MiR-29b antagonizes the pro-inflammatory tumor-promoting activity of multiple myeloma-educated dendritic cells.

Dendritic cells (DCs) have a key role in regulating tumor immunity, tumor cell growth and drug resistance. We hypothesized that multiple myeloma (MM) cells might recruit and reprogram DCs to a tumor-permissive phenotype by changes within their microRNA (miRNA) network. By analyzing six different miRNA-profiling data sets, miR-29b was identified as the only miRNA upregulated in normal mature DCs and significantly downregulated in tumor-associated DCs. This finding was validated in primary DCs co-cultured in vitro with MM cell lines and in primary bone marrow DCs from MM patients. In DCs co-cultured with MM cells, enforced expression of miR-29b counteracted pro-inflammatory pathways, including signal transducer and activator of transcription 3 and nuclear factor-κB, and cytokine/chemokine signaling networks, which correlated with patients' adverse prognosis and development of bone disease. Moreover, miR-29b downregulated interleukin-23 in vitro and in the SCID-synth-hu in vivo model, and antagonized a Th17 inflammatory response. All together, these effects translated into strong anti-proliferative activity and reduction of genomic instability of MM cells. Our study demonstrates that MM reprograms the DCs functional phenotype by downregulating miR-29b whose reconstitution impairs DCs ability to sustain MM cell growth and survival. These results underscore miR-29b as an innovative and attractive candidate for miRNA-based immune therapy of MM.

Development and validation of a bioanalytical method for quantification of LNA-i-miR-221, a 13-mer oligonucleotide, in rat plasma using LC-MS/MS.

LNA-i-miR-221, a 13-mer oligonucleotide, is a new miR-221 inhibitor that could be used as a novel drug for multiple myeloma. Herein, an ion-pair reversed phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of LNA-i-miR-221 in rat plasma. Plasma samples were prepared with an initial phenol/chloroform/isoamyl alcohol liquid-liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase at a flow rate of 0.4 mL/min. Under these conditions LNA-i-miR-221 and the analogue internal standard are co-eluted at 1.2 min. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The assay showed a good linearity within the calibration range 10-10000 ng/mL. The precision, accuracy, and recovery values were found to be <15% (<20% at LLOQ), 100 ±â€¯15%, and 97.6-103.7%, respectively. This method was successfully applied to measure the concentrations of LNA-i-miR-221 in plasma samples following the intravenous administration during a 4-week toxicity study in rats.

Anterior cruciate ligament reconstruction: MR imaging findings.

More than two million people tear their anterior cruciate ligament (ACL) each year, and ACL reconstruction occupies a significant proportion of everyday orthopedic practice, being one of the most commonly performed sports medicine surgical procedures. Patients with postoperative symptoms are frequently imaged to monitor ligament grafts and to identify complications. Given the number of patients undergoing ACL reconstruction, knowledge of the potential complications of this surgery is essential for radiologists. This article provides a review of imaging of ACL reconstruction procedures and the potential complications specific to this surgery.

A gene expression inflammatory signature specifically predicts multiple myeloma evolution and patients survival.

Multiple myeloma (MM) is closely dependent on cross-talk between malignant plasma cells and cellular components of the inflammatory/immunosuppressive bone marrow milieu, which promotes disease progression, drug resistance, neo-angiogenesis, bone destruction and immune-impairment. We investigated the relevance of inflammatory genes in predicting disease evolution and patient survival. A bioinformatics study by Ingenuity Pathway Analysis on gene expression profiling dataset of monoclonal gammopathy of undetermined significance, smoldering and symptomatic-MM, identified inflammatory and cytokine/chemokine pathways as the most progressively affected during disease evolution. We then selected 20 candidate genes involved in B-cell inflammation and we investigated their role in predicting clinical outcome, through univariate and multivariate analyses (log-rank test, logistic regression and Cox-regression model). We defined an 8-genes signature (IL8, IL10, IL17A, CCL3, CCL5, VEGFA, EBI3 and NOS2) identifying each condition (MGUS/smoldering/symptomatic-MM) with 84% accuracy. Moreover, six genes (IFNG, IL2, LTA, CCL2, VEGFA, CCL3) were found independently correlated with patients' survival. Patients whose MM cells expressed high levels of Th1 cytokines (IFNG/LTA/IL2/CCL2) and low levels of CCL3 and VEGFA, experienced the longest survival. On these six genes, we built a prognostic risk score that was validated in three additional independent datasets. In this study, we provide proof-of-concept that inflammation has a critical role in MM patient progression and survival. The inflammatory-gene prognostic signature validated in different datasets clearly indicates novel opportunities for personalized anti-MM treatment.

