Péter Ilonczai

Stem Cell Therapy: A Promising and Prospective Approach in the Treatment of Patients With Severe Buerger’s Disease

No effective blood-flow enhancement therapies are available for patients with severe peripheral arterial disease (SPAD), thus amputation remains the only option for relief of rest pain or gangrene. Autologous bone marrow—derived stem cell therapy (ABMSCT) is an emerging modality to induce angiogenesis from endothelial progenitors. A total of 5 patients with SPAD were treated by ABMSCT using isolated CD34+ cells with characterized phenotype administered by intramuscular injections. The follow-up before and 1, 3, 6, 9, and 12 months after ABMSCT was based on clinical (rest pain, walking distance without pain, nonhealing ulcers, ankle-brachial index [ABI]) and laboratory (angiography, duplex and laser ultrasonography, TcPO2) parameters. Significant improvement of pain and walking distance was observed in all patients. Nonhealing ulcers disappeared in 3 patients and became smaller and thinner in 1 patient. The average of ABI improved significantly on the treated limb but did not change on the contralateral limb. New collaterals were detected by angiography in 3 patients, but duplex ultrasonography detected improvement in one patient only. Laser ultrasonography showed a mild significant change, TcPO2 values improved mainly on the foot. Severe adverse events were not observed. We conclude that ABMSCT with isolated CD34+ cells is safe, effective, and results in sustained clinical benefit for patients with SPAD.

Founder effect is responsible for the p.Leu131Phe heparin-binding-site antithrombin mutation common in Hungary: phenotype analysis in a large cohort

Essentials Antithrombin Budapest3 (ATBp3; p.Leu131Phe) causing heparin‐binding‐site defect is common. We studied the clinical and laboratory phenotype of a large Hungarian ATBp3 cohort (n = 102). Founder effect of ATBp3 was confirmed by 12 genetic markers; anti‐FXa AT assay was 100% sensitive. The spectrum of thrombotic symptoms was wide in ATBp3 patients including arterial thrombosis.

Management and outcome of pregnancies in women with antithrombin deficiency: a single-center experience and review of literature.

Women with antithrombin (AT) deficiency have an increased risk for pregnancy-associated venous thromboembolism (VTE) and adverse pregnancy outcome. AT deficiency is a rare thrombophilia with heterogeneous genetic background. Owing to the few cases reported in the literature, management strategies of pregnancy with AT deficiency are inconsistent. Our aim was to examine the type of the genetic defect, management, maternal, and pregnancy outcome in patients with hereditary AT deficiency. Five expectant mothers with AT deficiency were followed in our center to evaluate thrombotic events, and maternal and pregnancy outcomes. AT gene sequencing was performed in all cases, and levels of AT and anti-activated factor X were regularly measured to guide the risk-adopted anticoagulant prophylaxis. Three mothers had homozygous type II heparin-binding site mutations and two had heterozygous type I mutations of the gene encoding AT. Two women had additional factor V Leiden heterozygous mutations. Three maternal VTEs – four healthy newborns and five pregnancy losses – were observed. The risk of patients to VTE and adverse pregnancy outcome was found to associate with the homozygous type II heparin-binding site mutation of the AT gene. High risk of maternal VTE and frequent pregnancy complications were observed to associate with AT deficiency. Our results support the need of individualized, risk-adopted anticoagulant therapy in patients with AT deficiency.

Autológ csontveloi eredetu ossejtterápia eredmńye elorehaladott perifériás artériás érbetegségben

BACKGROUND AND AIMS Amputation is the only current option for relief of rest pain or gangrene in patients with severe peripheral arterial disease. Up to now, no effective blood-flow enhancement therapies are available. Autologous bone marrow-derived stem cell transplantation is an arising therapy modality with an option of building new blood vessels through endothelial stem and/or progenitor cells. PATIENTS AND METHODS Five patients with severe peripheral arterial disorder were treated by autologous bone marrow-derived stem cell therapy. CD34+, CD133+ and CD45+/- cell number and ratio were determined. CD34+ cells were isolated by magnetic separation and collected into a 10 ml sample. The cell suspension was administered by local intramuscular injections (0.5-1.0 ml injections in the musculus gastrocnemius). The follow-up (before; 1, 3, 6, 9 and 12 months after the autologous bone marrow-derived stem cell therapy) based on clinical (rest pain, walking distance without pain, changes of non-healing ischaemic ulcers, ankle-brachial index) and laboratory (angiography, Color- and Laser-Doppler scan, measurement of transcutaneous oxygen tension and endothelial function test) parameters was documented and analyzed. RESULTS Improvement of pain and walking distance was observed in all five cases. In three cases the non-healing ischaemic ulcers disappeared, in one other case they became smaller and thinner, and in one case no change was realized. The average of ankle-brachial index improved significantly (before: 0.41, twelve months after: 0.83). New collaterals were detected by angiography in three patients, but duplex ultrasonography detected improvement in one patient only. Before and 1, 6 and 12 months after stem cell therapy the transcutaneous oxygen tension changed on the foot from 18.80/16.78/23.83/37.50 mmHg, and on the calf from 36.66/31.25/45.00/37.30 mmHg. The macro- and microcirculation parameters did not show improvement after 1 month, however, after 3, 6, 9 and 12 months improved parameters were recorded. Severe adverse events were not observed. In one case elevated level of serum creatinine phosphokinase, and in another case a mild form of vasculitis were detected. CONCLUSION autologous bone marrow-derived stem cell therapy with isolated CD34+ cells is effective, safe and results in sustained clinical benefit for patients with severe peripheral arterial disease.

