Boosting cytotoxicity of adoptive allogeneic NK cell therapy with an oncolytic adenovirus encoding a human vIL-2 cytokine for the treatment of human ovarian cancer.

Despite good results in the treatment of hematological malignancies, Natural killer (NK) cells have shown limited effectiveness in solid tumors, such as ovarian cancer (OvCa). Here, we assessed the potential of an oncolytic adenovirus expressing a variant interleukin-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, to enhance NK cell therapy efficacy in human OvCa ex vivo. Human OvCa surgical specimens were processed into single-cell suspensions and NK cells were expanded from healthy blood donors. OvCa sample digests were co-cultured ex vivo with NK cells and vIL-2 virus and cancer cell killing potential assessed in real time through cell impedance measurement. Proposed therapeutic combination was evaluated in vivo with an OvCa patient-derived xenograft (PDX) in mice. Addition of vIL-2 virus significantly enhanced NK cell therapy killing potential in treated OvCa co-cultures. Similarly, vIL-2 virus in combination with NK cell therapy promoted the best in vivo OvCa tumor control. Mechanistically, vIL-2 virus induced higher percentages of granzyme B in NK cells, and CD8+ T cells, while T regulatory cell proportions remained comparable to NK cell monotherapy in vivo. Ad5/3-E2F-d24-vIL2 virus treatment represents a promising strategy to boost adoptive NK cell therapeutic effect in human OvCa.

Improving the cytotoxic response of tumor-infiltrating lymphocytes towards advanced stage ovarian cancer with an oncolytic adenovirus expressing a human vIL-2 cytokine.

While the presence of tumor-infiltrating lymphocytes (TILs) associates with improved survival prognosis in ovarian cancer (OvCa) patients, TIL therapy benefit is limited. Here, we evaluated an oncolytic adenovirus coding for a human variant IL-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, as an immunotherapeutic strategy to enhance TIL responsiveness towards advanced stage OvCa tumors. Fragments of resected human OvCa tumors were processed into single-cell suspensions, and autologous TILs were expanded from said samples. OvCa tumor specimens were co-cultured with TILs plus vIL-2 virus, and cell killing was assessed in real time through cell impedance measurement. Combination therapy was further evaluated in vivo through a patient-derived xenograft (PDX) ovarian cancer murine model. The combination of vIL-2 virus plus TILs had best cancer cell killing ex vivo compared to TILs monotherapy. These results were supported by an in vivo experiment, where the best OvCa tumor control was obtained when vIL-2 virus was added to TIL therapy. Furthermore, the proposed therapy induced a highly cytotoxic phenotype demonstrated by increased granzyme B intensity in NK cells, CD4+ T, and CD8+ T cells in treated tumors. Our results demonstrate that Ad5/3-E2F-d24-vIL2 therapy consistently improved TILs therapy cytotoxicity in treated human OvCa tumors.

A Risk-scoring Model for Predicting Post-recurrence Survival in Patients With Endometrial Carcinoma.

AIMS: The survival time of patients with recurrent endometrial carcinoma is generally short. However, considerable interindividual variation exists. We developed a risk-scoring model for predicting post-recurrence survival in patients with endometrial carcinoma. MATERIALS AND METHODS: Patients with endometrial carcinoma treated at a single institution between 2007 and 2013 were identified. Pearson chi-squared analyses were used to compute odds ratios for the associations between risk factors and short survival after cancer recurrence. The results for biochemical analyses represented values at diagnosis of disease recurrence or values at initial diagnosis for those patients who had a primary refractory disease. Logistic regression models were constructed for the identification of variables that independently predict short post-recurrence survival. The models were used to assign points based on odds ratios for risk factors and risk scores were derived. RESULTS: In total, 236 patients with recurrent endometrial carcinoma were included in the study. Based on overall survival analysis, 12 months was selected as the cut-off for short post-recurrence survival. Factors associated with short post-recurrence survival were platelet count, serum CA125 concentration and progression-free survival. A risk-scoring model with an area under the receiver operating characteristic curve (AUC) of 0.782 (95% confidence interval 0.713-0.851) was developed in patients without missing data (n = 182). When patients with a primary refractory disease were excluded, age and blood haemoglobin concentration were identified as additional predictors of short post-recurrence survival. For this subpopulation (n = 152), a risk-scoring model with an AUC of 0.821 (95% confidence interval 0.750-0.892) was developed. CONCLUSIONS: We report a risk-scoring model that shows acceptable to excellent accuracy in predicting post-recurrence survival in patients with endometrial carcinoma, with primary refractory diseases included or excluded. This model has potential applications in precision medicine in patients with endometrial carcinoma.

