Reduced cerebral blood flow of lingual gyrus associated with both cognitive impairment and gait disturbance in patients with idiopathic normal pressure hydrocephalus.

BACKGROUND: Idiopathic normal pressure hydrocephalus (iNPH) is primarily characterized by cognitive impairment and gait disturbance. Our objective was to evaluate the clinical characteristics of iNPH and the association between cerebral blood flow (CBF), measured using single-photon emission computed tomography (SPECT), and both cognitive and gait disturbances in iNPH patients. METHODS: We compared cognitive and motor functions and neuroimaging findings between 29 iNPH patients and 35 age-matched Parkinson's disease (PD) patients. We examined the associations between cognitive and motor dysfunctions and CBF in iNPH patients using 99mTc-ECD SPECT subtraction imaging data from a database of healthy control subjects. RESULTS: The cognitive function of iNPH patients, as measured by the Mini-Mental State Examination (MMSE) and Frontal Assessment Battery (FAB), was significantly poorer than that of PD patients; however motor function of the legs based on the Unified PD Rating Scale (UPDRS) part III was similar across groups. Impairment in cognitive function based on the MMSE and FAB was significantly correlated with motor dysfunction of the legs on the UPDRS part III and the 3-m Timed Up and Go test. Furthermore, 99mTc-ECD SPECT subtraction imaging revealed lower CBF in the bilateral lingual gyrus of iNPH patients with severely impaired cognitive and motor functions than healthy control subjects. CONCLUSION: Patients with iNPH have severely impaired cognitive function; however, motor dysfunction of the legs is similar to PD patients. The cognitive and gait disturbances of iNPH are significantly interrelated, which may be associated with an impaired brain network that includes the bilateral lingual gyrus.

A Case of Coil Embolization with Crossing Y-Configuration Stenting for a Recurrent IC-PC Aneurysm after Clipping.

Objective: We report a case in which coil embolization using crossing Y-configuration stenting was effective for an internal carotid-posterior communicating artery (IC-PC) aneurysm with repeated recurrence after clipping. Case Presentation: The patient was a 57-year-old woman. Nine months after undergoing clipping for a ruptured right IC-PC aneurysm at 55 years of age, she developed a second subarachnoid hemorrhage (SAH) due to recurrence of the aneurysm and underwent clipping at the same site. A third SAH due to rupture of the left IC-PC aneurysm developed 1.5 years after the second clipping. Simultaneously, recurrence of a right IC-PC aneurysm was noted and she was referred to our department. The recurrent right IC-PC aneurysm was considered to have originated from the distal to the initial neck. It was 7 mm in size and had an irregularly shaped wide neck. As it was assumed that there would be marked adhesion due to repeated surgery, we decided to treat the aneurysm by coil embolization instead of direct surgery. Although the aneurysm neck partially involved the posterior communicating artery (Pcom), tight packing with a minimal residual neck was required. Therefore, crossing Y-configuration stenting was deployed on the internal carotid artery and Pcom using two Neuroform Atlas stents, and coil embolization was performed by the jail technique. The recurrent aneurysm was obliterated. There were no deficits or thrombotic complications after surgery. On DSA follow-up, no compaction or recurrence was observed, and the Pcom was well visualized one year later. Conclusion: Coil embolization by crossing Y-configuration stenting is a viable treatment option for a recurrent IC-PC wide neck aneurysm.

A case report: An increased oxygen extraction fraction in remote ischemic lesions after revascularization for moyamoya disease with a progression of posterior cerebral artery stenosis.

