Safety and efficacy of autologous hematopoietic stem-cell transplantation following natalizumab discontinuation in aggressive multiple sclerosis.

BACKGROUND AND PURPOSE: Natalizumab (NTZ) is a highly effective treatment for relapsing-remitting multiple sclerosis (MS), but its withdrawal is often followed by disease reactivation or rebound, even if other disease-modifying treatments (DMTs) are administered. In this study, for the first time, the safety and efficacy of autologous hematopoietic stem-cell transplantation (aHSCT) performed following NTZ discontinuation were retrospectively compared with conventional DMTs. METHODS: Patients with relapsing-remitting MS treated with NTZ who discontinued the drug after at least six administrations and with at least 6 months of follow-up were included. Patients underwent aHSCT after a minimum of 6 months following NTZ withdrawal, receiving meanwhile cyclophosphamide or corticosteroids, or other DMTs approved for MS (control group) after an adequate wash-out period. Both hematological and neurological follow-up were assessed according to standard policies. RESULTS: A total of 52 patients were included, 11 who received aHSCT and 41 who received DMTs. Baseline clinical and demographic characteristics were similar between the two groups. No fatality or life-threatening complications, including progressive multifocal leukoencephalopathy, were observed. At 3 years following NTZ discontinuation, no evidence of disease activity was reported in 54.5% of the patients in the aHSCT group compared with 11.5% of those in the DMT group (P = 0.0212). Disease reactivation in the patients with aHSCT was observed only during wash-out/bridging therapy and 100% of the cases were free from disease activity after aHSCT. CONCLUSIONS: These data suggest that an aggressive therapy should be established after NTZ with the shortest possible wash-out period. aHSCT after 6 months from NTZ withdrawal appears to be safe.

Profiling the onset of somatic embryogenesis in Arabidopsis.

BACKGROUND: Totipotency is the ability of a cell to regenerate a whole organism. Plant somatic embryogenesis (SE) is a remarkable example of totipotency because somatic cells reverse differentiation, respond to an appropriate stimulus and initiate embryo development. Although SE is an ideal system to investigate de-differentiation and differentiation, we still lack a deep molecular understanding of the phenomenon due to experimental restraints. RESULTS: We applied the INTACT method to specifically isolate the nuclei of those cells undergoing SE among the majority of non-embryogenic cells that make up a callus. We compared the transcriptome of embryogenic cells to the one of proliferating callus cells. Our analyses revealed that embryogenic cells are transcriptionally rather than metabolically active. Embryogenic cells shut off biochemical pathways involved in carbohydrate and lipid metabolism and activate the transcriptional machinery. Furthermore, we show how early in SE, ground tissue and leaf primordia specification are switched on before the specification of a shoot apical meristem. CONCLUSIONS: This is the first attempt to specifically profile embryogenic cells among the different cell types that constitute plant in vitro tissue cultures. Our comparative analyses provide insights in the gene networks regulating SE and open new research avenues in the field of plant regeneration.

Impairment of sperm DNA methylation in male infertility: a meta-analytic study.

Considering the widespread use of assisted reproductive techniques (ART), DNA methylation of specific genes involved in spermatogenesis achieves increasingly clinical relevance, representing a possible explanation of increased incidence of syndromes related to genomic imprinting in medically assisted pregnancies. Several trials suggested a relationship between male sub-fertility and sperm DNA methylation, although its weight on seminal parameters alteration is still a matter of debate. To evaluate whether aberrant sperm DNA methylation of imprinted genes is associated with impaired sperm parameters. Meta-analysis of controlled clinical trials evaluating imprinted genes sperm DNA methylation comparing men with idiopathic infertility to fertile controls. Twenty-four studies were included, allowing a meta-analytic evaluation for H19, MEST, SNRPN, and LINE-1. When a high heterogeneity of the results was demonstrated, the random effect model was used. H19 methylation levels resulted significantly lower in 879 infertile compared with 562 fertile men (7.53%, 95% CI: 5.14-9.93%, p < 0.001), suggesting a 9.91-fold higher risk ratio to show aberrant sperm DNA methylation (95% CI: 5.55-17.70, p < 0.001, I2  = 19%) in infertile men. The mean MEST methylation level was significantly higher in 846 infertile compared with 353 fertile men (3.35%, 95% CI: 1.41-5.29%, p < 0.001), as well as for SNRPN comparing 301 infertile men with 124 controls (3.23%, 95% CI: 0.75-5.72%, p < 0.001). LINE-1 methylation levels did not differ between 291 infertile men and 198 controls (0.44%, 95% CI: -2.04-1.16%, p = 0.63). The meta-analytic approach demonstrated that male infertility is associated with altered sperm methylation at H19, MEST, and SNRPN. Although its role in infertility remains unclear, sperm DNA methylation could be associated with the epigenetic risk in ART. In this setting, before proposing this analysis in clinical practice, an accurate identification of the most representative genes and a cost-effectiveness evaluation should be assessed in ad hoc prospective studies.

DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells.

Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated 'ex-vivo' system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.

Prediction of retail beef yield and fat content from live animal and carcass measurements in Nellore cattle.

Data from 156 Nellore males were used to develop equations for the prediction of retail beef yield and carcass fat content, expressed as kilograms and as a percentage, from live animal and carcass measurements. Longissimus muscle area and backfat and rump fat thickness were measured by ultrasound up to 5 d before slaughter and fasted live weight was determined 1 d before slaughter. The same traits were obtained after slaughter. The carcass edible portion (CEP in kg and CEP% in percentage; n = 116) was calculated by the sum of the edible portions of primal cuts: hindquarter, forequarter, and spare ribs. Trimmable fat from the carcass boning process, with the standardization of about 3 mm of fat on retail beef, was considered to be representative of carcass fat content. Most of the variation in CEP was explained by fasted live weight or carcass weight (R(2) of 0.92 and 0.96); the same occurred for CEP% (R(2) of 0.15 and 0.13), and for CEP, the inclusion of LM area and fat thickness reduced the equation bias (lower value of Mallow's Cp statistics). For trimmable fat, most variation could be explained by weight or rump fat thickness. In general, the equations developed from live animal measurements showed a predictive power similar to the equations using carcass measurements. In all cases, the traits expressed as kilograms were better predicted (R(2) of 0.39 to 0.96) than traits expressed as a percentage (R(2) of 0.08 to 0.42).

Pre-miR146a expression in follicular carcinomas of the thyroid.

INTRODUCTION: Micro-RNA, a new class of small, non-coding RNAs, have been shown to be deregulated in several human carcinomas. In particular, SNP rs2910164 in pre-miR146a appears to be correlated with papillary thyroid carcinoma and may be involved in its genetic predisposition. Since data on follicular thyroid carcinomas (FTC) are lacking, we evaluated the involvement of SNP rs2910164 in FTC. METHODS: Thirty-nine cases of FTC and 20 follicular adenomas, defined according to WHO criteria, were selected. DNA and RNA were extracted from formalin-fixed paraffin-embedded blocks of both neoplastic and non-neoplastic areas. The DNA region of pre-miR146a, containing SNP rs2910164, was sequenced. Total RNA including miRNAs was used for stem-loop RT reactions, and applying a standard TaqMan PCR kit protocol for real-time PCR. Wilcoxon signed-rank test and Friedman test were used for statistical analyses. RESULTS: In 31% of FTC, the G allele was observed in neoplastic tissues, compared with the non-neoplastic areas (p < 0.05), whereas the CC phenotype was completely absent in tumours. Moreover, the expression of pre-miR146a was found to be significantly down-regulated in neoplastic tissues from FTC cases (p = 0.043), although no significant differences were seen in follicular thyroid adenomas. DISCUSSION: The expression profile of pre-miR146a can be correlated with FTC tumourigenesis. The G allele in SNP rs2910164 appears to be correlated with the transition from normal to neoplastic tissue. The GG and GC alleles appear to be associated with an increased risk for FTC, while the CC allele seems to play a protective role.

Selection for feed efficiency traits and correlated genetic responses in feed intake and weight gain of Nellore cattle.

