Transcriptomic fingerprints in human peripheral blood mononuclear cells indicative of genotoxic and non-genotoxic carcinogenic exposure.

For evaluating genotoxic exposure in human populations a number of biomarkers has been successfully applied over the last 30 years to determine early biological effects due to exposure to carcinogens. Despite their success, these early biological effect markers provide limited mechanistic insight, and do not allow detection of exposure to non-genotoxic carcinogens. Gene expression profiling forms a promising tool for the development of new biomarkers in blood cells to overcome these limitations. The aim of our research was to identify novel genomics-based candidate markers for genotoxic and non-genotoxic carcinogen exposure in human peripheral blood cells (PBMC). Whole genome gene expression changes were investigated following 20 h of in vitro exposure to a high and low concentration of eight genotoxic and three non-genotoxic carcinogenic compounds using whole genome microarrays. Per condition, PBMC of five independent donors were exposed, all in the presence of human liver S9. Sets of genes, as well as biological pathways indicative of genotoxic exposure and of non-genotoxic carcinogenic exposure were identified. Furthermore, networks were built using the genotoxic and non-genotoxic gene sets, showing the majority of the genes to be interlinked and revealing distinctive transcription factors for both classes. The identification of these potential candidate marker genes might contribute to the development of genomic based biomarkers of carcinogen exposure.

Transcriptomic network analysis of micronuclei-related genes: a case study.

Mechanistically relevant information on responses of humans to xenobiotic exposure in relation to chemically induced biological effects, such as micronuclei (MN) formation can be obtained through large-scale transcriptomics studies. Network analysis may enhance the analysis and visualisation of such data. Therefore, this study aimed to develop a 'MN formation' network based on a priori knowledge, by using the pathway tool MetaCore. The gene network contained 27 genes and three gene complexes that are related to processes involved in MN formation, e.g. spindle assembly checkpoint, cell cycle checkpoint and aneuploidy. The MN-related gene network was tested against a transcriptomics case study associated with MN measurements. In this case study, transcriptomic data from children and adults differentially exposed to ambient air pollution in the Czech Republic were analysed and visualised on the network. Six genes from the network, i.e. BAX, DMNT1, PCNA, HIC1, p21 and CDC20, were retrieved. Based on these six genes and in combination with p53 and IL-6, a dedicated network was created. This dedicated network is possibly suited for the development of a reporter gene assay that could be used to screen populations complementary to the current MN test assay. In conclusion, we have shown that network analysis of transcriptomics data in relation to the formation of MN is possible and provides a novel mechanistic hypothesis by indicating which genes are regulated and influence others.

Novel point mutation in the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor in a case of severe congenital neutropenia hyporesponsive to G-CSF treatment.

Severe congenital neutropenia (SCN) is a heterogeneous condition characterized by a drastic reduction in circulating neutrophils and a maturation arrest of myeloid progenitor cells in the bone marrow. Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF). Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment. When this mutant G-CSF-R was expressed in myeloid cells, it was defective in both proliferation and survival signaling. This correlated with diminished activation of the receptor complex as determined by signal transducer and activator of transcription (STAT) activation, although activation of STAT5 was more affected than STAT3. Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor. This suggests that the mutation in the extracellular domain affects ligand-receptor complex formation with severe consequences for intracellular signal transduction. Together these data add to our understanding of the mechanisms of cytokine receptor signaling, emphasize the role of GCSFR mutations in the etiology of SCN, and implicate such mutations in G-CSF hyporesponsiveness.

Multiple pathways contribute to the hyperproliferative responses from truncated granulocyte colony-stimulating factor receptors.

