Dorota Jesionek-Kupnicka

Modified bacterial cellulose tubes for regeneration of damaged peripheral nerves

Introduction The subject of the experiment was bacterial nanocellulose, a natural polymer produced by bacteria – Gluconacetobacter xylinus. Following a specific modification process a cartilage-like material for restoration of damaged tissues may be produced. The obtained implants with excellent biocompatibility, mouldability, biophysical and chemical properties perfectly fit the needs of reconstructive surgery. The goal of the experiment was to develop and analyze cellulosic guidance channels in vivo for the reconstruction of damaged peripheral nerves. Material and methods The experiments were conducted on Wistar rats, femoral nerve. Cellulose was produced according to a self-patented method. In the experimental group tubulization was applied, whereas in the control traditional end-to-end connection was used. Observation time was 30, 60, 90, and 180 days. Results evaluation included histological analysis and postoperative observation of motor recovery. Results The overgrowth of connective tissue and disorganisation of neural structures was evident in 86.67% of control specimens, while for cellulosic group it was only 35% (p = 0.0022). Tubulization prevented the excessive proliferation of connective tissue and isolated from penetration with scar tissue. Autocannibalism, being probably an evidence of neurotrophic factors amassment, was observed in cellulosic group but not in the control one. Motor recovery did not differ significantly (p > 0.05). Biocompatibility of implants was affirmed by very small level of tissue response and susceptibility to vascularisation. Conclusions Cellulosic neurotubes effectively prevent the formation of neuromas. They are of very good biocompatibility and allow the accumulation of neurotrophic factors inside, thus facilitating the process of nerve regeneration.

Immune regulation of multiple sclerosis by transdermally applied myelin peptides

Antigen‐specific therapy targeting selective inhibition of autoreactive responses holds promise for controlling multiple sclerosis (MS) without disturbing homeostasis of the whole immune system. Key autoantigens in MS include myelin proteins, such as myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). In this study, we examined the effect of transdermal therapy with myelin peptides on immune responses in the skin, lymph nodes, and peripheral blood immune cells of MS patients.

New methods Modified bacterial cellulose tubes for regeneration of damaged peripheral nerves

Introduction The subject of the experiment was bacterial nanocellulose, a natural polymer produced by bacteria – Gluconacetobacter xylinus. Following a specific modification process a cartilage-like material for restoration of damaged tissues may be produced. The obtained implants with excellent biocompatibility, mouldability, biophysical and chemical properties perfectly fit the needs of reconstructive surgery. The goal of the experiment was to develop and analyze cellulosic guidance channels in vivo for the reconstruction of damaged peripheral nerves. Material and methods The experiments were conducted on Wistar rats, femoral nerve. Cellulose was produced according to a self-patented method. In the experimental group tubulization was applied, whereas in the control traditional end-to-end connection was used. Observation time was 30, 60, 90, and 180 days. Results evaluation included histological analysis and postoperative observation of motor recovery. Results The overgrowth of connective tissue and disorganisation of neural structures was evident in 86.67% of control specimens, while for cellulosic group it was only 35% (p = 0.0022). Tubulization prevented the excessive proliferation of connective tissue and isolated from penetration with scar tissue. Autocannibalism, being probably an evidence of neurotrophic factors amassment, was observed in cellulosic group but not in the control one. Motor recovery did not differ significantly (p > 0.05). Biocompatibility of implants was affirmed by very small level of tissue response and susceptibility to vascularisation. Conclusions Cellulosic neurotubes effectively prevent the formation of neuromas. They are of very good biocompatibility and allow the accumulation of neurotrophic factors inside, thus facilitating the process of nerve regeneration.

Expression of CD34 in gastric cancer and its correlation with histology, stage, proliferation activity, p53 expression and apoptotic index