The objectives of this study were to estimate genetic parameters for indicator traits of feed efficiency and to recommend traits that would result in better responses to selection for increased weaning weight (weaning weight adjusted to 210 d of age [W210]), ADG, and metabolic BW (BW(0.75)) and lower DMI. Records of W210 from 8,004 Nellore animals born between 1978 and 2011 and postweaning performance test records from 678 males and females born between 2004 and 2011 were used. The following feed efficiency traits were evaluated: G:F, partial efficiency of growth (PEG), relative growth rate (RGR), Kleiber's ratio (KR), residual feed intake (RFI), residual weight gain (RWG), and residual intake and gain (RIG). Covariance and variance components were estimated by the restricted maximum likelihood method using multitrait analysis under an animal model. Estimates of genetic gain and correlated responses were obtained considering single-stage and 2-stage selection. Heritability estimates were 0.22 ± 0.03 (W210), 0.60 ± 0.08 (DMI), 0.42 ± 0.08 (ADG), 0.56 ± 0.06 (BW(0.75)), 0.19 ± 0.07 (G:F), 0.25 ± 0.09 (PEG), 0.19 ± 0.07 (RGR), 0.22 ± 0.07 (KR), 0.33 ± 0.10 (RFI), 0.13 ± 0.07 (RWG), and 0.19 ± 0.08 (RIG). The genetic correlations of DMI with W210 (0.64 ± 0.10), ADG (0.87 ± 0.06), and BW(0.75) (0.84 ± 0.05) were high. The only efficiency traits showing favorable responses to selection for lower DMI were G:F, PEG, RFI, and RIG. However, the use of G:F, PEG, or RFI as a selection criterion results in unfavorable correlated responses in some growth traits. The linear combination of RFI and RWG through RIG is the best selection criterion to obtain favorable responses in postweaning growth and feed intake of Nellore cattle in single-stage selection. Genetic gains in feed efficiency are expected even after preselection for W210 and subsequent feed efficiency testing of the preselected animals.

Body chemical composition of Nellore bulls with different residual feed intakes.

Empty body and carcass chemical compositions, expressed as content of water, ether extract, protein, minerals, and energy, were evaluated in Nellore bulls with different residual feed intakes (RFI). Forty-nine not castrated males, with 343 kg of average initial BW and 398 kg of average slaughter BW, were studied. Animals were divided in two subgroups: reference group (RG) and ad libitum feeding group. At the end of the adaptation period, animals of subgroup RG were slaughtered and the other animals were finished in individual pens for approximately 100 d, until they reached a subcutaneous fat thickness over the LM of 4 mm, and were slaughtered at an average age of 540 d. Body composition was obtained after grinding, homogenizing, sampling, analyzing, and combining blood, hide, head + feet, viscera, and carcass. Tissue deposition rates and chemical composition of gain were also measured based on gains estimated by comparative slaughter technique. No significant differences in slaughter BW (P = 0.8639), empty BW (P = 0.7288), HCW (P = 0.6563), or empty body and carcass rates of gain were observed between RFI groups, demonstrating that the low (-0.331 kg DM/d) and high (+0.325 kg DM/d) RFI animals presented similar body sizes and growth rates. No significant differences in empty body or carcass content of water, ether extract, protein, minerals, and energy were observed between the low and high RFI animals. And also there were no significant differences in empty BW or carcass gain, demonstrating that low and high RFI animals had a similar growth potential. More efficient animals (low RFI) consumed less feed than less efficient animals (high RFI) but presented similar body sizes, growth rates, and empty body and carcass chemical composition.

Of blades and branches: understanding and expanding the Arabidopsis ad/abaxial regulatory network through target gene identification.

HD-ZIPIII and KANADI transcription factors have opposing and dramatic affects on plant development. Analysis of mutants shows these proteins to be master regulators of ad/abaxial (i.e., upper/lower) leaf polarity, leaf blade outgrowth, and branch formation. Because these factors do their work by regulating other genes, we have focused our attention on defining their targets. We have found overlap between the ad/abaxial regulatory pathway and hormone signaling pathways, especially pathways of abscisic acid and auxin signaling. This has led to the discovery that abscisic acid signaling acts upstream of HD-ZIPIII and KANADI in the control of germination and may ultimately explain how environmental stress pathways control new growth at the shoot apex. Auxin signaling conversely is downstream from HD-ZIPIII and KANADI action with these factors controlling targets at all steps of auxin action-biosynthesis, transport, regulation of transport, and signaling. Based on these findings, we propose a model in which the HD-ZIPIII and KANADI factors pattern auxin response in the embryo. Finally, many genes targeted for control by HD-ZIPIII and KANADI proteins are themselves transcription factors-indicating these master regulators call up tissue specific subprograms of transcriptional control to affect the many polar differences observed across tissues.