Mutations in the granulocyte colony-stimulating factor receptor (G-CSF-R) gene leading to a truncated protein have been identified in a cohort of neutropenia patients highly predisposed to acute myeloid leukemia. Such mutations act in a dominant manner resulting in hyperproliferation but impaired differentiation in response to G-CSF. This is due, at least in part, to defective internalization and loss of binding sites for several negative regulators, leading to sustained receptor activation. However, those signaling pathways responsible for mediating the hyperproliferative function have remained unclear. In this study, analysis of an additional G-CSF-R mutant confirmed the importance of residues downstream of Box 2 as important contributors to the sustained proliferation. However, maximal proliferation correlated with the ability to robustly activate signal transducer and activator of transcription (STAT) 5 in a sustained manner, whereas co-expression of dominant-negative STAT5, but not dominant-negative STAT3, was able to inhibit G-CSF-stimulated proliferation from a truncated receptor. Furthermore, a Janus kinase (JAK) inhibitor also strongly reduced the proliferative response, whereas inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) or phosphatidylinositol (PI) 3-kinase reduced proliferation to a lesser degree. These data suggest that sustained JAK2/STAT5 activation is a major contributor to the hyperproliferative function of truncated G-CSF receptors, with pathways involving MEK and PI 3-kinase playing a reduced role.

Genome-wide differential gene expression in children exposed to air pollution in the Czech Republic.

The Teplice area in the Czech Republic is a mining district where elevated levels of air pollution including airborne carcinogens, have been demonstrated, especially during winter time. This environmental exposure can impact human health; in particular children may be more vulnerable. To study the impact of air pollution in children at the transcriptional level, peripheral blood cells were subjected to whole genome response analysis, in order to identify significantly modulated biological pathways and processes as a result of exposure. Using genome-wide oligonucleotide microarrays, we investigated differential gene expression in children from the Teplice area (n=23) and compared them with children from the rural control area of Prachatice (n=24). In an additional approach, individual gene expressions were correlated with individual peripheral blood lymphocyte micronuclei frequencies, in order to evaluate the linkage of individual gene expressions with an established biomarker of effect that is representative for increased genotoxic risk. Children from the Teplice area showed a significantly higher average micronuclei frequency than Prachatice children (p=0.023). For considerable numbers of genes, the expression differed significantly between the children from the two areas. Amongst these genes, considerable numbers of genes were observed to correlate significantly with the frequencies of micronuclei. The main biological process that appeared significantly affected overall was nucleosome assembly. This suggests an effect of air pollution on the primary structural unit of the condensed DNA. In addition, several other pathways were modulated. Based on the results of this study, we suggest that transcriptomic analysis represents a promising biomarker for environmental carcinogenesis.

Peritoneal dialysis underscores its merits in portal hypertensionrelated and nephrogenic ascites.

In the Western world, peritoneal dialysis (PD) is less frequently applied as substitute therapy in end-stage renal disease (ESRD). In the Netherlands the use of PD has decreased from 30.3 to 13.5% due to several factors, but not due to lower PD-related outcome. The lower penetrance of PD diminishes experience with and exposure of young professionals to this treatment modality. This does not enhance a free and motivated choice among renal replacement therapies for patients who cannot be transplanted pre-emptively. To rejuvenate interest in PD and to underscore its merits, we would like to share the use of PD on two extraordinary occasions, where PD was the only way out. Ascites due to portal hypertension with profound gastrointestinal haemorrhage and nephrogenic ascites poses major management challenges in ESRD patients. In conclusion, PD came to the rescue and tremendously increased quality of life in the patients presented. To be readily available, a certain penetrance of and expertise in PD as renal replacement therapy is warranted.

STAT3-mediated differentiation and survival and of myeloid cells in response to granulocyte colony-stimulating factor: role for the cyclin-dependent kinase inhibitor p27(Kip1).

The signal transducer and activator of transcription (STAT) proteins have been implicated in cytokine-regulated proliferation, differentiation and cell survival. Granulocyte colony-stimulating factor (G-CSF), a regulator of granulocytic differentiation, induces a robust and sustained activation of STAT3. Here, we show that introduction of dominant negative (DN) forms of STAT3 interferes with G-CSF-induced differentiation and survival in murine 32D cells. G-CSF induces expression of the cyclin-dependent kinase (cdk) inhibitor p27(KiP1) (but not p21(CiP1)), which is completely blocked by DN-STAT3. The ability of tyrosine-to-phenylalanine substitution mutants of the G-CSF receptor to activate STAT3 strongly correlated with their capacity to induce p27 expression and their ability to mediate differentiation and survival, suggesting a causal relationship between STAT3 activation, p27 expression and the observed cellular responses. We identified a putative STAT binding site in the promoter region of p27 that showed both STAT3 binding in electrophoretic mobility shift assays and functional activity in luciferase reporter assays. Finally, we studied G-CSF-induced responses in primary bone marrow and spleen cells of p27-deficient mice. Compared with wild-type, myeloid progenitors from p27-deficient mice showed significantly increased proliferation and reduced differentiation in response to G-CSF. These findings indicate that STAT3 controls myeloid differentiation, at least partly, via upregulation of p27(Kip1).