The cerebral metabolism, such as the oxygen extraction fraction (OEF), in remote ischemic lesions following revascularization for moyamoya disease (MMD) has not yet been fully elucidated. We herein report a patient with an increased OEF in a remote ischemic lesion after revascularization in a case of adult-onset MMD. A 21-year-old woman suffered from a left parietal lobe infarction due to MMD. At 2 months after onset, left superficial temporal artery (STA)-middle cerebral artery (MCA) bypass and encephalo-myo-synangiosis (EMS) were performed. The postoperative course was uneventful. 15O-positron emission tomography (PET) performed at 2 months after the first operation revealed an increased OEF in the contralateral (right) frontal lobe that was suspected of being possible remote ischemia. The patient underwent right STA-MCA bypass and EMS. 15O-PET at 14 days after the second operation revealed an increased OEF in the contralateral (left) occipital lobe that was suspected of potentially being remote ischemia caused by a watershed shift. Two years after the second surgery, left occipital artery (OA)-posterior cerebral artery (PCA) anastomosis and EMS were performed due to transient right hemianopsia. Neither rebleeding nor ischemic complications occurred 2 years after the third surgery. We need to be alert for the possible progression of PCA stenosis in MMD after revascularization. It might induce remote ischemia after revascularization. OA-PCA bypass is therefore considered to be an effective treatment option in such cases.

Arterial spin labeling magnetic resonance imaging at short post-labeling delay reflects cerebral perfusion pressure verified by oxygen-15-positron emission tomography in cerebrovascular steno-occlusive disease.

BACKGROUND: Arterial transit time correction by data acquisition with multiple post-labeling delays (PLDs) or relatively long PLDs is expected to obtain more accurate imaging in cases of the cerebrovascular steno-occlusive disease. However, there have so far been no reports describing the significance of arterial spin labeling (ASL) images at short PLDs regarding the evaluation of cerebral circulation in ischemic cerebrovascular disease. PURPOSE: To clarify the role of short-PLD ASL in cerebrovascular steno-occlusive disease. MATERIAL AND METHODS: Fifty-three patients with cerebrovascular steno-occlusive disease were included in this study. All patients underwent ASL magnetic resonance imaging and 15O-PET within two days of each modality. To compare the ASL findings with each parameter of PET, the right-to-left (R/L) ratio, defined as the right middle cerebral artery (MCA) value/left MCA value, was calculated. RESULTS: There is a significant correlation between the ASL images at a short PLD and the ratio of cerebral blood flow and cerebral blood volume by 15O-PET, which may accurately reflect the cerebral perfusion pressure. A receiver operating characteristic curve analysis indicated that ASL images at PLD 1000 and 1500 ms were more accurate than at PLD 2000-3000 ms for the detection of a ≥10% change in the PET cerebral blood flow. CONCLUSION: ASL images at shorter PLDs may be useful at least as a screening modality to detect the changes in the cerebral circulation in cerebrovascular steno-occlusive disease. We must evaluate ASL images at multiple PLDs while considering the arterial transit time of each case at present.

Hybrid Surgical and Endovascular Approach via the Contralateral Superficial Middle Cerebral Vein to Occlude Cavernous Sinus Dural Arteriovenous Fistula in a Hybrid Operating Room: A Case Report.

Most cases of cavernous sinus dural arteriovenous fistula (CS-dAVF) are treated via the inferior petrous sinus (IPS) through the transfemoral vein approach, but there are cases where treatment through the superficial middle cerebral vein (SMCV) is required. A hybrid operating room (OR) is useful because it allows for smooth direct surgery and endovascular treatment in a clean surgical field. We herein report a case of simultaneous treatment for CS-dAVF by coil embolization via a contralateral SMCV and middle cerebral artery (MCA) aneurysm by clipping in a hybrid OR. A 68-year-old woman had been suffering from left chemosis and ptosis for 2 months before visiting our hospital. Digital subtraction angiography (DSA) revealed Borden type II and Cognard type II a+b CS dAVF with parenchymal hemorrhaging and an unruptured left M1/M2 junction aneurysm. Since passing through the CS via the femoral vein was unsuccessful, we decided to access the right CS via the left CS through the intercavernous sinus (ICS) via the left SMCV by the pterional approach in a hybrid OR equipped with a multi-axis working system angiography machine. Endovascular treatment via direct cannulation into the contralateral SMCV following craniotomy in a hybrid OR is an optional strategy for treating complicated CS-dAVF.

An acid-hydrolyzed wheat protein activates the inflammatory and NF-κB pathways leading to long TSLP transcription in human keratinocytes.