Systematic molecular and clinical analysis of uterine leiomyomas from fertile-aged women undergoing myomectomy.

STUDY QUESTION: What are the distributions and associated clinical characteristics of mediator complex subunit 12 (MED12), high mobility group AT-hook 2 (HMGA2) and fumarate hydratase (FH) aberrations in uterine leiomyomas from fertile-aged myomectomy patients? SUMMARY ANSWER: These driver mutations account for the majority (83%) of tumours in fertile-aged patients. WHAT IS KNOWN ALREADY: Alterations affecting MED12, HMGA2 and FH account for 80-90% of uterine leiomyomas from middle-aged hysterectomy patients, while the molecular background of tumours from young myomectomy patients has not been systematically studied. STUDY DESIGN, SIZE, DURATION: A retrospective series of 361 archival uterine leiomyoma samples from 234 women aged ≤45 years undergoing myomectomy in 2009-2014 was examined. Associations between the molecular data and detailed clinical information of the patients and tumours were analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA was extracted from formalin-fixed paraffin-embedded samples and MED12 exons 1 and 2 were sequenced to identify mutations. Level of HMGA2 expression was evaluated by immunohistochemistry. Biallelic FH inactivation was analysed with 2-succinylcysteine staining, which is an indirect method of assessing FH deficiency. All patients' medical histories were reviewed, and clinical information of patients and tumours was combined with molecular data. MAIN RESULTS AND THE ROLE OF CHANCE: The median age at operation was 34 years. The majority (58%) of patients were operated on for a single leiomyoma. Known driver mutations were identified in 83% of tumours (71% MED12; 9% HMGA2; 3% FH). In solitary leiomyomas, the MED12 mutation frequency was only 43%, and 29% were wild-type for all driver alterations. MED12 mutations were associated with multiple tumours, smaller tumour size and subserosal location. LIMITATIONS, REASONS FOR CAUTION: Although comprehensive, the study is retrospective in nature and all samples have been collected for routine diagnostic purposes. The use of paraffin-embedded samples and immunohistochemistry may have led to an underestimation of mutations. Due to the limited sample size and rarity of especially FH-deficient leiomyomas, the data are partly descriptive. WIDER IMPLICATIONS OF THE FINDINGS: The contribution of driver mutations in leiomyomas from young myomectomy patients is comparable to tumours obtained from hysterectomies of mostly middle-aged women. Our results support the earlier findings that MED12 mutations are associated with multiple tumours, smaller tumour size and subserosal location. The study emphasizes the distinct molecular background of solitary leiomyomas, and more research is needed to clarify the underlying causes of the notable proportion of wild-type leiomyomas. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the Academy of Finland (307773), the Sigrid Jusélius Foundation, the Cancer Foundation Finland and the iCAN Digital Precision Cancer Medicine Flagship. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Up-front rituximab maintenance improves outcome in patients with follicular lymphoma: a collaborative Nordic study.