Selective targeting of IRF4 by synthetic microRNA-125b-5p mimics induces anti-multiple myeloma activity in vitro and in vivo.

Interferon regulatory factor 4 (IRF4) is an attractive therapeutic target in multiple myeloma (MM). We here report that expression of IRF4 mRNA inversely correlates with microRNA (miR)-125b in MM patients. Moreover, we provide evidence that miR-125b is downregulated in TC2/3 molecular MM subgroups and in established cell lines. Importantly, constitutive expression of miR-125b-5p by lentiviral vectors or transfection with synthetic mimics impaired growth and survival of MM cells and overcame the protective role of bone marrow stromal cells in vitro. Apoptotic and autophagy-associated cell death were triggered in MM cells on miR-125b-5p ectopic expression. Importantly, we found that the anti-MM activity of miR-125b-5p was mediated via direct downregulation of IRF4 and its downstream effector BLIMP-1. Moreover, inhibition of IRF4 translated into downregulation of c-Myc, caspase-10 and cFlip, relevant IRF4-downstream effectors. Finally, in vivo intra-tumor or systemic delivery of formulated miR-125b-5p mimics against human MM xenografts in severe combined immunodeficient/non-obese diabetic mice induced significant anti-tumor activity and prolonged survival. Taken together, our findings provide evidence that miR-125b, differently from other hematologic malignancies, has tumor-suppressor activity in MM. Furthermore, our data provide proof-of-concept that synthetic miR-125b-5p mimics are promising anti-MM agents to be validated in early clinical trials.

From target therapy to miRNA therapeutics of human multiple myeloma: theoretical and technological issues in the evolving scenario.

The progress in the understanding of biological events underlying multiple myeloma (MM) development and progression has allowed the design of molecularly targeted therapies (MTTs) for this disease and several new compounds are presently under investigation in the preclinical and clinical settings. The recent discovery that miRNAs, and short non coding RNAs in general, are involved in the pathogenesis of cancer has raised the issue whether a novel therapeutic approach should be provided by selective interference with miRNA network. This review will focus on the rationale of miRNA-based therapeutics, providing the most relevant information on biogenesis and technical issues in miRNA analysis. Finally, a detailed overview of the recent findings on miRNA therapeutics of MM will be discussed.

miR-29b sensitizes multiple myeloma cells to bortezomib-induced apoptosis through the activation of a feedback loop with the transcription factor Sp1.

MicroRNAs (miRNAs) with tumor-suppressor potential might have therapeutic applications in multiple myeloma (MM) through the modulation of still undiscovered molecular pathways. Here, we investigated the effects of enforced expression of miR-29b on the apoptotic occurrence in MM and highlighted its role in the context of a new transcriptional loop that is finely tuned by the proteasome inhibitor bortezomib. In details, in vitro growth inhibition and apoptosis of MM cells was induced by either transient expression of synthetic miR-29b or its stable lentivirus-enforced expression. We identified Sp1, a transcription factor endowed with oncogenic activity, as a negative regulator of miR-29b expression in MM cells. Since Sp1 expression and functions are regulated via the 26S proteasome, we investigated the effects of bortezomib on miR-29b-Sp1 loop, showing that miR-29b levels were indeed upregulated by the drug. At the same time, the bortezomib/miR-29b combination produced significant pro-apoptotic effects. We also demonstrated that the PI3K/AKT pathway plays a major role in the regulation of miR-29b-Sp1 loop and induction of apoptosis in MM cells. Finally, MM xenografts constitutively expressing miR-29b showed significant reduction of their tumorigenic potential. Our findings indicate that miR-29b is involved in a regulatory loop amenable of pharmacologic intervention and modulates the anti-MM activity of bortezomib in MM cells.

Promises and challenges of MicroRNA-based treatment of multiple myeloma.