Hydrolyzed wheat proteins (HWPs) contained in cosmetics have occasionally caused immediate-type hypersensitivity following repeated skin exposure. Although the Cosmetic Ingredient Review Expert Panel concluded that < 3,500 Da HWP is safe for use in cosmetics, it remains biologically unknown how allergenic HWPs evoke immediate-type allergy percutaneously. Keratinocyte-derived thymic stromal lymphopoietin (TSLP) induces type 2 immune responses, which play an essential role in the pathogenesis of immediate-type allergy. Previously, we demonstrated that protein allergens in cultured human keratinocytes strongly induced long-form TSLP (loTSLP) transcription. However loTSLP-regulating signaling by HWP is poorly understood. In this study, we performed global gene expression analysis by microarray to investigate how the allergenic HWP acts on epidermal keratinocytes and the induction of loTSLP. Compared to human serum albumin (HSA), allergenic HWP induced a distinct gene expression pattern and preferentially activated various inflammatory pathways (High Mobility Group Box 1, Interleukin [IL]-6, IL-8, and acute phase response signaling). We identified 85 genes as potential nuclear factor-kappa B (NF-κB) target genes in GP19S-treated cells, compared with 29 such genes in HSA-treated cells. In addition, HWP specifically altered IL-17 signaling pathways in which transcription factors, NF-κB and activator protein-1, were activated. NF-κB signaling may be an important factor for HWP-induced inflammatory loTSLP transcription via inhibition assay. In conclusion, allergenic HWP caused an easily sensitizable milieu of activated inflammatory pathways and induced NF-κB-dependent loTSLP transcription in keratinocytes.

Predicting the results of a 24-hr human patch test for surfactants: utility of margin-setting in a reconstructed human epidermis model.

To predict the results of a 24-hr closed human patch test, we previously recommended the use of in vitro test with a reconstructed human epidermis (RhE) model adopted in OECD TG 439, and proposed the margin method, which includes evaluation of twice the concentration to avoid a false positive for surfactants. Therefore, in this study, we used LabCyte EPI-MODEL as a RhE model, and confirmed the reproducibility of this method using five surfactants, including benzalkonium chloride (BC), sodium lauryl sulfate (SLS), and lauryl betaine (LB), for which false negative results have previously been reported, and three different surfactants. For all surfactants, prediction of patch test results using a margin of two revealed that human tests could be performed safely, confirming the utility of the margin method. In addition, we examined the relationship with critical micellar concentration (CMC). The IC50 for cell viability in the RhE model for three types of surfactants (BC, SLS, and LB) was 2.7- to 49.7-times the CMC. Therefore, the range of concentrations in which tests were performed with the present method was within the range of concentrations with high cleansing. Furthermore, we examined the relationship between cell viability and release of the inflammatory mediator interleukin-1α (IL-1α). IL-1α release was associated with cell viability, supporting the results of the human patch test.

Eliminating the contribution of lipopolysaccharide to protein allergenicity in the human cell-line activation test (h-CLAT).

We previously developed a test for detecting naturally occurring protein-induced skin sensitization based on the markers and criteria of the human cell-line activation test (h-CLAT) and showed that the h-CLAT was useful for assessing the allergenic potency of proteins. However, test proteins were contaminated with varying amounts of lipopolysaccharide (LPS), which might have contributed to the stimulation of CD86 and CD54 expression. In this study, we developed a method to exclude the effects of LPS in the assessment of skin sensitization by naturally occurring proteins. We tested two inhibitors [the caspase-1 inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk; hereafter referred to as YVAD), which can mitigate the LPS-induced increases in CD54 expression, and polymyxin B (PMB), which suppresses the effect of LPS by binding to its lipid moiety (i.e., the toxic component of LPS)]. After a 24 hr exposure, YVAD and PMB reduced LPS-induced CD86 and CD54 expression. In particular, the effect of PMB was dependent upon pre-incubation time and temperature, with the most potent effect observed following pre-incubation at 37°C for 24 hr. Moreover, only pre-incubation with cell-culture medium (CCM) at 37°C for 24 hr showed an inhibitory effect similar to that of PMB, with this result possibly caused by components of CCM binding to LPS. Similar effects were observed in the presence of ovalbumin (with 1070 EU/mg LPS) and ovomucoid, and lysozyme (with 2.82 and 0.234 EU/mg LPS, respectively) in CCM. These results indicated that PMB and CCM effectively eliminated the effects of LPS during assessment of protein allergenicity, thereby allowing a more accurate evaluation of the potential of proteins to induce skin sensitization.

