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Background The phase 3 KATHERINE trial demonstrated significantly improved invasive disease–free survival with adjuvant trastuzumab emtansine (T‐DM1) versus trastuzumab in patients with HER2‐positive early breast cancer and residual invasive disease after neoadjuvant chemotherapy plus HER2‐targeted therapy. Methods Patients who received taxane‐ and trastuzumab‐containing neoadjuvant therapy (with/without anthracyclines) and had residual invasive disease (breast and/or axillary nodes) at surgery were randomly assigned to 14 cycles of adjuvant T‐DM1 (3.6 mg/kg intravenously every 3 weeks) or trastuzumab (6 mg/kg intravenously every 3 weeks). The European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire–Core 30 (QLQ‐C30) and breast cancer module (QLQ‐BR23) were completed at screening, at day 1 of cycles 5 and 11, within 30 days after study drug completion, and at 6‐ and 12‐month follow‐up visits. Results Of patients who were randomly assigned to T‐DM1 (n = 743) and trastuzumab (n = 743), 612 (82%) and 640 (86%), respectively, had valid baseline and ≥1 postbaseline assessments. No clinically meaningful changes (≥10 points) from baseline in mean QLQ‐C30 and QLQ‐BR23 scores occurred in either arm. More patients receiving T‐DM1 reported clinically meaningful deterioration at any assessment point in role functioning (49% vs 41%), appetite loss (38% vs 28%), constipation (47% vs 38%), fatigue (66% vs 60%), nausea/vomiting (39% vs 30%), and systemic therapy side effects (49% vs 36%). These differences were no longer apparent at the 6‐month follow‐up assessment, except for role functioning (23% vs 16%). Conclusion These data suggest that health‐related quality of life was generally maintained in both study arms over the course of treatment., Patient‐reported outcomes are reported from the randomized, phase 3 KATHERINE trial, which demonstrated significantly improved invasive disease–free survival with adjuvant T‐DM1 compared with trastuzumab in patients who had residual invasive disease following neoadjuvant chemotherapy plus HER2‐targeted therapy. Patients who are treated with T‐DM1 have a greater incidence of any grade and grade ≥3 adverse events compared with trastuzumab‐treated patients; however, these adverse events appear to have a minimal impact on patient‐reported quality of life.

Glioblastoma multiforme (GBM) is an aggressive primary brain tumor, with poor prognosis. Major obstacles hampering effective therapeutic response in GBM are tumor heterogeneity, high infiltration of immunosuppressive myeloid cells, and the presence of the blood-brain barrier. The C-X-C Motif Chemokine Ligand 12/ C-X-C Motif Chemokine Receptor 4 (CXCL12/ CXCR4) signaling pathway is implicated in GBM invasion and cell cycle progression. While the CXCR4 antagonists (AMD3100) has a potential anti-GBM effects, its poor pharmacokinetic and systemic toxicity had precluded its clinical application. Moreover, the role of CXCL12/ CXCR4 signaling pathway in anti-GBM immunity, particularly in GBM-mediated immunosuppression has not been elucidated. Here, we developed a synthetic protein nanoparticle (SPNPs) coated with the cell-penetrating peptide iRGD (AMD3100 SPNPs) to target the CXCR4/CXCL12 signaling axis in GBM. We showed that AMD3100 SPNPs effectively blocked CXCR4 signaling in mouse and human GBM cells in vitro as well as in GBM model in vivo. This results in inhibition of GBM proliferation and induction of immunogenic tumor cell death (ICD) leading to inhibition of GBM progression. Our data also demonstrate that blocking CXCR4 sensitizes GBM cells to radiation, eliciting enhanced release of ICD ligands. Combining AMD3100 SPNPs with radiotherapy inhibited GBM progression and led to long-term survival; with 60% of mice remaining tumor-free. This was accompanied by an anti-GBM immune response and sustained immunological memory that prevented tumor recurrence without further treatment. Finally, we showed that systemic delivery of AMD3100 SPNPs decreased the infiltration of CXCR4+ monocytic myeloid-derived suppressor cells to the tumor microenvironment. With the potent ICD induction and reprogrammed immune microenvironment, this strategy has significant potential for future clinical translation.Graphical abstractImmunological mechanism targeting Glioblastoma (GBM) upon blocking CXCR4 signaling pathway with AMD3100-conjugated nanoparticles (SPNPs).(1) Radiotherapy induces glioma cell death, followed by Damage-associated molecular patterns (DAMPs) release. Dendritic cells (DC) are activated by DAMPs and migrate to the regional lymph node where they prime cytotoxic T lymphocyte immune response. Tumor-specific cytotoxic T cells infiltrate the tumor and attack glioma cells. (2) Glioma cells express CXCR4, as well its ligand CXCL12. CXCL12 induces glioma cell proliferation and, (3) as well as mobilization in the bone marrow of CXCR4 expressing myeloid MDSC, which will infiltrate the tumor, and inhibit tumor-specific cytotoxic T cells activity. GEMM of glioma when treated systemically with SPNPs AMD3100 SPNPs plus radiation, nanoparticles block the interaction between CXCR4 and CXCL12, thus (4) inhibiting glioma cell proliferation and (5) reducing mobilization in the bone marrow of CXCR4 expressing myeloid MDSC, (6) generating a reduced MDSC tumor infiltration, as well as releasing MDSC inhibition over tumor specific cytotoxic T cell response.

Accumulating evidence suggest that cerebral microvascular disease increases with advancing age and is associated with lacunar stroke, leukoaraiosis, vascular dementia and Alzheimer disease. Increased blood brain barrier (BBB) permeability/leakage takes “center stage” in ongoing age-related vascular/brain parenchymal injury. Although significant effort has been made in defining the gene mutations and risk factors involved in microvascular alterations in vascular dementia and Alzheimer disease, the intra- and intercellular pathogenic mechanisms responsible for vascular hyperpermeability are still largely unknown. The present study aimed to reveal the ongoing senescence process in brain endothelial cells and its effect on BBB integrity in healthy/non-disease conditions. An analysis of BBB integrity during the life span of C56Bl6 mice (young, 2–6 months; middle-aged, 6–12, months; old, 16–22 months) showed increased BBB permeability for different molecular sized tracers (sodium fluorescein, inulin and 20 kDa dextran) in aged mice which was accompanied by modifications in tight junction (TJ) complex organization, manifested as altered TJ protein expression (particularly claudin-5). A gene screening analysis of aging associated markers in brain microvessels isolated from “aged” mice (C56Bl6, 18-20 months) and human brain samples showed a significant decline in sirtuin-1 expression (Sirt1; ~2.8-fold) confirmed at mRNA and protein levels and by activation assay. Experiments in Sirt1 transgenic mice and brain endothelial cell-specific Sirt1 knockout mice indicated that Sirt1 affects BBB integrity, with loss increasing permeability. Similarly, in vitro, overexpressing Sirt1 or increasing Sirt1 activity with an agonist (Sirt1720) protected against senescence-induced brain endothelial barrier hyperpermeability, stabilized claudin-5/ZO-1 interactions and rescued claudin-5 expression. These findings reveal a novel role of Sirt1 in modulating aging-associated BBB persistent leakage.

We performed a retrospective assessment of patient- and transplant-specific characteristics and outcomes for 4142 patients undergoing allogeneic haematopoietic cell transplant for myelofibrosis between 1995 and 2018 across 278 centres. Activity increased steadily across the four analysed eras (

The complexity of the blood–brain barrier (BBB) and neurovascular unit (NVU) was and still is a challenge to bridge. A highly selective, restrictive and dynamic barrier, formed at the interface of blood and brain, the BBB is a “gatekeeper” and guardian of brain homeostasis and it also acts as a “sensor” of pathological events in blood and brain. The majority of brain and cerebrovascular pathologies are associated with BBB dysfunction, where changes at the BBB can lead to or support disease development. Thus, an ultimate goal of BBB research is to develop competent and highly translational models to understand mechanisms of BBB/NVU pathology and enable discovery and development of therapeutic strategies to improve vascular health and for the efficient delivery of drugs. This review article focuses on the progress being made to model BBB injury in cerebrovascular diseases in vitro.

