A method for screening groundwater vulnerability from subsurface hydrocarbon extraction practices.

This paper describes a new screening method for assessing groundwater vulnerability to pollution from hydrocarbon exploitation in the subsurface. The method can be used for various hydrocarbon energy sources, including conventional oil and gas, shale gas and oil, coal bed methane and underground coal gasification. Intrinsic vulnerability of potential receptors is assessed at any particular location by identifying possible geological pathways for contaminant transport. This is followed by an assessment of specific vulnerability which takes into account the nature of the subsurface hydrocarbon activity and driving heads. A confidence rating is attached to each parameter in the assessment to provide an indication of the confidence in the screening. Risk categories and associated confidence ratings are designed to aid in environmental decision making, regulation and management, highlighting where additional information is required. The method is demonstrated for conventional gas and proposed shale gas operations in northern England but can be adapted for use in any geological or hydrogeological setting and for other subsurface activities.

Site-specific clinical disease onset in multiple sclerosis.

BACKGROUND AND PURPOSE: Multiple sclerosis is a chronic inflammatory disorder of the central nervous system characterized by acute episodes of neurological dysfunction thought to reflect focal areas of demyelination occurring in clinically eloquent areas. These symptomatic relapses are generally considered to be random clinical events occurring without discernible pattern. The hypothesis that relapses may follow a predetermined sequence and may provide insights into underlying pathological processes was investigated. METHODS: Employing prospective clinical database data from 1482 patients who had experienced one or more consecutive relapses were analysed. Using regression analysis, site and symptom of index event were compared with those of first relapse. RESULTS: It is demonstrated that following disease ignition subsequent relapses may not be random events but dependent on characteristics of the index event. All anatomical sites were more likely to be affected in the first relapse if that site had been involved in the index event with a similar association observed when comparing by symptoms. CONCLUSION: These findings have importance in understanding the evolution of the disease and predicting individual disease progression and may aid with patient counselling and management.

Systemic complement profiling in multiple sclerosis as a biomarker of disease state.

BACKGROUND: There is increasing evidence of significant and dynamic systemic activation and upregulation of complement in multiple sclerosis (MS), which may contribute to disease pathogenesis. OBJECTIVE: We aimed to investigate the pathological role of complement in MS and the potential role for complement profiling as a biomarker of MS disease state. METHODS: Key components of the classical, alternative and terminal pathways of complement were measured in plasma and cerebrospinal fluid (CSF) of patients with MS in different clinical phases of disease and in matched controls. RESULTS: Increased plasma levels of C3 (p<0.003), C4 (p<0.001), C4a (p<0.001), C1 inhibitor (p<0.001), and factor H (p<0.001), and reduced levels of C9 (p<0.001) were observed in MS patients compared with controls. Combined profiling of these analytes produced a statistical model with a predictive value of 97% for MS and 73% for clinical relapse when combined with selected demographic data. CSF-plasma correlations suggested that source of synthesis of these components was both systemic and central. CONCLUSION: These data provide further evidence of alterations in both local and systemic expression and activation of complement in MS and suggest that complement profiling may be informative as a biomarker of MS disease, although further work is needed to determine its use in distinguishing MS from its differential.

Multiple sclerosis and COVID-19: Assessing risk perception, patient behaviors and access to disease-modifying therapies.

BACKGROUND: Following the outbreak of COVID-19, global healthcare systems have had to rapidly adapt. People with multiple sclerosis (pwMS) were required to make decisions about their individual risk and consequent work and social behaviors. This study aimed to evaluate risk perception and patterns of shielding behavior amongst pwMS at the onset of the COVID-19 pandemic and the subsequent impact on patients' employment and access to disease modifying therapies (DMTs). METHODS: Postal surveys were sent to 1690 people within a UK population-based MS cohort during the first wave of the COVID-19 pandemic. Patients were surveyed on: (i) perceived vulnerability to COVID-19; (ii) isolation behavior; (iii) interruption to DMT; (iv) employment status; (v) level of satisfaction with their current working arrangement. RESULTS: Responses were received from 1000 pwMS. Two thirds of patients reported isolating at home during the first wave of the pandemic. This behavior was associated with increased age (p<0.0001), higher disability (p<0.0001) and use of high-efficacy DMTs (p = 0.02). The majority of patients reported feeling vulnerable (82%) with perceived vulnerability associated with higher EDSS (p<0.0001) and receiving a high-efficacy DMT (p = 0.04). Clinician-defined risk was associated with shielding behavior, with those at high-risk more likely to self-isolate/shield (p<0.0001). Patients on high-efficacy DMTs were more likely to have an interruption to their treatment (50%) during the first wave of the pandemic. Most pwMS experienced a change to their working environment, and most were satisfied with the adjustments. CONCLUSION: This study highlights the risk perception, social behavioral practices and changes to treatment experienced by pwMS during the first wave of the COVID-19 pandemic in a large, well-described UK cohort. The results may help inform management of pwMS during future pandemic waves.

