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ΣΚΟΠΟΣ: Παρά τις εκτεταμένες μελέτες δεν είναι γνωστό εάν οι ποικίλες μη-φυσιολογικές λειτουργίες των Β κυττάρων που παρατηρούνται σε ασθενείς με Συστηματικό Ερυθηματώδη Λύκο (ΣΕΛ) είναι δευτεροπαθείς ή πρωτογενώς συμβάλλουν στην παθογένεια της νόσου. Η στοχευμένη θεραπεία της παροδικής εξάλειψης των Β κυττάρων μπορεί να είναι ωφέλιμη σε ασθενείς με ενεργό υπερπλαστική νεφρίτιδα ΣΕΛ. Συγκριτικές μελέτες προ- και μετά παροδική εξάλειψη δίνουν την ευκαιρία να διερευνηθεί ο παθογενετικός ρόλος των Β κυττάρων. Σκοπός μας ήταν η μελέτη της σύνθεσης του ρεπερτορίου των βαριών αλυσίδων των ανοσοσφαιρινών (Ig-VH/DH/JH) των Β κυττάρων και η ανεύρεση ενδεχόμενων αλλαγών σε πρώϊμη φάση της αναγέννησης μετά χορήγηση Rituximab (anti-CD20 αντίσωμα), καθώς και εάν οι αλλαγέςσυνδέονται με ενεργότητα νόσου.ΥΛΙΚΟ-ΜΕΘΟΔΟΙ: Μελετήθηκαν 7 ασθενείς με ενεργό υπερπλαστική νεφρίτιδα ΣΕΛ οι οποίοι έλαβαν ως θεραπεία Rituximab, παράλληλα με 4 υγιή άτομα που χρησίμευσαν ως μάρτυρες. Σε 3 ασθενείς έγιναν επιπλέον μελέτες στο χρονικό σημείο του 33-35% της ανάκτησης των Β κυττάρων σε σχέση με το ποσοστό τους πριν τη θεραπεία. Η μελέτη έγινε σε γενωμικό DNA, που αποκτήθηκε από περιφερικό αίμα, με τη μέθοδο ενίσχυσης και κλωνοποίησης των ανασυνδυασμένων γονιδίων Ig-VH/DH/JH.ΑΠΟΤΕΛΕΣΜΑΤΑ: Ανάλυση 1246 Ig-VH/DH/JH αλληλουχιών έδειξε ότι, σε αντίθεση με τους 4 υγιείς μάρτυρες, και στους 7 ασθενείς που εξετάσθηκαν στη φάση ενεργού νεφρίτιδος εντοπίσθηκαν ομάδες κλωνικά σχετιζόμενων αλληλουχιών. Επιπλέον, διαπιστώθηκαν υπερμεταλλάξεις στη VH DNAαλληλουχία ενδεικτικές της ύπαρξης κλωνικής εξέλιξης/διαφοροποίησης συγκεκριμμένων Β κυττάρων. Ανάλυση του ρεπερτορίου (χρησιμοποιούμενα γονίδια VH/DH/JH, CDR3 περιοχή) τόσο στους ασθενείς, όσο και στους μάρτυρες δεν έδειξε διαφορές. Αν και το ποσοστό των υπερμεταλλαγμένων DNA αλληλουχιών δεν διέφερε μεταξύ ΣΕΛ και μαρτύρων, οι ασθενείς είχαν περισσότερες υπερμεταλλάξεις/VH αλληλουχία. Σε 3 ασθενείς που εξετάσθηκαν μετά Rituximab (1ος πλήρη ύφεση, 2ος μερική ύφεση, 3ος ενεργό νόσο/θεραπευτική αποτυχία) το «νέο» ρεπερτόριο των Β κυττάρων ήταν συγκρίσιμο με αυτό των μαρτύρων, αλλα περιείχε περισσότερες ολιγοκλωνικές εκπτύξεις. Το φαινόμενο ήταν ανεξάρτητο από την κλινική ενεργότητα της νόσου και από την εξαφάνιση των αυτοαντισωμάτων, υποδεικνύοντας εμμένουσα ανωμαλία του ρεπερτορίου των Β κυττάρων σε ΣΕΛ. Περαιτέρω ανάλυση με πολυνομιακό στατιστικό πρότυπο για την εντόπιση αντιγονικής επιλογής, έδειξε ότι μόνο 25% των ολιγοκλωνικών εκπτύξεων προήλθαν από επιλογή. ΣΥΜΠΕΡΑΣΜΑ: Η παροδική εξάλειψη των Β κυττάρων από το Rituximab,διατηρεί στο «νέο» Ig-VH/DH/JH ρεπερτόριο την αναμενόμενη ετερογένεια, ωστόσο οι ολιγοκλωνικές εκπτύξεις δεν διορθώνονται. Η ανωμαλία αυτή, που περιγράφεται για πρώτη φορά, φαίνεται ότι είναι πρωτογενής και μπορεί να συμβάλλει στην παθογένεια της νόσου προσφέροντας ιδιαίτερο πλεονέκτημα επιβίωσης/πολλαπλασιασμού σε συγκεκριμμένα Β κύτταρα που ήδη έχουν,ή/και δυνατόν να αποκτούν αυτοδραστικές ιδιότητες.

