Assessment of cardiac load-responsiveness in veno-arterial extracorporeal life support: A case series.

INTRODUCTION: Well-timed explant of veno-arterial extracorporeal life support (V-A ECLS) depends on adequate assessment of cardiac recovery. Often, evaluation of cardiac recovery consists of reducing support flow while visualizing cardiac response using transoesophageal echocardiography (TEE). This method, however, is time consuming and based on subjective findings. The dynamic filling index (DFI) may aid in the quantitative assessment of cardiac load-responsiveness. The dynamic filling index is based on the relationship of support flow and pump speed, which varies with varying hemodynamic conditions. This case series intends to investigate whether the DFI may support TEE in facilitating the assessment of cardiac load-responsiveness. METHODS: Measurements for DFI-determination were performed in seven patients while simultaneously assessing ventricular function by measuring the aortic velocity time integral (VTI) using TEE. Measurements consisted of multiple consecutive transient speed manipulations (∼100 r/min) during weaning trials, both at full support and during cardiac reloading at reduced support. RESULTS: The VTI increased between full and reduced support in six weaning trials. In five of these trials DFI decreased or remained equal, and in one case DFI increased. Of the three trials in which VTI decreased between full and reduced support, DFI increased in two cases and decreased in one case. Changes in DFI, however, are mostly smaller than the detection threshold of 0.4 mL/rotation. CONCLUSION: Even though current level of accuracy of the parameter requires further investigation to increase reliability and possibly predictability, DFI seems likely to be a potential parameter in supporting TEE for the assessment of cardiac load-responsiveness.

Reliability of diagnosis from Mohs slides: interpersonal and intrapersonal agreement on basal cell carcinoma presence and histological subtype.

BACKGROUND: The success of Mohs micrographic surgery (MMS) depends partly on the correct diagnosis of slides. OBJECTIVES: To determine reliability of diagnosis from Mohs slides. METHODS: This was a prospective study evaluating the reliability of diagnosis from Mohs slides of basal cell carcinoma (BCC) presence, BCC location on the slide and BCC subtype among six raters who independently assessed 50 Mohs slides twice with a 2-month interval. Slides were randomly selected whereby difficult-to-diagnose slides were oversampled. For each slide, a reference diagnosis was established by an expert panel. Cohen's kappa (κ) was calculated to determine levels of agreement interpersonally (rater vs. reference diagnosis) and intrapersonally (rater at T1 vs. T2). Multivariable logistic regression was used to determine independent risk factors for slides with interpersonal discordant diagnosis. The variables studied were BCC presence, whether a slide was scored as easy or difficult to diagnose, review duration of the 50 slides, profession and years of experience in diagnosis from Mohs slides. RESULTS: Interpersonal and intrapersonal agreement were substantial on BCC presence (κ = 0·66 and 0·68) and moderate on BCC subtype (κ = 0·45 and 0·55). Slides that were scored as difficult to diagnose were an independent risk factor for interpersonal discordant diagnosis on BCC presence (odds ratio 3·54, 95% confidence interval 1·81-6·84). CONCLUSIONS: Reliability of diagnosis from Mohs slides was substantial on BCC presence and moderate on BCC subtype. For slides that are scored difficult to diagnose, a second opinion is recommended to prevent misinterpretation and thereby recurrence of skin cancer.

Determination of three-dimensional muscle architectures: validation of the DTI-based fiber tractography method by manual digitization.

In the last decade, diffusion tensor imaging (DTI) has been used increasingly to investigate three-dimensional (3D) muscle architectures. So far there is no study that has proved the validity of this method to determine fascicle lengths and pennation angles within a whole muscle. To verify the DTI method, fascicle lengths of m. soleus as well as their pennation angles have been measured using two different methods. First, the 3D muscle architecture was analyzed in vivo applying the DTI method with subsequent deterministic fiber tractography. In a second step, the muscle architecture of the same muscle was analyzed using a standard manual digitization system (MicroScribe MLX). Comparing both methods, we found differences for the median pennation angles (P < 0.001) but not for the median fascicle lengths (P = 0.216). Despite the statistical results, we conclude that the DTI method is appropriate to determine the global fiber orientation. The difference in median pennation angles determined with both methods is only about 1.2° (median pennation angle of MicroScribe: 9.7°; DTI: 8.5°) and probably has no practical relevance for muscle simulation studies. Determining fascicle lengths requires additional restriction and further development of the DTI method.