Recent evidence suggests that blood–brain barrier (BBB) recovery and reestablishment of BBB impermeability after stroke is incomplete. This could influence stroke recovery, increase the risk of repeat stroke, and be a solid substrate for developing vascular dementia. Although accumulating evidence has defined morphological alterations and underlying mechanisms of tight junction (TJ) changes during BBB breakdown in acute stroke, very little is known about the type of alterations and mechanisms in BBB “leakage“ found subacutely or chronically. The current study examined BBB structural alterations during the “BBB leakage” associated with the chronic phase of stroke in male mice and both genders of humans. We found significant upregulation of claudin-1 mRNA and protein, a nonspecific claudin for blood vessels, and downregulation in claudin-5 expression. Morphological and biochemical as well as fluorescence resonance energy transfer and fluorescence recovery after photobleaching analysis of postischemic brain endothelial cells and cells overexpressing claudin-1 indicated that newly synthesized claudin-1 was present on the cell membrane (∼45%), was incorporated into the TJ complex with established interaction with zonula occludens-1 (ZO-1), and was building homophiliccis- andtrans-interactions. The appearance of claudin-1 in the TJ complex reduced claudin-5 strands (homophilic claudin-5cis- andtrans-interactions) and claudin-5/ZO-1 interaction affecting claudin-5 incorporation into the TJ complex. Moreover, claudin-1 induction was associated with an endothelial proinflammatory phenotype. Targeting claudin-1 with a specific C1C2 peptide improved brain endothelial barrier permeability and functional recovery in chronic stroke condition. This study highlights a potential “defect” in postischemic barrier formation that may underlie prolonged vessel leakiness.SIGNIFICANCE STATEMENTAlthough rarely expressed at the normal blood–brain barrier (BBB), claudin-1 is expressed in pathological conditions. Analyzing poststroke human and mouse blood microvessels we have identified that claudin-1 is highly expressed in leaky brain microvessels. Our results reveal that claudin-1 is incorporated in BBB tight junction complex, impeding BBB recovery and causing BBB leakiness during poststroke recovery. Targeting claudin-1 with a claudin-1 peptide improves brain endothelial barrier permeability and consequently functional neurological recovery after stroke.

Objective: Streamlining the diagnosis is a key factor in improving the treatment outcomes for breast cancer. The aim of this study was to determine factors influencing time to seeking medical advice and treatment onset in women who are diagnosed with breast cancer in Serbia. Methods: The study was a multicenter, cross-sectional national survey, performed at 10 oncology centers in Serbia. Time intervals spent throughout the complex diagnostic pathway were evaluated using a validated questionnaire administered to women with breast cancer (n = 800). Total interval (TI) was determined using predefined time scales, including one referring to patient interval (PI), and several related to health care system interval (SI). Results: Mean PI, SI, and TI were 4.5, 9.2, and 12.9 weeks, respectively; 20% of patients had a PI>12 weeks. Based on the multivariate regression model, longer PI was associated with perceived lack of time and personal disregard or trivialization of detected symptoms and signs. Women who were supported by family members or friends and had at least a secondary level education tended to have a shorter PI. Longer PI was correlated with a longer SI, while regular self-examination, having been diagnosed by an oncologist, and living in a major city were associated with shorter SI. Conclusions: Several factors, related to psychological, demographic, behavioral, and health system characteristics, determined both the time to seeking medical advice and treatment onset for breast cancer. These findings support review and refining of national strategies and policies to promote early detection, diagnosis, and treatment of breast cancer.

Background/Aim. Stem cells (SCs) guarantee complete/longterm bone marrow (BM) repopulation after SC-transplants. The aim of the study was to evaluate absolute count of total SCs (determined by ISHAGE-sequential-gating protocol – SCish) and relative frequency of immature CD34+/CD90+ (CD90+SCish) subset in peripheral blood (PB) as predictive factors of mobilization and apheresis product (AP) quality. Methods. Mobilization included chemotherapy and granulocytegrowth- factor (G-CSF). Harvesting was performed by Spectra- Optia-IDL-system. The SCsish were determined as a constitutional part of CD34+ cells in the “stem-cell-region” using FC- 500 flow-cytometer. In this study, the original ISHAGEsequential- gating protocol was modified by introduction of anti-CD90-PE monoclonal-antibody into the analysis of CD90 expression on SCish (CD90+SCish). The results were presented as a percentage of SCish per nucleated-cell count, absolute SCish count in μL of the PB or the AP, percentage of the CD90+SCish expressed to SCish and absolute CD90+SCish count in μL of the PB or the AP. Results. The absolute count of total SCish and CD90+SCish was significantly higher (p = 0.0007 and p = 0.0266, respectively) in the AP than in the PB samples. The CD90+SCish/total SCish indexes from PB were higher than indexes from the AP (p = 0.039). The relative frequency of CD90+SCish showed a highly significant inverse correlation with the absolute count of total SCish in both, the PB and AP (p = 0.0003 and p = 0.0013 respectively). The relative frequency of CD90+SCish from the PB also showed a significant (p = 0.0002) inverse relationship with total SCish count in the AP. Patients with less than 10% CD90+SCish in the PB had evidently higher (p = 0.0025) total SCish count in the AP. Conclusion. We speculate that lower CD90+SCish yield in the AP is not a consequence of an inferior collection efficacy, but most likely a result of several still not fully resolved immature SC cytomorphological/ biophysical features. Therefore, following the mobilization by chemotherapy G-CSF, some logical questions appear – whether we should follow the absolute count of total SCish, or, whether we should test for relative frequency of CD90+SCish prior to harvesting. To reach the final conclusions, it is essential to conduct further controlled and larger investigations concerning the correlation of circulating and harvested SCs with patients' hematopoietic recovery. [Project of the Ministry of Defence of the RS, Project MF/VMA 9/17-19 and Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 41031]

Introduction. Bence-Jones myeloma multiplex is a progressive disease characterized by excessive numbers of abnormal plasma cells in the bone marrow and overproduction of incomplete immunoglobulins, containing only the light chain portion of the immunoglobulins. This type of myeloma occurs 15-20%. The median overall survival is approximately 4 years. Aim of this study was to define prognostic factors affecting overall survival in Bence-Jones multiple myeloma patients. Material and Methods. From 1995 to 2015, we treated 75 patients (49 men and 26 female), average age 55.9 years (range 31-85). Results. Conventional chemotherapy introductory clinical response was achieved in 54 patients (72%), while in 21 patients (28%) the established disease was resistant. Transplantation was done in 45 patients (60%), while 30 patients (40%) were treated with conventional chemotherapy. In the group of patients with transplantation done, tandem was carried out in 11 patients and secondary stem cell transplantation was done in 5 relapsed patients. With 1 patient with tandem stem cell transplantation allogenic (singen) stem cell transplantation was done. Transplant related mortality is 1.5%. The transplanted patients had significantly longer PFS (mediana 13 months vs 7 months, p

Neutrophils are considered key participants in post-ischemic stroke inflammation. They are the first white blood cells to arrive in ischemic brain and their presence in the brain tissue positively correlates with post-ischemic injury severity. CXCL1 is a neutrophil attractant chemokine and the present study evaluates whether redirecting neutrophil migration using a peripherally implanted CXCL1-soaked sponge can reduce brain inflammation and improve outcomes in a novel mouse model of thromboembolic (TE) stroke. TE stroke was induced by injection of a platelet-rich microemboli suspension into the internal carotid artery of adult C57BL/6 male mice. The model induced neuroinflammation that was associated with increases in multiple brain and serum cytokines/chemokines at the mRNA and protein levels, including very marked increases in CXCL1. In other groups of animals, an absorbable sterile hemostatic sponge, previously immersed in either saline (0.9%NaCl) or CXCL1, was implanted into subcutaneous pockets formed in the inguinal region on the left and right side following stroke surgery. Mice implanted with the sponge soaked with CXCL1 had significantly reduced neuroinflammation and infarct size after TE stroke compared to mice implanted with the sponge soaked with 0.9%NaCl. There was also reduced mortality and improved neurological deficits in the TE stroke + CXCL1 sponge group compared to the TE stroke +0.9%NaCl sponge group. In conclusion: redirecting bloodstream leukocytes toward a peripherally-implanted neutrophil chemokine CXCL1-soaked sponge improves outcomes in a novel mouse model of thromboembolic stroke. The present findings suggest a novel therapeutic strategy for patients with acute stroke.

Vascular disruption is the underlying cause of cerebral hemorrhage, including intracerebral, subarachnoid and intraventricular hemorrhage. The disease etiology also involves cerebral hemorrhage-induced blood–brain barrier (BBB) disruption, which contributes an important component to brain injury after the initial cerebral hemorrhage. BBB loss drives vasogenic edema, allows leukocyte extravasation and may lead to the entry of potentially neurotoxic and vasoactive compounds into brain. This review summarizes current information on changes in brain endothelial junction proteins in response to cerebral hemorrhage (and clot-related factors), the mechanisms underlying junction modification and potential therapeutic targets to limit BBB disruption and, potentially, hemorrhage occurrence. It also addresses advances in the tools that are now available for assessing changes in junctions after cerebral hemorrhage and the potential importance of such junction changes. Recent studies suggest post-translational modification, conformational change and intracellular trafficking of junctional proteins may alter barrier properties. Understanding how cerebral hemorrhage alters BBB properties beyond changes in tight junction protein loss may provide important therapeutic insights to prevent BBB dysfunction and restore normal function.