MicroRNAs (miRNAs) recently emerged with a key role in multiple myeloma (MM) pathophysiology and are considered important regulators of MM cell growth and survival. Since miRNAs can act either as oncogenes or tumour suppressors, the potential of targeting the miRNA network arises as a novel therapeutic approach for human cancer. Potential strategies based on miRNA therapeutics basically rely on miRNA inhibition or miRNA replacement approaches and take benefit respectively from the use of antagomirs or synthetic miRNAs as well as from lipid-based nanoparticles which allow an efficient miRNA-delivery. The availability of experimental in vivo platforms which recapitulate the growth of MM cells within the specific human bone marrow microenvironment in immunocompromised mice (SCID-hu and SCID-synth-hu) provides powerful systems for development of miRNA-based therapeutics in MM. Preliminary findings on the anti-MM activity of synthetic miRNAs in such experimental models offer a proof-of-principle that miRNA therapeutics is a promising opportunity for this still incurable disease representing the rationale for a new venue of investigation in this specific field.

Mouse models as a translational platform for the development of new therapeutic agents in multiple myeloma.

Mouse models of multiple myeloma (MM) are basic tools for translational research and play a fundamental role in the development of new therapeutics against plasma cell malignancies. All available models, including transplantable murine tumors in syngenic mice, xenografts of established human cell lines in immunocompromised mice and transgenic models that mirror specific steps of MM pathogenesis, have demonstrated some weaknesses in predicting clinical results, particularly for new drugs targeting the human bone marrow microenvironment (huBMM). The recent interest to models recapitulating the in vivo growth of primary MM cells in a human (SCID-hu) or humanized (SCID-synth-hu) host recipient has provided powerful platforms for the investigation of new compounds targeting MM and/or its huBMM. Here, we review and discuss strengths and weaknesses of the key in vivo models that are currently utilized in the MM preclinical investigation.

Psychrophilic superoxide dismutase from Pseudoalteromonas haloplanktis: biochemical characterization and identification of a highly reactive cysteine residue.

A psychrophilic superoxide dismutase (SOD) has been characterized from the Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph). PhSOD is a homodimeric iron-containing enzyme and displays a high specific activity, even at low temperature. The enzyme is inhibited by sodium azide and inactivated by hydrogen peroxide; it is also very sensitive to peroxynitrite, a physiological inactivator of the human mitochondrial Mn-SOD. Even though PhSOD is isolated from a cold-adapted micro-organism, its heat stability is well above the maximum growth temperature of P. haloplanktis, a feature common to other Fe- and Mn-SODs. The primary structure of PhSOD was determined by a combination of mass spectrometry and automated Edman degradation. The polypeptide chain is made of 192 amino acid residues, corresponding to a molecular mass of 21251 Da. The alignment with other Fe- and Mn-SODs showed a high amino acid identity with Fe-SOD from Vibrio cholerae (79%) and Escherichia coli (70%). A significant similarity is also shared with human mitochondrial Mn-SOD. PhSOD has the unique and highly reactive Cys57 residue, located in a variable region of the protein. The three-dimensional model of the PhSOD monomer indicates that Cys57 is included in a region, whose structural organization apparently discriminates between dimeric and tetrameric SODs. This residue forms a disulfide adduct with beta-mercaptoethanol, when this reducing agent is added in the purification procedure. The reactivity of Cys57 leads also to the formation of a disulfide bridge between two PhSOD subunits in specific denaturing conditions. The possible modification of Cys57 by physiological thiols, eventually regulating the PhSOD functioning, is discussed.

Chronomodulated infusion of cisplatin, 5-fluorouracil and folinic acid: lack of activity in advanced colorectal cancer.

BACKGROUND: The chronomodulated infusion of 5-FU, FA and oxaliplatin allows a significant increase in dose intensity and antitumor efficacy in patients with metastatic colorectal cancer. Here we investigated if substitution of oxaliplatin with cisplatin produced a similar antitumor activity in previously untreated patients with advanced colorectal cancer. METHODS: We enrolled 21 consecutively evaluated ambulatory patients with metastatic colorectal cancer. Each treatment cycle consisted of a 5-day course of continuous chronomodulated venous infusion of drugs. Daily doses were 600 mg/m2 5-FU, 150 mg/m2 FA (L-form), and 12 mg/m2 cisplatin. The cycles were repeated every 21 days. RESULTS: All patients completed at least 3 cycles. Overall a total number of 105 cycles were administered. One partial response (lasting 3 months) and 13 stable disease (lasting from 3 to 12 months) were observed. The remaining 7 patients had progression of the disease. Hematologic and gastrointestinal toxicity was always < or = G2 in all cycles. CONCLUSIONS: The results of this study discourage the substitution of cisplatin for the more active compound, oxaliplatin, in a chronomodulated schedule of infusion with 5-FU and FA in patients with metastatic colorectal cancer.