Inhibiting connexin channels protects against cryopreservation-induced cell death in human blood vessels.

OBJECTIVES: Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation. MATERIALS AND METHODS: Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination. RESULTS: We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins. CONCLUSIONS: We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability.

Variability of microcirculatory measurements in healthy volunteers.

Reliable assessment of the microcirculation is important to investigate microcirculatory properties in various disease states. The GlycoCheck system automatically analyzes sublingual sidestream dark field images to determine the perfused boundary region (PBR; a measure of glycocalyx thickness), red blood cell filling percentage, and microvascular vessel density. Although GlycoCheck has been used to study the microcirculation in patients, little is known about the reproducibility of measurements in healthy volunteers. We assessed intra- and interobserver agreement by having two experienced observers perform three consecutive microcirculation measurements with the GlycoCheck system in 49 healthy volunteers. Intraobserver agreement of single measurements were poor (intraclass correlation coefficients (ICCs) < 0.4) for PBR, red blood cell filling percentage and microvascular vessel density. ICCs increased to values > 0.6 (indicating good reproducibility) for all parameters when performing and averaging three consecutive measurements. No systematic differences were observed between observers for any parameter. Interobserver variability was fair for PBR (ICC = 0.53) and red blood cell filling percentage (ICC = 0.58) and poor for perfused vessel density (ICC = 0.20). In conclusion, GlycoCheck software can be used with acceptable reliability and reproducibility for microcirculation measurements on a population level when averaging three consecutive measurements. Repeated measurements are preferably performed by the same observer.

3D finite element models from serial section histology of skeletal muscle tissue - The role of micro-architecture on mechanical behaviour.

In this contribution we create three-dimensional (3D) finite element models from a series of histological sections of porcine skeletal muscle tissue. Image registration is performed on the stained sections by affinely aligning them using auxiliary markers, followed by image segmentation to determine muscle fibres and the extracellular matrix in each section, with particular regard to the continuity of the fibres through the stack. With this information, 3D virtual tissue samples are reconstructed, discretised, and associated with appropriate non-linear elastic anisotropic material models. While the gross anatomy is directly obtained from the images, the local directions of anisotropy were determined by the use of an analogy with steady state diffusion. The influence of the number of histological sections considered for reconstruction on the numerically simulated mechanical response of the virtual tissue samples is then studied. The results show that muscle tissue is fairly heterogeneous along the fascicles, and that transverse isotropy is inadequate in describing their material symmetry at the typical length scale of a fascicle. Numerical simulations of different load cases suggest that ignoring the undulations of fibres and their non-uniform cross-sections only moderately affects the passive response of the tissue in tensile and compressive modes, but can become crucial when predicting the response to generic loads and activation.

[A man with an anomaly in his ear]. / Een man met een afwijking in het oor.

A 65-year-old man presented with an anomaly in his left ear. He had no complaints, but was ashamed of the lesion. On physical examination a pigmented, pedunculated, polypoid tumour of approximately 2 x 2.5 cm was seen, which filled the whole cavum conchae. After excision, histopathological examination showed a verruca seborrhoica.

Improved objectivity of grading of T(A,1) transitional cell carcinomas of the urinary bladder by quantitative nuclear and proliferation related features.