Collagen is a major constituent of the gingival extracellular matrix, which crucially affects the histoarchitecture of the gingival tissue. Collagen type I dominates the gingival tissue, while type II, III and IV are present to a lesser extent. Changes in distribution and density occur in the inflamed gingiva. The aim of the study was to examine the organization and density of the collagen in healthy and inflamed gingiva. The material for the study consisted of the gingival biopsies performed in 96 patients aged from 13 to 70 years. The gingival specimens were classified into 4 groups: healthy gingiva, gingivitis, moderate periodontal disease and severe periodontal disease. The samples of the gingival tissues were stained by the method of Van Gieson's Stain, while the VD of the collagen was measured bz the use of multipurpose testing system M42. In healthy gingival collagen are thick and receptive to color. In gingivitis collagen is mostly preserved structure, but reduced volume; in the periodontitis collagen bundles are reduced, short, thin, disorganized, often fragmented and less susceptibility to color. In healthy gingiva collagen fibers occupy 58.6 ± 5.1% of the volume of lamina propria; in gingivitis significantly lower (44.2 ± 6.2%); in moderate periodontitis 32.7 ± 8.5% and in the progressed periodontitis only 28.7 ± 9.7%. Based on our results, we concluded that in periodontal disease occurs collagenolysis. Quantitative evaluation of gingival collagen volume density may reflect the clinical severity of periodontal disease.

Background: Reducing treatment delay improves outcomes in breast cancer. The aim of this study was to determine factors influencing patient- and system-related delays in commencing breast cancer treatment in different countries. Methods: A total of 6588 female breast cancer patients from 12 countries were surveyed. Total delay time was determined as the sum of the patient-related delay time (time between onset of the first symptoms and the first medical visit) and system-related delay time (time between the first medical visit and the start of therapy). Results: The average patient-related delay time and total delay time were 4.7 (range: 3.4–6.2) weeks and 14.4 (range: 11.5–29.4) weeks, respectively. Longer patient-related delay times were associated with distrust and disregard, and shorter patient-related delay times were associated with fear of breast cancer, practicing self-examination, higher education level, being employed, having support from friends and family and living in big cities. The average system-related delay time was 11.1 (range: 8.3–24.7) weeks. Cancer diagnosis made by an oncologist versus another physician, higher education level, older age, family history of female cancers and having a breast lump as the first cancer sign were associated with shorter system-related delay times. Longer patient-related delay times and higher levels of distrust and disregard were predictors of longer system-related delay times. Conclusions: The delay in diagnosis and treatment of breast cancer remains a serious problem. Several psychological and behavioural patient attributes strongly determine both patient-related delay time and system-related delay time, but their strength is different in particular countries.

Objective: The aim of this study was to investigate the differences between the salivary levels of IL-8 in patients with Type 1 diabetes mellitus (DM) with (DM+P) or without (DM-P) concomitant periodontitis and healthy subjects. The correlations between the levels of these cytokines and clinical periodontal parameters were also established. Methods: Twenty children and adolescents with Type 1 DM (10 diagnosed with periodontitis, 10 presenting no signs of periodontitis) and a control group consisting of 20 healthy children and adolescents aged 7-18 years were recruited for this study. Results: The Salivary IL-8 level was statistically significantly (p

Background/Aim. Peri-implantitis presents inflammatory process that affects soft and hard supporting tissues of osseointegrated implant based on inflammatory osteoclastogenesis. The aim of this study was to investigate whether receptor activator of nuclear factor kappa B (RANK) concentrations in peri-implant crevicular fluid could be associated with clinical parameters that reflect inflammatory nature of peri-implantitis. Methods. The study included 67 patients, 22 with diagnosed peri-implantitis, 22 persons with healthy peri-implant tissues and 23 patients with periodontitis. Clinical parameters from each patient were recorded and samples of peri-implant/gingival crevicular fluid were collected for the enzyme-linked immunosorbent assay (ELISA) analysis. Results. RANK concentration was significantly increased in samples from the patients with periimplantitis when compared to healthy implants (p lt 0.0001), where the average levels were 9 times higher. At the same time RANK concentration was significantly higher in periimplantitis than in periodontitis sites (p lt 0.0001). In implant patients pocket depths and bleeding on probing values were positively associated with high RANK concentrations (p lt 0.0001). Conclusion. These results revealed association of increased RANK concentration in samples of periimplant/ gingival crevicular fluid with peri-implant inflammation and suggests that RANK could be a pathologic determinant of peri-implantitis, thereby a potential parameter in assessment of peri-implant tissue inflammation and a potential target in designing treatment strategies., Uvod/Cilj. Periimplantitis predstavlja inflamatorni proces koji zahvata meko i tvrdo potporno tkivo osteointegrisanog implantata, i zasnovan je na inflamatornoj osteoklastogenezi. Cilj studije bio je da se utvrdi povezanost koncentracije receptora aktivatora nuklearnog faktora kapa-B (RANK), kao glavnog receptora osteoklastnog metabolizma, sa kliničkim parametrima periimplantitisa. Metode. Studija je uključila 67 sistemski zdravih pacijenata (22 sa periimplantitisom, 22 sa zdravim implantatima i 23 sa periodontopatijom). Pacijentima su mereni klinički parametri i uziman je uzorak periimplantne/gingivalne tečnosti za određivanje koncentracije RANK-a ELISA metodom. Rezultati. Koncentracija RANK-a bila je značajno povišena kod periimplantitisa u odnosu na zdrave implantate (p lt 0,0001), gde je srednja vrednost koncentracije bila 9 puta veća. Istovremeno, RANK je bio značajno viši kod periimplantitisa nego kod parodontopatije (p lt 0,0001). U grupi sa implantatima dubina periodontalnog džepa i krvarenje na probu bili su pozitivno udruženi sa visokim vrednostima RANK-a (p lt 0,0001). Zaključak. Rezultati istraživanja pokazuju udruženost povišenosti koncentracije RANK-a sa periimplantnom inflamacijom i navodi na zaključak da bi RANK mogao da bude patološka determinanta periimplantitisa, a time i potencijalni parametar za praćenje inflamacije periimplantnog tkiva i potencijalni cilj za pravljenje terapijskih strategija.

Outcome of patients with chronic myeloid leukemia and a low-risk score: allogeneic hematopoietic stem cell transplantation in the era of targeted therapy. A report from the EBMT Chronic Malignancies Working Party

Background/Aim. Introducing tyrosine kinase inhibitors (TKIs) has essentially changed curative approach, to be precise, clearly improved treatment efficacy for chronic myeloid leukemia (CML). Thus, the place and usage of allogeneic stem cell transplant (SCT) in CML treatment - as a former "nearly monopolistic" therapeutic manner - is nowadays controversial. The objective of this retrospective study was to evaluate the results obtained in the treatment of CML patients, with a particular attempt to define parameters critical for clinical benefit and superior overall outcome following allogeneic SCT. Methods. A total of 32 CML patients (27 in chronic phase and 5 with advanced disease), with female/male ratio 11/21, aged from 9 to 54 (32 in average) years, underwent allogeneic SCTs (1993 to 2009). The initial treatment for 25 patients was interferon alpha (IFN-α) with or without ARA-C, and additional 7 patients with no response to imatinib mesylat (IM). The time from diagnosis to SCT was approximately 12 (range 3- 37) months. The patient were categorized according to the risk for the disease, transplant-related mortality (TRM) scoring system, and stem cell (SC) source. The basic conditioning regimen was a combination of busulphan and cyclophosphamide (BuCy-2). Graft-versus-host disease (GvHD) was typically prevented with cyclosporine-A (CsA) and methotrexate (MTX). Results. Engraftment was observed in 26 (84.4%) patients, with polymorphonuclear (PMNs) and platelet (Plt) recovery on the 15th (range 10-22) and 19th (range 11-29) posttranspalnt days, respectively. Acute GvHD (aGvHD) had 13/26 (50%), and chronic GvHD (cGvHD) 10/21 (47.1%) patients. The incidence of overall TRM was 46.8% (15/32), while early death was noticed in 4 (12.5%) patients. A cause of death in 9 (28.1%) patients was cGvHD, in 2 (6.25%) patients infection, and in 3 (9.35%) cases disease-relapse was occurred. Fourteen (43.7%) of the patients are still alive, 9 from the low-risk group for TRM, with long-term survival from 1 to 16 years. Patients who received SCs from peripheral blood (PB) vs bone marrow (BM) had significantly faster engraftment (p < 0.05), lower oropharingeal mucositis rate (25% vs 70%; p < 0.05), but more frequent cGvHD (83.3% vs 30.3%; p < 0.05). A significantly improved (log-rank = 2.39; p < 0.01) overall survival (OS) was obtained in BM-setting. Conclusion. The results obtained in this study are in accordance with data from analogous clinical trials. Exactly, in the era of the new target therapy (TKI application), allogeneic SCT can be still a convenient therapeutic approach for well-selected CMLpatients, especially for those with initial high-risk disease and lower probability of TRM.