The introduction of the anti-CD20 antibody rituximab in combination with chemotherapy (R-chemo) has improved the prognosis of patients with follicular lymphoma (FL). During the last decade, the addition of a maintenance treatment with rituximab (MR) after R-chemo has been tested with the hope of further improving the outcome of these patients. Using 2 independent population-based cohorts, we investigated the effect of up-front MR on time related end points as well as the risk of histological transformation (HT). FL patients were included if they: (1) completed first-line induction treatment with R-chemo, (2) were alive after induction treatment and eligible for MR, and (3) had no evidence of HT at this time point. The training cohort consisted of 733 Danish patients of whom 364 were consolidated with MR; 369 were not. Patients receiving MR more often had advanced clinical stage (90% vs 78%), high Follicular Lymphoma International Prognostic Index (FLIPI) score (64% vs 55%), and bone marrow infiltration (49% vs 40%). Those consolidated with MR had an improved 5-year overall survival (OS; 89% vs 81%; P = .001) and progression-free survival (PFS; 72% vs 60%; P < .001). In the training cohort, MR was associated with a reduction of HT risk (P = .049). Analyses of an independent validation cohort of 190 Finnish patients confirmed the favorable impact of MR on 5-year OS (89% vs 81%; P = .046) and PFS (70% vs 57%; P = .005) but did not find a reduced risk of HT. The present population-based data suggest that the outcome of patients with FL has improved after consolidation of R-chemo with MR.

Evaluation of fungal extracts to determine immunomodulatory properties.

BACKGROUND: Immunoglobulin E-mediated allergies have doubled in prevalence during recent decades in developed countries.This increase has been attributed, in part, to high hygiene standards, which have reduced exposure to microbes. The capacity of microbes to induce type 1 helper T cell (TH1) responses may imply suppression of TH2 responses. However, little research has been performed with fungal extracts. OBJECTIVES: To evaluate the TH1-inducing properties of fungal extracts. METHODS: A total of 24 fungal extracts, including Cetavlon-precipitated polysaccharides from different yeasts, molds, and mushrooms were prepared.The extracts were screened for production of interferon (IFN)gamma in human peripheral blood mononuclear cells. The active compounds were further purified by mild acid hydrolysis and by column chromatography and studied in human peripheral blood mononuclear cells. RESULTS: Expression of IFN-gamma was induced by several extracts. The strongest expression of IFN-gamma was induced by Candida albicans. The Cetavlon-precipitated mannans of fungi induced cytokine responses that were similar or superior to those induced by whole extracts, C albicans being the most potent inducer of IFN-gamma. Column chromatography-fractionated mild acid hydrolysis of Calbicans mannan was performed. Fractions containing oligosaccharides of 12-16 mannoses induced production of tumor necrosis factor. CONCLUSIONS: Several fungal extracts induce IFN-gamma. The most promising preparations were yeast-derived oligosaccharides. Further research should be focused on purification and eventual synthesis of the extracts.

Volatile organic compounds and formaldehyde as explaining factors for sensory irritation in office environments.

This study's database comprised results of volatile organic compound (VOC) measurements from 176 office buildings. In 23 of the 176 buildings, formaldehyde measurements were also conducted. It was suspected that the buildings had indoor air problems, but a walk-through inspection did not reveal any clear, abnormal contaminant sources. The 50 most abundant VOCs and their concentrations in 520 air samples were analyzed. The irritation potency was estimated for 33 out of the 50 common VOCs and their mixtures, as well as for formaldehyde. This information was used to calculate the recommended indoor air levels (RILs) for the VOCs. The RILs were considerably higher than the measured mean indoor air concentrations in the buildings. However, the RIL for formaldehyde was exceeded in most of the 23 buildings studied. According to the evaluation of irritation potency, formaldehyde was a more likely cause of sensory irritation than the mixture of common nonreactive VOCs at the concentrations that occurred in the buildings without abnormal indoor sources. Furthermore, environmental symptoms of office workers were characterized in 20 office buildings (including the database of 176 office buildings) with the aid of an indoor air questionnaire. The most frequent symptoms related to the indoor environment were involved the upper respiratory tract. However, no relationship could be shown between the reported symptoms and the occurrence of VOC and formaldehyde concentrations in these buildings. Generally, the study results indicated that formaldehyde was the more likely agent causing sensory irritation than the mixture of the common nonreactive VOCs at the concentrations occurring in the buildings without abnormal indoor sources.

Comparison of VOC and ammonia emissions from individual PVC materials, adhesives and from complete structures.