AIM: To analyse whether the mean nuclear area of the 10 largest nuclei (MNA-10), the mitotic activity index (MAI), and Ki-67 immunoquantitative features have additional value to discriminate different grades of T(A,1) transitional cell carcinoma (TCC) of the urinary bladder. MATERIALS/METHODS: One hundred and fifty of 200 consecutive cases (75%) showing interobserver agreement on duplicate blind grade assessment by independent pathologists were studied. Using random numbers, the 150 cases were divided into sets for learning (n = 75) and testing (n = 75). Single and multivariate analyses were applied to discriminate the different grades in the learning set. The multivariate classifier developed in this way was evaluated in the test set (n = 75). RESULTS: With the MNA-10 alone, using the classification MNA-10 < 80 microm(2) = grade 1, 80 microm(2) < MNA-10 < 130 microm(2) = grade 2, MNA-10 > 130 microm(2) = grade 3, 71% of all 150 cases were correctly classified (69% of grade 1 v grade 2 and 76% of grade 2 v grade 3). With multivariate analysis, the best discriminating features in the learning set (17 grade 1, 30 grade 2, and 28 grade 3) between grades 1 and 2 were MNA-10 and MAI, and between grades 2 and 3 MAI and Ki-67. With these features, 94% of grade 1 v grade 2 and 97% of grade 2 v grade 3 were correctly classified in the learning set (overall, 95% correct, none of the grade 3 cases misclassified). In the test set the classification results were similar. When the three grades were entered at the same time for discrimination, Ki-67 area % and MAI was the best discriminating combination, both in the sets for learning and testing. Overall correct classification results in the sets for learning and testing were slightly lower, but still 94% and 92%. Most importantly, none of the grade 3 cases was misclassified; the classification shifts all occurred between grades 1 and 2. CONCLUSIONS: The combination of MNA-10, MAI, and Ki-67 gives much better discrimination between grades 1, 2, and 3 in T(A,1) TCC of the urinary bladder than MNA-10 alone. The similarity of the classification results of the learning set and test set are encouraging and this quantitative pathological grading model should be applied in a prospective study.

Proliferation markers and DNA content analysis in urinary bladder TaT1 urothelial cell carcinomas: identification of subgroups with low and high stage progression risks.

AIMS: To evaluate whether in situ biomarkers Ki67, mitotic activity index (MAI), p53, mean area of the 10 largest nuclei (MNA10), and whole genome DNA ploidy by flow and image cytometry (FCM and ICM, respectively) have independent prognostic value in urinary bladder urothelial cell carcinomas (UCs). METHODS: Ki67 and p53 immunoquantitation was performed in TaT1 consensus diagnosis UCs. MAI and MNA10 were also determined. Single cell suspensions were stained (DAPI for FCM; Feulgen for ICM). There was enough material for all measurements in 171 cases. Kaplan-Meier curves and multivariate survival analysis (Cox) were used to assess the prognostic value of all features (including classic clinicopathological risk factors, such as stage, grade, multicentricity, carcinoma in situ). RESULTS: Thirteen (7.6%) patients progressed. Of the classic factors, grade was strongly prognostic in univariate analysis, as were all the biomarkers. In multivariate analysis, the strongest independent combinations for progression were MNA10 (threshold (T) = 170.0 micro m(2)) plus MAI (T = 30), or MNA10 (T = 170.0 micro m(2)) plus Ki67(T = 25.0%). p53 (T = 35.2%) plus Ki67 (T = 25.0%) also predicted progression well, with high hazard ratios, but p53 measurements were not as reproducible as the other features. The prognostic value of the quantitative biomarkers exceeded that of the classic risk factors and DNA ploidy. The sensitivity, specificity, positive, and negative predictive values of MNA10/MAI or MNA10/Ki67 at the thresholds mentioned were 100%, 79%, 57%, and 100%, respectively. These feature combinations were also strongest prognostically in the high risk treatment subgroup. CONCLUSIONS: The combined biomarkers MNA10/Ki67 or MNA10/MAI are more accurate and reproducible predictors of stage progression in TaT1 UCs than classic prognostic risk factors and DNA ploidy.

The vehicle modulates cellular and humoral responses in contact hypersensitivity to oxazolone.

