Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis.

Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis), but may also contribute to vascular thrombosis. Hereditary deficiency of factor XII (FXII), the protease that triggers the intrinsic pathway of coagulation in vitro, is not associated with spontaneous or excessive injury-related bleeding, indicating FXII is not required for hemostasis. We demonstrate that deficiency or inhibition of FXII protects mice from ischemic brain injury. After transient middle cerebral artery occlusion, the volume of infarcted brain in FXII-deficient and FXII inhibitor-treated mice was substantially less than in wild-type controls, without an increase in infarct-associated hemorrhage. Targeting FXII reduced fibrin formation in ischemic vessels, and reconstitution of FXII-deficient mice with human FXII restored fibrin deposition. Mice deficient in the FXII substrate factor XI were similarly protected from vessel-occluding fibrin formation, suggesting that FXII contributes to pathologic clotting through the intrinsic pathway. These data demonstrate that some processes involved in pathologic thrombus formation are distinct from those required for normal hemostasis. As FXII appears to be instrumental in pathologic fibrin formation but dispensable for hemostasis, FXII inhibition may offer a selective and safe strategy for preventing stroke and other thromboembolic diseases.

Vitamin D3 receptor is highly expressed in Hodgkin's lymphoma.

BACKGROUND: Hodgkin lymphoma (HL) is one of the most frequent lymphoma in the western world. Despite a good overall prognosis, some patients suffer relapsing tumors which are difficult to cure. Over a long period Vitamin D has been shown to be a potential treatment for cancer. Vitamin D acts via the vitamin D receptor, a nuclear receptor, acting as an inducible transcription factor. We aimed to investigate the expression of vitamin D receptor as a possible diagnostic marker and potential therapeutic target in HL as well as in B-cell derived non-Hodgkin lymphoma (B-NHL). METHODS: We used a panel of 193 formalin fixed tissues of lymphoma cases consisting of 55 cases of HL and 138 cases on several B-NHL entities. RESULTS: Vitamin D receptor is strongly expressed in tumor cells of HL, regardless of the sub entity with an overall positivity of 80% of all HL cases. In contrast, only about 17% of the analyzed origin-NHL showed positivity for vitamin D receptor. The detection of nuclear localization of vitamin D receptor in the tumor cells of HL suggests activated status of the vitamin D receptor. CONCLUSIONS: Our study suggests VDR as a specific marker for tumor cells of HL, but not of B-NHL subtypes. Further, the observed nuclear localization suggests an activated receptor status in tumor cells of HL. Further investigations of mutational status and functional studies may shed some light in functional relevance of vitamin D receptor signaling in HL.

Inactivating SOCS1 mutations are caused by aberrant somatic hypermutation and restricted to a subset of B-cell lymphoma entities.

STATs are constitutively activated in several malignancies. In primary mediastinal large B-cell lymphoma and Hodgkin lymphoma (HL), inactivating mutations in SOCS1, an inhibitor of JAK/STAT signaling, contribute to deregulated STAT activity. Based on indications that the SOCS1 mutations are caused by the B cell-specific somatic hypermutation (SHM) process, we analyzed B-cell non-HL and normal B cells for mutations in SOCS1. One-fourth of diffuse large B-cell lymphoma and follicular lymphomas carried SOCS1 mutations, which were preferentially targeted to SHM hotspot motifs and frequently obviously inactivating. Rare mutations were observed in Burkitt lymphoma, plasmacytoma, and mantle cell lymphoma but not in tumors of a non-B-cell origin. Mutations in single-sorted germinal center B cells were infrequent relative to other genes mutated as byproducts of normal SHM, indicating that SOCS1 inactivation in primary mediastinal large B-cell lymphoma, HL, diffuse large B-cell lymphoma, and follicular lymphoma is frequently the result of aberrant SHM.

Autocrine NGFbeta/TRKA signalling is an important survival factor for Hodgkin lymphoma derived cell lines.