Objective: Detailed analyses of clinical characteristics of myocardial infarction (MI) as an essential thrombocythemia (ET)- and polycythemia vera (PV)-related complication have been so far presented mostly as case reports. Therefore, the aim of this retrospective analysis was to evaluate the main cardiological and hematological characteristics for better understanding myocardial complications in ET/PV. Methods: A retrospective analysis was carried out involving 263 patients diagnosed with ET or PV (155/108) between 1998 and 2014. Fourteen patients suffered MI during the hematological follow-up. Their clinical characteristics were compared to 162 patients (97 ET and 65 PV patients) who did not exhibit any major thrombotic complications (MI, stroke/transient ischemic attack, and venous events) before or after hematological diagnosis of ET/PV. Results: Fourteen MI events occurred among the 263 patients (5.3%). Vascular risk factors were found in 92.9% (13/14) of analyzed cases. In all, 71.4% of the MI complications developed within 12 months after the diagnosis of ET/PV. The coronary angiography findings revealed ST-elevation MI in four cases and non-ST-elevation MI in 10. Significant stenosis of coronary arteries requiring percutaneous coronary intervention with a stent implantation was present in seven cases, while three had complex stenoses or previous grafts/stents. All of them had undergone coronary artery bypass graft operations. Conclusion: The results of the present study suggest that early detection and consideration of individual management of vascular risk factors in ET/PV patients are also important. Furthermore, a better theoretic understanding of platelet activation and role of leukocytes in myeloproliferative neoplasm-related thrombosis could open new perspectives in thrombosis prediction and prevention.

Infectious complications remain one of the most challenging concerns for the burn team. The goal of this study was to evaluate the diagnostic and therapeutic approaches and prognosis in burn patients with septic shock. This retrospective study included patients with severe burn injury who were admitted to a burn intensive care unit (ICU). Demographic and clinical data were recorded for each patient. The diagnostic and prognostic value of a number of clinical and laboratory parameters and various treatment options were evaluated. Sixty-four of the 378 patients (16.9%) were identified as having experienced a septic shock during ICU stay. The mortality rate of patients with septic shock was 46.9%. The main bacterial strains responsible for infection were Gram-negative bacteria (78.1%). Factors associated with outcome of septic shock on logistic regression analysis were presence of stage III of acute kidney injury (odds ratio [95% confidence interval] 2.03 [1.06-3.84]; P = 0.019), and lactate levels > 4 mmol/L during the first 48 hours of shock (odds ratio 1.92; 95% confidence interval: 1.02-3.62; P = 0.043). Prognosis of septic shock remains poor in burn patients with septic shock. The main causative pathogens of septic shock identified in our burn ICU were Gram-negative species. The main prognostic factors identified in this study were the presence of AKI, stage III, and high lactate levels early after the onset of septic shock.