The development of contact hypersensitivity (CHS) greatly depends on the allergenicity of the inducing agent. However, various cofactors are known to influence the outcome of the response as well. From this perspective, we have compared the effects of five different vehicles: acetone, ethanol, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), and a 4 to 1 mixture of acetone and olive oil (AOO) on the cellular and humoral immune responses to epicutaneously applied oxazolone in female BALB/c mice. A single application of 0.2% oxazolone dissolved in acetone or ethanol induced stronger proliferative responses and higher lymph node cell numbers than the other three vehicles. Moreover, both vehicles led to higher numbers of oxazolone-specific Ab forming cells in the draining lymph nodes of sensitized animals. When the IgG2a/IgG1 ratios were determined to indicate the type of T helper cell involved, the highest values were obtained with AOO and lowest with DMF and DMSO, while acetone and ethanol were in between. Moreover, no correlation was found between oxazolone-specific antibody production and cellular responses, measured as [3H]thymidine incorporation of draining lymph node cells after sensitization and increased ear thickness after challenge. From this study it can be concluded that cellular and humoral responses in CHS to oxazolone are dissimilarly affected by the vehicles used.

Immunotoxic organotins as possible model compounds in studying apoptosis and thymocyte differentiation.

In the mid-seventies it appeared that some organotin compounds selectively caused thymus atrophy. From that time onward efforts were made to reveal molecular and cellular mechanisms involved. In this review recent studies into organotin-sensitive stages and processes of thymocyte maturation are discussed. Together these studies resulted in the recognition of organotin compounds as possible model compounds in studying immature thymocyte differentiation and protein synthesis-independent apoptotic cell death of thymocytes.

Predictive testing for autoimmunity.

Many chemicals, in particular drugs, cause systemic allergy or autoimmune-like disorders. Due to complex pathogenesis and strong dependence on genetic make-up, these immunotoxicological effects are usually missed in standard toxicity testing. Besides, animal studies that demonstrate chemically induced systemic allergy or autoimmune-like disorders are scarce. Here, animal models are presented that would fit into a predictive two-tiered strategy, designed to allow screening for immunostimulatory potential in the first tier, and more elaborate testing for allergenic or autoimmunogenic potential of selected chemicals in the second tier. The popliteal lymph node assay (PLNA), with or without reporter antigens, would fit in the first tier, and relevant route of exposure protocols with selected strains of mice or rats may be further developed to compose the second tier. To date, the relevant route of exposure models mentioned here (with 'normal' inbred mice and/or Brown Norway rats) has been tested with only a few chemicals, and the PLNA, although tested with over 100 chemicals, is not validated as yet. Conceivably, a major challenge in immunotoxicology is to incorporate the present knowledge on chemical-induced systemic allergy and autoimmunity in further development and validation of predictive models and strategies.

Cellular and molecular aspects of organotin-induced thymus atrophy.

1. Organotin compounds, di-n-butyltin dichloride (DBTC) in particular, have been shown to cause thymus atrophy in the rat. 2. DBTC-induced thymus atrophy results from a depletion of small CD4+CD8+ thymocytes which is caused by a diminished production of immature CD4-CD8+ and CD4+CD8+ thymoblasts. 3. DBTC inhibits the activation, but not the differentiation of immature CD4-CD8+ thymocytes in vitro and in vivo suggesting a selective antiproliferative activity of DBTC. 4. DBTC inhibits the adhesion molecule-mediated binding of thymocytes to thymic epithelial cells. 5. DBTC enhances the Ca2+ release elicited by cross-linking of the T cell receptor complex (TcR alpha beta-CD3) on thymocytes and moreover delays cap formation of the TcR alpha beta-CD3 receptor. 6. It is concluded that DBTC possibly interferes with the functioning of the cytoskeleton. The relation of the in vitro findings to the inhibition of immature CD4-CD8+ thymocyte activation and the induction of thymus atrophy is unknown as yet.

Transfer of IP3 through gap junctions is critical, but not sufficient, for the spread of apoptosis.

Decades of research have indicated that gap junction channels contribute to the propagation of apoptosis between neighboring cells. Inositol 1,4,5-trisphosphate (IP3) has been proposed as the responsible molecule conveying the apoptotic message, although conclusive results are still missing. We investigated the role of IP3 in a model of gap junction-mediated spreading of cytochrome C-induced apoptosis. We used targeted loading of high-molecular-weight agents interfering with the IP3 signaling cascade in the apoptosis trigger zone and cell death communication zone of C6-glioma cells heterologously expressing connexin (Cx)43 or Cx26. Blocking IP3 receptors or stimulating IP3 degradation both diminished the propagation of apoptosis. Apoptosis spread was also reduced in cells expressing mutant Cx26, which forms gap junctions with an impaired IP3 permeability. However, IP3 by itself was not able to induce cell death, but only potentiated cell death propagation when the apoptosis trigger was applied. We conclude that IP3 is a key necessary messenger for communicating apoptotic cell death via gap junctions, but needs to team up with other factors to become a fully pro-apoptotic messenger.

