Balázs Csernus

Selective increase in TNFα permeation across the blood–spinal cord barrier after SCI

Abstract We generated a novel mouse model of spinal cord injury (SCI) by hemisection of the right L1 lumbar spinal cord, measured the permeability of the blood–spinal cord barrier (BSCB), and tested the hypothesis that tumor necrosis factor α (TNFα) penetrates the injured BSCB by an enhanced transport system. SCI produced stereotypical sensorimotor deficits resembling the classically described Brown–Sequard syndrome. Disruption of the BSCB was reflected by increased spinal cord uptake of radiolabeled albumin from blood; this was transient (immediately after SCI) and confined to the lumbar spinal cord. By contrast, specific increase in the entry of TNFα was detected in brain, cervical, thoracic, and lumbar spinal cord at 1 week after SCI, in addition to its immediate and transient increase consistent with barrier disruption. Lack of a second peak of increase in the entry of IL1β further supported the specificity of the TNFα response. Moreover, enhanced uptake of radiolabeled TNFα was suppressed by excess non-radiolabeled TNFα, indicating competition of entry via the known transport system for TNFα. Therefore, upregulation of the transport system after SCI probably mediates the increased permeation of TNFα across the BSCB. Enhanced entry of TNFα at 1 week after SCI was concurrent with sensorimotor and gait improvement of the mouse. We conclude that SCI by lumbar hemisection activates the transport system for TNFα at the BBB and suggest that selective permeation of TNFα may facilitate functional recovery.

Microsatellite instability and hMLH1 promoter hypermethylation in Richter's transformation of chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is an indolent B cell non-Hodgkin lymphoma (NHL) that may transform into diffuse large B cell lymphoma (DLBL). This transformation is referred to as Richter’s syndrome or transformation. To analyze whether microsatellite instability (MSI) and DNA mismatch repair defects are associated with Richter’s transformation, we have performed microsatellite analysis, mutational analysis of hMLH1 and hMSH2 genes and methylation status analysis of CpG island of the hMLH1 promoter on serial biopsy specimens from 19 patients with CLL. Ten cases of CLL showed no histologic alteration in the second biopsy, and nine cases of CLL underwent morphologic transformation to DLBL in the second biopsy. Using eight microsatellite loci, high level of MSI was associated with Richter’s transformation in four cases of CLL, but none of the CLLs displayed this level of MSI without transformation. Mutations of the hMLH1 or hMSH2 genes were not detected in any of the lymphoma samples. In five cases of Richter’s transformation the hMLH1 promoter was hypermethylated in both CLL and DLBL samples. Hypermethylation of the hMLH1 promoter associated with high-level of MSI in four cases, and low-level of MSI in one case. These results suggest that in certain cases of Richter’s transformation the DNA mismatch-repair defect-initiated genetic instability may play a role in tumor progression.

Relationship between the mutational status of VH genes and pathogenesis of diffuse large B-cell lymphoma in Richter's syndrome.

Patients with chronic lymphocytic leukemia (CLL) may develop diffuse large B-cell lymphoma (DLBL), also known as Richters syndrome. Mutational status of immunoglobulin (Ig) heavy-chain variable region (VH) genes have prognostic impact in CLL. Patients with mutated VH genes have a stable disease, whereas patients with unmutated VH gene have more aggressive disease. The mutational status of CLLs that transform to DLBL is unknown. To reveal whether Richters syndrome occurs in CLLs with mutated or unmutated VH genes, we have performed mutational analysis on serial specimens from eight patients. CLL and DLBL tumorclones were identical in five cases and they were different in three cases. Six CLLs expressed unmutated and two cases expressed mutated VH genes. In five of the six unmutated CLLs, the DLBL clones evolved from CLL tumorclones and the VH genes expressed by DLBLs were also unmutated. In one unmutated and two mutated CLLs, the DLBLs expressed mutated VH genes, but in these three cases the DLBL tumorclones developed as independent secondary neoplasm. These results suggest that Richters syndrome may develop in both mutated or unmutated CLLs, but clonal transformation of CLL to DLBL occur only in the unmutated subgroup of CLL.

Luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix down-regulates the mRNA expression of pituitary receptors for LH-RH by counteracting the stimulatory effect of endogenous LH-RH.

The mechanisms through which LH-RH antagonists suppress gonadotroph functions and LH-RH receptor (LH-RH-R) production are incompletely understood. To elucidate these mechanisms, we investigated the effects of Cetrorelix on the mRNA expression of pituitary LH-RH-R and luteinizing hormone (LH) secretion in three experimental systems with different pituitary LH-RH environments. Ovariectomy induced 3.61-fold and 6.34-fold increases in the mRNA expression of pituitary LH-RH-R in rats after 11 and 21 days, respectively. After (5 h) a single injection of 100 microg Cetrorelix, no significant decrease occurred in the mRNA levels of pituitary LH-RH-R in ovariectomized (OVX) rats with high pituitary exposure to LH-RH, but there was a significant 23.2% reduction in cycling rats with normal hypophysial LH-RH environment. Prolonged treatment for 10 days with a Cetrorelix depot formulation releasing 100 microg/day decreased the concentration of mRNA for pituitary LH-RH-R by 72.6% in OVX rats, but only by 32.9% in normal rats. The decline in serum LH was 98.7% in OVX rats and 63.2% in normal rats, resulting in a minimal 0.1--0.2 ng/ml LH concentration in both groups. A continuous exposure of pituitary cells to 100 nM Cetrorelix in the superfusion system, which is devoid of LH-RH, did not cause any significant changes in LH-RH-R mRNA level. These studies demonstrate that prolonged exposure to Cetrorelix in vivo, but not in vitro, down-regulates the mRNA expression of the pituitary receptors for LH-RH. Our findings indicate that LH-RH antagonists exert their inhibitory effects on the gene expression of pituitary LH-RH-R by counteracting the stimulatory effect of endogenous LH-RH.

ROR1 expression is not a unique marker of CLL.

Recent studies have identified receptor tyrosine kinase‐like orphan receptor 1 (ROR1) on the surface of chronic lymphoid leukaemia (CLL) cells. In order to determine whether ROR1 expression is a suitable surrogate marker for the diagnosis of CLL we analysed the mRNA level of ROR1 in different types of non‐Hodgkin lymphomas (NHL), and detected elevated levels of ROR1 compared to control peripheral mononuclear cells in several entities (CLL ≥ mantle cell lymphoma (MCL) > marginal zone lymphoma (MZL) >> diffuse large B‐cell lymphoma > follicular lymphoma). ROR1 protein was expressed intensely on the cell surface of lymphoma cells with leukaemic blood count detected by three colour immunofluorescence. Our results indicate that ROR1 expression is not limited to CLL cases, but it is more prevalent in NHLs, mainly in MCL where it is expressed intensely and MZL where it is expressed moderately, suggesting a general role of ROR1 in lymphoma genesis and/or maintenance. Copyright

Upregulation of p55 and p75 receptors mediating TNF-α transport across the injured blood-spinal cord barrier