KIF5A and the contribution of susceptibility genotypes as a predictive biomarker for multiple sclerosis.

There is increasing interest in the development of multiple sclerosis (MS) biomarkers that reflect central nervous system tissue injury to determine prognosis. We aimed to assess the prognostic value of kinesin superfamily motor protein KIF5A in MS by measuring levels of KIF5A in cerebrospinal fluid (CSF) combined with analysis of single nucleotide polymorphisms (SNPs; rs12368653 and rs703842) located within a MS susceptibility gene locus at chromosome 12q13-14 region. Enzyme-linked immunosorbent assay was used to measure KIF5A in CSF obtained from two independent biobanks comprising non-inflammatory neurological disease controls (NINDC), clinically isolated syndrome (CIS) and MS cases. CSF KIF5A expression was significantly elevated in progressive MS cases compared with NINDCs, CIS and relapsing-remitting MS (RRMS). In addition, levels of KIF5A positively correlated with change in MS disease severity scores (EDSS, MSSS and ARMSSS), in RRMS patients who had documented disease progression at 2-year clinical follow-up. Copies of adenine risk alleles (AG/AA; rs12368653 and rs703842) corresponded with a higher proportion of individuals in relapse at the time of lumbar puncture (LP), higher use of disease-modifying therapies post LP and shorter MS duration. Our study suggests that CSF KIF5A has potential as a predictive biomarker in MS and further studies into the potential prognostic value of analysing MS susceptibility SNPs should be considered.

Interleukin 1-induced, T cell-mediated regression of immunogenic murine tumors. Requirement for an adequate level of already acquired host concomitant immunity.

Intraperitoneal injection of human rIL-1 in a dose of 0.5 microgram daily for 5 d, or 1 microgram daily for 3 d, was capable of causing complete regression of immunogenic SA1 sarcoma growing subcutaneously in syngeneic or semisyngeneic mice. Higher doses of IL-1 were not more therapeutic against the SA1 sarcoma, but needed to be given to cause complete regression of the immunogenic L5178Y lymphoma. On the other hand, the P815 mastocytoma was much less responsive to IL-1 therapy, in that it failed to undergo complete regression in response to doses of IL-1 capable of causing regression of the L5178Y lymphoma. IL-1 caused regression of the SA1 sarcoma when given on days 6-8 of tumor growth, but not when given on days 1-3. This refractoriness of a small tumor to IL-1 therapy suggests that the antitumor action of IL-1 is based on an underlying host-immune response that is not generated until after day 3 of tumor growth. Direct evidence for the participation of host immunity in IL-1-induced tumor regression was supplied by results showing that IL-1 was not therapeutic against the SA1 sarcoma growing in T cell-deficient (TXB) mice, unless these mice were first infused with Ly-2+ and L3T4+ T cells from donor mice bearing an established SA1 sarcoma. In contrast, normal T cells, or T cells from donor mice bearing a YAC-1 lymphoma, failed to provide TXB recipients with the ability to cause regression of their SA-1 sarcoma in response to IL-1 treatment. The results are in keeping with the interpretation that exogenous IL-1, by augmenting the production of tumor-sensitized T cells, converts a subtherapeutic level of host immunity to a therapeutic level. The results suggest, in addition, that IL-1 only stimulates the replication of T cells that are already engaged in the antitumor immune response.

Genetics of species differences in sailfin and shortfin mollies.