Hodgkin-Reed/Sternberg cells, the tumor cells of Hodgkin lymphoma, aberrantly express several receptor tyrosine kinases, among them TRKA whose stimulation supports B cell survival. We show here high expression of TRKA in Hodgkin-Reed/Sternberg cell lines as compared to normal B cells and other B cell lymphomas, without major increases in TRKA gene dosage. A fraction of TRKA is constitutively activated, likely due to the coexpression of NGFbeta, the TRKA high affinity ligand. The TRK inhibitor K-252a decreased survival of Hodgkin-Reed/Sternberg cell lines accompanied by decreased AKT activation. Inclusion of TRK inhibitors in therapeutic regimens may thus be an interesting possibility.

The effects of COX-2 selective and non-selective NSAIDs on the initiation and progression of atherosclerosis in ApoE-/- mice.

In this study, we investigated the effects of prolonged administration of the selective COX-2 inhibitors celecoxib and rofecoxib and the non-selective NSAID naproxen on the initiation and progression of atherosclerosis. ApoE(-/-) mice, as well as corresponding wild-type mice, were fed either a normal chow or a high fat Western diet with or without addition of the respective drugs over a period of 16 weeks. Thereafter, aortic lesion size, plasma lipid levels, and COX-2 expression in the plaques were determined. The results showed that neither the COX-2 selective inhibitors nor naproxen had a significant impact on the initiation and progression of atherosclerosis in diet-fed ApoE(-/-) mice, although both celecoxib and rofecoxib showed a tendency to reduce plaque size. This slight effect may be due to selective inhibition of COX-2 activity because the COX-2 expression was not altered in the plaque. Plasma lipid levels were also not significantly influenced by these drugs. Interestingly, in ApoE(-/-) mice that have been fed with normal chow, we found an increased incidence of plaque formation after treatment with celecoxib and rofecoxib, indicating that coxibs may promote the initiation of atherosclerosis. This effect was probably masked in diet-fed mice by the more pronounced effects of the high cholesterol diet. In conclusion, the reduction in diet-induced plaque size in animals fed a high fat diet and the promotion of atherosclerosis in mice on a normal diet indicate a dual role of the coxibs. In advanced stages of atherosclerosis, they may exert antithrombotic properties due to their COX-2 inhibiting activity, whereas in very early stages they may favor the initiation of atherogenesis. However, because these results were only observed in ApoE(-/-) and not in wild-type animals, coxibs may increase the risk of thrombosis in patients with a predisposition for thrombotic complications.

Host DNases prevent vascular occlusion by neutrophil extracellular traps.

Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

Common features and differences in the transcriptome of large cell anaplastic lymphoma and classical Hodgkin's lymphoma.

BACKGROUND AND OBJECTIVES: Anaplastic large cell lymphoma (ALCL) and classical Hodgkin's lymphoma (HL) are derived from different cell types, namely T cells and B cells, respectively. However, both lymphomas share a similar cytological and immunohistochemical tumor cell phenotype with little resemblance to their cells of origin. DESIGN AND METHODS: In this study, the transcriptional profiles of ALCL cell lines, primary ALCL tumor cells from peripheral blood and HL cell lines were compared to each other and to normal B-cell subsets, B non-Hodgkin's lymphomas (NHL) and B NHL- and Epstein-Barr virus (EBV)-transformed B-cell lines in order to establish their relationship at the transcriptional level and to identify genes with possible pathobiological impact. Expression of some of the genes identified was confirmed in microdissected primary tumor cells by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: HL samples clustered separately from ALCL samples, but HL and ALCL were found to be more closely related to each other than to any normal or malignant B-cell sample in the dataset. Their relationship was determined to a large extent, but not exclusively, by lack of expression of B-cell antigens and by the over-expression of mRNA encoding activation markers and structural proteins. Apart from established differences between HL and ALCL, further genes of interest could be identified that distinguish both entities from each other and from the other samples. The differential expression of PRAME, DDR2, SOCS3 and CEBPD in HL and ALCL was confirmed in primary tumor tissue by immunohistochemistry and/or RT-PCR. INTERPRETATION AND CONCLUSIONS: At a transcriptional level HL is more closely related to Alk+ ALCL than to the B-NHL or B-cell samples investigated, although it is a B-cell derived lymphoma. The newly identified genes discriminating HL and ALCL may be pathobiologically important and may serve as possible therapeutic targets.