Tumor necrosis factor (TNF-α) is involved in the inflammation and tissue regeneration occurring after spinal cord injury (SCI). This study tests the specific role of p55 and p75 receptors in mediating the transport of TNF-α across the blood-spinal cord barrier (BSCB) after SCI by compression. Transcytosis of 125I-TNF-α across a monolayer of the cerebral endothelial cells that compose the blood-brain barrier was significantly reduced in the absence of functional p55 and p75 receptors. At 3 d after SCI, double receptor knockout mice had a significantly reduced increase in TNF-α uptake from blood to injured lumbar spinal cord as compared with their inbred controls, despite the similar extent of BSCB disruption (measured by 99mTc-albumin). The p75 single receptor knockout mice had a reduced increase in 125I-TNF-α uptake, whereas the p55 receptor knockout mice had no significant increase of 125I-TNF-α uptake after SCI, suggesting that the p55 receptor plays a major role. Hence, the increased uptake of TNF-α 3 d after SCI is not explained by nonspecific barrier disruption but by receptor-mediated upregulation of transport. Quantitative RT-PCR analysis further showed that upregulation of TNF-α transport was related to increased expression of mRNA for p55 and p75 receptors. The increase of p55 receptor expression was more robust and seen between 12 h and 1 wk after SCI, whereas the increase of p75 receptor expression occurred later and involved fewer regions. Thus, the differential upregulation of p55 and p75 receptors indicates that permeation of TNF-α across the injured BSCB remains a regulated process. Knowledge of receptor-mediated regulation could facilitate effective therapeutic manipulation of BSCB permeation of vascular cytokines important to CNS regeneration.

Clonal selection in the bone marrow involvement of follicular lymphoma

To characterize the pathways of bone marrow (BM) involvement of follicular lymphoma (FL), we performed morphological and immunophenotypical analysis of tumor cells from lymph nodes (LNs) and corresponding BMs in 21 patients with FL. In three cases, genealogical trees were constructed based on the immunoglobulin variable region heavy chain (IgVH) gene sequences of tumor clones from LNs and BMs. Results showed that FLs within the BMs display identical or lower cytological grades than in the LNs. In the majority of cases, different proportions of tumor cells expressed bcl-2, CD10 and Ki67 in LNs and BMs. Tumor cells in the BM showed ongoing somatic hypermutation of the IgVH genes; the distribution of these mutations was highly consistent with antigen selection. The topology of the genealogical trees revealed that different subclones populate the LN and BM and BM infiltration may occur at different points of the clonal evolution of FL. Early descendants of the original tumor clone and derivatives of diversified tumor clones may invade the BM. These results suggest that the BM involvement of FL is associated with intensive clonal selection of tumor cells, and the BM provides a microenvironment similar to the germinal centers of LNs, where tumor cells retain their biological nature.

Antiproliferative actions of growth hormone-releasing hormone antagonists on MiaPaCa-2 human pancreatic cancer cells involve cAMP independent pathways.

We evaluated the effects of GHRH antagonists on the proliferation of MiaPaCa-2 human pancreatic cancer cells and cAMP signaling in vitro. GHRH antagonists inhibited the proliferation of MiaPaCa-2 cells in vitro in a dose-dependent way and caused a significant elevation in cAMP production. In a superfusion system, short-term exposure of the cells to GHRH antagonists evoked an acute, dose-dependent release of cAMP into the medium. Native GHRH, which stimulates cAMP efflux from pituitary at nanomolar doses, did not influence cAMP release from cultured or superfused MiaPaCa-2 cells even at 10-30 microM. VIP, PACAP, secretin and glucagon also did not influence cell proliferation or cAMP production. Adenylate cyclase activator forskolin (FSK) caused a greater cAMP response, but a smaller antiproliferative effect than GHRH antagonists. Combined treatment with FSK and GHRH antagonist JV-1-38 potentiated the cAMP-inducing effect of FSK, but did not produce a greater inhibition of cell proliferation than JV-1-38 alone. A selective accumulation of radiolabeled GHRH antagonist [(125)I]JV-1-42 in vivo in MiaPaCa-2 carcinoma xenografted into nude mice was also observed. In conclusion, second messengers other than cAMP participate in the signal transduction pathways of GHRH analogs mediated by tumoral GHRH receptors.