Unusual case of bifocal leptomeningeal melanocytoma in the posterior fossa with seeding in the spinal canal.

A 26-year-old man presented with signs of raised intracranial pressure. CT and MRI of the head demonstrated two separate lesions in the posterior fossa. The radiological differential diagnoses included multiple meningiomas, schwannomas, neurofibromas and subependymomas. Both lesions were surgically resected. Histopathological examination revealed localisations of a leptomeningeal melanocytoma. Leptomeningeal melanocytoma is a rare tumour of the central nervous system. Generally, it has a good prognosis if radical resection can be performed. In cases of subtotal resection, adjuvant radiotherapy should be considered. Local recurrences are common. Less frequently, leptomeningeal metastases and, on rare occasions, distant metastases or progression to malignant melanoma have been described. We describe an unusual case with multiple localisations of melanocytoma in the posterior fossa and spinal canal, with the emphasis being on the radiological findings and diagnosis of this rare tumour. After surgery of the brain, this patient was irradiated on the craniospinal axis.

Ultrafine but not fine particulate matter causes airway inflammation and allergic airway sensitization to co-administered antigen in mice.

BACKGROUND: Airborne particulate matter (PM) is an important factor associated with the enhanced prevalence of respiratory allergy. The PM adjuvant activity on allergic sensitization is a possible mechanism of action involved, and the induction of airway inflammation is suggested to be of importance in PM-induced adjuvant activity. OBJECTIVE: Because differently sized PM have different toxic potentials, we studied the role of particle size in the induction of airway inflammation and allergic sensitization. This was done using fine (0.250 and 0.260 micro m) and ultrafine (0.029 and 0.014 micro m) titanium dioxide (TiO(2)) and carbon black particles (CBP) with known differences in airway toxicity. METHODS: Mice were intranasally exposed to ovalbumin (OVA) alone or in combination with one of the different particles. The induction of airway inflammation and the immune adjuvant activity were studied in the lungs and lung-draining peribronchial lymph nodes (PBLN) at day 8. OVA-specific antibodies were measured at day 21, and the development of allergic airway inflammation was studied after OVA challenges (day 28). RESULTS: When administered at the same total particle mass (200 micro g), exposure to ultrafine TiO(2) and CBP-induced airway inflammation, and had immune adjuvant activity. The latter was shown by increasing both the PBLN cell numbers and the production of OVA-specific T-helper type 2 (Th2) cytokines (IL-4, IL-5, IL-10 and IL-13). Whereas OVA-specific IgE and IgG1 levels in serum were only increased in animals exposed to the ultrafine TiO(2), allergic airway inflammation could be detected in both ultrafine TiO(2)-and CBP-treated groups after challenges with OVA. CONCLUSION: Our data show that only the ultrafine particles, with a small diameter and a large total surface area/mass, cause airway inflammation and have immune adjuvant activity in the current model supporting the hypothesis that particle toxicity is site-dependent and related to adjuvant activity.

The organotin-induced thymus atrophy, characterized by depletion of CD4+ CD8+ thymocytes, is preceded by a reduction of the immature CD4- CD8+ TcR alpha beta-/low CD2high thymoblast subset.

