Lunaemycins, New Cyclic Hexapeptide Antibiotics from the Cave Moonmilk-Dweller Streptomyces lunaelactis MM109T.

Streptomyces lunaelactis strains have been isolated from moonmilk deposits, which are calcium carbonate speleothems used for centuries in traditional medicine for their antimicrobial properties. Genome mining revealed that these strains are a remarkable example of a Streptomyces species with huge heterogeneity regarding their content in biosynthetic gene clusters (BGCs) for specialized metabolite production. BGC 28a is one of the cryptic BGCs that is only carried by a subgroup of S. lunaelactis strains for which in silico analysis predicted the production of nonribosomal peptide antibiotics containing the non-proteogenic amino acid piperazic acid (Piz). Comparative metabolomics of culture extracts of S. lunaelactis strains either holding or not holding BGC 28a combined with MS/MS-guided peptidogenomics and 1H/13C NMR allowed us to identify the cyclic hexapeptide with the amino acid sequence (D-Phe)-(L-HO-Ile)-(D-Piz)-(L-Piz)-(D-Piz)-(L-Piz), called lunaemycin A, as the main compound synthesized by BGC 28a. Molecular networking further identified 18 additional lunaemycins, with 14 of them having their structure elucidated by HRMS/MS. Antimicrobial assays demonstrated a significant bactericidal activity of lunaemycins against Gram-positive bacteria, including multi-drug resistant clinical isolates. Our work demonstrates how an accurate in silico analysis of a cryptic BGC can highly facilitate the identification, the structural elucidation, and the bioactivity of its associated specialized metabolites.

Cell therapy in critical limb ischemia: A comprehensive analysis of two cell therapy products.

BACKGROUND: Cell therapy has been proposed as a salvage limb procedure in critical limb ischemia (CLI). In spite of the fact that clinical trials found some efficacy, the mechanism of action remains elusive. The objective of this study was to characterize two autologous cell therapy products (CTPs) obtained from patients with advanced peripheral arterial disease. METHODS: Bone marrow (BM-CTPs) (n = 20) and CTPs obtained by non-mobilized cytapheresis (peripheral blood [PB]-CTPs) (n = 20) were compared. CTPs were characterized by their cell composition, by the quantification of endothelial progenitor cells (EPCs) and mesenchymal stromal cells (MSCs) and by transcriptomic profiling. The angiogenic profile and the 6-month outcome of CLI patients are described. RESULTS: Patients presented inflammation syndrome and high levels of CXCL12, soluble stem cell factor and granulocyte colony-stimulating factor, whereas granulocyte macrophage colony-stimulating factor was low. Circulating CD34+ cells represented rare events. BM and PB-CTPs were heterogeneous. Mature cells and colony-forming unit-endothelial cells were in higher concentration in PB-CTPs, whereas CD34+ stem cells and EPCs were more abundant in BM-CTPs. MSCs were identified in both CTPs. Transcriptomic profiling revealed the strong angiogenic potential of BM-CTPs. Transcutaneous partial pressure of oxygen, C-reative protein and neutrophil content in CTPs are predictive of the clinical outcome at 6 months. DISCUSSION: Transcriptomic allows an accurate characterization of CTPs. BM-CTPs have the richest content in terms of stem cells and transcriptome. The high content of mature cells in PB-CTPs means that they work via a paracrine mechanism. The clinical outcome indicates the deleterious influence the patients' status and the limits of an autologous approach. In this respect, MSCs may allow an allogenic strategy.

Autologous Bone Marrow Mononuclear Cell Implantation and Its Impact on the Outcome of Patients With Critical Limb Ischemia　- Results of a Randomized, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Cell therapy is a therapeutic option for patients presenting with nonrevascularizable critical limb ischemia (CLI). However there is a lack of firm evidence on its efficacy because of the paucity of randomized controlled trials.Methods and Results:The BALI trial was a multicenter, randomized, controlled, double-blind clinical trial that included 38 patients. For all of them, 500 mL of bone marrow were collected for preparation of a BM-MNC product that was implanted in patients assigned to active treatment. For the placebo group, a placebo cell-free product was implanted. Within 6 months after inclusion, major amputations had to be performed in 5 of the 19 placebo-treated patients and in 3 of the 17 BM-MNC-treated patients. According to a classical logistic regression analysis there was no significant difference. However, when using the jackknife analysis, 6 months after inclusion BM-MNC implantation was associated with a lower risk of major amputation (odds ratio (OR): 0.55; 95% confidence interval (CI): 0.52-0.58; P<0.0001) and of occurrence of any event (major or minor amputation, or revascularization) (OR: 0.30; 95% CI: 0.29-0.31; P<0.0001). The secondary endpoints (i.e., pain, ulcers, TcPO2, and ankle-brachial index value) were not statistically different between groups. CONCLUSIONS: Our results suggested that cell therapy reduced the risk of major amputation in patients presenting with nonrevascularizable CLI.