The tumor microenvironment: biology of a complex cellular and tissue society.

The tumor microenvironment is a "complex society" of many cell types and their extracellular matrix. All these cell types and the matrix take part to the generation of a tumor "tissue". It is well established that preneoplastic proliferating cells cannot give origin to a tumor without an appropriate blood supply. In fact, angiogenesis could be considered the rate limiting step of tumor growth. In this context microenvironment components play a pivotal role in the regulation of the angiogenic switch and in cancer progression. For these reasons the comprehension of biological and molecular mechanisms involved in the relationship between tumor cells and the microenvironment could unveil new therapeutic and preventive approaches to cancer. In this complex scenario molecular imaging of the microenvironment is crucial to dissect cellular and stromal dynamic contributions.

Genome sequence, comparative analysis, and population genetics of the domestic horse.

We report a high-quality draft sequence of the genome of the horse (Equus caballus). The genome is relatively repetitive but has little segmental duplication. Chromosomes appear to have undergone few historical rearrangements: 53% of equine chromosomes show conserved synteny to a single human chromosome. Equine chromosome 11 is shown to have an evolutionary new centromere devoid of centromeric satellite DNA, suggesting that centromeric function may arise before satellite repeat accumulation. Linkage disequilibrium, showing the influences of early domestication of large herds of female horses, is intermediate in length between dog and human, and there is long-range haplotype sharing among breeds.

Comparative study of methodologies for pulse wave velocity estimation.

Arterial stiffness, estimated by pulse wave velocity (PWV), is an independent predictor of cardiovascular mortality and morbidity. However, the clinical applicability of these measurements and the elaboration of reference PWV values are difficult due to differences between the various devices used. In a population of 50 subjects aged 20-84 years, we compared PWV measurements with three frequently used devices: the Complior and the PulsePen, both of which determine aortic PWV as the delay between carotid and femoral pressure wave and the PulseTrace, which estimates the Stiffness Index (SI) by analyzing photoplethysmographic waves acquired on the fingertip. PWV was measured twice by each device. Coefficient of variation of PWV was 12.3, 12.4 and 14.5% for PulsePen, Complior and PulseTrace, respectively. These measurements were compared with the reference method, that is, a simultaneous acquisition of pressure waves using two tonometers. High correlation coefficients with the reference method were observed for PulsePen (r = 0.99) and Complior (r = 0.83), whereas for PulseTrace correlation with the reference method was much lower (r = 0.55). Upon Bland-Altman analysis, mean differences of values +/- 2s.d. versus the reference method were -0.15 +/- 0.62 m/s, 2.09 +/- 2.68 m/s and -1.12 +/- 4.92 m/s, for PulsePen, Complior and Pulse-Trace, respectively. This study confirms the reliability of Complior and PulsePen devices in estimating PWV, while the SI determined by the PulseTrace device was found to be inappropriate as a surrogate of PWV. The present results indicate the urgent need for evaluation and comparison of the different devices to standardize PWV measurements and establish reference values.

Postoperative analgesia after cesarean section by continued administration of levobupivacaine with the On-Q Painbuster system over the fascia vs ketorolac + morphine i.v.

OBJECTIVE: This study aimed to detect if continuous local infusion of levobupivacaine with the On-Q Painbuster system provided postoperative analgesia of similar quality to morphine + ketorolac i.v. in patients undergoing cesarean section. MATERIALS AND METHODS: Using a randomized prospective double-blind study, 20 women undergoing cesarean section with a standardized spinal technique were randomly assigned into two groups to receive either 10 mg morphine + 120 mg ketorolac + saline solution up to 96 ml with an elastomeric pump i.v. (group A) or local infusion of levobupivacaine 0.2% with the On-Q PAINBUSTER system (group B). Both groups were administered ketorolac i.v. in bolus in case of pain. RESULTS: The two groups differed in their VAS scores with group A experiencing significantly less pain than group B; the consumption of analgesics was significantly lower in group A than in group B. CONCLUSIONS: The i.v. system with morphine and ketorolac is more effective than levobupivacaine subcutaneous infusion in reducing postoperative pain associated with cesarean section.

Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount.