Thymic changes in the rat induced by the thymus atrophy-inducing organotin compound di-n-butyltin dichloride (DBTC) were examined using FACS analyses. The number of CD4+CD8+ thymocytes was reduced by DBTC treatment from Day 2 onwards and reached minimum level on Days 4 and 5 after dosing. On these days the CD4-CD8- and both the CD4-CD8+ and CD4+CD8- subsets were not affected. On Day 2 we observed a reduced proportion of transferrin receptor (CD71)-positive CD4-OX44- cells, representing the cycling immature CD4-CD8+ cells, and of CD71+OX44- cells, representing the cycling CD4+CD8+ cells, but not of CD71+CD4-CD8- cells. When compared to controls, the FSChigh cell population of DBTC-treated rats contained less CD4-OX44- and OX44- cells, which were further characterized as CD2high and T-cell receptor (TcR)alpha beta- low. Moreover, fewer TcR alpha beta high cells were detected in the OX44- thymoblast subset of DBTC-treated rats. The number of CD4-CD8- thymoblasts appeared marginally decreased while the numbers of CD4+OX44+ cells, representing mature CD4+ cells, were not affected. These data indicate that DBTC causes a preferential initial depletion of immature CD4-CD8+CD2high TcR alpha beta-low thymoblasts. This initial event may result in a decreased formation of CD4+CD8+ thymoblasts and of small CD4+CD8+ thymocytes. These characteristics of the initially depleted subset indicate a specific anti-proliferative effect of DBTC and may give clues for the mechanism involved in the induction of thymus atrophy.

Stress proteins (HSP) and chemical-induced autoimmunity.

Various environmental and iatrogenic chemicals have been implicated in the induction of autoimmune responses and biomarkers for identification of such chemicals are imperative. The present study was initiated to examine whether induction of stress protein (HSP) synthesis is a common effect of immunoactive chemicals. This would be interesting because HSP-induction could result in the presentation of HSP-derived T cell epitopes, and recognition of these epitopes by HSP-reactive T cells could facilitate the initiation of (auto)immune responses. Such a role for HSP would clarify early aspects of chemical induction of immune responses and could provide a valuable biomarker for the identification of potentially immunoactive chemicals. It was found that of eight immunoactive chemicals, only HgCl2, dinitrochlorobenzene, and dibutyltin dichloride induced synthesis of HSC73/HSP72 and HSP90 in murine splenocytes in vitro. The induction by HgCl2 was identical in splenocytes from mice susceptible or not for Hg-induced autoimmunity. Following footpad injection of HgCl2, but not diphenylhydantoin, a marginal induction of HSC73 and possibly HSP72 but not HSP90 was found to precede the chemical-induced lymphoproliferation in draining lymph nodes of BALB/c mice. Finally, using stimulation of the IgG1 response to TNP-ficoll as a model for non-antigen-linked, T cell-dependent B cell stimulation, it was found that stimulation of this response by chemicals is independent of HSP induction. From these results, we conclude that it is unlikely that HSP function as general initiating neoantigens in chemically induced autoimmune responses.

Selective immunomodulation by the autoimmunity-inducing xenobiotics streptozotocin and HgCl2.

Exposure to certain drugs and environmental chemicals can provoke the onset of autoimmune disease in susceptible individuals by releasing (self) epitopes for which tolerance has not been established, while simultaneously providing the necessary adjuvant activity. The resulting response type is influenced by the genotype of exposed individuals and relates to susceptibility to the adverse immune effects of the chemicals. Here, we assessed the modulatory role of the chemical compounds themselves. A single injection of streptozotocin (STZ) increased the number of CD8+ cells, macrophages, apoptotic cells, and IFN-gamma-producing T helper and T cytotoxic cells, whereas the number of CD4+ cells and B cells was reduced in the draining lymph node. Coinjection with the reporter antigen TNP-OVA resulted in primary and secondary production of TNP-specific antibodies that were predominantly of IgG2a and IgG2b isotype, whereas STZ did not enhance priming for delayed-type hypersensitivity (DTH) responses to TNP-OVA. Injection of HgCl2 on the other hand, reduced the number of IFN-gamma-producing cells, induced accumulation of B cells and CD4+ and CD8+ T cells, enhanced IgG1 and IgE production to TNP-OVA, and primed for secondary IgG1 and IgE production as well as for DTH reactions. Together these results indicate that a single injection of STZ stimulates type-1 responses, whereas HgCl2 enhanced mixed type-1 and -2 responses in BALB/c mice. These response types match the (auto)immune effects elicited to unknown (auto)antigens following multiple injections of these chemicals.