Emission rates of volatile organic compounds (VOCs) and ammonia measured from six PVC materials and four adhesives in the laboratory were compared to the emission rates measured on site from complete structures. Significantly higher specific emission rates (SERs) were generally measured from the complete structures than from individual materials. There were large differences between different PVC materials in their permeability for VOCs originating from the underlying structure. Glycol ethers and esters from adhesives used in the installation contributed to the emissions from the PVC covered structure. Emissions of 2-ethylhexanol and TXIB (2,2,4-trimethyl-1,3-pentanediol diisobutyrate) were common. High ammonia SERs were measured from single adhesives but their contribution to the emissions from the complete structure did not appear as obvious as for VOCs. The results indicate that three factors affected the VOC emissions from the PVC flooring on a structure: 1) the permeability of the PVC product for VOCs, 2) the VOC emission from the adhesive used, and 3) the VOC emission from the backside of the PVC product.

A multi-faceted approach to risk assessment of laboratory animal allergens at two facilities.

BACKGROUND: Risk assessment of exposure to allergens is difficult because the relationship between exposure, sensitization, and symptoms has not been fully established. Laboratory animal allergens (LAA) are an important occupational health risk factor; 10-32% of workers exposed to these allergens develop allergic diseases. This article introduces a versatile approach to assessing the risks posed by LAA at two laboratory animal facilities. METHODS: The risk assessment approach that was used at the laboratory animal facilities included questionnaires for management and employees, a hazard identification visit and measurements in the workplaces, as well as the creation of a list of generally recommended procedures to reduce allergen exposure. RESULTS: The prevalence of work-related allergic symptoms was 17%. Suggested countermeasures at the sites included changes in ventilation and work practices, reduction of unnecessary exposure, recommendations for more comprehensive use of personal protective equipment, and wider communication about LAA risks. CONCLUSIONS: The approach managed to identify critical points and potential means for controlling LAA exposure.

Acute effects of Aspergillus versicolor aerosols on murine airways.

Mice were exposed to aerosols from Aspergillus versicolor extract by inhalation (for 15-20 min). Changes in respiratory function parameters were monitored during the exposure to evaluate acute effects on the upper and lower respiratory tracts and the conductive airways. The single inhalation exposure to A. versicolor aerosols at the range of 12-151 mg/m3 provoked upper respiratory tract irritation in the airways of mice. The higher the exposure concentration the higher was the increase in the sensory irritation (SI) response. No bronchoconstriction or pulmonary irritation was observed. The causative agents for the SI response in the fungal extract are not known.

Slight respiratory irritation but not inflammation in mice exposed to (1-->3)-beta-D-glucan aerosols.

Airway irritation effects after single and repeated inhalation exposures to aerosols of beta-glucan (grifolan) were investigated in mice. In addition, the effects on serum total immunoglobulin E (IgE) production and histopathological inflammation in the respiratory tract were studied. The beta-glucan aerosols provoked slight sensory irritation in the airways, but the response was not concentration dependent at the levels studied. Slight pulmonary irritation was observed after repeated exposures. No effect was found on the serum total IgE levels, and no signs of inflammation were seen in the airways 6 h after the final exposure. The results suggest that, irrespective of previous fungal sensitization of the animals, inhaled beta-glucan may cause symptoms of respiratory tract irritation but without apparent inflammation. Respiratory tract irritation reported after inhalation of fungi may not be entirely attributed to beta-glucan.

The effects of wood dusts on the redox status and cell death in mouse macrophages (RAW 264.7) and human leukocytes in vitro.

