Selumetinib Activity in Thyroid Cancer Cells: Modulation of Sodium Iodide Symporter and Associated miRNAs.

BACKGROUND: The MEK (mitogen-activated protein kinase)⁻inhibitor selumetinib led to increased radioiodine uptake and retention in a subgroup of patients suffering from radioiodine refractory differentiated thyroid cancer (RR-DTC). We aimed to analyse the effect of selumetinib on the expression of sodium iodide symporter (NIS; SLC5A5) and associated miRNAs in thyroid cancer cells. METHODS: Cytotoxicity was assessed by viability assay in TPC1, BCPAP, C643 and 8505C thyroid cancer cell lines. NIS, hsa-let-7f-5p, hsa-miR-146b-5p, and hsa-miR-146b-3p expression was determined by quantitative RT-PCR. NIS protein was detected by Western blot. Radioiodine uptake was performed with a Gamma counter. RESULTS: Selumetinib caused a significant reduction of cell viability in all thyroid cancer cell lines. NIS transcript was restored by selumetinib in all cell lines. Its protein level was found up-regulated in TPC1 and BCPAP cells and down-regulated in C643 and 8505C cells after treatment with selumetinib. Treatment with selumetinib caused a down-regulation of hsa-let-7f-5p, hsa-miR-146b-5p and hsa-miR-146b-3p in TPC1 and BCPAP cells. In 8505C cells, a stable or down-regulated hsa-miR-146b-5p was detected after 1h and 48h of treatment. C643 cells showed stable or up-regulated hsa-let-7f-5p, hsa-miR-146b-5p and hsa-miR-146b-3p. Selumetinib treatment caused an increase of radioiodine uptake, which was significant in TPC1 cells. CONCLUSIONS: The study shows for the first time that selumetinib restores NIS by the inhibition of its related targeting miRNAs. Further studies are needed to clarify the exact mechanism activated by hsa-miR-146b-5p, hsa-miR-146b-3p and hsa-let7f-5p to stabilise NIS. Restoration of NIS could represent a milestone for the treatment of advanced RR-DTC.

Pretherapeutic drug evaluation by tumor xenografting in anaplastic thyroid cancer.

BACKGROUND: Despite various attempts at modifying usual treatment modalities, anaplastic thyroid cancer (ATC) is still associated with unfavorable prognosis. Results of preclinical investigations are often of limited transferability to clinical tumor biology. Individualized multimodal treatment regimens, including novel growth-inhibiting drugs, might be a future option. METHODS: Tumor tissue, freshly prepared from a patient operated for ATC, was xenotransplanted to nude mice. While the patient obtained a hyperfractionated external beam radiation, mice carrying xenotransplanted tumors were randomized (n = 6) and treated by multikinase inhibitors (sorafenib [S]: vascular endothelial growth factor receptor [VEGF-R], platelet derived growth factor receptor, RET; vandetanib [V]: VEGF-R, endothelial growth factor receptor [EGF-R]; and MLN8054 [M]: Aurora kinases [AK]). Antiproliferative, antiangiogenic, and proapoptotic effects were evaluated. RESULTS: Treatment of successfully xenotransplanted fresh ATC tumor tissue by multikinase inhibitors and aurora kinase inhibitor reduced the tumor volume up to 61% depending on the drug and time of application (3 wk of treatment: 46% [M], 34% [V], 30% [S]; 5 wk of treatment: 61% [S]). Tumor cell proliferation (BrdU) was reduced between 34% and 58% [S] and [V]. Reduction of tumor vascularity was between 67% [V] and 33% [S] and was accompanied by decreased EGF-R/VEGF-R2 receptor activity [V/V,S]. Tumor cell apoptosis (caspase 3 activity) increased up to 2.4-fold [S]. CONCLUSIONS: Successful in vivo evaluation of novel drugs in xenotransplanted fresh tumor tissue allows in-time (while patient receives standard treatment) prospective analysis for possible additional clinical application. However, technical specifications have to be taken into account to obtain stable in vivo tumor growth. Based on the individual results, a tailored clinical drug application seems possible.