The SH2 domain-containing protein tyrosine phosphatase SHP-1 is induced by granulocyte colony-stimulating factor (G-CSF) and modulates signaling from the G-CSF receptor.

The SH2 domain-containing protein tyrosine phosphatase SHP-1 is expressed widely in the hematopoietic system. SHP-1 has been shown to negatively control signal transduction from many cytokine receptors by direct docking to either the receptor itself, or to members of the Jak family of tyrosine kinases which are themselves part of the receptor complex. Motheaten and viable motheaten mice, which are deficient in SHP-1, have increased myelopoiesis and show an accumulation of morphologically and phenotypically immature granulocytes, suggesting a role for SHP-1 in granulocytic differentiation. Here, we report that SHP-1 protein levels are up-regulated during the granulocyte colony-stimulating factor (G-CSF)-mediated granulocytic differentiation of myeloid 32D cells. Enforced expression of SHP-1 in these cells leads to decreased proliferation and enhanced differentiation, while introduction of a catalytically inactive mutant produces increased proliferation and results in a delay of differentiation. In vitro binding revealed that the SH2 domains of SHP-1 are unable to associate directly with tyrosine-phosphorylated G-CSF receptor (G-CSF-R). Furthermore, over-expression of SHP-1 in Ba/F3 cells expressing a G-CSF-R mutant lacking all cytoplasmic tyrosines also inhibited proliferation. Together, these data suggest that SHP-1 directly modulates G-CSF-mediated responses in hematopoietic cells via a mechanism that does not require docking to the activated G-CSF-R.

IL-12-secreting CD19-targeted cord blood-derived T cells for the immunotherapy of B-cell acute lymphoblastic leukemia.

Disease relapse or progression is a major cause of death following umbilical cord blood (UCB) transplantation (UCBT) in patients with high-risk, relapsed or refractory acute lymphoblastic leukemia (ALL). Adoptive transfer of donor-derived T cells modified to express a tumor-targeted chimeric antigen receptor (CAR) may eradicate persistent disease after transplantation. Such therapy has not been available to UCBT recipients, however, due to the low numbers of available UCB T cells and the limited capacity for ex vivo expansion of cytolytic cells. We have developed a novel strategy to expand UCB T cells to clinically relevant numbers in the context of exogenous cytokines. UCB-derived T cells cultured with interleukin (IL)-12 and IL-15 generated >150-fold expansion with a unique central memory/effector phenotype. Moreover, UCB T cells were modified to both express the CD19-specific CAR, 1928z, and secrete IL-12. 1928z/IL-12 UCB T cells retained a central memory-effector phenotype and had increased antitumor efficacy in vitro. Furthermore, adoptive transfer of 1928z/IL-12 UCB T cells resulted in significantly enhanced survival of CD19(+) tumor-bearing SCID-Beige mice. Clinical translation of CAR-modified UCB T cells could augment the graft-versus-leukemia effect after UCBT and thus further improve disease-free survival of transplant patients with B-cell ALL.

Hepatopulmonary syndrome and venous emboli causing intracerebral hemorrhages after liver transplantation: a case report.

Increasing experience has fostered the acceptance of liver transplantation as a treatment for patients with hepatopulmonary syndrome. Morbidity and mortality is most commonly attributed to progressive arterial hypoxemia postoperatively. A cerebral hemorrhage has been reported in one patient with hepatopulmonary syndrome after transplantation. However, a postmortem examination of the brain was not performed and the pathogenesis or type of cerebral hemorrhage was undefined. We report on a patient with severe hepatopulmonary syndrome who developed multiple intracranial hemorrhages after transplantation. The intracerebral hemorrhages were most consistent with an embolic etiology on postmortem examination. We postulate that venous embolization, caused by the manipulation of a Swan Ganz catheter in a thrombosed central vein, resulted in pulmonary emboli that passed through dilated intrapulmonary vessels into the cerebral microcirculation. Special attention to central venous catheters and avoidance of manipulation may be warranted in subjects with severe hepatopulmonary syndrome after liver transplantation.

