation, kinetic and dynamic data have been used in order to reduce uncertainties when establishing exposure guidelines for examples of chemicals representing four classes of pesticides (warfarin, lindane, carbaryl and parathion). An intensive search of databases was conducted for these pesticides, and toxicokinetic and toxicodynamic parameters in inter- and intra-species were evaluated. The kinetic and dynamic subfactors were less than the proposed values of Renwick and the International Programme on Chemical Safety (IPCS). The composite factors for all the examined pesticides were less than 100. The present study indicated that in setting exposure levels it is important to incorporate kinetic and dynamic data, as they become available, rather than rely on default uncertainty factors, which are imprecise in many cases.     

U.S. EPA Reference Dose/Reference Concentration Methodology: Update on a Review of the Process

The U.S. Environmental Protection Agency (USEPA) has been reviewing several approaches to testing and risk assessment related to implementation of the Food Quality Protection Act (FQPA) and the Amendments to the Safe Drinking Water Act (SDWA), both signed into law in 1996. Based on recommendations from a review of issues related to children's health protection under these laws, the USEPA established the RfD Technical Panel to evaluate in depth the current reference dose (RfD) and reference concentration (RfC) process in general, and in particular with respect to how well children and other potentially sensitive subpopulations are protected. The RfD Technical Panel also was asked to consider scientific issues that have become of greater concern in RfD and RfC derivation (e.g., neurotoxicity, immunotoxicity), and to raise issues that should be explored or developed further for application in the RfD/RfC process. This paper provides the current status of the activities of the RfD Technical Panel. The Technical Panel has recommended that acute, short- term, and intermediate reference values should be set for chemicals, where possible, and that these values should be incorporated into the USEPA's Integrated Risk Information System (IRIS) Database. A review of current testing procedures is underway, including the endpoints assessed, life stages covered by exposure and outcome evaluation, and information that can be derived from current protocols on various durations of exposure. Data gaps identified for risk assessment include the types of pharmacokinetic data that should be collected, especially for developmental toxicity studies, the impact of aging on toxic responses occurring after early exposure as well as concomitant with exposure in old age, and information available on latency to response. The implications of the RfD Technical Panel's recommendations for various uncertainty factors are also being explored.     

Studies on the use of uncertainty factors in deriving RfDs

Risk assessment of exposure to chemicals having a toxic end point routinely uses the reference dose (RfD) approach based on uncertainty factors of 10. RfD model can be used with widely different databases. However, the quality of individual risk assessment is unequal among chemicals, often resulting in either an over‐ or underestimation of adverse health risk. The purpose of this investigation was to evaluate whether the magnitude of the 10X uncertainty factors has scientific merit against data from recent human and animal experimental studies. Although we assessed the use of uncertainty factors for representative chemicals from various classes of compounds, such as volatile organics, alcohols, gasoline components, and pesticides, we are presenting our findings for 24 chemicals.

A compilation and comparison of ratios between LOAEL/NOAEL (Lowest Observed Adverse Effect Level/No Observed Adverse Effect Level), and subchronic/chronic values were made. Although a 10X uncertainty factor is most commonly used in the risk assessment processes, an examination of the datasets which have been used to calculate RfDs suggests different values which are scientifically justifiable.     

A path forward for improved noncancer assessment: the truly adverse dose (the TAD)

Conventional noncancer assessment involves the computation of hazard quotients (HQ), simple ratios of an individual’s or a population’s estimated chemical intake, and a reference dose (the RfD) that is held to reflect a safe level of exposure. HQs?>?1, indicating RfD exceedance, have considerable uncertainty for two reasons. It is not known that the critical effect of the animal study that supports the RfD is adverse (i.e., harmful), and it is unclear at what dose higher than an RfD, truly health compromising effects are triggered. Through methodically reviewing critical studies of the U.S. EPA’s IRIS database, we investigated the efficacy of the present assessment scheme with its considerable emphasis on RfD development. Aside from noted inconsistencies in reporting, and a substantial percentage of oral noncarcinogens having a less than ideal toxicological review source, our analysis found numerous instances of absent toxicological information to fuel HQ computation, with this largely a function of the dated nature of the supporting studies. In light of our analysis, we propose a recommended replacement scheme for noncancer assessment. In place of estimating the degree to which RfDs are exceeded, we recommend determining whether Truly Adverse Doses (TADs), to be newly developed, are approached.     