Wood dusts are classified as carcinogenic to humans and also produce other toxic, allergic, and acute effects in woodworkers. However, little is known about causative agents in wood dusts and their mechanisms of action. The effects of different tree species and particle size for biological activity were studied. The differences in the production of reactive oxygen species (ROS) and cell death (necrotic and apoptotic) between mouse macrophage (RAW 264.7) cells and human polymorphonuclear leukocytes (PMNL) for pine, birch, and beech dust exposures were investigated in vitro. The pine and birch dust exposure (1-100 microg/ml) produced concentration-dependent ROS production in both the cells, which was one order of magnitude higher with pine dust. The ROS production was faster in human PNML than murine RAW cells. The higher concentrations (500 and/or 1000 microg/ml) decreased ROS formation. With pine and birch dust exposure, this was probably due to the necrotic cell death. The pine dust concentrations of 500 and 1000 microg/ml were cytotoxic to human PMNL. The beech dust exposure activated the ROS production and decreased the cell viability only at the highest concentrations, being least potent of the three dusts. A sign of the apoptotic cell death in the murine RAW cells was observed at the pine dust concentration of 100 microg/ml. The exposure to the birch and beech dusts with a smaller particle size (<5 microm) produced greater ROS production than exposure to the corresponding dust with a wide range of particle sizes. However, changing the particle size did not affect the cell viability. The results indicate that the type of wood dust (tree species and possibly particle size) has a significant impact on the function and viability of phagocytic cells.

The 3-year follow-up study in a block of flats - experiences in the use of the Finnish indoor climate classification.

Indoor climate of two new blocks of flats was investigated. The case building was built for people with respiratory diseases by following the instructions of the Finnish Classification of Indoor Climate, Construction and Finishing Materials, while the control building was built using conventional building technology. The main indoor air parameters (temperature, relative humidity and levels of CO, CO2, ammonia, total volatile organic compounds, total suspended particles, fungal spores, bacteria and cat, dog and house dust mite allergens) were measured in six apartments of both the buildings on five occasions during the 3-year occupancy. In addition, a questionnaire to evaluate symptoms of the occupants and their satisfaction with their home environment was conducted in connection with indoor air quality (IAQ) measurements. The levels of indoor air pollutants in the case building were, in general, lower than those in the control building. In addition, the asthmatic occupants informed that their symptoms had decreased during the occupancy in the case building. This case study showed that high IAQ is possible to reach by careful design, proper materials and equipment and on high-quality construction with reasonable additional costs. In addition, the study indicated that good IAQ can also be maintained during the occupancy, if sufficient information on factors affecting IAQ and guidance on proper use and care of equipment are available for occupants.

Effects of aerosols from nontoxic Stachybotrys chartarum on murine airways.

Acute effects on the upper and lower respiratory tract due to inhalation exposure to Stachybotrys chartarum (Sc) extract were investigated in mice. In addition, the capacity of the Sc exposure to activate immune system and cause inflammation in the respiratory tract was studied. The inhalation of Sc extract aerosols was observed to provoke sensory irritation in the airways of both naive and Sc-immunized mice. In contrast, exposure to aerosolized ovalbumin or phosphate buffered saline did not cause this effect. Exposure to Sc twice a week for 3 wk increased significantly the serum total immunoglobulin E (IgE) levels in BALB/c mice immunized with Sc as well as in nonimmunized mice. A slight presence of inflammatory cells was observed in the alveoli 3 days after the last exposure to Sc. In conclusion, Sc extract has the capacity to provoke sensory irritation in the murine airways and to activate the murine immune system.

A review: fungal exposure assessment in indoor environments.

While the fungal exposure assessment was based on the determination of fungal propagules for a long time, recent progress has led to the development of methodology for other fungal agents, e.g. the fungal cell wall components, metabolites, and allergens, that may be responsible for health effects caused by fungal exposure. This review includes a summary of the sampling techniques and analytical methods that are currently used or are in progress for the fungal exposure assessment. Prospects for the future trends are also discussed. In the future, the development will focus on sampling techniques that allow longer sampling times, a higher sampling efficiency for relevant particle sizes, and better possibilities for a wide range of analyses. In addition, new or modified methodology based on chemical, immunochemical, and molecular biological techniques to measure fungal agents related to health effects will improve the understanding of biological responses caused by fungal exposure.

Preliminary description of antigenic components characteristic of Stachybotrys chartarum.

