Prognostic value of apoptosis inducing factor in uveal melanoma.

In malignant tumors including uveal melanoma there is a continuous effort in search for additional and relevant factors with predictive value and possible therapeutic indications. In the present work we evaluated the 5-year mortality in a group of patients with surgically treated uveal melanoma and its relation to selected demographic, clinical and histopathological parameters, including the expression of apoptosis inducing factor (AIF) in the neoplastic tissue.We analyzed retrospectively the clinical data of patients with uveal melanoma treated surgically (enucleation, endoresection, exenteration) in the period from 2001 to 2007 (n=54). Immunohistochemical detection of AIF expression in formalin fixed and in paraffin embedded tissue samples was evaluated semiquantitatively, intensity and percentage multiplicative Quick Score (QS) was calculated and compared between patients with over 5 year (n=32) and less than 5 year (n=22) survival. In the analyzed group of 54 patients the 5 year mortality was 41 %. We confirmed the negative prognostic significance of some of the known prognostic factors as the tumor size and volume, T3 and T4 stage in the TNM classification and the mixed histological type of the tumor. Immunohistochemistry performed on 49 melanoma specimens showed AIF cytoplasmic positivity, no nuclear translocation was detected. The cut-off value of AIF expression QS ≥ 4 (18) in tumor cells separated the 5 year survival of patients (P = 0.018), odds ratio 5.2 (1.24 - 21.73). Moderate and strong expression of AIF in tumor cells also correlated with less favorable prognosis. Confocal microscopy proved colocalization of AIF with mitochondrial marker in neoplastic cells.The prognosis of patients with uveal melanoma can be more accurate with inclusion of immunohistochemical detection of AIF expression. Increased expression of the AIF protein appears as a new negative prognostic factor predicting the 5 year survival.

Effect of melatonin on blood pressure and nitric oxide generation in rats with metabolic syndrome.

Melatonin, a multitasking indolamine, seems to be involved in a variety of physiological and metabolic processes via both receptor-mediated and receptor-independent mechanisms. The aim of our study was to find out whether melatonin can affect blood pressure (BP), nitric oxide synthase (NOS) activity, eNOS and nNOS protein expressions in rats with metabolic syndrome (SHR/cp). Rats were divided into four groups: 6-week-old male WKY andSHR/cp and age-matched WKY and SHR/cp treated with melatonin (10 mg/kg/day) for 3 weeks. BP was measured by tail-cuff plethysmography. NOS activity, eNOS and nNOS protein expressions were determined in the heart, aorta, brain cortex and cerebellum. MT(1) receptors were analyzed in the brain cortex and cerebellum. In SHR/cp rats, BP was decreased after melatonin treatment. In the same group, melatonin did not affect NOS activity and eNOS protein expression in the heart and aorta, while it increased both parameters in the brain cortex and cerebellum. Interestingly, melatonin elevated MT1 protein expression in the cerebellum. Neuronal NOS protein expression was not changed within the groups. In conclusion, increased NOS activity/eNOS upregulation in particular brain regions may contribute partially to BP decrease in SHR/cp rats after melatonin treatment. Participation of MT(1) receptors in this melatonin action may be supposed.

Effect of deuterium-depleted water on selected cardiometabolic parameters in fructose-treated rats.

Deuterium-depleted water (DDW) has a lower concentration of deuterium than occurs naturally (less than 145 ppm). While effects of DDW on cancer started to be intensively studied, the effects on cardiovascular system are completely unknown. Thus, we aimed to analyze the effects of DDW (55+/-5 ppm) administration to 12-week-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) treated with 15 % fructose for 6 weeks. Blood pressure (BP) and selected biochemical parameters were measured together with determination of nitric oxide synthase (NOS) activity and iNOS and eNOS protein expressions in the left ventricle (LV) and aorta. Neither DDW nor fructose had any significant effect on BP in both strains. DDW treatment decreased total cholesterol and triglyceride levels in WKY, but it was not able to prevent increase in the same parameters elevated due to fructose treatment in SHR. Both fructose and DDW increased insulin level in WKY. Fructose did not affect NOS activity either in WKY or SHR. DDW increased NOS activity in LV of both WKY and SHR, while it decreased NOS activity and iNOS expression in the aorta of SHR with or without fructose treatment. In conclusion, DDW treatment significantly modified biochemical parameters in WKY together with NOS activity elevation in the heart. On the other hand, it did not affect biochemical parameters in SHR, but decreased NOS activity elevated due to iNOS upregulation in the aorta.