Noncancer Risk Assessment: A Probabilistic Alternative to Current Practice

Based on imperfect data and theory, agencies such as the United States Environmental Protection Agency (USEPA) currently derive “reference doses” (RfDs) to guide risk managers charged with ensuring that human exposures to chemicals are below population thresholds. The RfD for a chemical is typically reported as a single number, even though it is widely acknowledged that there are significant uncertainties inherent in the derivation of this number.

In this article, the authors propose a probabilistic alternative to the EPA's method that expresses the human population threshold as a probability distribution of values (rather than a single RfD value), taking into account the major sources of scientific uncertainty in such estimates. The approach is illustrated using much of the same data that USEPA uses to justify their current RfD procedure.

Like the EPA's approach, our approach recognizes the four key extrapolations that are necessary to define the human population threshold based on animal data: animal to human, human heterogeneity, LOAEL to NOAEL, and subchronic to chronic. Rather than using available data to define point estimates of “uncertainty factors” for these extrapolations, the proposed approach uses available data to define a probability distribution of adjustment factors. These initial characterizations of uncertainty can then be refined when more robust or specific data become available for a particular chemical or class of chemicals.

Quantitative characterization of uncertainty in noncancer risk assessment will be useful to risk managers who face complex trade-offs between control costs and protection of public health. The new approach can help decision-makers understand how much extra control cost must be expended to achieve a specified increase in confidence that the human population threshold is not being exceeded.     

Animal carcinogenicity studies: 1. Poor human predictivity

The regulation of human exposure to potentially carcinogenic chemicals constitutes society's most important use of animal carcinogenicity data. Environmental contaminants of greatest concern within the USA are listed in the Environmental Protection Agency's (EPA's) Integrated Risk Information System (IRIS) chemicals database. However, of the 160 IRIS chemicals lacking even limited human exposure data but possessing animal data that had received a human carcinogenicity assessment by 1 January 2004, we found that in most cases (58.1%; 93/160), the EPA considered animal carcinogenicity data inadequate to support a classification of probable human carcinogen or non-carcinogen. For the 128 chemicals with human or animal data also assessed by the World Health Organisation's International Agency for Research on Cancer (IARC), human carcinogenicity classifications were compatible with EPA classifications only for those 17 having at least limited human data (p = 0.5896). For those 111 primarily reliant on animal data, the EPA was much more likely than the IARC to assign carcinogenicity classifications indicative of greater human risk (p < 0.0001). The IARC is a leading international authority on carcinogenicity assessments, and its significantly different human carcinogenicity classifications of identical chemicals indicate that: 1) in the absence of significant human data, the EPA is over-reliant on animal carcinogenicity data; 2) as a result, the EPA tends to over-predict carcinogenic risk; and 3) the true predictivity for human carcinogenicity of animal data is even poorer than is indicated by EPA figures alone. The EPA policy of erroneously assuming that tumours in animals are indicative of human carcinogenicity is implicated as a primary cause of these errors.     

Evaluating the Relationship Between Variance in Enzyme Expression and Toxicant Concentration in Health Risk Assessment

The replacement of default uncertainty factors with those based on chemical-specific data is a topic of interest to a growing number of government-based organizations and those in affiliated professional societies. The division of the uncertainty factors for animal-to-human extrapolation and human interindividual variance (UF_A and UF_H, respectively) into their pharmacodynamic (PD) and pharmacokinetic (PK) components invites additional and specific considerations. Where data are available, or substantiated PK models have been developed, the animal-to-human chemical-specific differences have been quantified and utilized to replace the PK component of the uncertainty factor. The increasing degree to which the genome is being characterized has stimulated additional interest in describing the impact of genetic polymorphisms on susceptibility. Frequently, proteins for which the genes are being evaluated are the group of xenobiotic metabolizing enzymes. In-depth understanding of the genetic polymorphisms of genes coding for Aldehyde dehydrogenase, glucuronyl transferase and cytochrome P450 enzyme forms has been combined with information on the bioactivation or detoxication of environmental contaminants. The preliminary conclusion of some of these considerations is that alterations in enzyme content or enzyme activity result in a de facto alteration of risk. While this may be true of the “all-or-none” genetic alterations, the impact of more subtle changes in enzyme content and/or activity are more difficult to predict. The hepatotoxicity of trichloroethylene (TCE) is dependent upon an initial, cytochrome P450 2E1 (CYP2E1)-mediated oxidative step. Variance of CYP2E1 content of human liver has been characterized from a bank of tissues from human organ donors and combined with data describing the in vitro Michaelis-Menten kinetic parameters in order to extrapolate the metabolic capacity (and variance thereof) from in vitro to in vivo and assess its impact on PK through incorporation in a physiologically based pharmacokinetic (PBPK) model. This presentation summarizes that work, and demonstrates and discusses why extremes of CYP2E1-mediated metabolic capacity in adult humans has virtually no impact on the PK metric most closely related to hepatotoxic injury from TCE exposure.     

