Critical limb ischemia: thrombogenic evaluation of two autologous cell therapy products and biologic profile in treated patients.

BACKGROUND: Cell therapy has been proposed as a salvage limb procedure in critical limb ischemia (CLI). Autologous cell therapy products (CTP) are obtained from patients with advanced peripheral arterial disease to be injected at the site of ischemia. Thrombogenicity of CTPs has not yet been assessed. The objectives were: 1) to assess thrombotic risk in candidates for cell therapy, 2) to evaluate two different CTPs in terms of thrombogenic potential, and 3) to evaluate clinical thrombotic events. STUDY DESIGN AND METHODS: In this ancillary study of a Phase I and II clinical trial, bone marrow (BM)-CTPs (n = 20) and CTPs obtained by cytapheresis (peripheral blood [PB]-CTPs; n = 20) were compared. Inflammatory and coagulation markers were measured at baseline and 24 hours after CTP implantation. CTP cell content and tissue factor (TF) expression (mRNA and protein) were analyzed. Thrombin generation assessed CTP-related thrombogenicity. RESULTS: All patients presented cardiovascular risk factors. At baseline, the patients' biologic profile was characterized by high levels of fibrinogen, C-reactive protein (CRP), D-dimer, interleukin (IL)-6, and plasmatic TF, whereas IL-10 was low. Although different in terms of cell composition, both BM- and PB-CTPs support low thrombin generation. Twenty-four hours after implantation, biologic markers remained stable in the PB-CTP group, except for IL-6. In the BM-CTP group, a significant increase of IL-6 but also of CRP and D-dimer was observed. Clinically, one single patient developed deep vein thrombosis 24 hours after the implantation of autologous PB-CTP. CONCLUSION: CTPs supported low thrombin generation and were well tolerated after calf implantation.

Normal levels of peripheral CD19(+) CD5(+) CLL-like cells: toward a defined threshold for CLL follow-up -- a GEIL-GOELAMS study.

BACKGROUND: The development of flow cytometry as a useful tool for the detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) is potentially hampered by the fact that a normal subset of B-cells with a similar immunophenotype is present in the peripheral blood. This subset of CLL-like cells is not well defined in terms of frequency. METHODS: Here, we performed a multicenter study with a panel of four-color antibody combinations possibly useful for the detection of MRD in CLL, to establish the levels of normal CLL-like cells in 49 healthy controls. ROC curves established the upper level of such cells at 4 × 10(-4) . The two best combinations were further applied to 419 samples from 117 treated CLL patients. RESULTS: The combinations CD19/CD5/CD43/CD79b and CD19/CD5/CD81/CD22 appeared very robust and well correlated to enumerate normal CLL-like cells in a lysis no-wash approach. In follow-up samples from CLL patients, they disclosed only 9.8% of the samples within the normal range. In more than 90% of the cases, it was thus possible to report confidently on the absence or presence of MRD in these patients. CONCLUSIONS: This manuscript reports on the frequency of CD19(+) CD5(+) B-cells in normal peripheral blood and confirms the combinations recommended by the European research initiative on CLL as being performing to assess remaining CLL cells above a threshold of 4 × 10(-4) white blood cells.

Chain-dependent photocytotoxicity of tricationic porphyrin conjugates and related mechanisms of cell death in proliferating human skin keratinocytes.

Photodynamic therapy (PDT) is a poor treatment option for nodular basal cell carcinomas and squamous cell carcinomas. As a result, the search for new photosensitizers with better effectiveness is of current interest. The photocytotoxicity of conjugates (P-R) of a water-soluble tri-cationic porphyrin (P-H) having similar efficiency of production of singlet oxygen, the PDT cytotoxin, has been assessed in vitro. Links between uptake, intracellular localization, photooxidative stress, photocytotoxicity and ability to induce programmed cell death are established. Conjugates bearing methyl (P-Me), Di-O-isopropylidene-(-d-galactopyranosyl (P-OGal) or N,N'-dicyclohexylureidooxycarbonyl (P-DDC) chains are efficiently taken-up by proliferating NCTC 2544 keratinocytes. The relative order of photocytotoxicity is P-OGal >P-DDC=P-Me≫P-H. The photocytotoxic potential of P-Me, P-OGal and P-DDC equals that of endogenous protoporphyrin IX induced by δ-aminolevulinic acid or its esters, the pro-drugs currently employed for PDT of skin lesions. Microfluorometry shows that P-Me, P-OGal, and P-DDC localize in endocytotic or pinocytotic vesicles but not in mitochondria or nucleus. Absence of annexin V binding, caspase activation or chromatin condensation suggests that cell photosensitization by P-R does not induce apoptosis. On the other hand, P-OGal photocytotoxicity correlates with appearance of multiple vesicles that have hallmarks of autophagy compartments, being decorated with the marker LC3 in cells transfected with an expression vector encoding GFP-LC3. p38 and JNK phosphorylation and inhibition of ERK1/2 phosphorylation suggest close relationship between mortality of NCTC 2544 keratinocytes and MAPK pathway impairment. Given their potentially easy formulation, water-soluble P-R are promising powerful photosensitizers for PDT of skin lesions.