Targeting the proteasome as a promising therapeutic strategy in thyroid cancer.

BACKGROUND AND OBJECTIVES: Targeting the ubiquitin-proteasome system by using proteasome inhibitors represents a novel approach for cancer therapy. Anaplastic thyroid cancer (ATC), a subtype of thyroid cancer (TC), fails to respond to conventional TC treatment. Here we investigated the effects of bortezomib on TC in vitro. Further, the study aimed to evaluate its potential for TC treatment in vivo. METHODS: Three anaplastic (Hth74, C643, Kat4), one follicular (FTC133), and one papillary (TPC1) TC cell lines were used. Antiproliferative, proapoptotic, and transcriptional effects of bortezomib treatment were analyzed in vitro and growth inhibition of ATC xenografts in vivo. Tumor samples were analyzed by Ki67, CD31, caspase-3, and NF-κB immunohistochemistry. RESULTS: In vitro, bortezomib inhibited proliferation of TC cells (IC(50) 4-10 nM), increased caspase-3 activity and induced cell cycle arrest. NF-κB activity was affected differently. In vivo, bortezomib treatment was effective in reducing tumor volume (up to 74%), accompanied by reduced proliferation (Ki67) and 57% reduced tumor vascularity. CONCLUSION: Proteasome inhibition is effective in reducing cell growth and inducing apoptosis of ATC in vitro and inhibiting tumor growth and vascularity in vivo. However, the impact on nuclear transcription remains controversial. Clinical evaluation of bortezomib treatment in ATC is warranted.

Evaluation of Aurora kinase inhibition as a new therapeutic strategy in anaplastic and poorly differentiated follicular thyroid cancer.

Due to an unfavorable prognosis using the usual therapy, patients with anaplastic thyroid cancer (ATC) are in desperate need of new therapeutic strategies. The objective of this study was to evaluate the effects of MLN8054, an inhibitor of the Aurora serine/threonine kinases, on ATC cells in vitro and on ATC xenografts as a new therapeutic strategy for ATC. Three anaplastic (Hth74, C643, Kat4) and one follicular (FTC133) thyroid cancer cell lines were evaluated in vitro and Kat4 xenografts in vivo. The antiproliferative effect of MLN8054 (0.1-10 µM) on thyroid cancer cells was quantified by sulphorhodamine B-assay. The proapoptotic effect and the effects on the cell cycle were evaluated by flow cytometry after Annexin-V-FITC staining. Further Histone H3 phosphorylation was analysed. In vivo, antiproliferative and antiangiogenic effects were assessed by tumor volume and morphometric analysis following immunohistochemical staining (Ki-67, pHisH3, CD31). Treatment of the different TC cells with MLN8054 inhibited proliferation in a time- and dose-dependent manner, with IC(50) values between 0.1 and 10 µM. Administration of MLN8054 resulted in an increase of apoptotic cells, decreased Histone H3 phosphorylation and induced cell cycle arrest. In vivo, treatment of ATC by MLN8054 resulted in an up to 86% reduced tumor volume and 89% reduced tumor vascularity. In conclusion, our data demonstrated that Aurora kinase inhibition is effective in reducing cell growth and inducing apoptosis of ATC in vitro and tumor growth and vascularity in vivo. Controlled clinical studies on MLN8054 or comparable compounds would be worthwhile to evaluate its potential therapeutic value for treatment of ATC.

Effect of Kinase Inhibitors on the Technetium-99m Uptake into Thyroid Carcinoma Cells In Vitro.

BACKGROUND/AIM: We evaluated the potential of the kinase inhibitors sorafenib, lenvatinib and selumetinib on increasing the uptake of technetium-99m into thyroid cancer cells. MATERIALS AND METHODS: Four established cell lines and three patient's cell cultures were treated with 0.1, 1 and 5 µM of sorafenib, lenvatinib and selumetinib for 72 hours. After incubation with 1 MBq of technetium-99m, the radioactivity uptake was measured. RESULTS: The experiments showed heterogeneous results. Maximum technetium-99m uptake increases of 312% (sorafenib), 326% (lenvatinib) and 759% (selumetinib) were obtained using the highest applied concentrations. In some tests, an uptake reduction or no effect was observed. CONCLUSION: Kinase inhibitors have a positive effect on technetium-99m uptake. Due to study limitations, a redifferentiating effect of the drugs could not be definitely proven. Unspecific cytotoxicity might have a confounding effect.