Critical limb ischaemia and the response to bone marrow-derived cell therapy according to tcPO2 measurement.

BACKGROUND: Cell therapy is an emerging potential biotherapy for critical limb ischaemia (CLI) patients who are not eligible for revascularization. However, the findings on this technique's efficacy are inconsistent. Trials investigating this topic focused on the more severe CLI patients who were often beyond any therapy. Therefore, identifying those who may truly benefit from cell transplantation is now warranted. To this end, we studied the prognostic value of tcPO2 for major amputation after 1 year in patients treated with bone marrow-derived cells. PATIENTS AND METHODS: CLI patients ineligible for revascularization were included in a cell-therapy pilot study. On inclusion, patients underwent tcPO2 measurement in supine and sitting positions. For a tcPO2 < 10 mmHg in the supine position, the vascular reserve was defined by tcPO2 > 30 mmHg in the sitting position. Patients were administered intramuscular injections of mononuclear cells derived from aspirated bone marrow. RESULTS: In total, 25 patients (a lower limbs) were included for analysis. At inclusion, 11 lower limbs had tcPO2 at rest > 10 mmHg, and 16 lower limbs had a tcPO2 < 10 mmHg. The success probability for cell therapy was 0.79 (95 % CI 0.38-0.94) and 0.44 (95 % CI 0.18-0.67), respectively (p = 0.1). Of the 16 limbs with tcPO2 < 10 mmHg, the success rate was considerably higher in patients demonstrating a tcPO2 increase in a sitting position of over 30 mmHg (6/8, success probability 0.71, 95 % CI 0.26-0.92) compared to those without (2/8, success probability 0.15, 95 % CI 0.01-0.48, p = 0.03). CONCLUSIONS: For patients with chronic CLI for whom cellular therapy is a therapeutic option, a tcPO2 < 10 mmHg at rest, without vascular reserve (i. e. < 30 mmHg when sitting), is a prognostic indicator for poor outcome.
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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.

Portal hypertension with extensive fibrosis and plasma cell infiltration in multiple myeloma.

Plasma cell infiltration of the liver has been described in about 45% of patient with multiple myeloma in autopsy review; however, it is usually not associated with significant liver dysfunction. Indeed, only rare cases of massive plasma cell infiltration leading to non-obstructive cholestasis and hepatic failure have been described. Here, we report a case with a history of 8 years of MM with extensive liver fibrosis and portal hypertension with no other evidence aetiology unless massive plasma cell infiltration who presented a significant regression of both biological liver abnormalities and liver stiffness after ten months of chemotherapy concomitantly to a significant decrease of the IgG serum monoclonal band.

Factors useful for predicting survival of myeloma patients in everyday practice. A 10-year study of 148 patients older than 55 years.

OBJECTIVE: The objective of this study was to identify prognostic factors in a uniform population of older patients with myeloma. METHODS: Thirty-one study centers in France included 148 patients who were older than 55 years at diagnosis and were followed up until death or for at least 10 years. The following tests were available for all patients: blood cell counts; serum, and urinary protein electrophoresis; and serum levels of creatinine, calcium, beta2 microglobulin (beta2m), lactic dehydrogenase (LDH), and C-reactive protein (CRP). RESULTS: Mean age was 71.9 years, median survival was 34 months, and mean survival was 47 months. In the univariate analysis, factors significantly associated with higher mortality were male gender (odds ratio [OR], 1-2.12), age older than 70 years (OR, 1.10-2.28), serum albumin<30 g/l (OR, 1.16-3.28), serum creatinine>100 micromol/l (OR, 1.34-2.81), beta2m>6 mg/l (OR, 1.78-4), CRP>6 mg/l (OR, 1.44-3.06), hemoglobin<10 g/dl (OR, 1.8-2.23). In the multivariate analysis, only two factors significantly predicted a higher risk of death: beta2m>6 mg/l (OR=2.439 [1.59-3.76]) and CRP>6 mg/l (OR=1.76 [1.18-2.63). beta2m level was >6 mg/l in 41 (27.7%) patients and CRP was >6 mg/l in 61 (43.6%) patients. Other potential prognostic factors such as chromosome 13 deletion were not investigated because they were not available for all study patients. CONCLUSIONS: The strength of this study is the 10-year follow-up in a uniform patient cohort. beta2m and CRP independently predicted the risk of death.

Intensive therapy before or during the conditioning regimen of allogeneic marrow transplantation in adult acute lymphoblastic leukemia patients: we must choose to reduce Toxicity--a Groupe Ouest-Est d'Etude des Leucemies et Autres Maladies du Sang study.