Premating reproductive isolation is a strong barrier to hybridization in natural populations, but little is known about the genetic mechanisms that allow changes in mating signals to develop and whether different components of a mating signal can evolve in concert when sexual selection favors phenotypic associations between them. In this study, we report results suggesting that changes in a behavioural trait (courtship display) and multiple phenotypically associated morphological traits (dorsal fin characters and length of the gonopodium) have contributed to divergence in mating signals used by sailfin mollies. Through the use of reciprocal F1 and backcross hybrids, we show that morphological traits important in separating sailfin from shortfin molly species have a genetic basis and are inherited in an autosomal, additive manner. We also report significant associations between the size of certain morphological traits (length of the dorsal fin and length of the gonopodium) and the tendency of males to perform courtship displays or gonopodial thrusts. In particular, higher courtship display rates were associated with increased dorsal fin length but decreased gonopodium length, characteristics most similar to sailfin species. Such phenotypic associations between different components of a mating signal suggest that selective forces can act in concert on multiple aspects of the signal, hence, promoting divergence and speciation in sailfin mollies.

Anti-EBNA-1 IgG is not a reliable marker of multiple sclerosis clinical disease activity.

BACKGROUND: Sero-epidemiological studies have demonstrated the association between multiple sclerosis (MS) and prior Epstein-Barr virus (EBV) infection. It has been hypothesized that intermittent peripheral EBV reactivation may drive continuing central inflammation. Recent investigation has shown significant differences in median serum levels of anti-EBV nuclear antigen-1 (EBNA-1) IgG between disease subgroups and positive correlation with disease activity reflected by number of Gd-enhancing lesions and T2 lesion volume. These important data have led to hopes that anti-EBNA-1 IgG may be useful as an easily accessible and effective biomarker of disease activity. METHODS: We examined the applicability of these findings in routine clinical practice, assessing a well-characterized cohort of 100 subjects (25 primary progressive, 25 stable relapsing remitting, 25 active relapsing remitting seen in acute relapse and 25 controls) for serum anti-EBNA-1 IgG using both the Liaison quantitative chemiluminescent assay and Biotest ELISA. RESULTS: We were unable to show a difference in quantitative analysis of serum anti-EBNA-1 IgG levels between disease subgroups and no correlation with phenotypic characteristics including age at onset (r = -0.17, P = 0.16), disease duration (r = 0.03, P = 0.78), EDSS (r = 0.03, P = 0.78) or MSSS (r = 0.02, P = 0.9). In addition, there was only moderate correlation between the two test methods used (intraclass correlation coefficient 0.67; 0.56-0.78) suggesting potential problems with test interpretation. CONCLUSIONS: We have been unable to determine a clinical value for serum anti-EBNA-1 IgG levels in MS or to confirm reported association with disease course and clinical disease activity.

Potency values from the local lymph node assay: application to classification, labelling and risk assessment.

Hundreds of chemicals are contact allergens but there remains a need to identify and characterise accurately skin sensitising hazards. The purpose of this review was fourfold. First, when using the local lymph node assay (LLNA), consider whether an exposure concentration (EC3 value) lower than 100% can be defined and used as a threshold criterion for classification and labelling. Second, is there any reason to revise the recommendation of a previous ECETOC Task Force regarding specific EC3 values used for sub-categorisation of substances based upon potency? Third, what recommendations can be made regarding classification and labelling of preparations under GHS? Finally, consider how to integrate LLNA data into risk assessment and provide a rationale for using concentration responses and corresponding no-effect concentrations. Although skin sensitising chemicals having high EC3 values may represent only relatively low risks to humans, it is not possible currently to define an EC3 value below 100% that would serve as an appropriate threshold for classification and labelling. The conclusion drawn from reviewing the use of distinct categories for characterising contact allergens was that the most appropriate, science-based classification of contact allergens according to potency is one in which four sub-categories are identified: 'extreme', 'strong', 'moderate' and 'weak'. Since draining lymph node cell proliferation is related causally and quantitatively to potency, LLNA EC3 values are recommended for determination of a no expected sensitisation induction level that represents the first step in quantitative risk assessment.

Maleic vinyl ether activation of murine macrophages against lung-metastasizing tumors.