Some non-sensitizers upregulate CD54 expression by activation of the NLRP3 inflammasome in THP-1 cells.

The human cell line activation test (h-CLAT) is a skin sensitization test that measures the expression of cell surface proteins CD86 and CD54 to evaluate the skin sensitization potential of test chemicals. However, some skin irritants have been reported to induce dramatically high CD54 expression leading to false-positive h-CLAT results. Furthermore, CD54 expression is strongly induced by cytokines, such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, or danger signals that activate its signaling pathways. In this study, we focused on the relationship between CD54 expression and the Nucleotide binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome, a protein complex that plays a pivotal role in intra-cellular inflammation. We observed the activation of caspase-1 and production of IL-1ß after exposure of THP-1 cells to 2,4-dinitrochlorobenzene (DNCB, sensitizer), octanoic acid (OA, non-sensitizer), and salicylic acid (SA, non-sensitizer), implying NLRP3 activation. These observations confirmed the activation of the inflammasome by CD54-only positive chemicals. CD54 expression, induced by OA and SA, was suppressed by potassium chloride, a typical inhibitor of NLRP3 inflammasome activation. These results suggested that the NLRP3 inflammasome may be activated in THP-1 cells resulting in the expression of CD54, and subsequently leading to false-positive results.

[A Surgical Case of Anaplastic Ependymoma Involving a Bridging Vein that Drained Directly into the Occipital Sinus].

INTRODUCTION: The draining veins of the brain stem and cerebellum commonly drain into the petrosal vein and sigmoid sinus, and often drain into the marginal sinus in the caudal part of the posterior fossa. Here, we report a rare case of anaplastic ependymoma involving a bridging vein that drained directly into the occipital sinus. CASE DESCRIPTION: A 6-year-old boy was admitted to our hospital with a 1-month history of nausea, headache, and dizziness. Magnetic resonance imaging(MRI)revealed a markedly enhanced fourth ventricular tumor and obstructive hydrocephalus. Surgical removal was performed via a midline suboccipital approach. When opening the dura, we observed a bridging vein that directly connected the brain stem and the tumor with the occipital sinus. Therefore, the Y-shaped dura mater incision was not inverted, and the tumor was totally removed while preserving the draining vein. After the operation, the patient's clinical course was uneventful. The pathological diagnosis was anaplastic ependymoma(WHO grade III). Subsequently, the patient received radiotherapy and was discharged without any neurological deficits 9 weeks after the operation. At 10 months after the initial surgery, the tumor recurred on the fourth ventricle floor. Thus, we performed a second surgical procedure and noted that the bridging vein had regressed. CONCLUSION: We report a rare draining vein that directly connected the brain stem to the occipital sinus. The tumor was removed without sacrificing this vein. Since the draining system of the posterior fossa is sometimes very complicated, we need to pay attention to it during the pre- and intra-operative periods.

Assessment of the skin sensitizing potential of chemicals, contained in foods and/or cosmetic ingredients, using a modified local lymph node assay with an elicitation phase (LLNA:DAE) method.

We evaluated the skin sensitizing potential of 10 natural organic chemicals, or their derivatives, which are included in foods and/or skin products, using a modified local lymph node assay (LLNA), with an elicitation phase (LLNA:DAE). The following compounds were tested: carminic acid, esculetin, 4-methyl esculetin, coumarin, quercetin, curcumin, naringenin, chlorogenic acid, isoscopoletin, and shikonin. Esculetin, 4-methyl esculetin, isoscopoletin, and shikonin yielded positive results. In particular, shikonin at a very low concentration (0.05%) induced an elicitation response. In conclusion, four of the 10 natural organic chemicals tested had a skin sensitization potential, with shikonin producing serious reaction even at a very low concentration.

Expansion of the applicability domain for highly volatile substances on the Short Time Exposure test method and the predictive performance in assessing eye irritation potential.