Results of pancreaticoduodenectomy for ampullary carcinoma and analysis of prognostic factors for survival.

BACKGROUND: Results of pancreaticoduodenectomy for ampullary carcinoma were evaluated, and prognostic factors for survival were analyzed. METHODS: During the period from 1984 to 1992 67 patients underwent subtotal or total pancreaticoduodenectomy for ampullary carcinoma. All clinicopathologic data and their influence on survival were studied. RESULTS: Subtotal pancreaticoduodenectomy was performed in 62 of 67 patients with a mortality of 6% and a morbidity of 65%; the remaining five patients underwent total pancreaticoduodenectomy. Intraabdominal infection was the most important complication. Resection margins were tumor free in 75% of 67 patients. The overall 5-year survival was 50%. Survival was significantly influenced by the involvement of resection margins. After resection with involved margins 5-year survival was 15% and 60% after resection with free margins (p < 0.001). Tumor size, lymph node involvement, and differentiation grade had limited and not significant influence on survival. CONCLUSIONS: Subtotal pancreaticoduodenectomy is the type of resection of first choice for ampullary carcinoma. Involvement of resection margins was the strongest prognostic factor for survival. Patients with a tumor size larger than 2 cm, with lymph node involvement, or with a poorly differentiated tumor still had a 5-year survival rate greater than 40%. Patients with involved margins might be candidates for studies on adjuvant therapy.

Benign fibrosing disease at the hepatic confluence mimicking Klatskin tumors.

BACKGROUND: Hilar obstructions remain a challenge with regard to diagnosis and treatment. METHODS: In the period from 1984 to 1990, 82 patients underwent resective surgery under the presumptive diagnosis of hilar cholangiocarcinoma (Klatskin tumor). The diagnosis was based on the combined appearances on direct cholangiography and ultrasonography in all cases, with the use of various other imaging modalities in some cases. RESULTS: The perioperative findings from an experienced surgical team were usually thought to be compatible with bile duct carcinoma. However, histologic examination of the resected specimens revealed benign fibrosing or localized sclerosing lesions in 11 patients (13.4%). CONCLUSIONS: The current state of diagnostic imaging fails as yet to discriminate reliably between benign and malignant hilar lesions. Whereas the immediate therapeutic consequences may be equal (resection followed by hepaticojejunostomy), the late consequences differ in a major way because benign disease has a much better prognosis. In the presence of suspicious hilar obstruction, operable lesions should not be treated by "palliative" intubational techniques and radiation therapy without a firm diagnosis of malignancy. However, overtreatment (extended liver resection, vascular reconstruction, and liver transplantation) should be avoided as well when a benign lesion has not been ruled out.

Expression and characterisation of human and rat CRF2alpha receptors.

Rat and human CRF2alpha receptors were expressed in CHO-pro5 cells and stable cell lines generated. Each receptor was characterised using [125I][tyr0]sauvagine and results compared to CRF1 receptors expressed in the same parental cell line. Under identical assay conditions, [125I][tyr0]sauvagine labelled both CRF1 and CRF2alpha receptors with high affinity. The level of expression varied from 103 fmol/mg membrane protein to 1842 fmol/mg membrane protein (rat CRF1 receptors and rat CRF2 receptors, respectively). It was possible to establish robust scintillation proximity assays (SPA) using wheat germ agglutinin (WGA) SPA beads to trap membrane protein. The success of the SPA assay format was dependent on the level of receptor expression observed. The rank order of affinities of a series of peptide CRF receptor agonists and antagonists was similar to that described in the literature for the two receptor subtypes as determined using radioligand binding and cAMP accumulation. No pharmacological differences were apparent between rat and human cloned receptors with the exception of alpha-helical CRF-(9-41). This peptide exhibited 10-fold higher affinity for rat CRF2alpha receptors as compared to human CRF2alpha receptors. PD 173307, PD 173602 and PD 174239 exhibited high affinity and selectivity for human CRF1 receptors, and as such represent useful tools for probing CRF receptor function.