Early Versus Delayed Autologous Stem Cell Transplantation and Interferon Maintenance in Multiple Myeloma: Single-Center Experience of 18 Years.

BACKGROUND: Autologous stem cell transplantation (ASCT) has become the mainstay of 1st-line treatment in younger patients with multiple myeloma (MM), but statistical confirmation of its superiority over other therapies, especially in the era of novel agents, is still lacking. METHODS: We reviewed the results of all 548 myeloma ASCTs performed in our institute over the past 18 years. RESULTS: More than one-half of the patients had access to novel agents before their transplantations. Although the age of the transplanted patients increased significantly over the years, treatment-related mortality (TRM) was remarkably low, especially in 1st-line transplanted patients (100-day TRM, 0.3%). The median overall survival (OS) of the entire cohort was 98.4 months. Patients transplanted within 12 months from the start of their therapy had significantly better responses than those having delayed ASCT (complete response rate, 58.1% vs 46.8%; P = .016) and significant post-ASCT progression-free survival (PFS) benefit (30.2 [26.1-34.3] mo vs 23.3 [16.8-29.8] mo; P = .036), but we found no significant overall survival difference. The results were similar in patients treated with or without novel agents before ASCT. During a period of time, interferon maintenance was our standard approach to post-ASCT maintenance. Our analysis showed not only a significant PFS advantage with interferon, but also a highly significant overall survival benefit (150.4 [105.1-195.8] mo vs 86.1 [72.5-99.7] mo; P = .003). CONCLUSIONS: Our findings demonstrate that delayed ASCT can be feasible in selected patients.

The potential role of HLA-DRB1*11 in the development and outcome of haematopoietic stem cell transplantation-associated thrombotic microangiopathy.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a serious complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT) with high mortality rate. We retrospectively studied the frequency, clinical and genetic associations and prognostic effect of TA-TMA, in a total of 425 consecutive adult patients, who underwent allo-HSCT for a malignant haematological condition between 2007 and 2013 at our single centre. TA-TMA developed in 19% of the patients. Unrelated donor type (P<0.001), acute GvHD grades II-IV (P<0.001), myeloablative conditioning regimens (P=0.003), tacrolimus-based GvHD prophylaxis (P=0.003), CMV infection (P=0.003) and carriership for HLA-DRB1*11 (P=0.034) were associated with the development of TA-TMA. Survival was adversely affected by the presence of TA-TMA (P<0.001). Among patients with TA-TMA, the outcome of HLA-DRB1*11 carriers was significantly better compared with non-carriers (P=0.003). As a new finding, our observations suggest that the presence of HLA-DRB1*11 antigen contributes to the development of TA-TMA and affects the outcome.

Biliary atresia - a new derivative method?

BACKGROUND: Biliary atresia is a progressive cholangiopathy in neonates of unknown origin. Surgical intervention - Kasai portoenterostomy - is the only treatment possible. However, only liver transplantation can be considered a definitive solution even in cases of favourable post-operative course, i.e., after bile passage has been recreated. OBJECTIVES: The authors set out to identify ways to optimize the operative treatment in biliary atresia patients. In particular, their objective was to identify a suitable bile derivation area outside porta hepatis, thereby simplifying the surgical procedure of the following liver transplantation. METHODS: The research was conducted from 2006 to 2010 on a set of 30 corrosive casts prepared by the Institute of Anatomy, Medical Faculty, Comenius University Bratislava. The research consisted of an analysis of individual tubular structures of the liver parenchyma, particularly the bile vessels. The authors explored the latter's position and branching out, as well as their relationships in different segments, and their availability in surgical exploration. RESULTS: The analysis of the corrosive casts resulted in selecting an area in liver segments II and III suitable for creating a new type of hepatoenterostomy. The area is sufficiently distant from porta hepatis, conveniently accessible to the surgeon, and has a broad bile duct branch. This type of anastomosis would preserve the anatomic situation in the porta hepatis area intact for the following transplantation. CONCLUSION: While the results of the experiment warrant some optimism, authors will have to wait until this type of derivation operation is applicable in practice. The authors continue their research by performing anastomosis in an animal model (Fig. 5, Ref. 18)

Protective effect of captopril, olmesartan, melatonin and compound 21 on doxorubicin-induced nephrotoxicity in rats.