The formation of new blood vessels is essential for tumor growth and progression. Until today there are only few studies of the immunohistochemical assessment of angiogenesis in gastric cancer by the evaluation of the expression of CD34 antigen. The aim of this study was to analyze the relationship between microvessel density (MVD) expressed as the mean count of CD34 immunostained vessels and clinicopathologic features of gastric tumors (the histological type according to the Lauren classification, tumor grade - G; presence of lymph node metastases - N; depth of tumor invasion; stage of disease (UICC-AJCC 1988–1992), p53 expression, tumor cell proliferative activity described as the Ki67 labelling index and apoptotic index of tumor cells - TUNEL method). We assessed formalin-fixed, paraffin-embedded tissue samples obtained during potentially radical gastrectomy from 58 patients with primary gastric adenocarcinoma. The representative tissue blocks from each tumor were used for the immunohistochemical assay and examined by two pathologists independently. MVD was counted in five tumor areas of the most intensive neovascularization (x 200 field by light microscopy) and the mean counts were recorded. The mean MVD (CD34 expression value ± SD) in this study was 43,15 ± 19,8 per x 200 field. The study demonstrated the statistically significant correlation between MVD and two main histological parameters: tumor grading (p < 0.001) and tumor histological type according to Lauren’s classification (p<0.05). In well - and moderately - differentiated tumors (G1/2) MVD was significantly lower in comparison to the group of poorly differentiated cancer - G3 (mean value: 31,62 vs. 49,89). MVD was higher in diffuse type of gastric cancer comparing to intestinal type (50.05 ± 19,03 vs. 39.17 ±20,09). However, the authors failed to find a significant correlation between MVD and other investigated histopathological features in malignant gastric tumors. The close relationship between CD34 immunostaining, gastric cancer tumor vascularity and main histological parameters was shown in this study. It can be stated that analysis of expression of angiogenesis in gastric cancer may be helpful for better estimation of hematogenous recurrence and the selection of the group of patients for adjuvant antiangiogenic treatment.

Molecular analysis of WWOX expression correlation with proliferation and apoptosis in glioblastoma multiforme

Glioblastoma multiforme is the most common type of primary brain tumor in adults. WWOX is a tumor suppressor gene involved in carcinogenesis and cancer progression in many different neoplasms. Reduced WWOX expression is associated with more aggressive phenotype and poor patient outcome in several cancers. We investigated alternations of WWOX expression and its correlation with proliferation, apoptosis and signal trafficking in 67 glioblastoma multiforme specimens. Moreover, we examined the level of WWOX LOH and methylation status in WWOX promoter region. Our results suggest that loss of heterozygosity (relatively frequent in glioblastoma multiforme) along with promoter methylation may decrease the expression of this tumor suppressor gene. Our experiment revealed positive correlations between WWOX and Bcl2 and between WWOX and Ki67. We also confirmed that WWOX is positively correlated with ErbB4 signaling pathway in glioblastoma multiforme.

High incidence of MGMT promoter methylation in primary glioblastomas without correlation with TP53 gene mutations

O(6)-methylguanine DNA methyltransferase (MGMT) reduces cytotoxicity of therapeutic or environmental alkylating agents. MGMT promoter methylation has been associated with TP53 G: C to A:T transition mutations in various types of cancers, and with poor prognosis in patients who did not receive chemotherapy. Mutations of TP53 are more frequent in secondary than in primary glioblastoma, thus the expected MGMT promoter methylation was low in primary glioblastoma. Glioblastoma patients with MGMT promoter methylation showed better response to chemotherapy based on alkylating agents and longer survival than patients without MGMT methylation. We examined 32 primary glioblastomas, treated with radiotherapy and surgery, for TP53 mutation by direct sequencing and MGMT promoter methylation by methylation-specific PCR. MGMT promoter methylation and TP53 mutations were detected in 72% and 31% of primary glioblastoma, respectively. Although not statistically significant, the frequency of TP53 G:C to A:T mutations were higher in cases with (26%) than without (11%) MGMT promoter methylation (p=0.376). MGMT promoter methylation had no impact on patient survival. Our data indicate that MGMT promoter methylation occurs frequently in primary glioblastoma, but does not lead to G:C to A:T TP53 mutations, has no independent prognostic value and is not a predictive marker unless glioblastoma patients are treated with chemotherapy.

CD38 Gene Polymorphisms Contribute to Genetic Susceptibility to B-Cell Chronic Lymphocytic Leukemia: Evidence from Two Case-Control Studies in Polish Caucasians

Given the recent findings on the importance of CD38 signaling in the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL), we hypothesized that single nucleotide polymorphisms (SNP) in the CD38 gene may be related to B-CLL risk. We evaluated two potentially functional CD38 SNPs, intronic rs6449182 (184C>G) and missense rs1800561 (418C>T, Arg140Trp) in two hospital-based case-control studies (study A and validation study B). Genotyping was done using PCR-based assays in a total of 460 Polish Caucasian patients with B-CLL and 503 age-matched and gender-matched controls. We found that frequencies of both variant alleles (rs6449182 G and rs1800561 T) were significantly higher in B-CLL. In study A, logistic regression analysis revealed an association between B-CLL and genotypes: rs6449182 CG [odds ratio (OR), 3.57; 95% confidence interval (95% CI), 2.4-5.3], rs6449182 GG (OR, 5.2; 95% CI, 2.36-11.5), and rs1800561 CT (OR, 6.72; 95% CI, 1.5-30.1), although no homozygous rs1800561 TT genotype was detected in either study. These results were confirmed in study B, which showed an association between B-CLL and genotypes rs6449182 CG (OR, 4.00; 95% CI, 2.7-6.0), rs6449182 GG (OR, 12.84; 95% CI, 4.3-38.7), and rs1800561 CT (OR, 10.12; 95% CI, 1.3-81.6), and in the combined analysis of both studies. We also observed that rs6449182 G carriers had more advanced clinical stage (P = 0.002) and tended to be younger at diagnosis (P = 0.056). Furthermore, we found higher CD38 transcript levels and higher proportions of CD38-positive cells in carriers of rs6449182 G and rs1800561 T alleles (P < 0.05 for all comparisons). In conclusion, our data show that CD38 SNPs may affect CD38 expression and contribute to the increased risk of B-CLL carcinogenesis. (Cancer Epidemiol Biomarkers Prev 2009;18(3):945–53)