The Short Time Exposure (STE) test method is an in vitro method for assessing the eye irritation potential of chemicals and is used to classify the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Category 1 and No Category (NC). The method has been adopted by the Organisation for Economic Co-operation and Development (OECD) as test guideline (TG) 491 since 2015. While this method can be used to classify GHS NC, it is not suitable for testing highly volatile substances and solids other than surfactants. Here we evaluated highly volatile substances to expand the applicability domain. According to TG 491, acetone, ethanol, iso-propanol, and methyl acetate as highly volatile substances resulted in false negatives. Saline was selected as a solvent of these false negatives. In this study, mineral oil was used as the solvent, because these false negatives were amphiphilic. Based on this change, four highly volatile substances were correctly evaluated. The predictive performance for classifying GHS NC was then verified using a substance dataset constructed in reference to the Draize eye test Reference Database and STE Summary Review Document. The accuracy and false-negative rate were 86.6% (194/224) and 3.8% (3/80), respectively. Collectively, the applicability domain was expanded by changing the solvent to mineral oil for highly volatile substances, and the predictive performance for the new applicability domain including highly volatile substances was excellent. The STE test method is suitable to classify GHS NC, indicating its applicability as a test method in a bottom-up approach.

A case report: C2 radiculopathy induced by neck flexion due to the cord compression of C2 segmental type vertebral artery relieved by microvascular decompression.

BACKGROUND: Decompression of an anomalous vertebral artery (VA) may effectively treat cervical myelopathy/radiculopathy due to resultant spinal cord or nerve compression. Here we report a case of C2 radiculopathy induced by neck flexion due to cord compression of the C2 segmental type VA relieved by microvascular decompression. CASE DESCRIPTION: A 30-year-old female presented with left occipitalgia, sensory abnormalities in the left upper and lower extremities, and neck pain induced by neck flexion. The magnetic resonance imaging (MRI) revealed an abnormal flow void, confirming that the VA was compressing the spinal cord at the C1 level. Three-dimensional computed tomography (3D-CT) showed an anomalous course of the left VA, which entered the spinal canal between the axis and atlas. Microvascular decompression was performed by transposing the artery (e.g., anchoring it to the dura using PTEF): this effectively relieved cord compression. CONCLUSION: An anomalous VA rarely causes cervical radiculopathy induced by neck flexion. When it occurs, microvascular decompression effectively relieves pressure resulting in a resolution of symptoms.

Acidic conditions induce the suppression of CD86 and CD54 expression in THP-1 cells.

To evaluate the sensitization potential of chemicals in cosmetics, using non-animal methods, a number of in vitro safety tests have been designed. Current assays are based on the expression of cell surface markers, such as CD86 and CD54, which are associated with the activation of dendritic cells, in skin sensitization tests. However, these markers are influenced by culture conditions through activating danger signals. In this study, we investigated the relationship between extracellular pH and the expression of the skin sensitization test human cell line activation test (h-CLAT) markers CD86 and CD54. We measured expression levels after THP-1 cells were exposed to representative contact allergens, i.e., 2,4-dinitrochlorobenzene and imidazolidinyl urea, under acidic conditions. These conditions were set by exposure to hydrochloric acid, lactic acid, and citric acid. An acidic extracellular pH (6-7) suppressed the augmentation of CD86 and CD54 levels by the sensitizer. Additionally, when the CD86/CD54 expression levels were suppressed, a reduction in the intracellular pH was confirmed. Furthermore, we observed that Na+/H+ exchanger 1 (NHE-1), a protein that contributes to the regulation of extracellular/intracellular pH, is involved in CD86 and CD54 expression. These findings suggest that the extracellular/intracellular pH has substantial effects on in vitro skin sensitization markers and should be considered in evaluations of the safety of mixtures and commercial products in the future.

Development of an in vitro photosafety evaluation method utilizing intracellular ROS production in THP-1 cells.