Chronic kidney disease (CKD) represents a serious public health problem with increasing prevalence and novel approaches to renal protection are continuously under investigation. The aim of this study was to compare the effect of melatonin and angiotensin II type 2 receptor agonist compound 21 (C21) to angiotensin converting enzyme inhibitor captopril and angiotensin II type 1 receptor blocker olmesartan on animal model of doxorubicin nephrotoxicity. Six groups of 3-month-old male Wistar rats (12 per group) were treated for four weeks. The first group served as a control. The remaining groups were injected with a single dose of doxorubicin (5 mg/kg i.v.) at the same day as administration of either vehicle or captopril (100 mg/kg/day) or olmesartan (10 mg/kg/day) or melatonin (10 mg/kg/day) or C21 (0.3 mg/kg/day) was initiated. After four week treatment, the blood pressure and the level of oxidative stress were enhanced along with reduced glomerular density and increased glomerular size. Captopril, olmesartan and melatonin prevented the doxorubicin-induced increase in systolic blood pressure. All four substances significantly diminished the level of oxidative burden and prevented the reduction of glomerular density and modestly prevented the increase of glomerular size. We conclude that captopril, olmesartan, melatonin and C21 exerted a similar level of renoprotective effects in doxorubicin-induced nephrotoxicity.

Plant SR proteins and their functions.

SR proteins are a family of splicing factors important for splice site recognition and spliceosome assembly. Their ability to bind to RNA and to interact with proteins as well identifies them as important players in splice site choice and alternative splicing. Plants possess twice as many SR proteins as animals, and some of the subfamilies are plant specific. Arabidopsis SR proteins are involved in different aspects of plant growth and development as well as in responses to environmental cues. The plant-specific subfamilies have been shown to be regulated by alternative splicing events, which are highly conserved in evolution. The tight regulation of splicing factors by alternative splicing might allow coordinated responses of their target genes.

A plethora of plant serine/arginine-rich proteins: redundancy or evolution of novel gene functions?

Precursor-mRNA (pre-mRNA) processing is an important step in gene expression and its regulation leads to the expansion of the gene product repertoire. SR (serine-arginine)-rich proteins are key players in intron recognition and spliceosome assembly and significantly contribute to the alternative splicing process. Due to several duplication events, at least 19 SR proteins are present in the Arabidopsis genome, which is almost twice as many as in humans. They fall into seven different subfamilies, three of them homologous with metazoan splicing factors, whereas the other four seem to be specific for plants. The current results show that most of the duplicated genes have different spatiotemporal expression patterns indicating functional diversification. Interestingly, most of the SR protein genes are alternatively spliced and in some cases this process was shown to be under developmental and/or environmental control. This might greatly influence gene expression of target genes as also exemplified by ectopic expression studies of particular SR proteins.

First Report of Soybean dwarf virus on Soybean in Wisconsin.