Gastric MALT lymphoma presenting as Waldenström's macroglobulinemia without bone marrow involvement.

We report a case of gastric mucosa-associated lymphoid tissue (MALT) lymphoma with macroglobulinemia in a 59-year-old man who presented with melena. A computed tomography scan of the abdomen showed irregular thickening of the wall of the stomach, and endoscopic examination disclosed enlarged and inflammatory folds of the fundus. Histopathologic examination of gastric samples showed mucosal infiltration by small lymphocytes, which were positive for CD20 and negative for CD10 and CD23, confirming the diagnosis of gastric MALT lymphoma. Serum electrophoresis detected a monoclonal peak and immunoelectrophoresis revealed an immunoglobulin M kappa component. Bone marrow aspirate and biopsy results were normal. The patient received chemotherapy. After treatment, he was in complete remission, and the serum monoclonal component had disappeared. Our observation is uncommon because of important macroglobulinemia occurring in gastric MALT lymphoma without bone marrow involvement.

Quantification of different human alpha interferon subtypes and pegylated interferon activities by measuring MxA promoter activation.

Alpha interferons (alpha-IFNs) are potent biologically active proteins synthesized and secreted by somatic cells during viral infection. Quantification of alpha-IFN concentrations in biological samples is used for diagnosis. More recently, recombinant IFNs have been used as antiviral, antiproliferative, and immunomodulatory therapeutic agents, and particularly for the treatment of chronic hepatitis C virus infection. For this purpose, IFN has recently been coupled to polyethylene glycol (PEG) to improve the pharmacokinetic properties. The measure of alpha-IFN in biological samples from treated patients could be useful to ensure compliance to therapy and the true IFN activity in relation to viral decay during follow-up. In particular, it could be used to monitor the PEG-IFN concentration in patients treated for hepatitis C virus infection. The most frequently used test is a bioassay based on the antiviral property of the IFN, but the assay is not highly reproducible. Here, we present a reporter test based on MxA promoter activation of chloramphenicol acetyltransferase expression (Mx-CAT). MxA is an antiviral protein induced and tightly regulated by alpha-IFN. The Mx-CAT assay showed good reproducibility of 15% and was suitable to quantify PEG-IFN and numerous other alpha-IFN subtypes as well, despite a differential MxA promoter activation in relation with the subtype. A good correlation was obtained with the reporter assay and a commercial enzyme-linked immunosorbent assay on samples from treated patients. This test could be useful for monitoring IFN therapy of chronically infected hepatitis C virus-infected patients treated with the standard IFN, PEG-IFN, and probably forthcoming recombinant IFNs.

ZAP-70 tyrosine kinase is constitutively expressed and phosphorylated in B-lineage acute lymphoblastic leukemia cells.

BACKGROUND AND OBJECTIVES: Zeta-associated protein 70 (ZAP-70), a member of the Syk family of protein tyrosine kinases, is normally expressed in T and NK cells. While little is known about ZAP-70 expression in normal human B cells, it has been reported that ZAP-70 is expressed in a subset of patients with chronic lymphocytic leukemia (CLL) with a poor prognosis. In this study, we examined the expression and phosphorylation status of ZAP-70 in B-lineage acute lymphoblastic leukemia (Blin-ALL). DESIGN AND METHODS: First, ZAP-70 protein expression was assessed by Western blotting and flow cytometry and ZAP-70 mRNA transcripts were analyzed by reverse transcription polymerase chain reaction (RT-PCR) on human precursor B cell lines. Experiments were then carried out on cells obtained from 18 patients with Blin-ALL and from normal human bone marrow. RESULTS: ZAP-70 was constitutively expressed and phosphorylated on tyr319 in human precursor Blin-ALL cell lines as well as in primary B leukemic cells from all examined Blin-ALL patients with pro-B, pre-B and B phenotypes, but not in malignant myeloid cells. Importantly, analysis of normal human bone marrow revealed expression of ZAP-70 transcripts only in the CD34+ cell fraction (either CD19-CD10- or CD19+CD10+) but not in the CD34- cell fraction (CD19+sIgM- pre-B cells or CD19+sIgM+ immature B cells). INTERPRETATION AND CONCLUSIONS: ZAP-70 was found to be expressed in the CD34+ normal bone marrow compartment including earlier B-cell progenitors, but not in CD34- pre-B and immature B cells. By contrast, ZAP-70 was consistently expressed and phosphorylated in Blin-ALL cells. Further studies are required to determine whether ZAP-70 may play a pathophysiological role in Blin-ALL.