The effect of essentiality on risk assessment

Metals are ubiquitous in the human environment, making exposure inevitable and requiring scientifically sound risk assessment methodology to ensure adequate health protection. Within this area, as part of its ongoing efforts to improve and harmonize internationally approaches to risk assessments, the International Program on Chemical Safety (IPCS) has initiated work to improve the risk assessment procedures for essential trace elements (ETEs). Zn, Cu, Se, Cr, and MO are ETEs for humans, with increasing evidence of an essential role for boron (B). For ETEs, there is a range of daily intake within which the organism maintains homeostasis. At intakes below this range, there is an increased risk from deficiency, and at intakes above the range toxicity may develop. Obviously, for ETEs one cannot assume zero exposure is without risk. Adequate health protection will require the cooperative effort of scientists in nutrition and toxicology to develop the limits of the accepted range for ETEs considering such unique properties of metals as bioavailability, speciation, interactions, and biokinetics. Based on previous work by other groups and the recommendations of an IPCS consultation, a scientific monograph will be completed by IPCS. It will examine present risk assessment methodology for ETEs, and develop scientific principles supporting use of a homeostatic model for the development of dietary reference values and tolerable daily intakes. The objective is to develop an internationally accepted methodology for assessing ETEs as part of the IPCS effort to harmonize approaches to risk assessment worldwide. A recent IPCS Task Group on Zn highlighted some of the scientific issues that require resolution to avoid an overlap of the recommended daily intake based on nutritional needs with that based on toxicity and will serve as a case study.     

Determining “safe” levels of exposure: The validity of the use of 10X safety factors

The current guideline for risk assessment of chemicals having a toxic end point routinely uses the reference dose (RfD) approach based on uncertainty factors of 10. With this method the quality of individual risk assessment varies among chemicals, often resulting in either an over‐ or under‐estimation of adverse health risk. The purpose of this investigation is to evaluate whether the magnitude of the 10X uncertainty factors have scientific merit against data from published experimental studies. A compilation and comparison of ratios between LOAEL/NOAEL (Lowest Observed Adverse Effect Level/No Observed Adverse Effect Level), subchronic/chronic, and animal/human values were made. The results of the present investigation revealed that the use of default factors could be over‐conservative or unprotective. More reasonable estimates of the risk to human health would result in a reduction of unnecessary, and expensive over‐regulation. In addition to the LOAEL to NOAEL, and subchronic to chronic ratios, the adequacy of uncertainty factors for animal to human extrapolations were examined. Although a 10‐fold uncertainty factor (UF) is most commonly used in the risk assessment process, an examination of the literature for the compounds presented here suggests that the use of different values is scientifically justifiable.     

Sepp1UF forms are N-terminal selenoprotein P truncations that have peroxidase activity when coupled with thioredoxin reductase-1

Mouse selenoprotein P (Sepp1) consists of an N-terminal domain (residues 1–239) that contains one selenocysteine (U) as residue 40 in a proposed redox-active motif (-UYLC-) and a C-terminal domain (residues 240–361) that contains nine selenocysteines. Sepp1 transports selenium from the liver to other tissues by receptor-mediated endocytosis. It also reduces oxidative stress in vivo by an unknown mechanism. A previously uncharacterized plasma form of Sepp1 is filtered in the glomerulus and taken up by renal proximal convoluted tubule (PCT) cells via megalin-mediated endocytosis. We purified Sepp1 forms from the urine of megalin^−/− mice using a monoclonal antibody to the N-terminal domain. Mass spectrometry revealed that the purified urinary Sepp1 consisted of N-terminal fragments terminating at 11 sites between residues 183 and 208. They were therefore designated Sepp1^UF. Because the N-terminal domain of Sepp1 has a thioredoxin fold, Sepp1^UF were compared with full-length Sepp1, Sepp1^Δ240–361, and Sepp1^U40S as a substrate of thioredoxin reductase-1 (TrxR1). All forms of Sepp1 except Sepp1^U40S, which contains serine in place of the selenocysteine, were TrxR1 substrates, catalyzing NADPH oxidation when coupled with H₂O₂ or tert-butylhydroperoxide as the terminal electron acceptor. These results are compatible with proteolytic cleavage freeing Sepp1^UF from full-length Sepp1, the form that has the role of selenium transport, allowing Sepp1^UF to function by itself as a peroxidase. Ultimately, plasma Sepp1^UF and small selenium-containing proteins are filtered by the glomerulus and taken up by PCT cells via megalin-mediated endocytosis, preventing loss of selenium in the urine and providing selenium for the synthesis of glutathione peroxidase-3.     