Expression and secretion of endostatin in thyroid cancer.

BACKGROUND: In thyroid cancer (TC) endostatin was identified as a powerful negative regulator of tumor angiogenesis in vitro. It is currently being evaluated in phase I trials for antiangiogenic therapy in various solid tumors. The aim of this study was to evaluate endostatin expression in archival TC specimens and its secretion following stimulation with thyrotropin (TSH) and epidermal growth factor (EGF) in TC cell lines. METHODS: Tissue microarrays of 44 differentiated and 7 anaplastic TC and their metastasis were immunostained for endostatin protein expression and compared with corresponding non-neoplastic thyroid tissue (NT). In vitro, six differentiated (FTC133, FTC236, HTC, HTC-TSHr, XTC, and TPC1) and three anaplastic (C643, Hth74, Kat4.0) TC cell lines were evaluated for basal as well as TSH (1-100 mU/ml) and EGF stimulated (1-100 ng/ml) endostatin. RESULTS: Endostatin was detected in all TC and more than half of the NT. Endostatin expression was more frequent and intense in differentiated as compared to anaplastic TC. In vitro, basal endostatin secretion varied between 33 +/- 5 pg/ml (FTC236) and 549 +/- 65 pg/ml (TPC1) and was doubled in FTC, when the "primary" (FTC133) was compared with the metastasis (FTC236). Some cell lines showed TSH-induced (e.g., 60% in XTC) or EGF-induced (e.g., 120% in TPC1) upregulation of endostatin secretion, while others did not, despite documented receptor expression. CONCLUSION: This study demonstrates endostatin expression in TC, metastasis and--less frequently and intensely--in NT, suggesting a possible association to tumor progression. In vitro, endostatin secretion of some cell lines is regulated by TSH and EGF, however the individual differences deserve further functional studies. These results support rather tumor-specific than histotype-specific expression and regulation of endostatin in TC.

Epigenetic Modifications in Thyroid Cancer Cells Restore NIS and Radio-Iodine Uptake and Promote Cell Death.

Epigenetic modifications have been identified as being responsible for the de-differentiation of thyroid tissue and its malignant transformation. Cell proliferation inhibitory effects of the pan-deacetylase inhibitors panobinostat, SAHA and Trichostatin A (TSA), the modulation of the sodium iodide symporter (NIS; SLC5A5), thyroid transcription factor 1 (TTF1), high mobility group A2 (HMGA2), and H19 and their putative targeting miRNAs have been evaluated in vitro. The cell viability was measured in five thyroid cancer cell lines (FTC133, TPC1, BCPAP, 8505C, C643) by real time cell analyzer xCELLigence. Expression of the above mentioned markers was performed by RT-qPCR and Western Blot. Radioiodine up-take was detected by Gamma Counter with I131. Cell viability decreased after treatment in all five cell lines. 10 nM panobinostat; 1 µM TSA or 10 µM SAHA caused a significant over-expression of NIS transcript in all five cell lines, whereas NIS protein was up-regulated in FTC133, BCPAP, and C643 cell lines only. Radioiodine up-take increased in FTC133 and C643 cells after 48 h of treatment with 10 nM panobinostat and 1 µM TSA. A significant down-regulation of the oncogene HMGA2 was detected in all five cell lines; except for TPC1 cells that were treated with 1 µM TSA. In accordance, hsa-let-7b-5p and hsa-let-7f-5p were stable or significantly over-expressed in all of the cell lines, except for TPC1 cells that were treated with 10 µM SAHA. TTF1 was significantly down-regulated in FTC133, BCPAP, and 8505C cells; whereas, TPC1 and C643 showed an up-regulated or stable expression. TTF1 was over-expressed in samples of human anaplastic thyroid cancer; whereas, it was down-regulated in follicular and undetectable in papillary thyroid cancer. H19 was over-expressed after 48 h treatment, except for BCPAP cells that were treated with panobinostat and SAHA. H19 was differently expressed in human anaplastic, follicular and papillary thyroid tumor samples. Deacetylase inhibitors reduced cell viability, restored NIS and H19, and suppressed the oncogenes HMGA2 and TTF1 in thyroid cancer cells.