A 16,500 molecular weight fraction of maleic vinyl ether (MVE-2) induced tumoristatic and tumoricidal activity in peritoneal macrophages of BALB/c and C57BL/6 mice following i.p. administration. Growth of B16 melanoma cells in vitro was inhibited up to 85% by MVE-2-activated, but not resident, peritoneal macrophages. In a tritiated thymidine release assay, B16 melanoma cells, and to a lesser extent Madison 109 lung carcinoma cells, were also sensitive to the cytolytic action of MVE-2-activated peritoneal macrophages. Administration i.v. of MVE-2 resulted in tumoristatic and tumoricidal activity in alveolar macrophages against radiolabeled B16 and Madison 109 lung carcinoma target cells. MVE-2-activated alveolar macrophages significantly inhibited L5178Y lymphoma colony formation following a 48-hr macrophage-tumor cell coincubation. BALB/c mice bearing the lung-metastasizing Madison 109 lung carcinoma footpad tumor were given MVE-2 i.v., using the same dosing regimen that induced alveolar macrophages to be tumoricidal in vitro. Significant increases in life span were observed, suggesting that the antitumor activity of MVE-2 in this tumor system may be mediated by the activation of alveolar macrophages, with a resulting decrease in metastatic growth in the lung.

Mutagenic activity of tumor-associated macrophages in Salmonella typhimurium strains TA98 and TA 100.

Suspensions of cells from a series of strain BALB/cfC3H mouse mammary tumors, and adherent and nonadherent cells from the tumors, were tested for their ability to increase the mutation rate of Salmonella typhimurium tester strains TA98 and TA100. Significant increases were seen with cells from three of four tumor lines tested on the TA98 strain and with one of four tested on the TA100 strain. The mutagenic activity was due primarily to cells in the adherent fractions which were greatly enriched for macrophages.

Cytokine mRNA profiles for isocyanates with known and unknown potential to induce respiratory sensitization.

Isocyanates are low-molecular-weight chemicals implicated in allergic asthmatic-type reactions. Identification of chemicals likely to cause asthma is difficult due to the lack of a validated test method. One hypothesis is that differential cytokine induction (Th1 versus Th2 profiles) in the draining lymph node following dermal application can be used to identify asthmagens and distinguish them from contact allergens. In this study, we compared the cytokine mRNA profiles of six chemicals: toluene diisocyanate (TDI), diphenylmethane-4,4'-diisocyanate (MDI), dicyclohexylmethane-4,4'-diisocyanate (HMDI), isophorone diisocyanate (IPDI), p-tolyl(mono)isocyanate (TMI), and meta-tetramethylene xylene diisocyanate (TMXDI). Whereas TDI and MDI are well-known respiratory sensitizers, documentation for HMDI, IPDI, TMI, and TMXDI is limited, but suggests that HMDI and IPDI may have respiratory sensitization potential in humans and TMI and TMXDI do not. Following dermal exposure of BALB/c mice, all six isocyanates induced cytokines characteristic of a Th2 response. Although LLNAs suggested that the doses chosen for the RPA were immunologically equivalent, the isocyanates tested differentiated into two groups, high responders and low responders. However, two of the low responders (TMI and TMXDI) were further tested and induced higher levels of Th2 cytokine message than dinitrochlorobenzene (not an asthmagen). Further study of these chemicals is needed to determine whether the Th2 cytokine responses observed for these low responders is predictive of asthmagenic potential or represents an insufficient signal.

A critique of the World Resources Institute's report "Pesticides and the immune system: the public health risks".

A recent World Resources Institute (WRI) report concluded that pesticides are a likely cause of immune suppression for millions of people throughout the world. The gravity of this conclusion motivated us to review the scientific evidence cited in the report. The predominant human evidence came from cross-sectional studies conducted in the former Soviet Union. These studies were difficult to evaluate due to incomplete reporting and had obvious limitations in terms of subject selection, exposure assessment,lack of quality control, statistical analysis, adequacy of the comparison group, and confounding. The toxicologic evidence was comprised mainly of acute high-dose studies in which the exposure conditions resulted in systemic toxicity. The relevance of these studies to effects at typical human exposure levels is questionable. We did not find consistent, credible evidence to support the conclusion of widespread pesticide-related immune suppression. Nonetheless, the WRI report is an important document because it focuses attention on a potentially important issue for future research and brings a substantial literature of foreign language studies to the attention of Western scientists.

Adverse effects of drugs used in the management of opportunistic infections associated with HIV infection.