The objective of this study was to characterize preliminarily immunogenic components characteristic of Stachybotrys chartarum to be used later for the development of a detection method for the fungus in environmental samples. The procedure for S.chartarum extract preparation was first optimized related to the age of the culture, culture type, and growth medium, and the antigenic composition of S. chartarum cultured in two different media was then characterized by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting method. Cross-reactivity of S. chartarum antigenic components with 10 other fungal species was identified by the inhibition immunoblotting method. The 10-day-old S. chartarum culture extract cultured in malt extract broth revealed a wider selection of proteins and antigenic components than the 30-day-old culture extract or the culture medium extracts. When cultured in cellulose broth, S. chartarum produced a higher number of proteins and antigenic components than in malt extract broth. The most dominant immunogenic components of S. chartarum cultured in cellulose broth were those of 65, 50, 37, and 27 kDa. The components of 65 and 50 kDa proved to be the most characteristic of this fungus according to the inhibition immunoblotting analyses. Several of the S. chartarum components were identified as glycoproteins. Carbohydrate moieties of the S. chartarum components also possessed an antibody binding activity.

The relationship between measured moisture conditions and fungal concentrations in water-damaged building materials.

We determined the moisture levels, relative humidity (RH) or moisture content (MC) of materials, and concentrations of culturable fungi, actinomycetes and total spores as well as a composition of fungal flora in 122 building material samples collected from 18 moisture problem buildings. The purpose of this work was to clarify if the is any correlation between the moisture parameters and microbial levels or generic composition depending on the type of materials and the time passed after a water damage. The results showed an agreement between the concentrations of total spores and culturable fungi for the wood, wood-based and gypsum board samples (r > 0.47). The concentrations of total spores and/or culturable fungi correlated with RH of materials particularly among the wood and insulation materials (r > 0.79), but not usually with MC (r < 0.45). For the samples collected from ongoing damage, there was a correlation between RH of materials and the concentrations of total spores and culturable fungi (r > 0.51), while such a relationship could not be observed for the samples taken from dry damage. A wide range of fungal species were found in the samples from ongoing damage, whereas Penicillia and in some cases yeasts dominated the fungal flora in the dry samples. This study indicates that fungal contamination can be evaluated on the basis of moisture measurements of constructions in ongoing damage, but the measurements are not solely adequate for estimation of possible microbial growth in dry damage.

Sensory irritating potency of some microbial volatile organic compounds (MVOCs) and a mixture of five MVOCs.

The authors investigated the ability/potencies of 3 microbial volatile organic compounds and a mixture of 5 microbial volatile organic compounds to cause eye and upper respiratory tract irritation (i.e., sensory irritation), with an animal bioassay. The authors estimated potencies by determining the concentration capable of decreasing the respiratory frequency of mice by 50% (i.e., the RD50 value). The RD50 values for 1-octen-3-ol, 3-octanol, and 3-octanone were 182 mg/m3 (35 ppm), 1359 mg/m3 (256 ppm), and 17586 mg/m3 (3360 ppm), respectively. Recommended indoor air levels calculated from the individual RD50 values for 1-octen-3-ol, 3-octanol, and 3-octanone were 100, 1000, and 13000 microg/m3, respectively-values considerably higher than the reported measured indoor air levels for these compounds. The RD50 value for a mixture of 5 microbial volatile organic compounds was also determined and found to be 3.6 times lower than estimated from the fractional concentrations and the respective RD50s of the individual components. The data support the conclusion that a variety of microbial volatile organic compounds may have some synergistic effects for the sensory irritation response, which constrains the interpretation and application of recommended indoor air levels of individual microbial volatile organic compounds. The results also showed that if a particular component of a mixture was much more potent than the other components, it may dominate the sensory irritation effect. With respect to irritation symptoms reported in moldy houses, the results of this study indicate that the contribution of microbial volatile organic compounds to these symptoms seems less than previously supposed.

Evaluation of sensory irritation of delta3-carene and turpentine, and acceptable levels of monoterpenes in occupational and indoor environment.