Photoreactive compounds that may experience exposure to ultraviolet (UV) radiation can lead to the intracellular production of reactive oxygen species (ROS), which may cause phototoxic and photoallergenic responses. Here, we developed a novel in vitro photosafety assay and investigated whether it could be used to predict phototoxicity and photosensitivity by measuring changes in intracellular ROS production. THP-1 cells that had previously taken up 5-(and-6)-carboxy-2',7'-difluorodihydrofluorescein diacetate (carboxy-H2DFFDA), a ROS-sensitive fluorescent reagent, were exposed to photoreactive substances such as phototoxic and photoallergenic materials and then subjected to with UV-A irradiation (5 J/cm2). The fluorescence intensity was subsequently measured using a flow cytometer, and the intracellular ROS production was calculated. A statistically significant increase in ROS following treatment with photoreactive substances was observed in cells irradiated with UV-A. In contrast, no significant increase was observed for non-photoreactive substances in comparison to the control solution. Next, to confirm the impact of intracellular ROS on the photosensitive response, changes in CD86 and CD54 expression were measured following quencher addition during the photo human cell line activation test (photo h-CLAT). The results confirmed the reduction of CD86 and CD54 expression in response to photoallergenic substances following quencher addition. Together, these findings suggest that intracellular ROS production is involved in photosensitizing reactions. Therefore, we suggest that the developed method utilizing intracellular ROS production as an index may be useful as a novel in vitro evaluation tool for photoreactive substances.

Improvement of human cell line activation test (h-CLAT) using short-time exposure methods for prevention of false-negative results.

Recently, animal testing has been affected by increasing ethical, social, and political concerns regarding animal welfare. Several in vitro safety tests for evaluating skin sensitization, such as the human cell line activation test (h-CLAT), have been proposed. However, similar to other tests, the h-CLAT has produced false-negative results, including in tests for acid anhydride and water-insoluble chemicals. In a previous study, we demonstrated that the cause of false-negative results from phthalic anhydride was hydrolysis by an aqueous vehicle, with IL-8 release from THP-1 cells, and that short-time exposure to liquid paraffin (LP) dispersion medium could reduce false-negative results from acid anhydrides. In the present study, we modified the h-CLAT by applying this exposure method. We found that the modified h-CLAT is a promising method for reducing false-negative results obtained from acid anhydrides and chemicals with octanol-water partition coefficients (LogKow) greater than 3.5. Based on the outcomes from the present study, a combination of the original and the modified h-CLAT is suggested for reducing false-negative results. Notably, the combination method provided a sensitivity of 95% (overall chemicals) or 93% (chemicals with LogKow > 2.0), and an accuracy of 88% (overall chemicals) or 81% (chemicals with LogKow > 2.0). We found that the combined method is a promising evaluation scheme for reducing false-negative results seen in existing in vitro skin-sensitization tests. In the future, we expect a combination of original and modified h-CLAT to be applied in a newly developed in vitro test for evaluating skin sensitization.

Lipopolysaccharide interferes with the use of the human Cell Line Activation Test to determine the allergic potential of proteins.

It was believed that high molecular weight molecules including proteins cannot penetrate the skin. However, protein penetration through disrupted/ruptured skin has been reported recently, thus carrying the potential for inducing an allergic response. We used the human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, to assess the allergic potential of proteins by measuring levels of CD86 and CD54 in the human monocytic leukemia cell line THP-1. Six allergens including ovalbumin (OVA) and human serum albumin (HSA; negative control) upregulated CD86 and/or CD54; a false-positive result was obtained using HSA. This was caused by lipopolysaccharide (LPS) contamination. Naturally derived materials often include LPS at various concentrations and may influence protein induction of CD86 and CD54. Additionally, polymyxin B, an LPS inhibitor, could not completely overcome the effect of LPS. Therefore, if test proteins contain ≥0.1 EU/mL LPS, their allergenic potency will not be assessed accurately using h-CLAT. These data show that naturally occurring materials or those derived from living organisms should be evaluated for their LPS content. It is important to confirm the applicability of in vitro methods such as h-CLAT for assessing the allergenic potency of naturally occurring proteins; our findings can be a foundation for future studies.

Predictive performance and inter-laboratory reproducibility in assessing eye irritation potential of water- and oil-soluble mixtures using the Short Time Exposure test method.