Soybean dwarf virus (SbDV) causes widespread economic losses on soybean (Glycine max (L.) Merr.) in Japan (4), and has been reported on soybean in Virginia (2), in various legumes in the southeastern United States (1), and in peas in California (3). During late July and early August of 2003, soybean plants in Wisconsin were surveyed for SbDV. In 286 soybean fields at the R2-R4 growth stage, the uppermost fully unfurled leaf was collected from 10 plants at each of five sites. Samples were collected at random without regard to symptoms. SbDV symptom information was not recorded. Samples were stored on ice until frozen at -80°C. Five fields in four Wisconsin counties (Columbia, Lafayette, Sauk, and Waushara) tested positive for SbDV using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). DAS-ELISA testing was conducted with reagents from Agdia, Inc (Elkhart, IN) following the manufacturer's protocol. Absorbance was read at 405 nm with a Stat Fax 2100 microplate reader (Awareness Technology, Inc., Palm City, FL) or visually evaluated. DAS-ELISA did not discriminate between strains of SbDV. The presence of SbDV was confirmed, and strain identity was inferred as dwarfing strain using reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from homogenized leaf tissue, reverse transcribed, and amplified with the SuperScript One Step RT-PCR System (Invitrogen, Carlsbad, CA) and SbDV-specific primers (5'-CTGCTTCTGGTGATTACACTGCCG-3' and 5'-CGCTTTCATTTAACGYCATCAAAGGG-3'). Size of the RT-PCR products (110 bp) was consistent with the dwarfing strain, SbDV-D. All locations that tested positive for SbDV showed soybean aphids, Aphis glycines Matsumura (Homoptera: Aphididae), on 100% of soybean plants. Several aphid species have been reported to vector SbDV, but at this time, vector relations in the Wisconsin infections are unknown. To our knowledge, this is the first report of SbDV infecting soybean in Wisconsin. References: (1) V. D. Damsteegt et al. Plant Dis. 79:48, 1995. (2) A. Fayad et al. Phytopathology (Abstr.) 90(Suppl.):S132, 2000. (3) G. R. Johnstone et al. Phytopathology (Abstr.) 74:795(A43), 1984. (4) T. Tamada et al. Ann. Phytopathol. Soc. Jpn. 35:282, 1969.

Molecular aspects of the ribosomal peptidyl transferase.

The proteins in a living cell are synthesized on a large bipartite ribonucleoprotein complex termed the ribosome. The peptidyl transferase, which polymerizes amino acids to yield peptides, is localized on the large subunit. Biochemical investigations over the past 35 years have led to the hypothesis that rRNA has a major role in all ribosomal functions. The recent high resolution X-ray structures of the ribosomal subunits clearly demonstrated that peptidyl transfer is an RNA-mediated process. As all ribosomal activities are dependent on bivalent metal ions, as is the case for most ribozymes, we investigated metal-ion-binding sites in rRNA by metal-ion-cleavage reactions. Some cleavage sites are near active sites and are evolutionarily highly conserved. The structure of the active site is flexible and undergoes changes during translocation and activation of the ribosome. Using modified P-site substrates, we showed that the 2'-OH group of the terminal adenosine is important for peptidyl transfer. These substrates were also used to investigate the metal ion dependency of the peptidyl transferase reaction.

Acute myeloid leukaemia of donor cell origin developing 5 years after allogeneic bone marrow transplantation for chronic myeloid leukaemia.

We report the case of a male patient with Ph-positive CML who developed AML 5 years after allogeneic BMT. Clinically, the AML seemed to develop on the basis of a myelodysplasia. The myeloid origin of blasts has been proven by immunophenotyping. The variable number of tandem repeats (VNTRs) and short tandem repeat (STR) showed donor-type haemopoiesis. The interphase FISH showed the XX genotype directly in the morphologically identifiable blasts and in the CD34-positive sorted bone marrow cells. This proved the new leukaemia to be of donor origin. The necessity of using multiple techniques and the advantage of combined immunophenotyping and FISH methods in this case is emphasized.

Autologous hematopoietic stem cell transplantation in chronic myeloid leukemia with different clinical stages.

Seven patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) were treated with an ICE-based regimen plus G-CSF with the aim of mobilizing and collecting Ph-negative peripheral stem cells in the setting of an autologous transplant program. Five patients had CML in the first chronic phase and 2 in the accelerated phase. All patients had been previously treated with interferon-alpha. Median value and ranges for harvested mononuclear cells, CD34+ cells and CFU-GM, respectively: 5.65 x 10(8)/kg (2.61-11.38); 1.48 x 10(6)/kg (0.216-3.5), and 3.43 x 10(4)/kg (0.243-11.6). FISH was the only useful method for detection of minimal residual disease on apheresis product showing <5% t(9;22) positive cells in 2 cases and <10% positive cells in 4 other cases. Four of seven autologous grafts have been transplanted to date. Busulfan conditioning was used in 1 case and TBI/Cy conditioning in 3 other cases. All patients are alive and well following transplantation and are on interferon-alpha therapy.