Background/Aim. Peripheral blood (PB) is used more frequently as a source of stem cells (SCs) for allogeneic transplantation. However, the influence of cell source on the clinical outcome of SC transplantation is not yet well established. The aim of this study was to compare the results of PBSC transplantation (PBSCT) with bone marrow transplantation (BMT) on the basis of engraftment, frequency and severity of immediate (mucositis, acute Graft versus Host Disease - aGvHD) and delayed (chronic GvHD - cGvHD) complications, as well as transplant-related mortality (TRM), transfusion needs, relapses and overall survival (OS). Methods. We analyzed 158 patients, women/men ratio 64/94 median age 29 (range 9-57), who underwent allogeneic SC transplantation between 1989 and 2009. All included patients had diseases as follows: acute myeloid leukemia (AML) - 39, acute lymphoblastic leukemia (ALL) - 47, chronic myeloid leukemia (CML) - 32, myelodysplastic syndrome (MDS) - 10, Hodgkin?s lymphoma (HL) - 2, multiple myeloma (MM) - 3, granulocytic sarcoma (GrSa) - 3, severe aplastic anemia (sAA) - 22. The patients underwent transplantations were divided into two groups: BMT group (74 patients) and PBSCT group (84 patients). Each recipient had HLA identical sibling donor. SCs from bone marrow were collected by multiple aspirations of iliac bone and from PB by one ?Large Volume Leukapheresis? (after recombinant human granulocyte colony stimulating factor, rHuG-CSF) application (5-12 ?g/kgbm, 5 days). Conditioning regimens were applied according to primary disease, GvHD prophylaxis consisted of combination of a cyclosporine A and methotrexate. Results. Engraftment, according to the count of polymorphonuclear and platelets, were significantly (p < 0.001) faster in the PBSCT vs BMT group. The needs for transfusion support were significantly (p < 0.01) higher in the BMT group. Those patients had more frequently oropharingeal mucositis grade 3/4 (33.3% vs 10.0%, p < 0.05). There were no significant differences in the incidence of aGvHD and cGvHD between the two groups. The patients who underwent PBSCT had more frequently extensive cGvHD in comparison with the BMT group (29.1% vs 11.29%, p < 0.05). SC source (SCS) had no significant influence on the TRM (21.62% vs 23.8%, p = 0.64) and the incidence of relapses (21.6% vs 29.7%, p = 0.32). Finally, the patients treated by BMT had a significantly better OS (logrank 2.33, p < 0.05). Conclusion. SCs harvesting from PB resulted in improved cell yield, faster engraftment, as well as in a decrease of immediate transplantation related complications with a reduced treatment cost. Allogeneic PBSCT were associated with more frequent extensive cGvHD, while the influence of SCS in TRM and relapses was not observed. Finally, the longterm OS was better in the patients treated by BMT. To verify impact of SC source on transplantation (PBSCT vs BMT) overall efficacy, more larger randomized clinical studies are needed.

Analysis of peri-implant crevicular fluid (PICF) offers a non-invasive means of studying the host response in peri-implant disease and may provide an early indication of patients at risk for active disease. This study examined the PICF levels of interleukin-1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8) and macrophage inflammatory protein-1alpha (MIP-1alpha) in patients with non-manifesting inflammation, early and late stages of mucositis. The study group comprised 90 adult healthy volunteers with endosseal titanium implants inserted. Samples were taken from peri-implant sulcus using a filter paper technique. Implant tissues were categorized clinically as healthy, early mucositis or advanced mucositis. Clinical manifestations were determined by: gingival index and bleeding on probing, plaque index and radiographic analyses. Cytokine concentrations were assesed using commercially available enzyme-linked immunosorbent assay kits. Patients from the control group (healthy patients) have significantly lower concentrations of IL-1beta, TNF-alpha, IL-8 and MIP-1alpha in PICF compared with both groups with mucositis. Positive correlation was noted in the control group between IL-1beta and TNF-alpha and between MIP-1alpha and IL-8 in the group with early mucositis. The results suggest that cytokines could be prognostic markers of implant failure.

In our study we investigated the role of the estrogen receptor (ER), progesterone receptor (PR) and clinicohistological parameters in breast cancer patients treated with tamoxifen during the early (2.5 years) vs. late (2.5-5 years) follow-up. The negative status of both ER and PR and tumors equal to or bigger than 2 cm defined the phenotypes and consequently the groups of patients with the worst clinical course of the disease: ER-negative PR-negative, ER-negative pT2 and PR-negative pT2. These high-risk subgroups were related to early follow-up indicating de novo resistance. It is relevant to point out that examined predictive indicators did not show significant importance in the late follow-up study.

Background/Aim. More than 90% of worldwide population is infected with human cytomegalovirus (CMV), one of the most common agents which complicate immunocompromised patients. Viral infections, in particular CMV ones are still a major cause of moratality and morbidity after stem cell transplantation (SCT). Monitoring is performed by detecting CMVAg or virus DNA in peripheral blood. Risk factors are donor/ recipient CMV status, type of transplant and acute graft versus host disease. The aim of the study was to determine the extent of validity of CMV infection monitoring after transplantation as a reliable parameter of further CMV replication course in patients with hematopoietic stem cell transplantation. Methods. A total of 49 patients with stem cell transplantation were studied prospectively during a 2-year period after transplantation for the presence of CMV DNA. Polymerase chain reaction (PCR) CMV DNA was performed on 222 full blood samples using Cobas Amplicor assay. Results. Activation of CMV was detected in 10/49 (20.48%) of the patients. The median posttransplantation time for the first positive PCR result was 6 weeks for the stem cell transplant patients. Viremia became negative in all the cases after the antiviral therapy with ganciclovir. Conclusion. Our data show that the level of CMV-DNA load at the time of initial CMV detection after transplantation could be a possible predictor for further course of CMV replication in patients receiving hematopoietic stem cell.

Background/Aim. Periodontitis is a chronic inflammatory disease of periodontal tissues with consequential is bone loss as a result of host immunological reactions caused by periopathogens. The aim of the study was to investigate if there is a correlation between clinical parameters and the presence of two most aggressive periopathogens (Aggregatibacter actinomycetemcomitans - Aa and Porphyromonas gingivalis - Pg) in patients with progressive periodontal lesions. Methods. A total of 34 systemic healthy people, 23 to 70 years old, were included in the study. The patients were clinically and radiologically examined, and after that, the representative pocket with greatest pocket depth was chosen and the sample was collected from that place. The measured clinic parameters were: gingival index, index of gingival bleeding, pocket depth and plaque indices. The multiplex Polymerase Chain Reaction (PCR) method was used for detection of periopathogens. After obtaining results, appropriate statistical tests were used to correlate the clinical and microbiological results. Results. Aa and Pg were detected in the same percentage of samples. Aa and Pg were detected in 35.29% samples alone, and in 29.41% both were detected. The values of measured clinical parameters did not show a statistical significance between the groups. In analysis of correlations among clinical parameters inside the groups, a statistical significance was found only between gingival and plaque index in the group with Aa. Conclusion. Clinical course of periodontitis in the developed stage does not differ in relation to the presence of different periopathogens as the major inductors of immunologically guided destructive processes., Uvod/Cilj. Parodontopatija je hronično inflamatorno oboljenje parodontalnih tkiva koje za krajnji ishod ima gubitak potpornog koštanog tkiva zuba usled imunoloških reakcija izazvanih parodontopatogenim bakterijama. Cilj studije bio je korelisanje kliničkih parametara i prisustva dve najagresivnije parodontopatogene bakterije (Aggregatibacter actinomycetemcomitans - A.a. i Porphyromonas gingivalis - P.g.) kod bolesnika sa progresivnim parodontalnim lezijama. Metode. U studiju su bila uključena 34 sistemski zdrava ispitanika, starost 23-70 godina. Ispitanici su klinički i radiološki pregledani i uzorak je uziman iz reprezentativnog parodontalnog džepa sa najvećom dubinom sondiranja. Od kliničkih parametara mereni su gingivalni indeks, indeks krvarenja gingive, dubina parodontalnog džepa i indeks plaka. Prisustvo parodontopatogena dokazivano je multipleks metodom PCR (Polymerase Chain Reaction), a rezultati su korelisani sa kliničkim parametrima primenom odgovarajućih statističkih testova. Rezultati. Ista procentualna zastupljenost oba mikroorganizma dokazana je u uzorcima, naime i A.a. i P.g. bili su prisutni u po 35,39% uzoraka, a u 29,41% dokazana su oba mikroorganizma. Rezultati su korelisani po grupama formiranim u odnosu na prisustvo bakterija. Vrednosti merenih kliničkih parametara nisu se statistički značajno razlikovale u zavisnosti od prisustva parodontopatogena. Međusobne korelacije kliničkih parametara unutar grupe nisu pokazale statističku značajnost, osim korelacije gingivalnog i plak indeksa u grupi sa A.a. Zaključak. Klinički tok uznapredovale faze parodontopatije ne razlikuje se u odnosu na vrstu parodontalnih bakterija kao induktora imunološki posredovanih destruktivnih procesa.