The Short Time Exposure (STE) test method is an alternative method for assessing eye irritation potential using Statens Seruminstitut Rabbit Cornea cells and has been adopted as test guideline 491 by the Organisation for Economic Co-operation and Development. Its good predictive performance in identifying the Globally Harmonized System (GHS) No Category (NC) or Irritant Category has been demonstrated in evaluations of water-soluble substances, oil-soluble substances, and water-soluble mixtures. However, the predictive performance for oil-soluble mixtures was not evaluated. Twenty-four oil-soluble mixtures were evaluated using the STE test method. The GHS NC or Irritant Category of 22 oil-soluble mixtures were consistent with that of a Reconstructed human Cornea-like Epithelium (RhCE) test method. Inter-laboratory reproducibility was then confirmed using 20 water- and oil-soluble mixtures blind-coded. The concordance in GHS NC or Irritant Category among four laboratories was 90%-100%. In conclusion, the concordance in comparison with the results of RhCE test method using 24 oil-soluble mixtures and inter-laboratory reproducibility using 20 water- and oil-soluble mixtures blind-coded were good, indicating that the STE test method is a suitable alternative for predicting the eye irritation potential of both substances and mixtures.

Development of a modified 3T3 Neutral Red Uptake Phototoxicity Test protocol for evaluation of poorly water-soluble substances.

The 3T3 neutral red uptake phototoxicity test (OECD TG432) is an alternative phototoxicity test method that is relatively easy and rapid to implement, with results obtainable in a short time, and is reported to have high reproducibility compared with in vivo assay methods. However, this method has been shown to be unsuitable for testing poorly water-soluble substances, which tend to separate out when mixed with the assay buffer solution. This causes difficulties in determining the dose dependency of substances and subsequent determination of the photoirritation factor because the ratio of cell viability, expressed as the half-maximal inhibitory concentration (IC50) in the presence or absence of light, is not calculable. In this study, we investigated the optimum conditions for the evaluation of poorly water-soluble substances. In the conventional method, the final solvent concentration was 1% and the pre-incubation time was 60 min, but in the modified method, 10% and 5 min were used, respectively. Next, the results from the conventional method were compared with those of our modified method, which was found to be viable and comparable with the conventional method. Moreover, the false positive results frequently obtained with poorly water-soluble substances in the conventional method were not evident with the modified method, thus confirming its usefulness for the evaluation of such substances. We therefore propose that the modified method can be used for the in vitro testing of poorly water-soluble substances in phototoxicity evaluations.

Long form of thymic stromal lymphopoietin of keratinocytes is induced by protein allergens.

A growing body of evidence suggests that epicutaneous sensitization of protein allergens induces immediate-type hypersensitivity (IHS) following induction of Type 2 immune responses in animals and humans. Thymic stromal lymphopoietin (TSLP) derived from keratinocytes is a cytokine that can activate dendritic cells and has been implicated in development of inflammatory Type 2 helper T-cells. However, there is no direct evidence that allergens directly regulate TSLP expression in keratinocytes. This study aimed to evaluate the response of TSLP to protein allergens in cultured human keratinocytes and to identify appropriate endpoints for IHS. The transcription of long-form TSLP (loTSLP) was strongly induced by ovalbumin, wheat gluten (WG), acid-hydrolyzed WG (acid-HWG), and extracts from feces of Dermatophagoides pteronyssinus and D. farina, and trypsin, but not by rare allergens, human serum albumin (HSA), or extracts of mite bodies. In acid-HWG, loTSLP mRNA was significantly augmented by acid hydrolysis of WG for 0.5 h compared to WG. However, prolonged acid hydrolysis attenuated this induction similarly to that reported in previous animal studies. These results suggested that intense loTSLP transcriptional induction was a characteristic of a high-allergenic protein. Additionally, TSLP production was induced by exposure to ovalbumin, WG, and acid-HWG in combination with a trio of cytokines, i.e. interleukin (IL)-4, IL-13, and tumor necrosis factor (TNF)-α. However, no TSLP protein was detected following exposure to HSA, even in the presence of these cytokines. With acid-HWG, TSLP protein release was consistent with loTSLP transcription. Thus, intense loTSLP transcriptional induction and TSLP protein expression are each effective indicators that can be used for in vitro screening of IHS.