Molecular cytogenetic analyses of immunoglobulin loci in nodular lymphocyte predominant Hodgkin's lymphoma reveal a recurrent IGH-BCL6 juxtaposition.

Chromosomal translocations juxtaposing different oncogenes to the immunoglobulin (IG) loci are the hallmark of various B-cell lymphomas. Because the tumor cells in nodular lymphocyte predominant Hodgkin's lymphoma (NLPHL) are also derived from B cells, we examined whether NLPHL harbors chromosomal translocations that affect IG loci. Fluorescence in situ hybridization was applied to 24 NLPHL cases using probes flanking the IGH, IGK, and IGL loci as well as the BCL6 gene. Fourteen of these cases were additionally analyzed by combined immunofluorescence and fluorescence in situ hybridization. Chromosomal breakpoints in the IGH locus were detected in five NLPHL. All these cases also contained a BCL6 breakpoint. Triple-color interphase cytogenetics demonstrated the presence of an IGH-BCL6 juxtaposition, indicating a t(3;14)(q27;q32) in all five cases. There was no evidence for breakpoints affecting the IGK or IGL loci. Our results show that translocations juxtaposing the BCL6 oncogene next to the IGH locus are recurrent in NLPHL.

In angioimmunoblastic T-cell lymphoma, neoplastic T cells may be a minor cell population. A molecular single-cell and immunohistochemical study.

The significance of T-cell proliferations in angioimmunoblastic lymphoma (AILD) is still enigmatic. Although classified as a malignant T-cell lymphoma in the World Health Organisation lymphoma classification, some cases of AILD lack dominant T-cell clones. In a previous study, based on single-cell polymerase chain reaction (PCR), we obtained similar results as studies of AILD using Southern blot or conventional PCR: some cases of AILD contained large T-cell clones, and, in other cases, T-cell clones were undetectable. As in single-cell studies, only a limited number of cells could be investigated; thus, we wanted to gain more insight into the amount and distribution of tumour cells. By applying triple immunofluorescent staining with antibodies directed against T-cell receptor Vbeta-family-specific epitopes, we investigated T-cell populations in AILD and their localisation in the tissue in relation to B cells (CD20) and follicular dendritic cells (CD21). In two of five cases investigated, only a minority of the T-cells compartment belonged to the tumour clone. Neoplastic T cells were found throughout the tissue, including areas dominated by B cells.

Thyroid fetal male microchimerisms in mothers with thyroid disorders: presence of Y-chromosomal immunofluorescence in thyroid-infiltrating lymphocytes is more prevalent in Hashimoto's thyroiditis and Graves' disease than in follicular adenomas.

The presence of fetal cells in a maternal compartment is defined as fetal-maternal microchimerism, which has been detected in thyroids of mothers suffering from autoimmunity. We analyzed the immunohistology of paraffin-embedded thyroid specimen taken at surgery from 49 women with Hashimoto's thyroiditis (n = 25), Graves' disease (n = 15), or nodular or diffuse follicular adenomas (n = 9), whose childbirth history was positive for sons. By fluorescence in situ hybridization we screened for X-chromosome- and Y-chromosome-specific staining and compared the finding with human leukocyte antigen (HLA) DQ types of the mothers and, where available, their offspring. In 23 thyroids we found Y-chromosome-specific staining, which was more frequent in thyroid autoimmune disease (60% Hashimoto's thyroiditis and 40% Graves' disease) than in follicular adenomas (22.2%). There was no significant difference for HLA DQ alleles among women whose thyroids showed Y-chromosome staining and those without. However, a subgroup of all investigated microchimerism-positive mother-child pairs and women with Hashimoto's thyroiditis and Graves' disease more often had the susceptibility alleles HLA DQA1*0501-DQB1*0201 or DQB1*0301. In conclusion, fetal microchimerism is observed in thyroids of mothers with sons, and this is found more frequently in thyroid autoimmune diseases.