Risk factors for acquisition of hepatitis C virus infection: a case series and potential implications for disease surveillance.

BACKGROUND: Transmission of hepatitis C virus (HCV) is strongly associated with use of contaminated blood products and injection drugs. Other "non-parental" modes of transmission including sexual activity have been increasingly recognized. We examined risk factors for acquiring HCV in patients who were referred to two tertiary care centers and enrolled in an antiviral therapy protocol. METHODS: Interviews of 148 patients were conducted apart from their physician evaluation using a structured questionnaire covering demographics and risk factors for HCV acquisition. RESULTS: Risk factors (blood products, injection/intranasal drugs, razor blades/ toothbrushes, body/ear piercing, occupational exposure, sexual activity) were identified in 141 (95.3%) of participants; 23 (15.5%) had one (most frequently blood or drug exposure), 41 (27.7%) had two, and 84 (53.4%) had more than two risk factors. No patient reported sexual activity as a sole risk factor. Body piercing accounted for a high number of exposures in women. Men were more likely to have exposure to street drugs but less exposure to blood products than women. Blood product exposure was less common in younger than older HCV patients. CONCLUSION: One and often multiple risk factors could be identified in nearly all HCV-infected patients seen in a referral practice. None named sexual transmission as the sole risk factor. The development of a more complete profile of factors contributing to transmission of HCV infection may assist in clinical and preventive efforts. The recognition of the potential presence of multiple risk factors may have important implications in the approach to HCV surveillance, and particularly the use of hierarchical algorithms in the study of risk factors.

Early postoperative complications after (sub)total pancreatoduodenectomy: the AMC experience.

The postoperative course is described in 70 patients who underwent pancreatic resection for a tumor of the head of the pancreas and peri-ampullary region: 64 patients had malignant disease. Subtotal pancreatoduodenectomy was carried out in 52 patients and total pancreatoduodenectomy in 18 patients. The overall 30-day mortality was 4.3% (3 patients died: one after subtotal, 2 after total pancreatoduodenectomy). Major complications required surgical reintervention in 15 patients. Another 24 patients developed minor complications, and responded well to conservative treatment. Infective complications were the main cause of post-operative morbidity, occurring in 29 patients. Dehiscence of the pancreatico-jejunostomy required surgical reintervention in one patient. This patient died. Leakage of the pancreatico-jejunostomy was radiologically demonstrated in 10 other patients. Four of these 10 patients presented with clinical symptoms: one needed surgical intervention and 3 responded to conservative management. The results of this study confirm the present trend of decreasing mortality after pancreatoduodenectomy. Postoperative morbidity remains high. Usually, leakage of the pancreatic anastomosis was not associated with serious postoperative complications and subsided without the need of treatment in the majority of cases.

Does additive radiotherapy after hilar resection improve survival of cholangiocarcinoma? An analysis in sixty-four patients.

Between September 1983 and January 1990, 64 patients with Klatskin tumors were treated with resective therapy in the Academic Medical Centre Amsterdam. Twenty-nine patients received postoperative adjuvant radiotherapy, 22 patients were treated with resective therapy only and there were 13 postoperative deaths. The longterm results in the patients treated with or without adjuvant radiotherapy were retrospectively assessed. Three patients had a curative resection. Comparison of both therapeutic modalities demonstrated a statistically significant difference in survival (P less than 0.001) in favour of the patients who were treated with additive radiotherapy. The radiotherapy was generally well tolerated.

Hepatocellular carcinoma in the Amsterdam area. A retrospective analysis in 61 patients.

In a retrospective study clinical features of hepatocellular carcinoma (HCC) in the Amsterdam area (1984-89) were assessed in 61 cases. The data obtained were used to discuss some of the current concepts on aetiology, diagnosis, and treatment of HCC. Presenting symptoms and findings usually reflected advanced (incurable) disease. Of the patients 25% had a normal alpha-1-foetoprotein (AFP), 55% had elevated levels, and in 20% data were absent. Platelet counts greater than 500 x 10(9)/l were found in 8% and hypercalcaemia in 10% of the patients. Treatment modalities were none (70%), surgery (16%), chemotherapy (8%), radiotherapy (3%), and endoprosthesis (2%). Sixty to 70% had died after 3 months and more than 90% after 1 year. Long survivors included 2 patients with the fibrolamellar type of HCC. The low incidence of HCC in The Netherlands probably precludes cost-effective screening programs to identify resectable small HCC. Unidentified masses are malignant until proven otherwise and should be resected if no firm diagnosis of benign disease can be made. Awareness of HCC and its risk factors may lead to earlier diagnosis and more selective use of diagnostic tests.