PCR-based clonality testing can be performed in all lymphoproliferations by analysing gene rearrangements of antigen receptors, rearrangements that are unique for each kind of lymphocyte. Reactive lymphoproliferations have polyclonally rearranged Ig/TCR genes, whereas malignant proliferations (leukaemias and lymphomas) show clonal rearrangements. The aim of this study was to assess the clinical benefits of clonality testing with previously evaluated consensus primers in leukaemia patients. The study included peripheral blood and bone marrow samples of 67 leukaemia patients (32 B-CLL, 24 B-ALL and 11 T-ALL). Clonality testing was based on PCR amplification of rearranged IgH and TCR genes. During diagnosis, monoclonal pattern was found in all analysed B-CLL and T-ALL samples. Testing in B-ALL patients showed positive results in all bone marrow and one peripheral blood samples. Results of clonality testing in B-CLL patients during follow-up were concordant between peripheral blood and bone marrow. Obtained results corresponded to clinical course in all but one patient. In B-ALL group, results of molecular testing in peripheral blood and bone marrow confirmed remission estimated according to clinical criteria in all except one patient. Before any clinical sign of relapse, monoclonal pattern was found in six/seven patients by bone marrow and in three/seven patients by peripheral blood analysis, respectively. Results of molecular monitoring in T-ALL patients did not confirme clinical evaluation in two patients. Obtained results indicate high accuracy of re-evaluated primers for clonality assessment in ALL and CLL patients at the time of diagnosis. Results of clonality testing in B-ALL patients indicate that bone marrow analysis has higher sensitivity compared to analysis of peripheral blood.

Brain edema (BE) is an uncommon but life-threatening complication of severe diabetic ketoacidosis (DKA) and its treatment. Despite advances in treatment of DKA, the pathogenesis of both initiation and progression of the associated BE is unclear. In the present study we examined the blood brain barrier (BBB) integrity and the potential involvement of the inflammatory mediators in BBB breakdown in two cases of fatal BE associated with DKA. In both cases there were typical signs of disruption of the BBB manifested by the absence of tight junction proteins (occludin, claudin-5, ZO-1 and JAM-1) in the parenchymal blood vessels, as well as albumin extravasation in examined brain areas. The neuroinflammatory markers chemokine CCL2, NF-kappaB and nitrotyrosine were localized in the perivascular areas of the disrupted BBB and diffusely distributed in the brain parenchyma. Our data indicate that neuroinflammation plays a role in the BBB disruption of the fatal BE of DKA.

INTRODUCTION Measurement of daily variability of peak expiratory flow (PEF) is widely accepted as an objective method to assess asthma severity. Recent investigations have proved nonlinearity of original Wright scale of peak flowmeter. All country members of European Union have been obliged to implement a new scale according to standard EN 13826 since 2005. This study examined whether the correction of PEF values for the inaccuracy of the scale would affect asthma management based on their daily variability. MATERIAL AND METHODS We analyzed PEF values (2352) in 34 children, aged 5-16, during 3-5 weeks of monitoring in order to establish the diagnosis of asthma by using peak flowmeters with Write scale. The correction of measured values for inaccuracy was managed with original Dr. M. Miller's predictive equation. The daily variability of PEF (amplitude percent mean) up to 20% was considered as "normal", 20-29.9% as "raised", and 30% and above as "high ". The assessment of daily variability was performed before and after correction. RESULTS There was no significant change in the number of days with airway lability as regarding the assessed whole study group (p = 0.475). However, 22 (64.7%) of children had at least one false clinical message about daily variability during the monitoring. It was overestimated in 12 (7%) days in younger (6.6 +/- 0.8 years) or of shorter stature (122.6 +/- 3.6 cm) and underestimated in 13 (4%) days in older (11.1 +/- 2.7) or taller ones (150.9 +/- 12.5 cm) (p < 0.001). CONCLUSION Usage of peak flowmeters with Wright scale may lead to an error in asthma severity assessment based on daily variability of PEF. It may cause overtreatment or undertreatment of asthmatic children.

Background and Purpose— The chemokine, monocyte chemoattractant protein-1 (CCL2), is a major factor driving leukocyte infiltration into the brain parenchyma in a variety of neuropathologic conditions associated with inflammation, including stroke. In addition, recent studies indicate that CCL2 and its receptor (CCR2) could have an important role in regulating blood-brain barrier (BBB) permeability. This study evaluated the role of the CCL2/CCR2 axis in regulating postischemic inflammation, BBB breakdown, and vasogenic edema formation. Methods— CCR2 −/− and CCR2 +/+ mice were subjected to focal transient cerebral ischemia. BBB permeability and brain edema formation were observed at days 1 and 5 of reperfusion by evaluating the product surface area for fluorescein isothiocyanate–albumin and measuring water and electrolyte contents. Immunohistochemistry was used to assess leukocyte infiltration. cDNA gene and protein arrays for inflammatory cytokines were used to assess inflammatory profiles in CCR2 +/+ and CCR2 −/− mice. Results— CCR2 −/− mice had reduced infarct sizes and significantly reduced BBB permeability and brain edema formation in the affected ischemic hemisphere compared with CCR2 +/+ mice. This reduction in injury was closely associated with reduced infiltration of not only monocytes but also neutrophils (7- and 4-fold decreases, respectively). In addition, CCR2 −/− mice had reduced expression/production of inflammatory cytokines during reperfusion. Conclusions— These data suggest that inhibiting the CCL2/CCR2 axis affects brain reperfusion outcome by reducing brain edema, leukocyte infiltration, and inflammatory mediator expression.

Introduction: Monitoring of peak expiratory flow (PEF) is recommended in numerous guidelines for management of asthma. Improvements in calibration methods have demonstrated the inaccuracy of original Wright scale of peak flowmeter. A new standard, EN 13826 that was applied to peak flowmeter was adopted on 1st September 2004 by some European countries. Correction of PEF readings obtained with old type devices for measurement is possible by Dr M. Miller?s original predictive equation. Objective. Assessment of PEF correction effect on the interpretation of measurement results and management decisions. Method. In children with intermittent (35) or stable persistent asthma (75) aged 6-16 years, there were performed 8393 measurements of PEF by Vitalograph normal-range peak flowmeter with traditional Wright scale. Readings were expressed as percentage of individual best values (PB) before and after correction. The effect of correction was analyzed based on The British Thoracic Society guidelines for asthma attack treatment. Results. In general, correction reduced the values of PEF (p

Background/Aim. Chronic myeloid leukemia (CML) represents a malignant myeloproliferative disease developed out of pluripotent hematopoietic stem cell that contains the fusion bcr-abl gene. Disorders that occur in the process of apoptosis represent one of the possible molecular mechanisms that bring about the disease progress. The aim of our study was to carry out the analysis of the presence of the amplification of the cmyc oncogene, as well as the analysis of the changes in the expression of Bcl-2 in the patients with CML. Methods. Our study included 25 patients with CML (18 in chronic phase, 7 in blast transformation). Using an immunohistochemical alkaline phosphatase-anti-alkaline phosphatase (APAAP) method, we analyzed the expression of cell death protein in the mononuclear bone marrow cells of 25 CML patients. By a differential PCR (polymerase chain reaction) method, we followed the presence of amplified c-myc gene in mononuclear peripheral blood cells. Results. The level of the expression of Bcl-2 protein was considerably higher in the bone marrow samples of the patients undergoing blast transformation of the disease. The amplification of c-myc gene was detected in 30% of the patients in blast transformation of the disease. Conclusion. The expression of Bcl-2 protein and the amplification of c-myc gene are in correlation with the disease progression.

The chemokine CCL2 is considered as one of the main effectors driving postischemic infiltration of monocytes into the brain parenchyma. New experimental data, however, suggest that CCL2 could also participate in blood–brain barrier (BBB) ‘opening’ during the transmigration of monocytes. The current study examines the role of CCL2 in regulating BBB permeability after ischemia in vitro. To address this issue, an in vitro BBB model (coculture of astrocytes and brain endothelial cells) was subjected to 5 h of oxygen glucose deprivation, followed by reoxgenation ( in vitro ischemia/reperfusion (I/R)) for 0 to 48 h. During reperfusion, there was a biphasic enhancement of barrier permeability, with a 200-fold increase in barrier permeability to FITC-albumin at 6 h and a further period of disruption around 24 h. The latter coincided with increased secretion of CCL2 by both astrocytes and brain endothelial cells and increased levels of the CCL2 receptor, CCR2. Applying antisense oligonucleotide or neutralizing antibody to block CCL2 significantly decreased I/R-induced enhancement of BBB permeability (approximately twofold) and redistribution of tight-junction (TJ) proteins (occludin, zonula occluden-1, 2, claudin-5). Similarly, absence of CCR2 from endothelial cells caused stabilization of TJ complexes and decreased the permeability of brain endothelial barrier during in vitro I/R. These data suggest CCL2/CCR2 has an important role in regulating brain endothelial permeability and might be a potential novel therapeutic target for stroke.