Pneumocystis carinii pneumonia (PCP) is one of the most common AIDS-defining diagnoses. First-line therapy is cotrimoxazole (trimethoprim-sulfamethoxazole), despite a high incidence of toxic effects, and a greater incidence of hypersensitivity reactions among HIV-positive patients compared with the seronegative population. Alternative agents such as intravenous pentamidine, or clindamycin with primaquine, and trimethoprim with dapsone, also have a wide range of serious adverse effects, but remain treatment options. Atovaquone appears promising for the treatment of both PCP and toxoplasmosis, and has a lower reported incidence of toxicity than the alternative agents. The most toxic antifungal drugs are reserved for serious infections, such as cryptococcal meningitis. Liposomal amphotericin B has less renal toxicity than standard formulations, and exemplifies that new formulations of existing drugs, although often expensive, may have a better adverse effect profile. There are 2 different drugs currently available for cytomegalovirus (CMV) infections, ganciclovir and foscarnet. Both have a high incidence of serious adverse effects; ganciclovir mainly causes bone marrow toxicity and foscarnet leads to renal toxicity. The drugs used for mycobacterial infection (including mycobacteria as well as tuberculosis) have a wide range of adverse effects, particularly skin rashes and drug-induced hepatitis. Some of these compounds are quite new, such as rifabutin and clarithromycin, and it is important to be ever vigilant for previously unreported adverse effects.

Evaluation of the primary humoral immune response following exposure of male rats to 17beta-estradiol or flutamide for 15 days.

There is a concern that certain industrial chemicals found in the environment may mimic or antagonize endogenous hormones and adversely affect the endocrine as well as the immune system. The objective of this study was to determine if exposure of Crl:CD (SD)BR male rats to 17beta-estradiol (17beta-E2), an estrogen receptor agonist, or flutamide (FLUT), an androgen receptor antagonist, would significantly alter the primary IgM humoral immune response to sheep red blood cells (SRBC). This study was conducted in the context of a male in vivo Tier I battery designed to identify endocrine-active compounds (EACs). The Tier I male battery consists of organ weights coupled with a comprehensive hormonal assessment. Rats were dosed by the intraperitoneal route for 15 days with vehicle or 0.001, 0.0025, 0.0075, or 0.050 mg/kg/day 17beta-E2 or 0.25, 1, 5, or 20 mg/kg/day FLUT. Six days prior to termination, selected rats were injected intravenously with SRBC for assessment of humoral immune function. Spleen cell number and spleen and thymus weights were obtained. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. At 0.050 mg/kg/day 17beta-E2, mean final body and absolute thymus weights were significantly decreased to 84 and 65% of control, respectively. 17beta-E2 did not significantly alter spleen weight, spleen cell number, or the primary IgM humoral immune response to SRBC. The no-observed-adverse-effect level (NOAEL) for immune system alteration was 0.050 mg/kg/day 17beta-E2 since the decrease in absolute thymus weight was judged to be secondary to the decrements in body weight. In the Tier I male battery, responses to 17beta-E2 included decreased absolute testis and epididymis weights, decreased relative accessory sex gland unit weights, hormonal alterations (decreased serum testosterone (T), dihydrotestosterone (DHT), and luteinizing hormone (LH), and increased serum prolactin and E2 levels). The lowest-observed-adverse-effect level (LOAEL) for the reproductive indices was 0.001 mg/kg/day 17beta-E2 based on the hormonal alterations seen at this level; no NOAEL was established. Exposure to FLUT did not significantly alter mean final body, spleen, or absolute thymus weights, spleen cell number, or the primary IgM humoral immune response to SRBC. A significant increase (118% of control) in relative thymus weight was observed at 20 mg/kg/day FLUT. The NOAEL for immune system alteration was 5 mg/kg/day FLUT based on the increased relative thymus weights that were judged to be compound-related. In the Tier I male battery, responses to FLUT included decreased absolute epididymis and relative accessory sex gland unit weights and hormonal alterations (increased serum T, DHT, E2, and LH, and decreased follicle stimulating hormone levels). The LOAEL for the reproductive indices was 0.25 mg/kg/day FLUT based on the hormonal alterations seen at this level; no NOAEL was established. Based on these data, the reproductive and not the immune system appears to be the primary target organ of toxicity in young adult male rats treated with either 17beta-E2 or FLUT.

Further evaluation of the incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats in a standard toxicology study.