Preoperative strength training for elderly patients awaiting total knee arthroplasty.

Objective. To investigate the feasibility and effects of additional preoperative high intensity strength training for patients awaiting total knee arthroplasty (TKA). Design. Clinical controlled trial. Patients. Twenty-two patients awaiting TKA. Methods. Patients were allocated to a standard training group or a group receiving standard training with additional progressive strength training for 6 weeks. Isometric knee extensor strength, voluntary activation, chair stand, 6-minute walk test (6MWT), and stair climbing were assessed before and after 6 weeks of training and 6 and 12 weeks after TKA. Results. For 3 of the 11 patients in the intensive strength group, training load had to be adjusted because of pain. For both groups combined, improvements in chair stand and 6MWT were observed before surgery, but intensive strength training was not more effective than standard training. Voluntary activation did not change before and after surgery, and postoperative recovery was not different between groups (P > 0.05). Knee extensor strength of the affected leg before surgery was significantly associated with 6-minute walk (r = 0.50) and the stair climb (r - = 0.58, P < 0.05). Conclusion. Intensive strength training was feasible for the majority of patients, but there were no indications that it is more effective than standard training to increase preoperative physical performance. This trial was registered with NTR2278.

Toxicogenomic profiles in relation to maternal immunotoxic exposure and immune functionality in newborns.

A crucial period for the development of the immune system occurs in utero. This results in a high fetal vulnerability to immunotoxic exposure, and indeed, immunotoxic effects have been reported, demonstrating negative effects on immune-related health outcomes and immune functionality. Within the NewGeneris cohort BraMat, a subcohort of the Norwegian Mother and Child Cohort Study (MoBa), immunotoxicity was demonstrated for polychlorinated biphenyls and dioxins, showing associations between estimated maternal intake levels and reduced measles vaccination responses in the offspring at the age of 3. The present study aimed to investigate this link at the transcriptomic level within the same BraMat cohort. To this end, whole-genome gene expression in cord blood was investigated and found to be associated with maternal Food Frequency Questionnaires-derived exposure estimates and with vaccination responses in children at 3 years of age. Because the literature reports gender specificity in the innate, humoral, and cell-mediated responses to viral vaccines, separate analysis for males and females was conducted. Separate gene sets for male and female neonates were identified, comprising genes significantly correlating with both 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and polychlorinated biphenyls (PCB) exposure and with measles vaccination response. Noteworthy, genes correlating negatively with exposure in general show positive correlations with antibody levels and vice versa. For both sexes, these included immune-related genes, suggesting immunosuppressive effects of maternal exposure to TCDD and PCB at the transcriptomic level in neonates in relation to measles vaccination response 3 years later.

In vitro cytokine release from human peripheral blood mononuclear cells in the assessment of the immunotoxic potential of chemicals.

Alternative methods to the use of animals in testing of chemicals are needed. We investigated if the immunotoxic potential of 12 dietary toxicants could be predicted from effects on cytokine release from human peripheral blood mononuclear cells (PBMC) after in vitro exposure. Nine cytokines were selected to reflect different types of immune responses. The toxicants were classified as immunotoxic or non-immunotoxic substances according to the published in vivo data. Isolated human PBMC were exposed for 20 h to three concentrations of each of the 12 substances in the presence of human liver S9 fraction. After further incubation of PBMC in fresh medium containing the mitogen phytohemagglutinin (PHA, 10 µg/ml) for 48 h, release of the nine selected cytokines into the supernatant as well as cell proliferation were measured by Luminex technology™ and the BrdU incorporation assay, respectively. All 12 substances investigated affected the release of one or more cytokines, and each of the substances showed different cytokine release patterns. Within the limitations of the study design, the present study suggests that the effect of the substances on mitogen-induced cytokine release from PBMC cannot predict their immunotoxic potential, but may be useful in mechanistic studies.