Induction of endoplasmic reticulum stress by sorafenib and activation of NF-κB by lestaurtinib as a novel resistance mechanism in Hodgkin lymphoma cell lines.

Hodgkin-Reed/Sternberg (HRS) cells of classical Hodgkin lymphoma show aberrant expression and activation of several receptor tyrosine kinases (RTK) in the majority of cases. Therefore, we tested whether tyrosine kinase inhibitors (TKI) already in clinical use or late stages of clinical trials have antiproliferative effects on HRS cell lines and evaluated the targets, affected signaling pathways, and mechanisms of cell death and resistance. Sorafenib and lestaurtinib had antiproliferative effects on HRS cell lines at concentrations achievable in patients. Sorafenib inhibited platelet-derived growth factor receptor (PDGFR) α, TRKA and RON, caused decreases in total and phosphorylated amounts of several signaling molecules, and provoked caspase-3-independent cell death, most likely due to endoplasmic reticulum stress as indicated by upregulation of GADD34 and GADD153 and phosphorylation of PERK. Lestaurtinib inhibited TRKA, PDGFRα, RON, and JAK2 and had only a cytostatic effect. Besides deactivation, lestaurtinib also caused activation of signaling pathways. It caused increases in CD30L and TRAIL expression, and CD30L/CD30 signaling likely led to the observed concomitant activation of extracellular signal-regulated kinase 1/2 and the alternative NF-κB pathway. These data disclose the possible use of sorafenib for the treatment of Hodgkin lymphoma and highlight NF-κB activation as a potential novel mechanism of resistance toward TKIs.

Clonality testing of malignant lymphomas with the BIOMED-2 primers in a large cohort of 1969 primary and consultant biopsies.

The introduction of the BIOMED-2 primers allowed for reliable comparisons of clonality testing data of malignant lymphomas from different laboratories. This study undertook a retrospective analysis of a large cohort of cases; 1862 cases involved the immunoglobulin heavy chain locus (IGH VH-JH), and 1527 cases involved the T cell receptor gamma locus (TCRG). We confirmed previously published clonality rates in various B cell, T cell, and Hodgkin lymphoma cases. In reactive lesions, clonality for the IGH locus was frequently accompanied by additional polyclonal background. Clonality for TCRG was found in a subgroup of diffuse large B cell lymphomas. On closer morphologic inspection, seven cases appeared to have arisen from an underlying peripheral T-cell lymphoma. Five cases with monoclonal TCRG rearrangements, originally diagnosed as Hodgkin lymphomas, were reclassified as T-cell lymphomas. TCRG clonality was very rarely only observed in Hodgkin lymphoma. In case of clear TCRG clonality a T-cell neoplasia must be ruled out on morphological grounds. By careful examination of the rearrangement patterns, including an assessment of a co-amplified polyclonal background, clonality testing provides a powerful tool which in concert with morphologic and immunohistochemical parameters can lead to a firm diagnosis.

Expression and functional relevance of cannabinoid receptor 1 in Hodgkin lymphoma.

BACKGROUND: Cannabinoid receptor 1 (CB1) is expressed in certain types of malignancies. An analysis of CB1 expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date. DESIGN AND METHODS: We examined the distribution of CB1 protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB1 signaling on cell survival was investigated. RESULTS: A predominant expression of CB1 was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB1-abundance and displayed a dose-dependent decline of viability under CB1 inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells. CONCLUSIONS: The present study identifies CB1 as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.