Individualised Multimodal Treatment Strategies for Anaplastic and Poorly Differentiated Thyroid Cancer.

The prognosis of anaplastic (ATC) and poorly differentiated thyroid cancer (PDTC) is poor, due to their radioiodine refractoriness (RAI-R), high metastatic potential and current lack of effective treatment strategies. We aimed to examine the efficacy of the tyrosine kinase inhibitors (TKIs) sorafenib and selumetinib and the histone deacetylase inhibitor (HDACI) panobinostat in patient-derived tumor tissue (PDTT) of ATCs/PDTCs, the expression of sodium iodide symporter (NIS) and radioiodine up-take (RAI-U). High Mobility Group AT-Hook 2 (HMGA2) and associated miRNAs expression was correlated with the clinical course of the patients. Inhibitory effects of panobinostat, sorafenib and selumetinib were measured by real time cell analyser xCELLigence in five PDTTs and human foreskin fibroblasts (HF) used as control. Expression of NIS, HMGA2 and associated miRNAs hsa-let-7f-5p, hsa-let-7b-5p, hsa-miR-146b-5p and hsa-miR-146b-3p was performed by RT-qPCR and Western blot. RAI-U was performed by Gamma Counter with I-131. Panobinostat showed the strongest cytotoxic effect (10 nM) in all PDTTs and HF and caused a significant over-expression of NIS transcript. TKIs were able to up-regulate NIS transcript in patient 5 and in HF. RAI-U was up-regulated after 24 h of treatment with TKIs and panobinostat in all PDTT and HF, except in patient 5. Selumetinib caused a significant suppression of HMGA2 in PDTT 1, 2, 4, 5 and HF; whereas sorafenib caused no change of HMGA2 expression. Panobinostat suppressed significantly HMGA2 in PDTT 2, 4 and HF. The expression of miRNAs hsa-let-7f-5p, has-let-7b-5p hsa-miR-146b-5p and hsa-miR-146b-3p was modulated heterogeneously. NIS protein level was over-expressed in three PDTTs (patients 1, 3 and 4) after 24 h of treatment with selumetinib, sorafenib and in particular with panobinostat. HF showed a stable NIS protein level after treatment. Panobinostat showed the strongest cytotoxicity in all treated PDTTs at the lowest dosage in comparison with TKI. All three compounds were able to modulate differently NIS, HMGA2 and related miRNAs. These factors represent valuable markers in PDTT for new treatment strategies for patients suffering from ATC/PDTC. Thus, the establishment of PDTT could be a useful tool to test the efficacy of compounds and to develop new and individualised multimodal treatment options for PDTCs and ATCs.

MicroRNAs let7 expression in thyroid cancer: correlation with their deputed targets HMGA2 and SLC5A5.