Human Leukocyte Antigen-G Polymorphisms Influence the Clinical Outcome in Diffuse Large B-Cell Lymphoma

The role of HLA‐G is extensively studied in cancer due to its inhibition of the immune response. Several polymorphisms in the HLA‐G gene have been reported to significantly affect its expression. We, therefore, investigated whether functionally relevant HLA‐G polymorphisms, HLA‐G‐725C/G/T, and HLA‐G 14‐base pair, have any influence on the susceptibility to diffuse large B‐cell lymphoma (DLBCL) and its clinical course. The polymorphisms were genotyped in 207 previously untreated patients with DLBCL and 150 unrelated controls. A significant difference in genotype distribution of HLA‐G polymorphic genotypes between the patients and controls was found. The frequencies of the HLA‐G−725GG or the HLA‐G−725GC genotype were lower, and those of the HLA‐G ins/ins genotype were higher in the patients compared with the controls. Patients carrying the HLA‐G‐725CC genotype presented a higher probability of overall survival (OS) than subjects with other genotype combinations of HLA‐G‐725C/G/T (P = 0.003). The homozygous HLA‐G del/del had a lower probability of OS than subjects carrying the HLA‐G deletion/insertion (del/ins) or the HLA‐G ins/ins genotype (P = 0.009). Two HLA‐G genotype‐based risk groups were defined according to the genotype distribution. The high‐risk (HR) group presented a shorter OS than low‐risk (LR) patients (P = 0.001). In a multivariate analysis adjusted for International Prognostic Index (IPI) factors, both the intermediate high/high IPI‐risk group (P < 0.0001) and the HR genotype group (P = 0.004) independently increased the risk of death. This is the first study indicating an important role of HLA‐G polymorphisms for the clinical course of DLBCL. The potential influence of HLA‐G polymorphisms on the susceptibility to DLBCL thus deserves further study.

Screening for EGFR Amplifications with a Novel Method and Their Significance for the Outcome of Glioblastoma Patients

Glioblastoma is a highly aggressive tumour of the central nervous system, characterised by poor prognosis irrespective of the applied treatment. The aim of our study was to analyse whether the molecular markers of glioblastoma (i.e. TP53 and IDH1 mutations, CDKN2A deletion, EGFR amplification, chromosome 7 polysomy and EGFRvIII expression) could be associated with distinct prognosis and/or response to the therapy. Moreover, we describe a method which allows for a reliable, as well as time- and cost-effective, screening for EGFR amplification and chromosome 7 polysomy with quantitative Real-Time PCR at DNA level. In the clinical data, only the patient’s age had prognostic significance (continuous: HR = 1.04; p<0.01). At the molecular level, EGFRvIII expression was associated with a better prognosis (HR = 0.37; p = 0.04). Intriguingly, EGFR amplification was associated with a worse outcome in younger patients (HR = 3.75; p<0.01) and in patients treated with radiotherapy (HR = 2.71; p = 0.03). We did not observe any difference between the patients with the amplification treated with radiotherapy and the patients without such a treatment. Next, EGFR amplification was related to a better prognosis in combination with the homozygous CDKN2A deletion (HR = 0.12; p = 0.01), but to a poorer prognosis in combination with chromosome 7 polysomy (HR = 14.88; p = 0.01). Importantly, the results emphasise the necessity to distinguish both mechanisms of the increased EGFR gene copy number (amplification and polysomy). To conclude, although the data presented here require validation in different groups of patients, they strongly advocate the consideration of the patient’s tumour molecular characteristics in the selection of the therapy.