Evaluation of flow cytometric HIT assays in relation to an IgG-specific immunoassay and clinical outcome.

Heparin‐induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)–heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT.

Acquired haemophilia: An often overlooked cause of bleeding - Experience from a Hungarian tertiary care centre

Acquired haemophilia is a potentially life-threatening bleeding disorder. Its early diagnosis and treatment is of major importance. We evaluated the elapsed time between the first presentation of the bleeding symptoms and the correct diagnosis in the cases of the acquired haemophilia patients referred to our centre between 1999 and 2011. The causes and consequences of the often delayed diagnosis were also examined. The clinical and laboratory data of 13 patients with acquired haemophilia were analysed. Eleven patients had inhibitors to factor VIII (FVIII), in one case the autoantibody developed to factor XIII (FXIII) and in one to factor V (FV). The median period between the onset of the bleeding symptoms and the correct diagnosis was 1.5 months (3.0 days–9.0 months). In four cases 4.0–9.0 months were needed to establish the diagnosis. The main reason of this delay was that either the prothrombin time was used exclusively to evaluate haemostasis in primary care and also in some secondary care centres, or the prolonged activated partial thromboplastin time went unnoticed despite the obvious bleeding symptoms. Our observation underlines the importance of early referral of patients with unexplained bleeding symptoms to centres with appropriate laboratory facilities and experience in the diagnosis of bleeding disorders.

[Autologous bone marrow-derived stem cell therapy in patients with severe peripheral arterial disorder].

BACKGROUND AND AIMS Amputation is the only current option for relief of rest pain or gangrene in patients with severe peripheral arterial disease. Up to now, no effective blood-flow enhancement therapies are available. Autologous bone marrow-derived stem cell transplantation is an arising therapy modality with an option of building new blood vessels through endothelial stem and/or progenitor cells. PATIENTS AND METHODS Five patients with severe peripheral arterial disorder were treated by autologous bone marrow-derived stem cell therapy. CD34+, CD133+ and CD45+/- cell number and ratio were determined. CD34+ cells were isolated by magnetic separation and collected into a 10 ml sample. The cell suspension was administered by local intramuscular injections (0.5-1.0 ml injections in the musculus gastrocnemius). The follow-up (before; 1, 3, 6, 9 and 12 months after the autologous bone marrow-derived stem cell therapy) based on clinical (rest pain, walking distance without pain, changes of non-healing ischaemic ulcers, ankle-brachial index) and laboratory (angiography, Color- and Laser-Doppler scan, measurement of transcutaneous oxygen tension and endothelial function test) parameters was documented and analyzed. RESULTS Improvement of pain and walking distance was observed in all five cases. In three cases the non-healing ischaemic ulcers disappeared, in one other case they became smaller and thinner, and in one case no change was realized. The average of ankle-brachial index improved significantly (before: 0.41, twelve months after: 0.83). New collaterals were detected by angiography in three patients, but duplex ultrasonography detected improvement in one patient only. Before and 1, 6 and 12 months after stem cell therapy the transcutaneous oxygen tension changed on the foot from 18.80/16.78/23.83/37.50 mmHg, and on the calf from 36.66/31.25/45.00/37.30 mmHg. The macro- and microcirculation parameters did not show improvement after 1 month, however, after 3, 6, 9 and 12 months improved parameters were recorded. Severe adverse events were not observed. In one case elevated level of serum creatinine phosphokinase, and in another case a mild form of vasculitis were detected. CONCLUSION autologous bone marrow-derived stem cell therapy with isolated CD34+ cells is effective, safe and results in sustained clinical benefit for patients with severe peripheral arterial disease.

Antithrombin Debrecen (p.Leu205Pro) – Clinical and molecular characterization of a novel mutation associated with severe thrombotic tendency