PURPOSE: Thyroid cancer (TC), the most common endocrine malignancy, increases its incidence worldwide. MicroRNAs have been shown to be abnormally expressed in tumors and could represent valid diagnostic markers for patients affected by TC. Our aim was to analyze the expression of tumorsuppressor hsa-let7b-5p and hsa-let7f-5p, together with their predicted targets SLC5A5 (NIS) and HMGA2, in papillary (PTC), follicular (FTC) and anaplastic (ATC). METHODS: 8 FTC, 14 PTC, 12 ATC and three normal thyroid tissue samples were analyzed for the expression of pre-let7b, hsa-let7b-5p and hsa-let7f-5p as SLC5A5 and HMGA2 by RT-qPCR. Data were analyzed by REST 2008. RESULTS: FTC patients showed a significant down-regulation of hsa-let7b-5p and its precursor. hsa-let7f-5p was overexpressed, and SLC5A5 was strongly suppressed. HMGA2 was overexpressed, reflecting no correlation with its regulatory let7 miRNAs. PTC samples were characterized by up-regulation of hsa-let7b-5p, its precursor and hsa-let7f-5p. SLC5A5 was strongly suppressed in comparison with normal thyroid tissue. HMGA2 was overexpressed, as shown in FTC, also. ATC samples showed a similar miRNAs profile as PTC. In contrast with FTC and PTC, these patients showed a stable or up-regulated SLC5A5 and HMGA2. CONCLUSIONS: Expression of HMGA2 is not correlated with the regulatory let7 miRNAs. Interestingly, SLC5A5 was down-regulated in FTC and PTC. Its expression could be modulated by hsa-let-7f-5p. ATC showed a loss of SLC5A5/hsa-let7f-5p correlation. SLC5A5, in ATC, needs further investigation to clarify the genetic/epigenetic mechanism altering its expression.

VEGF-mediated angiogenesis of human pheochromocytomas is associated to malignancy and inhibited by anti-VEGF antibodies in experimental tumors.

BACKGROUND: Pheochromocytomas are well-vascularized tumors of the adrenal medulla. In human pheochromocytomas, angiogenesis has been associated with tumor progression. The mechanisms, however, are unknown. METHODS: Surgical specimens of benign, invasive, and metastatic human pheochromocytomas (n = 10/5/5) were immunostained for vascular endothelial growth factor (VEGF) and CD34, to determine VEGF expression and microvessel density (vascular surface density, [VSD]). In PC12-pheochromocytoma cells, VEGF messenger RNA and protein were analyzed by Northern blotting and enzyme immunoassay; biologic activity by human umbilical vein endothelial cell-proliferation assay. Inhibition of angiogenesis of PC12 xenografts by 2 neutralizing anti-VEGF antibodies (C20-pAB, M461-mAB) was evaluated by VEGF expression, VSD, and mitotic activity. RESULTS: VEGF expression and VSD were significantly higher in metastatic pheochromocytomas (VEGF 37.1 +/- 10.9% vs 20.7 +/- 9%, VSD 26.2 +/- 8 vs 13.5 +/- 3.3 1/mm). VEGF messenger RNA and protein were confirmed in PC12 cells and stimulated by nerve growth factor. Conditioned PC12 medium increased human umbilical vein endothelial cell proliferation more than 2-fold. Xentrotransplanted PC12 cells had marked VEGF expression and angiogenesis, which was inhibited by anti-VEGF antibodies (VEGF-expression by 29 and 38%, VSD by 43 and 46%, P <.05). CONCLUSION: Higher VEGF expression and microvessel density in malignant pheochromocytomas suggest VEGF-mediated angiogenesis to be related to tumor progression. Angiogenesis of PC12 xenografts is mediated by VEGF. Neutralizing anti-VEGF antibodies inhibit angiogenesis in experimental pheochromocytomas and may have potential for treating nonresectable pheochromocytomas.

Expression of hedgehog signalling pathway in anaplastic thyroid cancer.

