Clinical and biological predictors of Cladribine effectiveness in Multiple Sclerosis: A real-world, single Centre study considering a two-year interval from year-2 dosing.

OBJECTIVES: Cladribine tablets (CLAD) for adult patients with highly active relapsing multiple sclerosis (RMS) have been available in Italy since 2018. We aimed to assess predictors of no-evidence-of-disease-activity-3 (NEDA-3) status after 24 months of the last dose of CLAD. RESULTS: We included 88 patients (70.5% female, mean age at CLAD start 35.4 ± 11.4). Eighteen patients were treatment naïve, 48 switched to CLAD from a First line Disease Modifying Drug (DMD), and 22 from Second line DMDs. All patients were observed for a median follow-up time of 2.4 (1-4) years after the last dose of CLAD. Forty-nine patients (55.7%) showed NEDA at the last available follow-up. Naïve patients (p = 0.001), those with a lower number of previous DMDs (p < 0.001) and, even though not significantly, those switching from first line DMDs (p = 0.069) were more likely NEDA3 at the last available follow-up. In a subgroup of 30 patients (34%), Serum Light Neurofilaments (sNFL) levels showed a decrease from baseline to the 24 months of follow-up, statistically significant from baseline to the sixth month, and from the first to the second year detection. sNFL levels at 12th month showed a strong inverse correlation with the time to NEDA3 loss. CONCLUSIONS: Our experience provides information for the 2-years after the last dose of CLAD, confirming a higher effectiveness of CLAD when placed early in the treatment algorithm. Given the ongoing expansion of the therapeutic landscape in MS, sNfL could support individualized decision-making, used as blood-based biomarker for CLAD responses in clinical practice.

Risk of multiple sclerosis relapses when switching from fingolimod to cell-depleting agents: the role of washout duration.

BACKGROUND: Fingolimod (FTY) induces sequestration of lymphocytes in secondary lymphoid organs and the average lymphocyte recovery following discontinuation takes 1-2 months. It has been hypothesized that the therapeutic effects of subsequent cell-depleting agents may be compromised if initiated before lymphocyte recovery has occurred. OBJECTIVE: To assess the risk of relapses following FTY discontinuation and the initiation of a B/T cell-depleting agent in relation to washout duration using data from the Italian MS Register. METHODS: The risk of relapses was assessed in relation to different washout durations (< 6, 6-11, 12-17 and > / = 18 weeks) in patients starting alemtuzumab, rituximab, ocrelizumab or cladribine following FTY discontinuation. RESULTS: We included 329 patients in the analysis (226F, 103 M; mean age 41 ± 10 years). During the cell-depleting treatment, the incidence rate ratio for a relapse was significantly greater in patients with a washout period of 12-17 and > / = 18 weeks compared to the reference period (< 6 weeks). The risk of a relapse was significantly influenced by the occurrence of relapses during FTY treatment and by washout length, with hazard ratios markedly increasing with the washout duration. CONCLUSION: The risk of relapses increases with the washout duration when switching from FTY to lymphocyte-depleting agents.

A sensitive and efficient detection method for bovine viral diarrhea virus (BVDV) in single preimplantation bovine embryos.

The objective was to develop a method to accurately and efficiently detect minute amounts of bovine viral diarrhea virus (BVDV) associated with a single embryo. There are two major challenges for BVDV detection in a single embryo: the test sensitivity and the efficiency of viral molecule recovery. These become even more critical when attempts are made to detect BVDV infections that occurred naturally, not through artificial exposure of the embryos to high affinity BVDV strains. We have developed a one-step sample preparation method that has increased the viral molecule recovery rate compared to the standard RNA isolation procedure by 7-100-fold. Instead of using the traditional virus exposure approach, we generated BVDV positive embryos via somatic cell nuclear transfer (SCNT) technology using BVDV positive donor cells. By combining the highly efficient sample preparation procedure with a sensitive one-step, real-time PCR system, we have developed a sensitive test that allows detection of as low as two copies of BVDV in a single embryo. This method will allow systematic risk assessment for BVDV transmission during in vitro embryo production via IVF or SCNT procedures.

An alternative LMI static output feedback control design for discrete-time nonlinear systems represented by Takagi-Sugeno models.

This paper presents a static output feedback controller design for discrete-time nonlinear systems exactly represented by Takagi-Sugeno models. By introducing past states in the control law as well as in the Lyapunov function, more relaxed results are obtained. Different conditions in terms of linear matrix inequalities are provided. The proposed conditions are less demanding than the ones in the literature. This is illustrated via numerical examples.

A velocity tracking approach for the data assimilation problem in blood flow simulations.