Impaired melanoma growth in VASP deficient mice.

Progression of tumors depends on interactions of cancer cells with the host environment. Expression of the cytoskeleton protein VASP is upregulated in various cancer entities. We analyzed the role of VASP for melanoma growth in murine allograft models. Growth of VASP expressing melanomas was retarded in VASP(-/-) versus wild-type animals. Over time tumor size was <50% in VASP(-/-) versus wild-type animals and independent of expression levels of Ena/VASP protein family members. Histological analyses showed smaller cells with impaired nutrition status and less vascularization in melanomas derived from VASP(-/-) versus counterparts from wild-type mice. Cumulatively, the data reveal a critical role of VASP in non-tumor cells in the tumor environment for melanoma growth in vivo.

A common human micro-opioid receptor genetic variant diminishes the receptor signaling efficacy in brain regions processing the sensory information of pain.

The single nucleotide polymorphism 118A>G of the human micro-opioid receptor gene OPRM1, which leads to an exchange of the amino acid asparagine (N) to aspartic acid (D) at position 40 of the extracellular receptor region, alters the in vivo effects of opioids to different degrees in pain-processing brain regions. The most pronounced N40D effects were found in brain regions involved in the sensory processing of pain intensity. Using the mu-opioid receptor-specific agonist DAMGO, we analyzed the micro-opioid receptor signaling, expression, and binding affinity in human brain tissue sampled postmortem from the secondary somatosensory area (SII) and from the ventral posterior part of the lateral thalamus, two regions involved in the sensory processing and transmission of nociceptive information. We show that the main effect of the N40D micro-opioid receptor variant is a reduction of the agonist-induced receptor signaling efficacy. In the SII region of homo- and heterozygous carriers of the variant 118G allele (n=18), DAMGO was only 62% as efficient (p=0.002) as in homozygous carriers of the wild-type 118A allele (n=15). In contrast, the number of [3H]DAMGO binding sites was unaffected. Hence, the micro-opioid receptor G-protein coupling efficacy in SII of carriers of the 118G variant was only 58% as efficient as in homozygous carriers of the 118A allele (p<0.001). The thalamus was unaffected by the OPRM1 118A>G SNP. In conclusion, we provide a molecular basis for the reduced clinical effects of opioid analgesics in carriers of mu-opioid receptor variant N40D.

Somatic hypermutation of SOCS1 in lymphocyte-predominant Hodgkin lymphoma is accompanied by high JAK2 expression and activation of STAT6.

Aberrant activities of JAK/STAT signaling pathways have been observed in several hematologic malignancies. Here, we show high expression of JAK2 in the tumor cells of lymphocyte-predominant Hodgkin lymphoma in 85% of cases and activation of JAK2 in 39% of cases. STAT6, which is a target of JAK2, was activated in 50% of the cases. SOCS1 controls JAK2 activity and degradation. Mutations in SOCS1 of either somatic or germ-line origin were observed in micromanipulated tumor cells of 50% of cases. Most mutations truncated SOCS1 or caused replacement of amino acids in functional important regions. Activating mutations in exon 12 of JAK2, which are frequent in myeloproliferative diseases, were not observed. In lymphocyte-predominant Hodgkin lymphoma SOCS1 function may thus be frequently impaired by mutations, and this may contribute to high JAK2 expression and activation of the JAK2/STAT6 pathway.

The aberrant coexpression of several receptor tyrosine kinases is largely restricted to EBV-negative cases of classical Hodgkin's lymphoma.