To improve the results in the treatment of adult acute lymphoblastic leukemia patients, different strategies have been proposed. The intensification could concern the induction and early consolidation phases, the conditioning regimen before allogeneic bone marrow transplantation (alloBMT), or both. We analyzed 2 consecutive trials for adult patients in first remission and with the same prognostic features. The Leucemie Aigue Lymphoblastique Paris-Ouest-France (LALPOF) protocol proposed alloBMT with standard conditioning after a classic induction and intensified consolidation scheme; the Groupe Ouest Est des Leucemies Aigues Lymphoblastiques (GOLEAL1) protocol tested an intensified induction and consolidation course before alloBMT with a reinforced conditioning regimen. The 4-year survival rates after alloBMT for LALPOF and GOELAL1 were, respectively, 71% +/- 12% and 36% +/- 13% ( P = .009). The 4-year disease-free survival reached 75% +/- 11% in the LALPOF study and 69% +/- 13% in the GOELAL1 study ( P = .30). The toxic death rate was significantly lower in the LALPOF (2/18) than in the GOELAL1 (6/15) group. Event-free survival at 4 years was significantly higher in LALPOF than in GOELAL1: 66% +/- 11% and 35% +/- 11%, respectively ( P = .02). For adult acute lymphoblastic leukemia patients in first remission, the intensification of chemotherapy before a reinforced conditioning regimen before alloBMT may lead to an increased toxic death rate.

Better outcome of adult acute lymphoblastic leukemia after early genoidentical allogeneic bone marrow transplantation (BMT) than after late high-dose therapy and autologous BMT: a GOELAMS trial.

Various transplantation strategies have been designed to improve the poor prognosis of adult (ages 15 to 60 years) acute lymphoblastic leukemia (ALL). The GOELAL02 trial evaluated the impact of early allogeneic bone marrow transplantation (alloBMT) or delayed unpurged autologous stem cell transplantation (ASCT) for patients who had no human leukocyte antigen (HLA)-matched sibling donor or who were older than 50 years. Inclusion criteria included at least one of the following: age older than 35 years; non-T-ALL; leukocytosis greater than 30 x 10(9)/L; t(9;22), t(4;11), or t(1; 19); or failure to achieve complete remission (CR) after one induction course. Among 198 patients, the median age was 33 years. The CR rate was 80% with vincristine, idarubicine, L-asparaginase, and randomized intravenous injection or oral steroids (P = nonsignificant [ns]). AlloBMT was performed after 2 consolidation courses while ASCT was delayed after 1 additional reinduction. Intensified conditioning regimen before transplantation included etoposide, cyclophosphamide, and total body irradiation (TBI). Median follow-up was 5.1 years. The median overall survival (OS) was 29 months, with a 6-year OS of 41%. On an intent-to-treat analysis for patients younger than 50 years, alloBMT significantly improved the 6-year OS (75% versus 40% after ASCT; P = .0027). Randomized interferon-alpha maintenance had no effect on relapse or survival after ASCT. In conclusion, the outcome of adult ALL is better after early alloBMT than after delayed ASCT.

Comparison of 200 mg/m(2) melphalan and 8 Gy total body irradiation plus 140 mg/m(2) melphalan as conditioning regimens for peripheral blood stem cell transplantation in patients with newly diagnosed multiple myeloma: final analysis of the Intergroupe Francophone du Myélome 9502 randomized trial.

High-dose therapy has become a common treatment for myeloma. The objective of this study (Intergroupe Francophone du Myélome [IFM] 9502 trial) was to compare in a prospective and randomized trial the 2 most widely used conditioning regimens before autologous stem cell transplantation in newly diagnosed symptomatic patients younger than 65 years old: 8 Gy total body irradiation plus 140 mg/m(2) melphalan (arm A) versus 200 mg/m(2) melphalan (arm B). A total of 282 evaluable patients were compared--140 in arm A and 142 in arm B. Baseline characteristics and disease response to 4 cycles of the VAD regimen performed before randomization and autologous stem cell transplantation were identical in the 2 treatment arms. In arm B, hematologic recovery was significantly faster for both the duration of neutropenia and thrombocytopenia, transfusion requirements were also significantly lower, and the median duration of hospitalization was significantly shorter. In arm A, the incidence of severe mucositis was significantly increased. The median duration of event-free survival was similar in both arms (21 vs 20.5 months, P =.6), but the 45-month survival was 65.8% in arm B versus 45.5% in arm A (P =.05). This difference might be attributed in part to better salvage regimens after relapse in arm B compared with arm A. We conclude that 200 mg/m(2) melphalan is a less toxic and at least as effective conditioning regimen when compared with 8 Gy total body irradiation with 140 mg/m(2) melphalan. This regimen should be considered as the standard of care before autologous stem cell transplantation in multiple myeloma.