USEtox human exposure and toxicity factors for comparative assessment of toxic emissions in life cycle analysis: sensitivity to key chemical properties

Purpose

The aim of this paper is to provide science-based consensus and guidance for health effects modelling in comparative assessments based on human exposure and toxicity. This aim is achieved by (a) describing the USEtox? exposure and toxicity models representing consensus and recommended modelling practice, (b) identifying key mechanisms influencing human exposure and toxicity effects of chemical emissions, (c) extending substance coverage.

Methods

The methods section of this paper contains a detailed documentation of both the human exposure and toxic effects models of USEtox?, to determine impacts on human health per kilogram substance emitted in different compartments. These are considered as scientific consensus and therefore recommended practice for comparative toxic impact assessment. The framework of the exposure model is described in details including the modelling of each exposure pathway considered (i.e. inhalation through air, ingestion through (a) drinking water, (b) agricultural produce, (c) meat and milk, and (d) fish). The calculation of human health effect factors for cancer and non-cancer effects via ingestion and inhalation exposure respectively is described. This section also includes discussions regarding parameterisation and estimation of input data needed, including route-to-route and acute-to-chronic extrapolations.

Results and discussion

For most chemicals in USEtox?, inhalation, above-ground agricultural produce, and fish are the important exposure pathways with key driving factors being the compartment and place of emission, partitioning, degradation, bioaccumulation and bioconcentration, and dietary habits of the population. For inhalation, the population density is the key factor driving the intake, thus the importance to differentiate emissions in urban areas, except for very persistent and mobile chemicals that are taken in by the global population independently from their place of emission. The analysis of carcinogenic potency (TD₅₀) when volatile chemicals are administrated to rats and mice by both inhalation and an oral route suggests that results by one route can reasonably be used to represent another route. However, we first identify and mark as interim chemicals for which observed tumours are directly related to a given exposure route (e.g. for nasal or lung, or gastrointestinal cancers) or for which absorbed fraction by inhalation and by oral route differ greatly.

Conclusions

A documentation of the human exposure and toxicity models of USEtox? is provided, and key factors driving the human health characterisation factor are identified. Approaches are proposed to derive human toxic effect factors and expand the number of chemicals in USEtox?, primarily by extrapolating from an oral route to exposure in air (and optionally acute-to-chronic). Some exposure pathways (e.g. indoor inhalation, pesticide residues, dermal exposure) will be included in a later stage. USEtox? is applicable in various comparative toxicity impact assessments and not limited to LCA.     

Challenges of including human exposure to chemicals in food packaging as a new exposure pathway in life cycle impact assessment

Purpose

Limiting exposure to potentially toxic chemicals in food packaging can lead to environmental impact trade-offs. No available tool, however, considers trade-offs between environmental impacts of packaging systems and exposure to potentially toxic chemicals in food packaging. This study therefore explores the research needs for extending life cycle impact assessment (LCIA) to include exposure to chemicals in food packaging.

Methods

The LCIA framework for human toxicity was extended for the first time to include consumer exposure to chemicals in food packaging through the product intake fraction (PiF) metric. The related exposure pathway was added to LCIA without other modifications to the existing toxicity characterization framework used by USEtox®, i.e., effect factor derivation. The developed method was applied to a high impact polystyrene (HIPS) container case study with the functional unit of providing 1 kg of yogurt in single servings. Various exposure scenarios were considered, including an evidence-based scenario using concentration data and a migration model. Human toxicity impact scores in comparative toxic units (CTU_h) for the use stage were evaluated and then compared to human toxicity impact scores from a conventional LCIA methodology.