Reliability of four simple, quantitative tests of balance and mobility in healthy elderly females.

Four common tests of balance and mobility were administered to 45 healthy women, aged 55-71 years: Sharpened Romberg (also defined as tandem stance), eyes open and closed; One-Legged Stance Test, eyes open and closed; Functional Reach; and Sit-To-Stand test. Two independent observers scored the tests, which were performed on two successive days. Inter-rater (IRR) and test-retest reliability (TRR) were good. Across the six different tests, Intraclass Correlation Coefficients ranged from 0.95 to 0.99 for scoring consistency between rates, and from 0.73 to 0.93 within raters. This is in line with previous findings based on similar subjects and similar, though not identical, testing procedures. Intercorrelations between the scores were moderate: r coefficients ranged 0.40-0.66. The results suggest that this simple test battery provides reliable scores, and that the different tests relate to a homogeneous construct, while not being redundant. It thus seems worthwhile to further investigate whether they represent and measure a unidimensional domain, rather than conceptually different dimensions, in view of achieving a unique measure of balance performance.

[Areas in Calabria with an elevated incidence of congenital hypothyroidism and iodine content of drinking water]. / Aree della Calabria con elevata incidenza di ipotiroidismo congenito e contenuto di iodio nelle acque potabili.

The screening of 128,442 newborns, carried out in our laboratory from 1/1/1991 to 31/12/1996, detected 51 cases of congenital hypothyroidism (CH). Due to a 98% coverage, the accurate regional map of CH distribution has been obtained. Two rural and/or mountainous areas showed high incidence, 1/1145 (area A) and 1/1193 (area B), respectively. The incidence of the remaining region is 1/4621, whereas the mean regional incidence is 1/2518. In order to verify a relationship between CH incidence and iodine deficiency, we analyzed the iodine concentration in drinkable water from 148 localities. The high incidence of CH in the A and B areas corresponds with a high percentage of water samples with iodine concentration < 2 micrograms/l (92% and 85% respectively). In 15 coastal localities with low CH incidence, iodine concentration was between 7 and 80 micrograms/l. For populations of the A and B areas showing dietary iodine deficiency due to local product diet, we suggest the iodine prophylaxis as soon as possible.

Length of stay of stroke rehabilitation inpatients: prediction through the functional independence measure.

A model for prediction of length of stay (LOS, in days) of stroke rehabilitation inpatients was developed, based on patients' age (years) and function at admission (scored on the Functional Independence Measure, FIMSM). One hundred and twenty-nine cases, consecutively admitted to three free-standing rehabilitation centres in Italy, were analyzed. A multiple linear regression using forward stepwise selection procedure was adopted. Median admission and discharge scores were: 57 and 75 for the total FIM score, 29 and 48 for the 13-item motor FIM subscore, 29 and 30 for the 5-item cognitive FIM subscore (potential range: 18-126, 13-91, 5-35, respectively). Median LOS was 44 days (interquartile range 30-62). The logLOS predictive model included three FIM items ("toilet transfer", TTr; "social interaction"; "expression") and patient's age (R2 = 0.48). TTr alone explained 31.3% of the variance of logLOS. These results are consistent with previous American studies, showing that FIM scores at admission are strong predictors of patients' LOS, with the transfer items having the greatest predictive power.

Trunk control test as an early predictor of stroke rehabilitation outcome.

BACKGROUND AND PURPOSE: The aim of this study was to investigate the construct and predictive validity of the Trunk Control Test (TCT) in postacute stroke patients by comparing TCT scores at admission and discharge with the Functional Independence Measure (FIM) scores. METHODS: Forty-nine patients participated in the study. The TCT examines four movements: rolling from a supine position to the weak side (T1) and to the strong side (T2), sitting up from a lying-down position (T3), and sitting balance (T4). The FIM is an 18-item scale (13 motor [motFIM] and 5 cognitive [cognFIM]) used to determine the level of dependence of patients in daily life. RESULTS: Thirty-six patients (73%) increased their TCT overall score at discharge. The TCT item-total correlations were high, both at admission and discharge (P < .0001). The individual TCT items were intercorrelated. Furthermore, the homogeneity of the TCT was confirmed by a high Cronbach's index. High correlations were found between admission and discharge scores in the different tests (TCT, FIM, and motFIM; P < .0001) and between TCT at admission and FIM (P < .0001) and motFIM (P < .0001) at admission. TCT at admission alone explained 71% of the variance in motFIM at discharge. CONCLUSIONS: The TCT showed a good sensitivity to change in assessing recovery of stroke patients. The high item-total correlation and Cronbach's alpha value of the TCT suggest that there is one homogeneous construct underlying the item list. The TCT construct validity was confirmed by the correlation between this test and the FIM scores. TCT at admission predicted motFIM at discharge even better than motFIM at admission alone. Possibly, the TCT captures basic motor skills that foreshadow the recovery of more complex behavioral skills described by the FIM.