Background. Factor V (FV) inhibitors are a rare disorder reported for the first time about fifty years ago, mostly with the unknown cause. The appearance of FV inhibitors is usually preceded by surgery, infections, administration of antibiotics or transfusions. Clinical manifestations of the presence of FV inhibitors vary from mild to severe and in some instances fatal hemorrhage. Case report. A healthy 51-year-old man with severe multiple injuries (traffic accident), and hemorrhage, which occurred during the orthopedic treatment, was admitted with hemoptysis, epistaxis and hematoma of the right upper leg, and with prolonged prothrombin time (PT), and activated partial thromboplastin time (aPTT). Treatment with vitamin K, fresh-frozen plasma and cryoprecipitate stopped the hemorrhage, but the results of coagulation tests were not normalized. The correction of aPTT and PT with normal plasma showed the decreased activity of FV (1%) due to the presence of inhibitors (titer 17.5 IU). The abnormal results of coagulation tests remained for three weeks, but without clinically manifested hemorrhagic syndrome. At the fourth week after the appearance of FV inhibitors PT, aPTT and the activity of FV became normal and antibodies disappeared spontaneously. Conclusion. Our patient with polytrauma developed a mild hemorrhagic syndrome due to the presence of FV inhibitors five weeks after the accident. Hemorrhage was treated with substitution therapy. The cause of the development of FV inhibitors was unclear (?fibrin glue? was not used during the orthopedic treatment). Factor V inhibitors disappeared spontaneously within four weeks. The fast spontaneous disappearance of FV inhibitors in our patient, confirmed the observations of some authors that they disappeared faster in those patients who were surgically treated prior to their appearance.

Ischemic preconditioning (PC) can markedly reduce ensuing ischemic damage. Although most attention has focused on the neuronal effects of PC, the authors have recently shown that ischemic PC reduces ischemia-induced cerebrovascular damage. In vivo, it is difficult to ascertain whether this is a direct cerebrovascular effect of PC. This study, therefore, examined whether cerebral endothelial cells can be preconditioned in vitro in the absence of other cell types. Experiments were performed on an immortalized mouse brain endothelial cell line or primary cultures of mouse brain microvessel endothelial cells. Cells were exposed to oxygen glucose deprivation (OGD) of either short duration, as a PC stimulus, or a long duration (5 hours) with or without reoxygenation to induce endothelial damage. Endothelial injury was assessed by measuring lactate dehydrogenase release and the expression of intercellular adhesion molecule-1 at the protein and mRNA levels. Experiments indicated that 1 hour of OGD was the optimal PC stimuli and that a 1 or 3 day interval was the optimal time interval between the PC stimulus and the injurious event. Preconditioned cells had less lactate dehydrogenase release during OGD (± reoxygenation) and reduced intercellular adhesion molecule-1 expression after OGD with reoxygenation. This study shows that cerebral endothelial cells can be directly preconditioned. The importance of this phenomenon in the overall effects of PC on the brain remains to be elucidated. Understanding the protective mechanisms elicited by PC may give insight into how to prevent ischemia-induced vascular damage (e.g., hemorrhagic transformation).

Kikuchi-Fujimoto disease (KFD), also know as histiocytic necrotizing lymphadenitis, is a benign disorder characterized histologically by necrotic foci surrounded by histiocytic aggregates, and with the absence of neutrophils. KFD was recognized in Japan, where it was first described in 1972. The disease is most commonly affecting young women. The cause of the disease is unknown, and its exact pathogenesis has not yet been clarified. Many investigators have postulated viral etiology of KFD, connecting it with Epstein Barr virus, human herpes simplex virus 6 parvo B 19, but also with toxoplasmic infection. Kikuchi-Fujimoto disease is usually manifested with lymphadenopathy and high fever, and is associated with lymphopenia splenomegaly, and hepatomegaly with abnormal liver function tests arthralgia, and weight loss. The disease has the tendency of spontaneous remission, with mean duration of three months. Single recurrent episodes of KFD have been reported with many years? pauses between the episodes. Kikuchi-Fujimoto disease may reflect systemic lupus erythematosus (SLE), and self-limited SLE-like conditions. Final diagnosis could only be established on the basis of typical morphological changes in the lymph node, and lymph node biopsy is needed for establishing the diagnosis. Lymphadenopathy in a patient with fever of the unknown origin could provide a clue to the diagnosis of lymphoma, tuberculosis, metastatic carcinoma, toxoplasmosis and infectious mononucleosis. As KFD does not have any classical clinical features and laboratory characteristics, it may lead to diagnostic confusion and erroneous treatment. We described a case of KFD, and suggested that this disease should be considered as a possible cause of fever of the unknown origin with lymphadenopathy.

The predictive value of Human Epidermal growth factor Receptor 2 (HER-2) on the response to chemotherapy and endocrine therapy in breast cancer patients has not yet been determined. The expression of other biomarkers in breast cancer can further influence the response to therapy. The aim of our study was to investigate if status of steroid receptors (SR) influenced the response to anthracycline-containing chemotherapy and tamoxifen in a group of HER-2 positive advanced breast cancer patients. Forty breast cancer patients, who were entered into the various prospective clinical trials conducted at the Institute of Oncology and Radiology of Serbia during their metastatic phase of disease, were involved into this analysis. Steroid receptors content were determined both by biochemical method and immunohistochemical (ICH) method, while HER-2 content were determined only by ICH method. Twelve out of 40 women were sequentially treated by anthracycline-containing chemotherapy and, always upon disease progression with antiestrogen tamoxifen. The objective response to anthracycline therapy was obtained in 4 out of 12 patients (RR=0.33, CI 95%=0.05-0.61). In three of them the response to tamoxifen was noticed, as well. Of 8 anthracycline resistant patients in this group, 7 patients also had disease progression as best response to tamoxifen despite the fact that most of them (5 out of 7 tamoxifen resistant women) had positive SR status. Our results showed a trend (Fisher test, p=0.06) that clinical response to anthracycline-containing chemotherapy might be of some predictive value for the response to subsequent tamoxifen therapy in HER-2 positive advanced breast cancer patients. However, these results were obtained on a small number of patients, so further investigation is warranted.

Introduction. Multiple myeloma (MM) is characterized by the presence of neoplastic proliferating plasma cells. The tumor is generally restricted to the bone marrow. The most common complications include renal insufficiency, hypercalcemia, anemia and reccurent infections. The spectrum of MM neurological complications is diverse, however, involvement of MM in the cerebrospinal fluid (CSF) and leptomeningeal infiltration are rare considered. In about 1% of the cases, the disease affects the central nervous system (CNS) and presents itself in the form of localized intraparenchymal lesions, solitary cerebral plasmocytoma or CNS myelomatosis (LMM). Case report. We presented the clinical course of a 55-year-old man with MM and LMM proven by malignant plasma cells in the CSF, hospitalized with the pain in the thoracic spine. His medical history was uneventful. There had been no evidence of mental or neurological impairment prior to the seizures. Physical examination showed no abnormalities. After a complete staging, the diagnosis of MM type biclonal gammopathia IgG lambda and free lambda light chains in the stage III was confirmed. The treatment started with systemic chemotherapy (with vincristine, doxorubicin plus high-dose dexamethasone - VAD protocol), radiotherapy and bisphosphonate. The patient developed weakness, nausea, febrility, dispnea, bilateral bronchopneumonia, acute renal insufficiency, confusions, headaches and soon thereafter sensomotor aphasias and right hemiparesis. The patient was treated with the adequate therapy including one hemodyalisis. His neurological status was deteriorated, so Multislice Computed Tomography (MSCT) of the head was performed and the findings were normal. Analysis of CSF showed pleocytosis, 26 elements/ mL and increased concentrations of proteins. Cytological analysis revealed an increased number of plasma cells (29%). Electrophoretic analysis of proteins disclosed the existance of monoclonal components in the serum, urine and CSF. Immunofixation electrophoretic and quantitative nephelometric tests confirmed Biclonal multiple myeloma of IgG lambda and light chain lambda isotypes. Analysis of neurothropic viruses with ELISA methods was negative. Once the presence of LMM was confirmed, the patient received intrathecal chemotherapy with methotrexate, cytosine arabinoside, dexamethasone three times a week, and systemic high doses of dexamethasone iv like a single agent without craniospinale irradiations. Despite the treatment, the patient died one month after the diagnosis. Autopsy was not performed. Conclusion. Presented patient, as well as most other patients with MM progressing to CNS infiltration was in the stage III. In addition to the detailed clinical examination, and all investigations required for MM diagnosis and staging of the disease, we introduced the additional CSF examination and calculation of kappa lambda ratio, that helped us make an early diagnosis and prognosis of MM with LMM. Although LMM had a low prevalence, it could be more frequent than expected especially in patients with high risk. CSF examination with positive plasma cells and abnormal morphology remains the hallmark for diagnosing CNS infiltration.