Changes of salivary amylase in serum and parotid gland during pharmacological and physiological stimulation.

Although serum amylase level is an important diagnostic factor in certain salivary and pancreatic diseases, little information is available regarding the mechanism by which parotid amylase reaches the circulatory system. The present study was carried out to investigate the relationship between parotid isoamylase concentrations in blood serum and in parotid tissue in response to various stimuli. Wistar rats were fed with standard laboratory rodent chow; water was supplied ad libitum. In the first experiment, after a 16-h fasting, rats received either 5 mg/kg pilocarpine or saline (control). In the second study, after fasting, half of the rats were fed for 1 h, the other half received no food. In the third experiment, the changes in serum and tissue enzyme levels were monitored in freely fed animals during the peak-food intake phase, the first 2 h of the dark period. Amylase concentration was determined by using starch as a substrate. Pancreatic and parotid isoamylase levels in serum were separated by gel-electrophoresis utilizing differences in ionic properties of the isoenzymes. As expected, pilocarpine strongly stimulated tissue amylase discharge and serum amylase elevation. Similar, but less pronounced changes were observed not only during refeeding of fasted animals, but also in nonfasted rats during their peak-feeding period. Our data suggest that pharmacological stimulation, such as with pilocarpine or feeding in fasted state, as well as a mild stimulation of parotid function by spontaneous food intake during nonfasted state results in a decrease in parotid tissue amylase activity and a proportional increase in serum levels of parotid isoamylase.

A conformational change in the ribosomal peptidyl transferase center upon active/inactive transition.

The ribosome is a dynamic particle that undergoes many structural changes during translation. We show through chemical probing with dimethyl sulfate (DMS) that conformational changes occur at several nucleotides in the peptidyl transferase center upon alterations in pH, temperature, and monovalent ion concentration, consistent with observations made by Elson and coworkers over 30 years ago. Moreover, we have found that the pH-dependent DMS reactivity of A2451 in the center of the 23S rRNA peptidyl transferase region, ascribed to a perturbed pKa of this base, occurs only in inactive 50S and 70S ribosomes. The degree of DMS reactivity of this base in the inactive ribosomes depends on both the identity and amount of monovalent ion present. Furthermore, G2447, a residue proposed to be critical for the hypothesized pKa perturbation, is not essential for the conditional DMS reactivity at A2451. Given that the pH-dependent change in DMS reactivity at A2451 occurs only in inactive ribosomes, and that this DMS reactivity can increase with increasing salt (independently of pH), we conclude that this observation cannot be used as supporting evidence for a recently proposed model of acid/base catalyzed ribosomal transpeptidation.

Remarkably reduced transplant-related complications by dibromomannitol non-myeloablative conditioning before allogeneic bone marrow transplantation in chronic myeloid leukemia.

A non-myeloablative conditioning protocol containing dibromomannitol (DBM/cytosine arabinoside/cyclophosphamide) has been applied to 36 chronic myeloid leukemia (CML) patients followed by bone marrow transplantation (BMT) from sibling donors. Risk factors include: accelerated phase (10 patients), older age (17 patients over >40 years) and long interval between diagnosis and BMT (27 months on average). Severe mucositis did not occur. Venoocclusive liver disease was absent. Infectious complications were rare. Although grade II-IV acute graft-versus-host disease (GVHD) was present in 9 (25%) cases, there were only 2 serious (III-IV) ones. Chronic GVHD occurred in 25 (69%) cases, preceded by acute GVHD in 9 of the 25 affected patients. Early hematological relapse, 7-29 weeks after BMT, developed in 6 patients (17.6%). No relapse was noted in the completely chimeric patients, however molecular genetic residual disease was observed in 6 patients, in most of them after transient short-term mixed chimeric state. Overall actual survival rate is 83.3% for the 36 cases, and leukemia-free survival is 72.2% for the 34 engrafted patients.