The purpose of this work is to study the activation of the hedgehog signalling pathway is associated with tumour progression in various types of cancer, hence the development of specific antagonists raises hope for new therapeutic strategies. Therefore, the expression of hedgehog pathway components in anaplastic thyroid cancer (ATC) and effects of the hedgehog inhibitor Cyclopamine on ATC cells were investigated in this study. Expression of the ligand Sonic Hedgehog (SHh), the transmembrane protein Smoothened (Smo), the receptor Patched (Ptc) and the target gene Gli-1 was evaluated in two ATC cell lines (Hth 74, C643) by RT-PCR and in tumour specimens by immunohistochemistry. The corresponding gene products were examined by western blotting analysis. After treatment with different concentrations of Cyclopamine the time-dependent course of cell viability in ATC cell lines was evaluated by MTT assay. SHh, Smo, Ptc and Gli were clearly expressed on mRNA and protein levels in both cell lines and in tumour samples (41 %SHh, 65 %Smo, 65 %Ptc and 65 %Gli). Treatment with Cyclopamine showed a time- and dose-dependent inhibition of cell numbers with IC50 values between 1 and 4 µM in both cell lines, comparable to other types of cancer. In conclusion, we believe that the hedgehog pathway is expressed in anaplastic thyroid carcinoma specimens and proliferation of ATC cell lines can be influenced by the Hh inhibitor Cyclopamine. Aberrant activation of this pathway might be involved in the aggressive biology of anaplastic cancer and further evaluation regarding a possible clinical impact of pathway inhibition is warranted.

Combined inhibition of cellular pathways as a future therapeutic option in fatal anaplastic thyroid cancer.

Conventional treatment by surgery, radioiodine, and thyroxin-suppressive therapy often fails to cure anaplastic thyroid cancer (ATC). Therefore several attempts have been made to evaluate new therapy options by use of "small molecule inhibitors". ATC was shown to respond to monotherapeutic proteasome and Aurora kinase inhibition in vitro as well as in xenotransplanted tumor cells. Aim of this study was to evaluate the effect of combined treatment targeting the ubiquitin-proteasome system by bortezomib and Aurora kinases by use of MLN8054. Three ATC cell lines (Hth74, C643, and Kat4.1) were used. The antiproliferative effect of combined treatment with bortezomib and MLN8054 was assessed by MTT-assay and cell cycle analysis (FACS). Proapoptotic effects were evaluated by measurement of Caspase-3 activity, and effects on VEGF secretion were analyzed by ELISA. Compared to mono-application combined treatment with bortezomib and MLN8054 resulted in a further decrease of cell density, whereas antagonizing effects were found regarding cell cycle progression. Caspase-3 activity was increased up to 2.7- and 14-fold by mono-application of MLN8054 and bortezomib, respectively. When the two drugs were used in combination, a further enhancement of Caspase-3 activity was achieved, depending on the cell line. VEGF secretion was decreased following bortezomib treatment and remained unchanged by MLN8054. Only in C643 cells, the bortezomib-induced down-regulation was enhanced when MLN8054 was applied simultaneously. In conclusion, our data demonstrate that targeting the proteasome and Aurora kinases simultaneously results in additional antitumoral effects in vitro, especially regarding cell growth and induction of apoptosis. The efficacy of this therapeutic approach remains to be revised by in vivo and clinical application.

Is rapid preparation for thyroidectomy in severe Graves' disease beneficial? The relationship between clinical and immunohistochemical aspects.

BACKGROUND: To evaluate the effect of Lugol's Iodine (LI) on angiogenic characteristics and postthyroidectomy morbidity in patients with Graves' Disease (GD). METHODS: Medical records of 153 patients, who underwent thyroidectomies for GD between 1987 and 2006 were reviewed. LI in addition to antithyroid drugs (ATD) was administered to 16 toxic Graves' cases (group I), while 137 received ATD only (group II). In order to evaluate thyroid vascularity, immunohistochemical staining (IHS) for VEGF and CD31 for assessing microvessel density (MVD) were performed in group I, 20 random cases of group II, and 7 stochastic patients with non-toxic goiter (group III). RESULTS: Early postoperative complications were significantly higher in group I compared to group II: (transient recurrent nerve palsy (P < 0.04), transient hypocalcemia (P < 0.02), and postoperative bleeding (P < 0.003)). Meanwhile permanent complications were not significantly different. VEGF-IHS revealed ten patients of group I (62.5%) to have positive staining compared to only 3 (15%) of group II. All patients of group III stained positive. The expression of VEGF was more intense in group I as compared to group II (P < 0.02). However, no significant difference of MVD among all groups was recorded. CONCLUSION: Preoperative LI treatment may be associated with increased early postoperative morbidity and did not affect thyroid vascularity.