A phase I study of 5-day continuous venous infusion of carboplatin at circadian rhythm-modulated rate compared with constant rate.

The circadian rhythm-modulated delivery of anticancer drugs has been shown to reduce toxicity and improve anticancer efficacy. The aim of this phase I trial was to compare the feasibility and tolerability of carboplatin (CBDCA) administered at circadian-modulated or flat infusion rate in 24 patients with advanced cancer. Each treatment cycle consisted of a 5-day continuous intravenous infusion of CBDCA, to be repeated at 28-day intervals. Three dose levels were determined, with a CBDCA dose 15%, 40% and 60% over that calculated using Calvert's formula. Two schedules were compared: schedule A (forty-four courses), with a at circadian rhythm-modulated rate (peak at 16.00 hr) and schedule B (fifty courses), at a constant rate. At the first and second dose level neither of the administered cycles were accompanied by hematologic toxicity higher than Grade 3. At the third dose level, two cycles out of 15 for schedule A and two out of 20 for schedule B were accompanied by Grade 4 thrombocytopenia. The repeat cycles were delayed from day 28 to 42 in some patients, with no differences between circadian-modulated and flat infusion. Three partial responses out of 9 evaluable patients were observed in schedule A and 2 out of 10 evaluable patients in schedule B. We showed no potential advantage of the chronomodulated 5-day CBDCA continuous infusion method over the flat rate method. Although antitumor effects were observed in this pilot study, this treatment cannot be assessed for efficacy relative to other schedules.

Chronomodulated infusion of 5-fluorouracil, folinic acid and carboplatin in colorectal cancer: a pilot study.

We investigated if chronomodulated infusion of fluorouracil, folinic acid in combination with carboplatin shows antitumor efficacy in patients advanced metastatic colorectal cancer. Thirteen patients entered into the study. Each treatment cycle consisted of a 5 day course of continuous venous infusion of 5-fluorouracil (5-FU; 500 mg/m2/die), folinic acid (FA; L-form, 150 mg/m2/die) and carboplatin (CBDCA; the dose being calculated according to the Calvert's formula). Patients received the drugs according to the circadian-modified infusion schedule with a sinusoidally modulated delivery with peak flow rate at 4.00 AM for 5-FU and FA and 4.00 PM for CBDCA. Overall a total number of 54 cycles were administered. Two partial responses and one minor response were observed among the 5 untreated patients. Five patients had progression of disease. Stabilization of previously progressive disease was obtained in the 5 remaining patients. These preliminary results show that the combination of 5-FU, FA and CBCDA infused at circadian-modulated rate has moderate activity in colorectal cancer, specially in previously untreated patients, with a mild and acceptable toxicity.

A phase I study of recombinant interferon-alpha administered as a seven-day continuous venous infusion at circadian-rhythm modulated rate in patients with cancer.

A Phase I trial of interferon-alpha (IFN-alpha) administered at circadian-rhythm modulated rate was carried out to evaluate maximum tolerated dose (MTD) and toxicity in patients with advanced malignancies. Recombinant IFN-alpha-2b was administered as a 7-day continuous venous infusion with maximum delivery between 6 p.m. and 3 a.m. Entry dose levels were 0.2, 2 and 4 MU/m2/day. The dose was escalated by an amount equal to the starting dose until a maximum of 6 entry dose levels was achieved, with a 1-week rest between each cycle. The maximal daily dose of IFN-alpha administered was 24 MU/m2/day. A programmable-in-time ambulatory pump was used, so that all patients could receive their treatment at home. Eighteen patients were entered in the study and 16 were evaluable for toxicity. Toxicity was mild to moderate except for two patients who developed WHO grade III toxicity. No significant decline in performance status was associated with treatment. Two objective responses were observed in patients with previously treated metastatic renal cell carcinoma. As compared to standard s.c./i.m. administration or continuous nonchronomodulated i.v. infusion of IFN-alpha, this circadian schedule has allowed to deliver high doses of drug with acceptable toxicity.