Poor prognosis of Hodgkin variant of Richter transformation in chronic lymphocytic leukemia treated with cladribine

It is with great interest that we read the comprehensive analysis of all 86 cases of Hodgkin variant of Richter transformation (Hodgkin-RT) in chronic lymphocytic leukemia (CLL) reported in the world literature during the last 35 years (Bockorny et al, 2012). A very interesting finding from this study was that patients in whom CLL had been treated with fludarabine had significantly shorter survival after transformation compared to the ones not treated with this agent (0·7 vs. 2·1 years, P = 0·019) (Bockorny et al, 2012). Very poor prognosis in the fludarabine-treated patients is in contrast with the general conviction that Hodgkin-RT outcome is superior to the outcome of more common Richter transformation to diffuse large B-cell lymphoma (DLBCL-RT) (Brecher & Banks, 1990). The authors hypothesized that shorter survival may reflect a more aggressive course of Hodgkin-RT in patients who had received fludarabine, and possibly also other purine nucleoside analogs (PNAs) (Bockorny et al, 2012). Besides fludarabine, PNAs used in CLL include cladribine and pentostatin (Karlsson et al, 2002; Kay et al, 2010). Unlike the practice of most European countries, in Poland cladribine is often used in CLL and has been included in large clinical trials of the Polish Adult Leukemia Study Group (PALG) coordinated by our centre (Robak et al, 2006). In order to evaluate the clinical course of HodgkinRT in patients treated previously with cladribine we performed a single-centre retrospective analysis to compare Hodgkin-RT incidence, characteristics and outcome with those of DLBCL-RT. We searched electronic databases of the Departments of Haematology and Pathology of the Medical University of Lodz to identify patients who developed biopsyor fine-needle aspiration–proven RT between January 2000 and November 2011. Hospital records and ambulatory charts were reviewed to determine clinical, laboratory and pathological features, treatment and outcome of CLL and RT. Hodgkin-RT and DLBCL-RT characteristics were compared by Fisher exact test or Mann–Whitney test. Survival after transformation was plotted using the Kaplan-Meier method and comparisons made by the log-rank test. Statistical analysis was performed using Predictive Analytics SoftWare (PASW) Statistics 18 (SPSS Inc., Chicago, IL, USA); P < 0·05 was considered significant. Among 786 patients with CLL admitted to our centre since January 2000, 40 (5·1%) patients developed RT including 33 (4·2%) DLBCL-RT, six (0·8%) Hodgkin-RT and one plasmablastic lymphoma. There were 23 male and 17 female patients with a median age of 67·2 years (range 35·8–84·9). RT was diagnosed after a median of 2·6 years (range 0–12·3) from CLL diagnosis. Median survival after transformation was 1·1 years, 95% confidence interval (95%CI) 0·4– 1·8 years. The patients with Hodgkin-RT comprised five men and one woman with a median age of 70·6 years (range 55·8– 76·3). One patient was reported earlier (Robak et al, 2003). All patients received cladribine-based regimens before transformation, namely five patients received CC (cladribine and cyclophosphamide) and one patient received RCC (CC plus rituximab). Histological subtypes of Hodgkin lymphoma (HL) were mixed cellularity (four patients), nodular sclerosis (one patient), and not determined in one patient. All patients presented with extensive disease involvement (Ann Arbor stage IIIB in three patients and IVB in three patients). Moreover, concomitant progression of CLL was documented in two patients. Treatment was mostly HL-type therapy including ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine) in three patients (in one combined with radiation) and COPP (cyclophosphamide, oncovin, procarbazine and prednisone) in one patient, while one patient received COP (cyclophosphamide, oncovin and prednisone) and one patient with poor performance status received steroids and vincristine. Two partial remissions were achieved, while four patients did not respond to chemotherapy. During the follow-up period, five patients died of disease progression at 0·5, 2, 4, 11 and 13 months from transformation, whilst one patient remained alive after 7 months of observation (Fig 1B). The comparison of patients with DLBCL-RT and Hodgkin RT is shown in Table I. Presenting characteristics and time-to-transformation were comparable in both types of RT. However, a trend suggesting more common preceding treatment with PNA in Hodgkin-RT was observed (P = 0·063). Most interestingly, we found significantly shorter survival after transformation in Hodgkin-RT compared to DLBCL-RT (P = 0·019) (Table I, Fig 1A). In contrast, no survival difference was seen between DLBCL-RT patients who had or had not received PNAs (P = 0·77), (Fig 1B). The results of this study indicate that Hodgkin-RT in cladribine-treated CLL patients is characterized by very short survival, even compared to DLBCL-RT. This is in line with the very poor prognosis of Hodgkin-RT patients treated previously with fludarabine (Bockorny et al, 2012), and suggests Correspondence