The standard mouse bioassay was used for obtaining the RD50 (i.e., the concentration that causes a 50% decrease in respiratory frequency) and for estimating the irritation properties of d-delta3-carene (i.e., (+)-delta3-carene) and commercial turpentine. The chemicals studied possess mainly sensory irritation properties similar to the previously studied monoterpenes, pinenes. The irritation potency of d-delta3-carene (RD50 = 1345 ppm) was almost equal to that of d-pinenes. Thus, d-delta3-carene was about four times more potent as a sensory irritant than I-beta-pinene, whereas the difference with I-alpha-pinene was more marked; as a sensory irritant, I-alpha-pinene is almost inactive. Based on sensory irritation potency and physicochemical and structural properties of pinenes and delta3-carene, the potency of a closely related monoterpene, limonene, is discussed. For commercial turpentine, a mixture of monoterpenes (mainly d-delta3-carene, I-beta-pinene, alpha-pinenes, and limonenes), the RD50 (1173 ppm) was the same order of magnitude as those of d-pinenes and d-delta3-carene. Apparently, d-monoterpenes are responsible for the sensory irritation caused by turpentine. In the wood industry and in the indoor air of nonindustrial environments, monoterpenes are thought to be one of the causative agents for irritation symptoms. The occupational exposure limit (OEL) of turpentine (100 ppm in Finland and the United States) is also used for individual monoterpenes, excluding limonene. Using results from this and our previous study, proposed OELs and recommended indoor levels (RILs) for selected monoterpenes and turpentine were determined based on their RD50 values. According to our studies, the present OEL of turpentine (100 ppm; 560 mg/m3) in Finland and in the United States seems to be suitable only for I-pinenes. For d-monoterpenes and turpentine, an OEL about three times lower is suggested. Our results show that recommended indoor levels (RILs) for monoterpenes are high compared to the concentrations measured indoors in nonindustrial environments. Thus, it is very unlikely that monoterpenes alone can cause irritation symptoms in homes or offices under normal conditions.

Stereospecificity of the sensory irritation receptor for nonreactive chemicals illustrated by pinene enantiomers.

To clarify the existence of a receptor protein for sensory irritants in trigeminal nerve endings, D- [i.e. (+)] and L- [i.e. (-)] enantiomers of alpha- and beta-pinene as models of nonreactive chemicals were evaluated for their potency in outbred OF1 and NIH/S mice using ASTM E981-84 bioassay. All pinenes possess sensory irritation properties and also induced sedation and signs of anaesthesia but had no pulmonary irritation effects. According to the ratio of RD50 (i.e. concentration which causes a 50% decrease in respiratory rate,f) and vapour pressure (Po), all pinenes are nonreactive chemicals. For nonreactive chemicals, Po and olive oil-gas partition (Loil) can be used to estimate their potency as sensory irritant. Thus, for enantiomers with identical physicochemical properties, the estimated RD50 values are the same. In addition, although alpha- and beta-pinene do not have identical Po and Loil values, their estimated potencies are quite close. However, the experimental results showed that D-enantiomers of pinenes were the most potent as sensory irritants and a difference in potency also exists between alpha- and beta-pinene. RD50 for D-enantiomers of alpha- and beta-pinene were almost equal, 1053 ppm and 1279 ppm in OF1 strain and 1107 ppm and 1419 ppm in NIH/S strain, respectively. Values differed by a factor of approximately 4 to 5 from L-beta-pinene for which the RD50 was 4663 ppm in OF1 and 5811 ppm in NIH/S mice. RD50 could not be determined for L-alpha-pinene; this pinene was almost inactive. D-alpha-pinene seems to best fit the receptor because its experimental RD50 was one-half of the estimated value while for D-beta-pinene those values were equal. On the contrary, L-beta-pinene was about 3 to 4 times less potent than estimated. L-alpha-pinene was only slightly active although it was estimated to be as potent as D-alpha-pinene. The remarkable difference in potency between L-enantiometers is most likely due to a structural difference between alpha- and beta-pinene: the more flexible beta-pinene can bend to fit into the receptor better than the rigid alpha-pinene. The results showed that the commonly used physicochemical descriptors cannot fully explain the potency of these chemicals; their three-dimensional structure should also be considered. Because of the stereospecificity of pinenes, a target site for nonreactive sensory irritants is most likely a receptor protein containing a chiral lipophilic pocket.