Several advances have been made in data assimilation techniques applied to blood flow modeling. Typically, idealized boundary conditions, only verified in straight parts of the vessel, are assumed. We present a general approach, on the basis of a Dirichlet boundary control problem, that may potentially be used in different parts of the arterial system. The relevance of this method appears when computational reconstructions of the 3D domains, prone to be considered sufficiently extended, are either not possible, or desirable, because of computational costs. On the basis of taking a fully unknown velocity profile as the control, the approach uses a discretize then optimize methodology to solve the control problem numerically. The methodology is applied to a realistic 3D geometry representing a brain aneurysm. The results show that this data assimilation approach may be preferable to a pressure control strategy and that it can significantly improve the accuracy associated to typical solutions obtained using idealized velocity profiles.

A microsatellite genetic linkage map for Xiphophorus.

Interspecies hybrids between distinct species of the genus Xiphophorus are often used in varied research investigations to identify genomic regions associated with the inheritance of complex traits. There are 24 described Xiphophorus species and a greater number of pedigreed strains; thus, the number of potential interspecies hybrid cross combinations is quite large. Previously, select Xiphophorus experimental crosses have been shown to exhibit differing characteristics between parental species and among the hybrid fishes derived from crossing them, such as widely differing susceptibilities to chemical or physical agents. For instance, genomic regions harboring tumor suppressor and oncogenes have been identified via linkage association of these loci with a small set of established genetic markers. The power of this experimental strategy is related to the number of genetic markers available in the Xiphophorus interspecies cross of interest. Thus, we have undertaken the task of expanding the suite of easily scored markers by characterization of Xiphophorus microsatellite sequences. Using a cross between Xiphophorus maculatus and X. andersi, we report a linkage map predominantly composed of microsatellite markers. All 24 acrocentric chromosome sets of Xiphophorus are represented in the assembled linkage map with an average intergenomic distance of 7.5 cM. Since both male and female F1 hybrids were used to produce backcross progeny, these recombination rates were compared between "male" and "female" maps. Although several genomic regions exhibit differences in map length, male- and female-derived maps are similar. Thus Xiphophorus, in contrast to zebrafish, Danio rerio, and several other vertebrate species, does not show sex-specific differences in recombination. The microsatellite markers we report can be easily adapted to any Xiphophorus interspecies and some intraspecies crosses, and thus provide a means to directly compare results derived from independent experiments.

Change in fluconazole susceptibility patterns and genetic relationship among oral Candida albicans isolates.

OBJECTIVE: To assess the genetic homogeneity or heterogeneity within each set of Candida albicans isolates colonizing/infecting the oral cavities of HIV-infected patients undergoing azole therapy when changes in susceptibility to fluconazole were detected. DESIGN: Fourteen HIV-positive patients suffering recurrent episodes of oral candidosis were prospectively followed from the first episode to the isolation of strains with decreased susceptibility to fluconazole. The strains of C. albicans isolated either from episodes or controls throughout the prospective study were analysed. METHODS: Electrophoretic karyotyping and hybridization with the repeated sequence probe 27A were used to delineate sequential isolates. In vitro susceptibility tests to fluconazole and ketoconazole were also performed. The results obtained by DNA fingerprinting with the probe combined with computer-assisted analysis were used to assess the genetic relationships amongst the strains. In addition, comparison with the genetic relatedness of a group of geographically unrelated strains was made. RESULTS: Isogenic populations of sequential isolates were observed only in two patients; 12 patients harboured heterogenic populations over time, although in 11 patients there was a predominant strain that was isolated more than once, and only one of these patients carried strains with a similarity index less than 80%. With the exception of two patients, each patient carried a major strain that became less susceptible to fluconazole. The similarity index for the unrelated strains was 59%. CONCLUSIONS: HIV-infected patients may carry a mixed population of strains, but the strains tend to be related to each other. The strains were maintained throughout the course of infection and at least one developed secondary resistance to fluconazole.

Molecular characterization by PCR-fingerprinting of Candida dubliniensis strains isolated from two HIV-positive patients in Spain.

Six Candida dubliniensis isolates were recovered from two HIV-infected individuals in the course of a prospective study of recurrent oral candidosis among HIV-positive patients in Spain. Candida albicans strains as well as non-albicans strains were also obtained from these two patients. C. dubliniensis strains were germ-tube-positive and produced abundant chlamydospores. Fingerprinting the genomic DNAs of these six C. dubliniensis with the C. albicans-specific probe 27A as well as karyotyping was performed to confirm the identification of these isolates. Further analysis of their genomic DNAs was performed by PCR-fingerprinting with the core sequence of phage M13, and they exhibited species-specific multilocus band patterns, clearly distinct from those of C. albicans isolates analyzed in this study and in a previous one (Diaz-Guerra 1997). Intraspecies variation was also seen among PCR patterns yielded by C. dubliniensis isolates from different patients. Although few strains have been analyzed, the use of this PCR-fingerprinting procedure is a promising tool for further epidemiologic studies with C. dubliniensis. The isolation of C. dubliniensis from Spanish HIV-infected patients contributes to the idea of widespread geographic distribution of this species.