A previous study (Ladics et al., 1995) conducted in our laboratory using the known immunosuppressant agent, cyclophosphamide, indicated that a functional assay for assessment of humoral immunity may be conducted in rats in a standard toxicology study. The objective of this study was to further examine the feasibility of conducting an immunotoxicological assay for assessing humoral immunity in rats in a standard toxicology study using a chemical, carbon tetrachloride (CCl4), whose principal target organ of toxicity is not the immune system. Specifically, the previous study and this study were done to determine whether the conduct of an assay for assessing humoral immune function would affect standard toxicological endpoints. Male CD rats were untreated or dosed orally for 30 or 90 days, excluding weekends, with vehicle or 12.5 or 25 mg/kg CCl4. Six days prior to sacrifice, selected rats were injected intravenously with sheep red blood cells (SRBC) for assessment of humoral immune function. One day prior to necropsy, blood for hematological and clinical chemical measurements was collected from each rat. On the day of necropsy standard protocol tissues were collected, weighed, processed to slides, and later examined microscopically. One-half of each spleen was used to assess spleen cell numbers and quantitate lymphocyte subsets (Thelper; Tcyt/sup; total T- and B-cells) by flow cytometry. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. Administration of 12.5 and 25 mg/kg CCl4 for 30 days decreased SRBC-specific serum IgM levels 42 and 45%, respectively, while 25 mg/kg CCl4 for 90 days increased SRBC-specific IgM levels by 50%. CCl4 did not alter splenic lymphocyte subset numbers nor the weight nor morphology of lymphoid organs. Exposure to 25 mg/kg CCl4 did increase liver weight and serum sorbitol dehydrogenase levels, as well as produce centrilobular fatty change. SRBC administration did not alter any hematological or clinical chemistry parameters, nor lymphocyte subset numbers. With the expected exception of the spleen (slight increase in number and size of germinal centers), administration of SRBC did not significantly alter the weights or morphology of routine protocol tissues. Furthermore, administration of SRBC did not mask the rather mild hepatotoxic effects of CCL4 exposure observed in this study. Based on these and previous findings, it appears that a functional assay for assessing humoral immunity may be conducted in animals on standard toxicology study without altering standard toxicological endpoints.

Possible incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats on standard toxicology study.

The objective of this study was to examine the feasibility of conducting an immunotoxicological assay for assessing humoral immunity in rats on standard toxicology study. Male CD rats were untreated or dosed intraperitoneally daily for 30 or 90 days, excluding weekends, with vehicle or 2 mg/kg cyclophosphamide (CY). Six days prior to sacrifice, selected rats were injected intravenously with sheep red blood cells (SRBC). One day prior to necropsy, blood samples for hematological and clinical chemical measurements were collected from each rat. On the day of necropsy standard protocol tissues were collected, weighed, processed to slides, and examined microscopically. One-half of each spleen was used to prepare a single cell suspension in order to assess spleen cell numbers. Serum was analyzed for anti-SRBC IgM antibody using an enzyme-linked immunosorbent assay. A second set of studies was performed to examine further the effect of SRBC administration on lymphoid organ weights using 30- and 90-day study age-equivalent naive male CD rats. Exposure of animals to 2 mg/kg CY for 30 or 90 days resulted in a 28% and 61% decrease, respectively, in SRBC-specific serum IgM levels. CY treatment also caused mild alterations in some leukocytic parameters, with significant decreases of 35% and 33% in white blood cell and lymphocyte counts, respectively, observed in 30-day CY-treated animals receiving SRBC. Injection of SRBC alone did not alter hematological or clinical chemistry parameters. With the expected exception of the spleen (increased number and size of germinal centers), administration of SRBC did not significantly alter the weights or morphology of routine protocol tissues. Furthermore, administration of SRBC did not mask the immunosuppressive effects of CY treatment under the conditions of this study. Based on our preliminary findings, a functional assay for assessing humoral immunity may be conducted in animals on standard toxicology study.

Identifying airway sensitizers: cytokine mRNA profiles induced by various anhydrides.