Results and discussion

Data allowed toxicity characterization of use stage exposure to only seven chemicals in HIPS out of fourty-four identified. Data required were the initial concentration of chemicals in food packaging, chemical mass transfer from packaging into food, and relevant toxicity information. Toxicity characterization demonstrated that the combined CTU_h for HIPS material acquisition, manufacturing, and disposal stages exceeded the toxicity scores related to consumer exposure to previously estimated concentrations of the seven characterizable chemicals in HIPS, by about two orders of magnitude. The CTU_h associated with consumer exposure became relevant when migration was above 0.1% of the European regulatory levels. Results emphasize missing data for chemical concentrations in food contact materials and a need to expand the current USEtox method for effect factor derivation (e.g., to consider endocrine disruption, mixture toxicity, background exposure, and thresholds when relevant).

Conclusions

An LCIA method was developed to include consumer exposure to chemicals in food packaging. Further study is required to assess realistic scenarios to inform decisions and policies, such as circular economy, which can lead to trade-offs between environmental impacts and potentially toxic chemicals in packaging. To apply the developed method, data regarding occurrence, concentration, and toxicity of chemicals in food packaging are needed. Revisiting the derivation of effect factors in future work could improve the interpretation of human toxicity impact scores.

     

Clinical features and serum biomarkers in HIV immune reconstitution inflammatory syndrome after cryptococcal meningitis: a prospective cohort study

Background

Although antiretroviral therapy (ART) improves survival in persons with cryptococcal meningitis (CM) and AIDS, ART frequently elicits HIV immune reconstitution inflammatory syndrome (IRIS), an exaggerated and frequently deadly inflammatory reaction that complicates recovery from immunodeficiency. The pathogenesis of IRIS is poorly understood and prediction of IRIS is not possible.

Methods and Findings

We prospectively followed 101 ART-naïve Ugandans with AIDS and recent CM for one year after initiating ART, and used Luminex multiplex assays to compare serum cytokine levels in participants who did or did not develop IRIS. IRIS occurred in 45% of participants with recent CM on ART, including 30% with central nervous system (CNS) manifestations. The median time to CM-IRIS was 8.8 wk on ART. Overall mortality on ART was 36% with IRIS and 21% without IRIS. CM-IRIS was independently associated with death (HR = 2.3, 95% CI 1.1–5.1, p = 0.04). Patients experiencing subsequent CM-IRIS had 4-fold higher median serum cryptococcal antigen (CRAG) levels pre-ART (p = 0.006). Higher pre-ART levels of interleukin (IL)-4 and IL-17 as well as lower tumor necrosis factor (TNF)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and vascular endothelial growth factor (VEGF) predicted future IRIS in multivariate analyses (area under the curve [AUC] = 0.82). An algorithm based on seven pre-ART serum biomarkers was a robust tool for stratifying high (83%), moderate (48%), and low risk (23%) for IRIS in the cohort. After ART was initiated, increasing levels of C-reactive protein (CRP), D-dimer, IL-6, IL-7, IL-13, G-CSF, or IL-1RA were associated with increasing hazard of IRIS by time-to-event analysis (each p≤0.001). At the time of IRIS onset, multiple proinflammatory cytokine responses were present, including CRP and IL-6. Mortality was predicted by pre-ART increasing IL-17, decreasing GM-CSF, and CRP level >32 mg/l (highest quartile). Pre-ART CRP level >32 mg/l alone was associated with future death (OR = 8.3, 95% CI 2.7–25.6, p<0.001).

Conclusions

Pre-ART increases in Th₁₇ and Th₂ responses (e.g., IL-17, IL-4) and lack of proinflammatory cytokine responses (e.g., TNF-α, G-CSF, GM-CSF, VEGF) predispose individuals to subsequent IRIS, perhaps as biomarkers of immune dysfunction and poor initial clearance of CRAG. Although requiring validation, these biomarkers might be an objective tool to stratify the risk of CM-IRIS and death, and could be used clinically to guide when to start ART or use prophylactic interventions. Please see later in the article for the Editors'' Summary     

Incidence,Clinical Spectrum,Risk Factors and Impact of HIV-Associated Immune Reconstitution Inflammatory Syndrome in South Africa

Background

Immune reconstitution inflammatory syndrome (IRIS) is a widely recognised complication of antiretroviral therapy (ART), but there are still limited data from resource-limited settings. Our objective was to characterize the incidence, clinical spectrum, risk factors and contribution to mortality of IRIS in two urban ART clinics in South Africa.