Sustained gastrointestinal colonization and systemic dissemination by Candida albicans, Candida tropicalis and Candida parapsilosis in adult mice.

The ability of nine clinical isolates of Candida species (three C. albicans, three C. tropicalis and three C. parapsilosis) to colonize and invade the gastrointestinal (GI) tract of adult male CD-1 (ICR) mice was determined. The effect of dietary tetracycline plus glucose supplementation on colonization was evaluated. Strains were intragastrically inoculated. Tetracycline and glucose altered substantially aerobic flora, especially streptococci (average fall 1.1 +/-0.3 log(10) CFU/g, p<0.01 by the Student's t test). At two weeks after oral challenge, sustained and high colonization of GI tract by Candida (mean 5,28 +/- 0.18 log(10) CFU/g, p<0.01) was achieved in all mice receiving glucose-tetracycline supplementation, excepting in animals inoculated with one of C. tropicalis isolates. Histologic sections of the stomachs revealed multiple intraepithelial micro-abscesses associated with hyphae in the region of the cardial-atrium fold. Under immunosuppression, systemic spread of C. albicans and C. tropicalis was observed in 62% and 24% of animals receiving dietary supplementation respectively. Dissemination was not noted for C. parapsilosis isolates. We have developed a simple and inexpensive murine model of sustained colonization of GI tract. This model could be useful for analyzing prophylaxis, treatment and diagnosis of systemic Candida infections and for evaluating virulence of strains.

Characterization of a possible nosocomial aspergillosis outbreak.

OBJECTIVE: To study the epidemiologic aspects of a suspected outbreak of nosocomial invasive aspergillosis. METHODS: Sixteen Aspergillus fumigatus strains were isolated from bronchoalveolar washings or sputa of 10 patients during a 9-month period. Furthermore, two environmental samples, isolated in a microbiological screening of the hospital, were also available for analysis. Random amplified polymorphic DNA analysis (RAPD) was carried out. RESULTS: The analysis performed by RAPD clearly demonstrated substantial genetic variation among the isolates. Both of the two different primers selected for RAPD analysis (R-108 and AP12h) were able to demonstrate that the strains isolated from all patients infected with the same fungal species and the environmental samples were genotypically distinct. The results by RAPD typing demonstrated that this technique could detect variability among isolates of Aspergillus fumigatus from different patients and even from the same patient. CONCLUSIONS: RAPD genotyping proved that the outbreak of invasive aspergillosis consisted of a series of events, non-related, and probably not coming from the same source within the hospital. This type of analysis is an easy, quick and highly discriminatory technique that may help in planning epidemiologic studies of aspergillosis.

[Frequency of sickle-cell anemia in the population of "Cuatro Bocas," Parroquia Ricaurte, Mara municipality, Zulia state, Venezuela]. / Frecuencia de anemia falciforme en la población de "Cuatro Bocas", Parroquia Ricaurte, Municipio Mara, Estado Zulia, Venezuela.

Isla de Toas is an island on the north of the Maracaibo Lake, it is known in the scientific community, for the high frequency of sickle cell disease, in a population with caucasoid phenotype. The purpose of the present work was to determine the frequency of sickle cell anemia in the population of Cuatro Bocas, situated 35 km from the southwest of Isla de Toas. The town is the center of confluence of a rural population constituted mainly of farmers. The sample consisted of 870 persons of both sexes, aged from 8 months to 66 years. The presence of the sickling phenomenon was determined in all the individuals, and hemoglobin electrophoresis in agarose was performed in all the positive samples. The following results were obtained: fifty-six cases (6.4%), showed drepanocytic changes, and forty-six of them were haemoglobin A/S, 8 were S/S and 2 were S/C. The higher frequency of hemoglobin S was in adolescents and adults. The family backgrounds suggest an insular origin of the sickle cell gene. About 75% of the affected population was ignorant of this condition. The hemoglobin values were lower in the individuals with the sickle cell disease (p < 0.05), than in the normal persons. Iron deficiency in adolescents was suspected because or their low hemoglobin values. The results indicate that the sickle cell gen is expanding to the nearest communities of the Mara county. It is important to consider that the findings of the present work should serve as an alert to the Public Health authorities, and that education of the population is important in order to prevent the spreading of the disease.