INTRODUCTION Hereditary antithrombin (AT) deficiency is a rare thrombophilic disorder with heterogeneous genetic background and various clinical presentations. In this study we identified a novel AT mutation. Genotype-phenotype correlations, molecular characteristics and thrombotic manifestations of the mutation were investigated. MATERIALS AND METHODS Thirty-one members of a single family were included. Clinical data was collected regarding thrombotic history. The mutation was identified by direct sequencing of the SERPINC1 gene. HEK293 cells were transfected with wild type and mutant SERPINC1 plasmids. Western blotting, ELISA and functional amidolytic assay were used to detect wild type and mutant AT. After double immunostaining, confocal laser scanning microscopy was used to localize mutant AT in the cells. Molecular modeling was carried out to study the structural-functional consequences of the mutation. RESULTS Unprovoked venous thrombotic events at early age, fatal first episodes and recurrences were observed in the affected individuals. The median AT activity was 59%. Genetic analysis revealed heterozygous form of the novel mutation p.Leu205Pro (AT Debrecen). The mutant AT was expressed and synthesized in HEK293 cells but only a small amount was secreted. The majority was trapped intracellularly in the trans‑Golgi and 26S proteasome. The mutation is suspected to cause considerable structural distortion of the protein. The low specific activity of the mutant AT suggested functional abnormality. CONCLUSIONS AT Debrecen was associated with very severe thrombotic tendency. The mutation led to misfolded AT, impaired secretion and altered function. Detailed clinical and molecular characterization of a pathogenic mutation might provide valuable information for individualized management.

Treatment of mantle cell lymphoma with autologous stem-cell transplantation in a patient with severe congenital haemophilia-A and chronic (B and C virus) hepatitis

Mantle cell lymphoma (MCL) is a highly malignant disease with a median survival of 3–5 years. Most of the patients have advanced stage disease at the time of diagnosis. Combined chemo-immunotherapy, mainly rituximab and cyclophosphamide, vincristine, doxorubicin, dexamethasone altering with methotrexate and cytarabine [hyper-CVAD (cyclophosphamide, vincristine, doxorubicine, dexamethasone altering with methotrexate and cytarabine) programme], autologous stem-cell transplantation (ASCT) in first remission and allogeneic stem-cell transplantation (in patients with relapsed or refractory MCL) improved the outcome, resulting in longterm disease control [1]. We present a case of MCL associated with severe congenital haemophilia A as well as chronic B and C virus infection. It is the first report of ASCT in such a patient. The first (and so far only) ASCT in a patient with severe haemophilia A and AIDS-related lymphoma was described in 2007 [2]. Our patient was not infected with HIV. A 38-year-old man, with severe congenital haemophilia A [factor VIII (FVIII) <1%, inhibitor was not detectable] and chronic (B and C virus) hepatitis, presented with generalized lymphadenopathy. Viral infections were acquired via factor substitutions. Lymph node biopsy revealed MCL (blastoid variant) with bone marrow involvement (stage IV/A). Viral loads were not investigated at this time. Eight cycles of CEOP (cyclophophamide, etoposide, oncovin and prednisone) therapy were administered in a county hospital. Follow-up PET-CT revealed only slight response. Treatment was continued with two cycles of dexamethasone, high-dose Ara-C and cisplatin (DHAP) with poor response. Hyper-CVAD programme was started at our department with good partial response. As a result of chronic viral infection and absence of appropriate virological investigation, rituximab was not added. Stem-cell collection was performed after the third cycle of therapy; however, some weeks later, rapid enlargement of abdominal lymph nodes was detected. Despite the lymph node progression, he proceeded to ASCT. At this time, a detailed virological investigation was carried out: HBV DNA, determined by RT-PCR method, was negative. Other HBV assays were performed as follows: HBeAg: negative, anti-HBe: positive, antiHBc IgM: negative. HCV RNA was positive at high titre (5 650 000 IU mL), measured by HCV Amplicor (TaqMan Roche, Rotkreutz, Switzerland) assay. Transaminase levels were normal. The therapy was supplemented with rituximab. The conditioning regimen included total body irradiation (TBI) (4 · 3 Gy, on days )7 to )4), cyclophosphamide (2 · 60 mg kg, on days )3 to )2) and rituximab (375 mg m on day )1), where treatment was supplemented with bortezomib (1.3 mg m on day )3), followed by infusion of 8.25 · 10 CD34+ cells bwkg (day 0). Recovery of neutrophils (‡1.0 · 10 L) occurred on day 9. As abdominal lymphadenopathy was still observed subsequent to transplantation, steroid therapy (32 mg methylprednisolone daily), alfa-2b interferon (1.5 MU three times weekly) and rituximab treatment (375 mg m once a month) were initiated. Before stem-cell transplantation, he was on prophylactic therapy (3.000 IU octanate/octapharma/ twice weekly). He had no bleeding complication during the year before transplantation. Once platelet count dropped below 50 · 10 L because of chemotherapy, 1 · 40 IU bwkg FVIII concentrate was administered daily to achieve 17–35% plasma FVIII levels (12 h after the FVIII substitution). There was Correspondence: Laszlo Rejto, 2nd Dept. of Medicine, Health Sciences Center, University of Debrecen, Nagyerdei 98, Debrecen 4012, Hungary. Tel.: +36 52 314 410; fax: +36 52 255 152; e-mail: lrejto@dote.hu

Temporarily successful eradication therapy in acquired haemophilia with high inhibitor titer: A case report with a new protocol

Temporarily successful eradication therapy in acquired haemophilia with high inhibitor titer: A case report with a new protocol -