We describe an optimized protocol for the transient transformation of tobacco protoplasts mediated by polyethylene-glycol (PEG). As expected, the quantitative beta-glucuronidase (Gus) activity driven by pCaMVGus was dependent on the amount of plasmid used. Nevertheless, we demonstrate by an immunodetection method that transformation efficiency did not depend on the amount of plasmid used but on the limitation imposed by cell competence. In fact, we obtained the same percentage of transformed cells (about 60%) using a wide range of plasmid concentrations (0.1-10 microg per test). Finally, we show that, when we used two plasmid types in a mixture at a concentration ranging from 0.1 to 10 microg for each, all transformed cells expressed proteins encoded by both plasmids. Transient expression and co-transformation experiments are routinely used methods and, probably, the major results from this work were assumed by many researchers in this field, but our data experimentally support this assumption.

Thalidomide: an old sedative-hypnotic with anticancer activity?

Thalidomide is a synthetic derivative of glutamic acid with sedative-hypnotic activity, which caused devastating teratogenic effects in the 1960s. This paper reviews the possible mechanisms of its teratogenic effect, its new therapeutic indications, the proposed mechanisms for its antitumor activity and, finally, reviews published studies of its application in oncology. Current data demonstrates that thalidomide is clinically promising in multiple myeloma, glioblastoma multiforme and renal cell cancer. Furthermore, a beneficial effect of the drug has been proposed in cancer-related cachexia, which merits further investigation. Well-designed, randomized studies are warranted to establish the possible indications of thalidomide as an antitumor compound.

Cisplatin and escalating doses of paclitaxel and epirubicin in advanced ovarian cancer. A phase I study.

PURPOSE: The combination of paclitaxel and cisplatin is considered the standard regimen for advanced ovarian cancer (AOC). A meta-analysis has shown that the incorporation of anthracyclines into first-line chemotherapy might improve long-term survival by 7-10%. We designed a phase I-II study in patients with AOC using a combination of a fixed dose of cisplatin with paclitaxel and epirubicin both given at escalating doses every 3 weeks. The objectives of this study were to determine both the maximum tolerated dose (MTD) and the antitumor activity of this combination. METHODS: Six different dose levels were planned. The starting doses were cisplatin 75 mg/m2, paclitaxel 140 mg/m2, and epirubicin 50 mg/m2. The doses of paclitaxel were escalated in 20-mg/m2 increments, alternating with 20-mg/m2 increments of epirubicin. Ten patients with AOC entered the phase I study. Three patients each were enrolled at level I and level II and four patients at level III, and at each level, 15 courses were administered. Patients received a median of five courses. RESULTS: Nonhematological toxicity was generally mild, except for grade 3 mucositis in one course at levels II and III, and grade 3 vomiting in one course at levels I and III. Hematological toxicities were grade 3-4 neutropenia in 60%, 47% and 60% of courses at levels I, II and III, respectively, and grade 3 anemia in one course at level III. At level III two of four patients developed a dose-limiting toxicity which was grade 4 neutropenia lasting more than 1 week. CONCLUSIONS: The MTD was reached at level II with cisplatin 75 mg/m2, paclitaxel 160 mg/m2, and epirubicin 50 mg/m2. The phase II part of the study is currently ongoing.

Resazurin detection of energy metabolism changes in serum-starved PC12 cells and of neuroprotective agent effect.

Resazurin is a dye, which becomes fluorescent when reduced by oxidoreductases within viable cells. Measurement of resazurin fluorescence is therefore an indicator of the cell's energy metabolism. Resazurin was used here to detect metabolic changes in PC12 cells following serum starvation. Serum withdrawal is a cytotoxic environmental change resulting in cell death in cultured cell lines as well as in primary cells of various tissue origins, including nerve cells. In particular, PC12 cells have been widely employed as a neuronal cell model and a large number of studies generated. Many molecular and morphological changes occur in PC12 cells after serum withdrawal, and apoptotic cell death is the final consequence. We show that resazurin can detect the metabolic impairment in serum-deprived PC12 cells and can measure the neuroprotective properties of PACAP 1-38, as early as day 1 after serum withdrawal. Resazurin constitutes an advantageous tool to discriminate between healthy and metabolically impaired cells, since fluorescence produced by the reduced dye can be measured in living cells without a lysis step. The experiment is fast, inexpensive, uses a small amount of cells and can easily be automated.