The initial use of immunosuppressive therapy (IST) in severe aplastic anemia (sAA) or reapplication of IST-centered methods following disease relapse is successful only in well-selected patients. The potential treatment by autologous stem cell (SC) transplant in sAA is still an innovative/pioneering therapeutic approach. To our best knowledge, this is the second published case of autologous SC transplant in sAA. The aim of this work was to optimize mobilization and timing for SC harvesting - using our own controlled-rate cryopreservation - with higher CD34(+)/CD90(+) subset yield and recovery in order to obtain complete and long-term hematopoietic reconstitution following autologous SC transplant. We report a 35 year-old sAA male patient who initially underwent IST using rabbit ATG and Cyclosporine A (CsA). He was supportive transfusion dependent for the whole period of IST-phase. After the second IST-cycle, polymorphonuclear (PMN) cell count increase (>2.0 × 10(9)/L) was observed, when SC mobilization, two large volume leukapheresis procedures and following autologous transplant were performed. The yields of harvested CD34(+) and CD34(+)/CD90(+) cells were 5.75 × 10(6)/kgbm and 1.7 × 10(6)/kgbm, respectively. The quantity of applied CD34(+) and CD34(+)/CD90(+) cells in autologous SC transplant were 5.45 × 10(6)/kgbm (7-AAD(CD34)(+)(viability)=95.42%) and 1.63 × 10(6)/kgbm (7-AAD(CD34)(+)(/CD90)(+)(viability)=95.42%), respectively. Hematopoietic reconstitution registered due to second month after autologous SC transplant and he is 24 months in complete medullar, hematological and clinical remission, with normal cytogenetic status - applying only continuous CsA therapy. The results obtained strongly confirm that in sAA, with no allogeneic SC donor, autologous transplant can result in a successful clinical outcome. We suggest that CD34(+)/CD90(+) subset count in peripheral blood and/or cell-harvest could be more valuable predictive factor than total CD34(+) quantity of optimized collection-timing and superior treatment efficacy of autologous SC transplant in sAA.

Background/Aim. In comparison to standard therapy autologous stem cell transplant (ASCT) with high doses melphalane has improved treatment of multiple myeloma (MM) patients. The aim of this study was to evaluate the results of treatment of MM patients in our center with ASCT conditioning with melphalane or combining busulphane, cyclophosphamide and melphalane. Methods. We performed 62 ASCT procedures in 47 patients from 1998 till 2008. Single ASCT were performed in 32 patients (68%), after 3-6 cycles of vincristine, adriamycin, dexamethasone (VAD) protocol and double in 9 patients (19%). Secondary ASCT was performed in 6 (13%) patients during progression of the disease after single ASCT. Mobilization of stem cells (SC) was either with a combination of etoposide and cyclophosphamide with granulocyte colony-stimulating factor (G-CSF) in 39 (83%) patients or cyclophosphamide plus G-CSF in 8 (17%) patients. All the patients received G-CSF five days after the ASCT and maintenance therapy afterwards in the patients with a complete remission (CR) or a partial remission (PR) was interferon alpha in 14 (30%) patients, thalidomide in 13 (28%) and others in 12 (26%) patients. Results. Median engraftment was on 12th day. In a 50-month follow-up period 64% patients were alive. The overall response rate (ORR), which was reached in 38 (80%) patients, was better in the group of patients treated in the early phase of MM. Totally 25 (53%) patients were without progression in a 25-month follow-up period. Twenty patients met criteria for CR + VGPR (very good partial remission), that was 5 patients more than in the period before ASCT. Fourteen (30%) patients died and median time till death was 17 months. Conclusion. The ASCT perfomed in early phase of MM after VAD induction had a significant influence on the treatment of MM patients. Reaching CR + VGPR before and after the ASCT is predictive factor for overall survival (OS) or prolongation of period till recidive appears, progression, therapy withdrowal or death.

A substantial body of evidence suggests that nicotine adversely affects cerebral blood flow and the blood-brain barrier and is a risk factor for stroke. The present study investigated the effect of nicotine on cerebrovascular endothelium under basal and ischemia/reperfusion injury under in vivo condition. Nicotine (2 mg/kg sc) was administered to mice over 14 days, which resulted in plasma nicotine levels of ∼100 ng/ml, reflecting plasma concentrations in average to heavy smokers. An analysis of the phenotype of isolated brain microvessels after nicotine exposure indicated higher expression of inflammatory mediators, cytokines (IL-1β, TNF-α, and IL-18), chemokines (CCL2 and CX3CL1), and adhesion molecules (ICAM-1, VCAM-1, and P-selectins), and this was accompanied by enhanced leukocyte infiltration into brain during ischemia/reperfusion ( P < 0.01). Nicotine had a profound effect on ischemia/reperfusion injury; i.e., increased brain infarct size ( P < 0.01), worse neurological deficits, and a higher mortality rate. These experiments illuminate, for the first time, how nicotine regulates brain endothelial cell phenotype and postischemic inflammatory response at the brain-vascular interface.

Brain edema is associated with a variety of neuropathological conditions such as brain trauma, ischemic and hypoxic brain injury, central nervous system infection, acute attacks of multiple sclerosis, and brain tumors. A common finding is an inflammatory response, which may have a significant impact on brain edema formation. One critical event in the development of brain edema is blood-brain barrier (BBB) breakdown, which may be initiated and regulated by several proinflammatory mediators (oxidative mediators, adhesion molecules, cytokines, chemokines). These mediators not only regulate the magnitude of leukocyte extravasation into brain parenchyma, but also act directly on brain endothelial cells causing the loosening of junction complexes between endothelial cells, increasing brain endothelial barrier permeability, and causing vasogenic edema. Here we review junction structure at the BBB, the effects of pro-inflammatory mediators on that structure, and focus on the effects of chemokines at the BBB. New evidence indicates that chemokines (chemoattractant cytokines) do not merely direct leukocytes to areas of injury. They also have direct and indirect effects on the BBB leading to BBB disruption, facilitating entry of leukocytes into brain, and inducing vasogenic brain edema formation. Chemokine inhibition may be a new therapeutic target to reduce vasogenic brain edema.

Background. Although the patients with diagnosed B-NHL are classified into the same disease stage on the basis of clinical, histopathological, and immunological parameters, they respond significantly different to the applied treatment. This points out the possibility that within the same group of lymphoma there are different diseases at molecular level. For that reason many studies deal with the detection of gene alterations in lymphomas to provide a better framework for diagnosis and treatment of these hematological malignancies. Aim. To define genetic alterations in the B-NHL with highest possibilities for diagnostic purposes and molecular detection of MRD. Methods. Formalin fixed and paraffin embedded lymph node tissues from 45 patients were examined by different PCR techniques for the presence of IgH and TCR ? gene rearrangement; K-ras and H-ras mutations; c-myc amplification and bcl-2 translocation. There were 34 cases of B-cell non-Hodgkin?s lymphoma (B-NHL), 5 cases of T-cell non-Hodgkin?s lymphoma (T-NHL) and 6 cases of chronic lymphadenitis (CL). The mononuclear cell fraction of the peripheral blood of 12 patients with B-NHL was analyzed for the presence of monoclonality at the time of diagnosis and in 3 to 6 months time intervals after an autologous bone marrow transplantation (BMT). Results. The monoclonality of B-lymphocytes, as evidenced by DNA fragment length homogeneity, was detected in 88 % (30/34) of B-NHL, but never in CL, T-NHL, or in normal PBL. Bcl-2 translocation was detected in 7/31 (22.6%) B-NHL specimens, c-myc amplification 9/31 (29%, all were more than doubled), K-ras mutations in 1/31 (3.23%) and H-ras mutations in 2/31 (6.45%) of the examined B-NHL samples. In the case of LC and normal PBL, however, these gene alterations were not detected. All the patients (12) with B-NHL had dominant clone of B-lymphocyte in the peripheral blood at the time of diagnosis while only in 2 of 12 patients MRD was detected 3 or 6 months after BMT. Conclusion. Because it is quick and simple, PCR analysis of clonal IgH rearrangements is very useful when diagnostic assistance is required. This technique is also very efficient for tracking minimal residual disease in lymphomas and leukemia's and for monitoring clonal evolution in acute and chronic lymphoblastic leukemia's and lymphomas. The presence of other genetic alterations, which we detected, should serve as an additional prognostic or predictive factor in the patients with B-NHL.

Hemorrhagic transformation upon reperfusion therapy has focused attention on ischemia-induced endothelial dysfunction. This study examined whether hyperglycemia may induce hemorrhagic transformation by enhancing endothelial mitochondrial damage during ischemia and whether preconditioning (PC) stimuli may limit ischemia-induced endothelial damage. In vivo, rats received 2.8 M D-glucose or arabinose (1 ml/100 g; i.p.) prior to undergoing two hours of middle cerebral artery occlusion and transcardiac fixation for electron microscopy. In vitro, brain endothelial cells were exposed to a PC impulse (short-term oxygen glucose deprivation; OGD) prior to an injurious event (5 hours OGD). Endothelial injury was assessed by measuring lactate dehydrogenase release. Hyperglycemia during cerebral ischemia resulted in marked changes in endothelial morphology and mitochondrial swelling. Thus, in the ischemic hemisphere, there was no evidence of endothelial mitochondrial swelling in normoglycemic rats (mean profile width 0.22 ± 0.04 vs. 0.17 ± 0.01 µm in contralateral hemisphere) but there was marked swelling in hyperglycemic rats (0.44 ± 0.02 µm). In vitro, cells preconditioned with one hour of OGD one day prior to 5 hours of OGD, showed reduced lactate dehydrogenase release (p < 0.05). In conclusion, hyperglycemia may have specific adverse effects on endothelial cell mitochondria during ischemia. Preventing those effects may help to ameliorate blood-brain barrier disruption on reperfusion. Insights into how to prevent endothelial injury may come from determining the mechanisms involved in endothelial preconditioning.