Myelodysplastic syndromes represent a group of heterogeneous hematopoietic neoplasms derived from an abnormal multipotent progenitor cell, characterized by a hyperproliferative bone marrow, dysplasia of the cellular hemopoietic elements and ineffective erythropoiesis. Anemia is a common finding in myelodysplastic syndrome patients, and blood transfusions are the only therapeutic option in approximately 40% of cases. The most serious side effect of regular blood transfusion is iron overload.Currently, cardiovascular magnetic resonance using T2 is routinely used to identify patients with myocardial iron overload and to guide chelation therapy, tailored to prevent iron toxicity in the heart. This is a major validated non-invasive measure of myocardial iron overloading and is superior to surrogates such as serum ferritin, liver iron, ventricular ejection fraction and tissue Doppler parameters.The indication for iron chelation therapy in myelodysplastic syndrome patients is currently controversial. However, cardiovascular magnetic resonance may offer an excellent non-invasive, diagnostic tool for iron overload assessment in myelodysplastic syndromes. Further studies are needed to establish the precise indications of chelation therapy and the clinical implications of this treatment on survival in myelodysplastic syndromes.

Polycythemia vera and essential thrombocythemia comprise myeloproliferative neoplasms, the main characteristic of which is an over-production of bone marrow myeloid lineage cells. The diseases typically involve thrombosis and their treatment, apart from the strictly hematological aspect, involves antithrombotic agents. In this review we aspire to present the possible role of the newly developed oral anticoagulants in the treatment scheme of polycythemia vera and essential thrombocythemia.

Myocardial injury due to acute carbon monoxide intoxication is often refractory to standard resuscitation methods. Levosimendan is a new inotropic agent that is used in the treatment of acute and chronic heart failure. We report a case of severe cardiopulmonary compromise after exposure to carbon monoxide in a 40-year-old woman who was monitored using transthoracic echocardiography and the arterial pulse contour analysis system (FloTrac) and successfully treated with levosimendan.

The incidence of FLT3 mutations (internal tandem duplication and Asp835) was investigated in bone marrow samples from 97 patients with myelodysplastic syndrome [(MDS); excluding cases with refractory anaemia with excess blasts in transformation] at the time of diagnosis and several time points thereafter. Three patients had FLT3 mutations at presentation. Forty-two patients progressed to acute myeloid leukaemia (AML), including the three patients with FLT3 mutations at MDS diagnosis. Three additional patients acquired FLT3 mutations and progressed to AML in 1 month. FLT3 mutations seem to be a critical additional genetic event that transforms a minority of MDS patients to AML.