Methods and Findings

498 adults initiating ART in Durban, South Africa were followed prospectively for 24 weeks. IRIS diagnosis was based on consensus expert opinion, and classified by mode of presentation (paradoxical worsening of known opportunistic infection [OI] or unmasking of subclinical disease). 114 patients (22.9%) developed IRIS (36% paradoxical, 64% unmasking). Mucocutaneous conditions accounted for 68% of IRIS events, mainly folliculitis, warts, genital ulcers and herpes zoster. Tuberculosis (TB) accounted for 25% of IRIS events. 18/135 (13.3%) patients with major pre-ART OIs (e.g. TB, cryptococcosis) developed paradoxical IRIS related to the same OI. Risk factors for this type of IRIS were baseline viral load >5.5 vs. <4.5 log₁₀ (adjusted hazard ratio 7.23; 95% confidence interval 1.35–38.76) and ≤30 vs. >30 days of OI treatment prior to ART (2.66; 1.16–6.09). Unmasking IRIS related to major OIs occurred in 25/498 patients (5.0%), and risk factors for this type of IRIS were baseline C-reactive protein ≥25 vs. <25 mg/L (2.77; 1.31–5.85), haemoglobin <10 vs. >12 g/dL (3.36; 1.32–8.52), ≥10% vs. <10% weight loss prior to ART (2.31; 1.05–5.11) and mediastinal lymphadenopathy on pre-ART chest x-ray (9.15; 4.10–20.42). IRIS accounted for 6/25 (24%) deaths, 13/65 (20%) hospitalizations and 10/35 (29%) ART interruptions or discontinuations.

Conclusion

IRIS occurred in almost one quarter of patients initiating ART, and accounted for one quarter of deaths in the first 6 months. Priority strategies to reduce IRIS-associated morbidity and mortality in ART programmes include earlier ART initiation before onset of advanced immunodeficiency, improved pre-ART screening for TB and cryptococcal infection, optimization of OI therapy prior to ART initiation, more intensive clinical monitoring in initial weeks of ART, and education of health care workers and patients about IRIS.     

Cryptococcus-Related Immune Reconstitution Inflammatory Syndrome (IRIS): Pathogenesis and its Clinical Implications

This review provides an overview of Cryptococcus neoformans immunology and focuses on the pathogenesis of Cryptococcus-related paradoxical immune reconstitution inflammatory syndrome (IRIS). Cryptococcal IRIS has three phases: (1) before antiretroviral therapy (ART), with a paucity of cerebrospinal fluid (CSF) inflammation and defects in antigen clearance; (2) during initial ART immune recovery, with pro-inflammatory signaling by antigen-presenting cells without an effector response; and (3) at IRIS, a cytokine storm with a predominant type-1 helper T-cell (Th₁) interferon-gamma (IFN-γ) response. Understanding IRIS pathogenesis allows for risk stratification and customization of HIV/AIDS care. In brief, persons at high IRIS risk may benefit from enhancing microbiologic clearance by use of adjunctive agents in combination with amphotericin, prolonging initial induction therapy, and/or increasing the initial consolidation antifungal therapy dose to at least 800 mg of fluconazole daily until the 2-week CSF culture is known to be sterile. Prophylactic anti-inflammatory therapies or undue delay of ART initiation in an attempt to prevent IRIS is unwarranted and may be dangerous.     

Antisteroidogenic effects of hydrogen peroxide on rat granulosa cells

Abstract

Rat granulosa cells (GCs) were treated with human chorionic gonadotropin (hCG), 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), forskolin, phorbol 12-myristate 13-acetate (PMA), A23187 or pregnenolone in the absence or presence of hydrogen peroxide (H₂O₂). Different doses of trilostane were applied to GCs treated with steroidogenic precursors, that is, 25-hydroxy-cholesterol (25-OH-C) in the absence or presence of H₂O₂. Results showed that all of the chemicals stimulated the progesterone (PG) release from rat GCs, but the stimulatory effects were inhibited by H₂O₂ dose-dependently. 25-OH-C stimulated the PG release, which was inhibited by H₂O₂ in the presence of trilostane. H₂O₂ attenuated steroidogenic acute regulatory (StAR) protein expression, but did not alter the expression of cytochrome P450 side chain cleavage (P450scc) in Western blotting. This study indicated that H₂O₂ inhibited PG production by GCs via cAMP pathway, protein kinase C (PKC) and the activities of intracellular calcium, P450scc and StAR protein.