It has been reported that cytarabine, acting by at least two different mechanisms, enhances the cytotoxic effect of cisplatin in in vitro systems. The aim of this open, prospective, randomised study was to estimate the eventual benefits from the inclusion of high-dose cytarabine in the cisplatin-5-fluorouracil (5-FU) regimen as first-line treatment of patients with advanced head and neck cancer. The study recruited successive patients with unresectable grade I/II head and neck cancer who were not suitable for irradiation treatment (T any N3 or T4 N2C), metastatic or previously irradiated. All patients gave their informed consent. A joint ear, nose and throat (ENT) oncological committee performed the selection. A total of 170 patients were included in the study. Patients randomised to arm A were given 1000 mg/m(2) cytarabine on day 1 preceding for 6 h cisplatin infusion, 30 mg/m(2)/24 h cisplatin intravenous (i.v.) bolus days 1-4 and 1000 mg/m(2)/24 h 5-FU in a 4-h infusion on days 1-4. Patients in arm B were given cisplatin and 5-FU in the same dosage and schedule as in arm A. Additional irradiation+/-surgery was performed if and when feasible. Patients in both arms were well balanced with regard to clinical variables. The following results were obtained: Arm A: 84 patients were included, 74 were evaluable for activity;complete response (CR) 8 (11%), partial response (PR) 40 (54%), stable disease (SD) 11 (15%), progressive disease (PD) 15 (20%). The overall response rate (RR) based on the evaluable patients was 48/74 (65%, 95% confidence interval (CI) 54-75%); The RR based on an intent-to-treat analysis was 57%, 95% CI 47-67%; Median survival was 13 months; There were 50 episodes of granulocytopenia grade IV and 15 of febrile neutropenia per 316 cycles. Arm B: 86 patients were included, 80 were evaluable for activity;CR 7 (9%), PR 29 (36%), SD 10 (12.5%), PD 34 (42.5%); The overall RR based on the evaluable patients was 36/80 (45%, 95% CI 35-56%); The RR based on an intent-to-treat analysis was 42%, 95% CI 32-52%; Median survival was 8 months; There were 14 episodes of granulocytopenia grade IV and 7 febrile neutropenias per 324 cycles. The RR was significantly higher in arm A (P=0.013), power (one-sided) 80%. The proportion of patients from the appropriate subset who achieved a clinical response making additional treatment feasible was higher in arm A (P=0.00015), as well as the proportion of patients with a performance status 2+3 achieving a response (P0.0001). Using the Log-rank test, patients from arm A achieved a significantly longer survival (P=0.009), with the probability of survival at 12 months of 0.58 for patients in arm A and 0.28 for patients in arm B. Grade IV granulocytopenia and thrombocytopenia were more frequent in arm A. Due to its haematological side-effects, cytarabine might not be the ideal drug to modulate the cytotoxicity of cisplatin. However, other modulators of its activity could be of interest for further studies in head and neck cancer patients.

Background and aims There is no much data in the literature about the effects of montelukast on the skin reactivity to inhaled allergens. We analysed whether the skin reactivity to allergens significantly changed after 30 days of daily application of montelukast. Methods Thirty children with asthma (7–14 y) and with skin reactivity to inhaled allergens were receiving 5 mg of montelukast daily for 30 days. Skin prick testing was done before and after therapy. Size of the papule was measured at twentieth minute after allergen application as quantitative (mean of the largest and normally set diameter on it) and qualitative-bimodal: positive/negative (cut-point: 3 mm). The control group consisted of children of the same age (n = 30) with positive skin reactivity who did not receive any medication and has been tested in the same way. The size of the papule, and the number of positive/negative tests for both groups were compared before and after therapy. The frequency of test conversion in both directions (crossing of positive to negative and vice versa) in the experimental group was compared to the control group. Results After thirty days of montelukast therapy the size of the papule in both groups was not significantly changed (p > 0.05). Compared to the control group, in the experimental group, there was no significant difference in the change of skin reactivity to allergens, either quantitatively (p > 0.05), or qualitatively (p > 0.05) evaluated.

Aim: Cancer suffering patients often delay going to the doctor, leading to the extension of the time elapsed from onset of symptoms to treatment of disease (total delay time; TDT), which can have a negative impact on disease progression and treatment outcomes. The aim of this study was to determine factors affecting two individual segments of TDT: time from the onset of symptoms to seeking medical advice (patient delay time; PDT) and time of first contact with the doctor to starting treatment (system delay time; SDT). Methods: 800 breast cancer patients from 10 centers residing in Serbia were investigated using a uniform questionnaire. TDT was determined using the 8 individual scales, including one referring to patient -related factors, and 7 related to subsequent steps in a typical diagnostic process, i.e. health system related factors. Regression models were performed using the variables from the questionnaire relating to the different personal characteristics and environmental features related to patients. Results: Mean PDT, SDT and TDT were 4.5, 9.2 and 12.9 weeks; 20 % of patients had PDT >12 weeks. In a multivariate regression model longer PDT and SDT were associated with distrust in the health care system and treatment success, lack of time to go to the doctor, and refusing to recognize the severity of the disease (p Conclusions: Several factors, mainly related to psychological and behavioral attributes, strongly determined both delay in seeking medical advice and treatment onset for breast cancer, indicating the need for implementing measures to raise awareness about the importance of early reporting to the doctor after noticing symptoms. Disclosure: L. Stamatovic: The study conducted in order to collect the data was sponsored by Roche; S.D. Mamula Masic: Full time employee of Roche; D. Vukotic: The study conducted in order to collect the data was sponsored by Roche; Z. Kovac: The study conducted in order to collect the data was sponsored by Roche; Z. Mihailovic: The study conducted in order to collect the data was sponsored by Roche; K. Zdravkovic: The study conducted in order to collect the data was sponsored by Roche; D. Petkovic: The study conducted in order to collect the data was sponsored by Roche; T. Bagi: The study conducted in order to collect the data was sponsored by Roche; N. Milic: The study conducted in order to collect the data was sponsored by Roche. I have been done statistical analysis for this abstract.

Aim: The aim of this study was to investigate difference in clinical outcome in patients with primary advanced or metastaitic endometrial cancer treated with standard chemotherapy doxorubicin-cisplatin (DOX-CDDP), regarding and focusing on histological subtype. Methods: Eligible patients had histologically-proven advanced and/or metastatic endometrial adenocarcinoma and were chemotherapy-naive. Treatment consisted of CDDP 50 mg/m2 added to DOX 60 mg/m2, every 3 weeks. Kaplan Maier method and Log rank test were used to assess survival prognostic factors. Median age was 64 years (range 34-77). Patients were divided into 2 groups according histopathologic type: Type 1 EC- endometrioid and Type 2 EC- serous/clear cell/ undifferentiated type. Between 2007 and 2012, we analysed 18 patients (60%) with Type 2 EC (6 papillary serous, 11 clear cell, 1 undifferentiated) and 12 patients (40%) with Type 1 EC. Results: FIGO stages were as follows: FIGO III = 18 (60%), FIGO IV = 12 (40%). Response for Type 1 EC was as follows: 12 (100%) patients had partial and complete remission (responders). Response for Type 2 was as follows: 7 patients (39%) had stable disease(SD) and 11 patients (61%) had partial and complete remission (responders).The combination DOX–CDDP provided a significantly greater therapeutic response in Type 1 EC compared to Type 2 EC ( Fischer Exact test; p =0.024). At time of analysis 76% of patients were still alive after median follow up of 21 months (9-58). Median progression free survival (PFS) for Type 1 EC was 18 months (range 8-56). Median progression free survival (PFS) for Type 2 EC was 11 months (5-26), so there was demonstrated significant difference between Type 1 and Type 2 EC according PFS (Log-Rank test; p = 0.0014). It was also shown trend in significance between Type 1 and 2 EC according OS (Log-Rank test; p = 0.062). Conclusions: Type 2 endometrial cancer is rare and differs from Type 1 especially for the high frequency of distant metastasis. Our results indicate that there is demonstrated worse response and outcomes to standard chemotherapy in Type 2 EC compared to Type 1 EC, so multicentric studies are needed to better define appropriate management for these kind of malignancies. Disclosure: All authors have declared no conflicts of interest.