Cathepsin Cs are key for the intracellular survival of the protozoan parasite, Toxoplasma gondii.

Cysteine proteases play key roles in apicomplexan invasion, organellar biogenesis, and intracellular survival. We have now characterized five genes encoding papain family cathepsins from Toxoplasma gondii, including three cathepsin Cs, one cathepsin B, and one cathepsin L. Unlike endopeptidases cathepsin B and L, T. gondii cathepsin Cs are exopeptidases and remove dipeptides from unblocked N-terminal substrates of proteins or peptides. TgCPC1 was the most highly expressed cathepsin mRNA in tachyzoites (by real-time PCR), but three cathepsins, TgCPC1, TgCPC2, and TgCPB, were undetectable in in vivo bradyzoites. The specific cathepsin C inhibitor, Gly-Phe-dimethylketone, selectively inhibited the TgCPCs activity, reducing parasite intracellular growth and proliferation. The targeted disruption of TgCPC1 does not affect the invasion and growth of tachyzoites as TgCPC2 is then up-regulated and may substitute for TgCPC1. TgCPC1 and TgCPC2 localize to constitutive secretory vesicles of tachyzoites, the dense granules. T. gondii cathepsin Cs are required for peptide degradation in the parasitophorous vacuole as the degradation of the marker protein, Escherichia coli beta-lactamase, secreted into the parasitophorous vacuole of transgenic tachyzoites was completely inhibited by the cathepsin C inhibitor. Cathepsin C inhibitors also limited the in vivo infection of T. gondii in the chick embryo model of toxoplasmosis. Thus, cathepsin Cs are critical to T. gondii growth and differentiation, and their unique specificities could be exploited to develop novel chemotherapeutic agents.

Toxopain-1 is critical for infection in a novel chicken embryo model of congenital toxoplasmosis.

We tested the hypothesis that cathepsins and specifically toxopain-1, a cathepsin B, play a critical role in the pathogenesis of toxoplasmosis. We found that inhibiting the expression of toxopain-1-specific mRNA and protein by >60% significantly decreased the capacity of the parasites to multiply and invade in vitro. To relate these in vitro results to the role of toxopain-1 in pathogenesis in vivo, we developed a novel chicken embryo model of congenital toxoplasmosis. Inhibiting either toxopain-1 expression or specific cysteine proteinase activity significantly reduced congenital infection of chicken embryos, as determined by histopathology and by the number of parasites quantified by real-time PCR. Our new model provides key in vivo validation for the hypothesis that toxopain-1 is a potential drug target in Toxoplasma gondii and also provides a new animal model for rapid, inexpensive screening of antiparasitic compounds.

Genetic manipulation of immunoglobulin binding proteins of Haemophilus somnus.

The relationship of the 76kDa immunoglobulin binding, surface antigen (p76) of Haemophilus somnus to the high molecular weight immunoglobulin binding proteins (HMW IgBPs) was investigated. The kanamycin resistance gene from pLS88 was used via homologous recombination with allelic exchange to replace a portion of the gene encoding IgBPs of H. somnus strain 8025. Recombinants were shown by Western immunoblotting to express and secrete truncated antigens of approximately 200kDa and not to produce p76. The truncated HMW IgBP variants retained the ability to bind bovine IgG2 by the Fc portion as demonstrated by Western immunoblotting against IgG2 anti-DNP. This data indicated that the deleted 1.8kb BglII fragment was not required for secretion or immunoglobulin Fc binding by the HMW IgBPs but was required for expression of the downstream p76 gene. Functional studies showed that, in addition to Fc binding of IgG2 to truncated HMW IgBPs, the mutant strain 8025 Kan1 was equally resistant to killing by mouse complement but less virulent than the wild type parent (8025) in a mouse septicemia model of H. somnus infection. However, mutant strain 8025 Kan1 did adhere less well than the wild type to bovine pulmonary artery endothelial cells. It is probable that p76 and the missing peptides of the HMW IgBPs play a role in this aspect of virulence and perhaps other aspects.