It has recently been demonstrated that a considerable subset of CLL patients (>50%) displays intraclonal diversification in their monoclonal Ig sequence, suggesting that the mechanism of ongoing somatic mutations is retained in a significant proportion of B CLL cells. The complexity of studying this phenomenon relies on the enclaved risk of misinterpreting superimposed mutations originating from experimental artifacts. We are still lacking a more detailed picture of inraclonal diversification regarding its frequency and developmental pattern in disease compartments other than blood. In order to clarify some of these questions, we analyzed a total of 675 Ig clones from 11 CLL patients. DNA was extracted from blood in 6 patients (1st group: pt.1 to 6) or from blood/marrow (BM)/lymph nodes (LN) obtained at the same in 5 patients (2nd group: pt.7-11). PCR was performed for 40 cycles with Taq Platinum polymerase and leader or FR1 VH1-6 primers and a consensus JH primer. Monoclonal PCR products were ligated to TA vector and 20 to 70 clones were sequenced. from both orientations. Polymerase mediated base error was 4.4x10−4 after 40 PCR cycles. Base differences observed in two or more clones out of 20, were verified twice and assigned as true intraclonal diversification. This was observed in 6 (pt 2, 6, 7, 9, 10, 11) out of 11 pts (54.5%). Pts 2, 6, 10, 11 were mutated and pt. 7, 9 unmutated in their Ig sequence. Intraclonal diversification was observed in pt. 2 and 6, whereas pt.7, 9, 10, 11 in the 2nd group, displayed intraclonal diversification in some or in all compartments. Base point mutations that were unique in one clone only were observed at the polymerase mediated error rate and were not verified by a second idependent experiment. In the 2nd group pt. 8 displayed lack of diversification both in blood and marrow. Pt.7 displayed diversification only in BM clones (2/64, 3.1%) but not in 71 blood derived or in 53 LN derived clones. Pt. 9 had diversified clones in blood only (2/26, 7.6%) and no diversified clones in 25 BM clones. Pt.10 had diversified clones both in blood and marrow with a distinct pattern: in blood (62 clones) 4 different patterns of diversification were observed in 54 clones (84%); 17/54 were evolving clones. In BM two of the 4 patterns of diversification present in blood were observed in 28/57 clones (51.7%); ten were evolving clones. In pt.11, no diversified clones were present in blood (50 clones) whereas marrow had 4/60 diversified clones (6,66%, two patterns of diversification) and LN had 20/40 diversified clones (50%, two patterns of diversification); 8/20 diversified were evolving clones. The patterns of diversification were different bettween LN and BM in this patient. Base changes in 110 diversified clones were in FRs rather than CDRs (16:1) and resulted in S(silent) rather than R(replacements), S/R 2:1. In conclusion, intraclonal diversification must be considered true if identical base substitutions are observed in minimum two clones. Pattern and frequency are not identical in B CLL cells from different sites (blood vs BM vs LN). Preferance of FRs v CDRs and SvR substitutions implies that diversification procces is not antigen driven. The presence of a functional mechanism of ongoing somatic mutations in leukemic clones and the ability of diversification to target with altered frequency/patterns various disease compartments implies a role of tissue millieu in CLL clone evolution.

The monoclonal Ig expressed on CLL cells was considered to be identical in all cells of the leukemic clone in a CLL patient. However, intraclonal VDJ gene diversification was recently reported in CLL B cells using SSCP analysis and sequencing of Ig PCR products exhibiting altered mobility. We used a different approach, by cloning directly monoclonal VHDHJH PCR bands from CLL patients and sequencing a large number of clones. Peripheral blood from five patients with typical CLL was used to extract DNA.. In one patient DNA from bone marrow and a lymph node biopsy was also analysed. PCR was performed with VH family specific primers and a consensus JH primer for 30 cycles. Monoclonal bands were ligated to TOPO vector and 25 to 70 insert containing clones were sequenced from both orientations. Sequenses contigs were aligned with DNASTAR and were compared with the germ line counterpart using BLAST and IMGT data bases. Intraclonal base variation was observed in 8–33% of the clones in all five patients. One to five base point-mutations /clone were found and there were mostly transitions (in 79% of mutations). In one patient with two monoclonal Ig sequences, (one hypermutated V3-7 and one unmutated VH3-23) intraclonal variation was observed in 69% and 8% of the clones respectivelly. In two patients clones with partially shared mutations were observed indicating the further diversification of a parental subclone. In the patient that VHDHJH clones from blood/bone marrow/lymph node were obtained, a difference in the frequency of diversified clones was noted. All samples were obtained from the patient within one month to exclude the possibility of time related mutations in the monoclonal Ig of the patient. In the patient’s VH1-69 unmutated sequence, 19 of 64 (29,6%) of bone marrow derived clones had additional mutations but they were found in only 12 of 71 (16%) peripheral blood derived clones. Five of 53 (9,4%) lymph node derived clones had additional point mutations. Low R/S ratio and targeting of FR rather than CDR regions is against the hypothesis of antigen driven somatic mutations in a fraction of the CLL cells in a given patient. The difference in diversification rate between different tissues in the same patient raises the question of the role of the patient microenvironment/stroma in partcipating in these mutations observed in CLL subclones. With the analysis of large number of clones it is possible that all CLL patients may have at least some degree of intaraclonal VHDHJH gene diversification.