[Restricted antibody diversity after bone marrow transplantation--homogeneous immunoglobulins]. / A csontveló-átültetés után kialakuló korlátozott ellenanyagkészlet--homogén immunglobulinok.

After bone marrow transplantation, a prolonged dysregulation of humoral immunity, including restricted electrophoretic heterogeneity of serum immunoglobulins and the appearance of homogeneous immunoglobulin components, can be observed. The current study was undertaken to characterize further and define the posttransplantational incidence of monoclonal and oligoclonal immunoglobulins, as well as the clinical and laboratory correlations of these phenomena. For this purpose, serial serum protein (IgM, IgG, IgA and CRP) quantification, electrophoresis and immunofixation were performed on 29 patients undergoing allogeneic bone marrow transplantation for chronic myeloid leukemia. 23 out of the 29 patients developed transient oligoclonal and/or monoclonal gammopathies that appeared between 20 and 1750 posttransplantational days. No correlation, however, between the development of graft versus host disease, EBV or CMV infections, or any other symptoms and development of homogeneous immunoglobulin components was seen. Therefore, the development of oligoclonal and monoclonal gammopathies after bone marrow transplantation may be an ubiquitous finding reflecting the inadequacy, i.e. oligoclonality of the recovering B-cell system.

[Regeneration of the immune system after bone marrow transplantation]. / Az immunrendszer regenerációja csontvelö-átültetés után.

After haematopoietic stem cell transplantation, reconstitution of bone marrow consists of two distinct phenomena, numerical recovery of bone marrow cellular elements on the one hand and functional recovery of cellular interactions on the other. Immune reactivity during the first month postgrafting is extremely low. Cytotoxic and phagocytic functions usually recover by day 100, while more specialized and cooperative functions of T and B cells remain impaired up to one year or more postgrafting. Regeneration of total CD4+ T cell number in adult (and especially in elderly) transplant recipients is severely limited and occurs largely by peripheral expansion of mature CD4+ T cells. While restoration of total CD8+ T cell number is commonly seen in adults, potentially important alterations in the subset composition of CD8+ populations remain. Contracted T cell repertoires for CD4+ and CD8+ T cells are consistently found in adults after T cell regeneration. This suggests that thymic function is frequently limiting in adults and that thymic-independent pathways are insufficient for restoring host immunocompetence. Although there are similarities in immune reconstitution after alllo- and autologous haematopoietic stem cell transplantations, allogeneic transplantation involves graft versus host disease and the use of immunosuppressive therapy to control it, both of which further interfere in the early developmental stages of immune reconstitution.

[Autologous hematopoietic stem cell transplantation in chronic myeloid leukaemia with different clinical stages] / Autológ hemopoetikus ôssejt-transzplantáció eredményei chronicus myeloid leukaemiában.

For most chronic myeloid leukaemia patients the option of a potentially curative allogeneic stem cell transplantation is not available because of age or lack of donor. Alternative therapy with interferon-alpha appears to prolong survival but is probably not curative. The aim of the study is to analyse the clinical results of the first Hungarian autologous transplantations in CML. METHODS: Seven patients were treated with ICE-based regimen plus G-CSF with the aim of mobilising and collecting Ph-negative peripheral stem cells in the setting of autologous transplant program. Five patients had CML in first chronic phase and two in accelerated phase. All patients have been previously treated with interferon-alpha. RESULTS: Median value and ranges for harvested mononuclear cells, CD34(+) cells and CFU-GM were: 5.65x10(8)/kg (2.61-11.38), 1.48x10(6)/kg (0.216-3.5) and 3.43x10(4)/kg (0.243-11.6), respectively. Four out of seven autologous grafts have been transplanted. Busulfan conditioning was used in one case and TBI/Cy conditioning in three patients. All patients are alive and well post-transplant being on interferon-alpha therapy. CONCLUSIONS: Based on the clinical advantages of autologous transplantation including long-term chronic phase, achievement of second chronic phase and improved response to interferon-alpha therapy, the procedure can offer an alternative treatment in CML in lack of HLA-identical donor.