Somatic hypermutation of IGVH genes and aberrant somatic hypermutation in follicular lymphoma without BCL-2 gene rearrangement and expression

The findings of this study suggest that follicular lymphoma with typical morphological and immunophenotypic features may develop even without the involvement of the BCL-2 gene. See related perspective article on page 1773. Background Follicular lymphoma is characterized by the t(14;18) translocation resulting in constitutive expression of BCL-2 protein; however approximately 10–15% of follicular lymphomas do not express BCL-2 protein, and a small fraction of these cases does not exhibit translocation of the BCL-2 gene either. It is highly debated whether cases of follicular lymphoma without BCL-2 gene rearrangement and expression represent a separate lymphoma entity with distinct biological characteristics, different from the BCL-2-positive cases. Design and Methods To further characterize follicular lymphomas without BCL-2 gene rearrangement and expression, we analyzed and compared the mutational status of IGVH genes as well as other genes (C-MYC, PAX-5 and RHOH) frequently involved in the specific type of genomic instability called aberrant somatic hypermutation in 11 cases of BCL-2-negative and 7 cases of BCL-2-positive follicular lymphomas. We also determined the levels of expression of activation-induced cytidine deaminase in these cases. Results The analyzed cases were grade 2 and grade 3A follicular lymphomas. Our findings demonstrate that follicular lymphomas without BCL-2 gene rearrangement and expression are associated with ongoing somatic hypermutation of the IGVH genes, low activity of aberrant somatic hypermutation and elevated activation-induced cytidine deaminase expression. These results were in concordance with the results found in the cases of BCL-2-positive follicular lymphoma. Conclusions Although, BCL-2 protein overexpression is considered to be a critical pathogenic event in the development of follicular lymphoma, our findings suggest that follicular lymphomas with the same morphology, immunophenotype, mutational pattern and activation-induced cytidine deaminase expression may develop without the involvement of BCL-2 gene. The present data support the hypothesis that BCL-2-positive and BCL-2-negative follicular lymphomas (grades 1-3A) represent a homogenous group with different initial but several common additional molecular pathways.

Targeted cytotoxic analogue of luteinizing hormone-releasing hormone (LH-RH) only transiently decreases the gene expression of pituitary receptors for LH-RH

A cytotoxic analogue of LH‐RH, AN‐207, consisting of 2‐pyrrolinodoxorubicin (AN‐201) linked to carrier [D‐Lys6]LH‐RH, was developed for targeted therapy of cancers expressing LH‐RH‐receptors. To determine its possible side‐effects on the pituitary gland, we investigated the gene expression of pituitary LH‐RH‐receptors and LH secretion in ovariectomized female and normal male rats after treatment with the maximum tolerated dose of AN‐207. The effect of AN‐207 on the gene expression of the pituitary GH‐RH‐receptors and GH secretion was also assessed in male rats. Five hours after a single i.v. injection of AN‐207 at 175 nmol/kg, there was a 39–51% decrease in mRNA expression for the pituitary LH‐RH‐receptors in male and female rats. The carrier, at an equimolar dose, caused a similar reduction (37–39%), whereas the cytotoxic radical AN‐201, at an equitoxic dose (110 nmol/kg), produced only a 12–24% decrease (NS) in the mRNA expression of LH‐RH‐receptors. AN‐207 and the carrier analogue induced a comparable 90–100‐fold increase in serum LH concentrations in male rats, and the same 12‐fold elevation in OVX rats at 5 h. Seven days after treatment with AN‐207, the mRNA levels for the LH‐RH receptors and the serum LH concentration were back to normal in both sexes. AN‐207, the carrier, and AN‐201 had no significant effect on the expression of mRNA for GH‐RH‐receptors in the pituitary. In vitro, a continuous perfusion of pituitary cells with 10 nM AN‐207 did not affect the hormone‐releasing function of the targeted LH cells or the nontargeted GH cells. Our results demonstrate that cytotoxic LH‐RH analogue AN‐207, at the maximum tolerated dose causes only a transient decrease in the gene expression of the pituitary LH‐RH receptors, and the levels of mRNA for LH‐RH receptor fully recover within 7 days. Moreover, the carrier hormone moiety, and not the cytotoxic radical in AN‐207 is responsible for this transient suppression. Our findings suggest that the therapy with cytotoxic LH‐RH analogues will not inflict permanent damage to pituitary function.