Exposure to low molecular weight (LMW) chemicals in the workplace has been linked to a variety of respiratory effects. Within the LMW chemicals, one of the major classes involved in these effects are the acid anhydrides. The immunological basis of respiratory hypersensitivity involves CD4+ cells. By virtue of their induction of cytokines typical of CD4+ T-helper type 2 (Th2) cells-interleukin (IL)-4, 10, and 13-respiratory sensitizers may be identified and differentiated from contact sensitizers which induce Th1 cytokines (IL-2 and IFN-gamma). Our previous work suggested that the ribonuclease protection assay (RPA) was useful in identifying the respiratory sensitizer, trimellitic anhydride (TMA), based on quantitative differences in Th2 cytokine mRNA as compared to the contact sensitizer dinitrochlorobenzene (DNCB). Therefore, the purpose of the studies described in this report was to expand the chemicals tested in the RPA. To this end, four acid anhydrides with known respiratory sensitization potential, TMA, maleic anhydride (MA), phthalic anhydride (PA) and hexahydrophthalic anhydride (HHPA), were tested. Although previously determined to induce immunologically equivalent responses in a local lymph node assay (LLNA), the initial dose chosen (2.5%) failed to induce Th2 cytokine mRNA expression. To determine if the lack of cytokine expression was related to dose, LLNAs were conducted at higher doses for each of the anhydrides. The highest doses evaluated (four- to six-fold higher than those used in the initial RPA) gave equivalent proliferative responses for the various anhydrides and were used for subsequent RPA testing. At these higher doses, significant increases in Th2 versus Th1 cytokine mRNA were observed for all anhydrides tested. These results suggest that the RPA has the potential to serve as a screen for the detection of LMW airway sensitizing chemicals. However, the basis for selecting immunologically equivalent doses may require some modification.

Further evaluation of the local lymph node assay in the final phase of an international collaborative trial.

The local lymph node assay (LLNA) is a method used for the prospective identification in mice of chemicals that have the potential to cause skin sensitization. We report here the results of the second and final phase of an international trial in which the performance of the assay has been evaluated using seven test materials in five independent laboratories. The additional chemicals examined here included compounds which are considered less potent allergens than some of those tested in the first phase of the investigation, and includes hexylcinnamic aldehyde (HCA), a chemical recommended by the Organization for Economic Cooperation and Development (OECD) as a positive control for skin sensitization studies. In each laboratory all skin sensitizing chemicals examined (2,4-dinitrochlorobenzene {DNCB}, HCA, oxazolone, isoeugenal and eugenol) elicited positive responses of comparable magnitude as judged by the derived lowest concentration of test chemical required to elicit a 3-fold or greater increase in the proliferative activity of draining lymph node cells compared with vehicle-treated controls. We observed that sodium lauryl sulphate, considered to be a non-sensitizing skin irritant, also induced a positive response in the assay. Para-aminobenzoic acid (pABA), a nonsensitizing chemical, was negative at all test concentrations in each laboratory. Some laboratories incorporated minor modifications into the standard assay procedure, including the evaluation of lymph nodes pooled from individual mice rather than treatment groups and the use of statistical analyses. The use of statistics did not markedly change the determination of the lowest concentration yielding a positive response. These data confirm that the local lymph node assay is robust and yields equivalent results when performed independently.

An international evaluation of the murine local lymph node assay and comparison of modified procedures.

The murine local lymph node assay is a predictive test for the identification of skin-sensitizing chemicals. The method has been the subject both of national inter-laboratory studies and of extensive comparisons with guinea pig tests. In the investigations reported here, the local lymph node assay has been evaluated further in the context of an international study comprising five independent laboratories. In addition, the influence of minor modifications to the standard assay procedure on the performance of the test has been examined. The modified procedures investigated were exposure of mice for 4 rather than 3 consecutive days, excision of lymph nodes 4 rather than 5 days after the initiation of exposure and the use of an alternative isotope. All five laboratories, irrespective of whether the standard or a modified protocol was used, were able to identify accurately, and with comparable sensitivity, potassium dichromate and 2,4-dinitrochlorobenzene as skin sensitizers. Using standard criteria, none of the laboratories recorded positive responses with methyl salicylate, a non-sensitizer. In the standard protocol, lymph nodes are pooled for each experimental group and the vigor of responses measured as a stimulation index relative to vehicle controls. A stimulation index of 3 or greater is considered to indicate skin-sensitizing potential. One further modification adopted by three of the laboratories was to analyze nodes from individual animals and, thereby, permit statistical evaluation. This allowed a direct comparison of statistical significance with the conventional stimulation index as criteria for a positive response. The data indicate that, while statistical evaluation may provide, in some instances, for small increases in sensitivity, this may be at the expense of some loss of selectivity. There are, however, insufficient data presently to draw firm conclusions regarding the relative value of statistical analysis. These studies demonstrate that the local lymph node assay is sufficiently robust to accommodate minor procedural and technical modifications without material changes in test performance.