The Hodgkin-Reed/Sternberg (HRS) cells of classical Hodgkin's lymphoma (HL) aberrantly express up to 7 different receptor tyrosine kinases (RTK) with extensive heterogeneity regarding the number and combinations of expressed RTKs in individual cases and a more prominent coexpression in nodular-sclerosis (ns) than mixed-cellularity (mc) HL. To investigate whether RTK expression patterns are related to other pathogenetic mechanisms and clinical behaviour, we analysed a large collection of EBV(+) and EBV(-) cases of ns and mc subtype and cases with relapses for expression of the 7 RTKs. No specific relation of any RTK to a specific group of cases was observed. The analysis of average numbers of expressed RTKs per case as a measure for strength of overall RTK signalling revealed a relation with the histological subtype and the EBV-status. RTK coexpression was significantly higher in EBV(-) nsHL cases compared to both EBV(-) and EBV(+) mcHL cases. Among mcHL cases RTK coexpression was significantly higher in EBV(-) compared to EBV(+) cases. Coexpression of 3 and more RTKs was largely restricted to EBV(-) cases. The inverse correlation between strong RTK signalling and presence of EBV may indicate that RTK signalling can at least partially replace the role of EBV in HRS cell pathogenesis. For cases with aberrant coexpression of several RTKs inclusion of RTK inhibitors in therapy regimens may be a novel option.

Molecular biology of Hodgkin's and Reed/Sternberg cells in Hodgkin's lymphoma.

Hodgkin's and Reed/Sternberg (HRS) cells, the tumour cells in classical Hodgkin's lymphoma (HL), represent transformed B cells in nearly all cases. The detection of destructive somatic mutations in the rearranged immunoglobulin (Ig) genes of HRS cells in classical HL indicated that they originate from preapoptotic germinal centre (GC) B cells that lost the capacity to express a high-affinity B-cell receptor (BCR). Several aberrantly activated signalling pathways and transcription factors have been identified that contribute to the rescue of HRS cells from apoptosis. Among the deregulated signalling pathways, activation of multiple receptor tyrosine kinases in HRS cells appears to be a specific feature of HL. In about 40% of cases of classical HL the HRS cells are infected by Epstein-Barr virus (EBV), indicating an important role of EBV in HL pathogenesis. Interestingly, nearly all cases of HL with destructive Ig gene mutations eliminating BCR expression (e.g. nonsense mutations) are EBV-positive, suggesting that EBV-encoded genes have a particular function to prevent apoptosis of HRS-cell precursors that acquired such crippling mutations. This idea is further supported by the recent demonstration that isolated human GC B cells harbouring crippled Ig genes can be rescued by EBV from cell death, giving rise to lymphoblastoid cell lines. The molecular analysis of composite Hodgkin's and non-Hodgkin's lymphomas indicated that many cases develop from a common GC B-cell precursor in a multistep transformation process with both shared and distinct oncogenic events.

Aberrant expression of ID2, a suppressor of B-cell-specific gene expression, in Hodgkin's lymphoma.

The global loss of B-cell-specific gene expression is a distinctive feature of the Hodgkin-Reed/Sternberg (HRS) cells of classical Hodgkin's lymphoma (HL). The reasons for this loss remained largely unknown as transcription factors with pleiotropic effects on B-cell-specific gene expression, namely E2A, EBF, and PAX5, are present in primary HRS cells. We show here that ID2, which can inactivate E2A and perhaps PAX5, is not detectable in normal B cells but is strongly and uniformly expressed in HRS cells of all cases of classical HL. Recurrent chromosomal gains of the ID2 gene might contribute to this aberrant expression. Co-immunoprecipitation of E2A with ID2 from HRS-derived cell lines together with the high amount of ID2 relative to the B-cell transcription factors E2A and PAX5 in HRS-derived cell lines and primary HRS cells indicated that aberrant ID2 expression contributes significantly to the loss of the B-cell-specific gene expression in HRS cells. ID2 was also expressed in lymphocyte-predominance HL, mediastinal large B-cell, diffuse large B-cell, and Burkitt's lymphoma, where lower amounts of ID2 relative to E2A and PAX5 compared with HRS cells might prevent a global down-regulation of B-cell-specific genes and ID2 may contribute to lymphomagenesis in other ways.