[Development of a milk beverage based on pumpkin flakes]. / Desenvolvimento de bebida láctea a base de flocos de abóbora.

Vitamin A deficiency is one of the majors public health problems in Brazilian Northeast, and among other causes may be concerned to precocious weaning. Aiming at reducing this problem, a dehydrated product based on powdered milk and pumpkin flakes was developed to act as a carotene source at weaning period. Pumpkin flakes were obtained by drum drying at a 6 atm steam pressure, 0.75 m2 contact surface and 1 rpm, and had their content of carotenoids, beta carotene, centesimal composition molds and yeast and faecal coliforms evaluated. The flakes were added to sugar and whole powdered milk according to FAO/WHO nutritional recommendation to 6-12 months old children and submitted to acceptability test by a 6 judges' panel. The results showed that the drying process had a 7% efficiency score and the flakes composition presented 4.84% moisture; 4.0% protein; 5.5% ash; 1.30% fat; 6.22% fiber; 78.14% carbohydrates; 115.08 micrograms/g total carotenois and 80.64 micrograms/g beta-carotene content. No coliforms were detected and molds and yeast content was 4.0 x 10(2) CFU/g. The powdered formulation properly diluted in water supports 45% of the RDA for children (calories, protein, carbohydrates and lipids) and 100% vitamin A and protein considering a 400 ml/day ingestion.

Nectar secretion dynamic links pollinator behavior to consequences for plant reproductive success in the ornithophilous mistletoe Psittacanthus robustus.

The mistletoe Psittacanthus robustus was studied as a model to link flower phenology and nectar secretion strategy to pollinator behaviour and the reproductive consequences for the plant. The bright-coloured flowers presented diurnal anthesis, opened asynchronously throughout the rainy season and produced copious dilute nectar as the main reward for pollinators. Most nectar was secreted just after flower opening, with little sugar replenishment after experimental removals. During the second day of anthesis in bagged flowers, the flowers quickly reabsorbed the offered nectar. Low values of nectar standing crop recorded in open flowers can be linked with high visitation rates by bird pollinators. Eight hummingbirds and two passerines were observed as potential pollinators. The most frequent flower visitors were the hummingbirds Eupetomena macroura and Colibri serrirostris, which actively defended flowering mistletoes. The spatial separation between anthers, stigma and nectar chamber promotes pollen deposition on flapping wings of hovering hummingbirds that usually probe many flowers per visit. Seed set did not differ between hand-, self- and cross-pollinated flowers, but these treatments set significantly more seeds than flowers naturally exposed to flower visitors. We suggest that the limitation observed in the reproductive success of this plant is not related to pollinator scarcity, but probably to the extreme frequency of visitation by territorial hummingbirds. We conclude that the costs and benefits of plant reproduction depend on the interaction strength between flowers and pollinators, and the assessment of nectar secretion dynamics, pollinator behaviour and plant breeding system allows clarification of the complexity of such associations.

Large scale in vivo risk assessment of bovine viral diarrhea virus (BVDV) transmission through transfer of bovine embryos produced via somatic cell nuclear transfer (SCNT).

The objective was to use the bovine viral diarrhea virus (BVDV) as a model to assess the risk of infectious disease transmission in the system of in vitro embryo production and transfer via somatic cell nuclear transfer (SCNT) technology. The risks of BVDV transmission in the SCNT embryo production were previously evaluated. In that in vitro study, following standard operating procedures (SOP), including pre-nuclear transfer donor cell testing, oocyte decontamination and virus-free cell and embryo culture conditions, SCNT embryos produced were free of detectable viral RNA. The current study focused on the evaluation of the potential risk of disease transmission from SCNT embryos to recipients, and the risk of producing persistently infected animals via SCNT embryo transfer. Blood samples were collected from 553 recipients of SCNT embryos and 438 cloned calves and tested for the presence of BVDV viral RNA via a sensitive real time PCR method. All samples tested were negative. These results, in conjunction with the previous in vitro study, confirmed that the established SCNT embryo production and transfer system is safe and presents no detectable risk of disease transmission.

Risk and prevention of bovine viral diarrhea virus (BVDV) transmission through embryo production via somatic cell nuclear transfer (SCNT) using oocytes from persistently infected donors.

The objective was to assess the risk of transmission of bovine viral diarrhea virus (BVDV) through embryo production via somatic cell nuclear transfer (SCNT), with oocytes obtained from persistently infected (PI) donors. Using ultrasound-guided follicular aspiration following superstimulation, oocytes were obtained from five female beef cattle, including three that were PI and two that were negative for BVDV. In the three PI cattle, seven aspirations yielded 32 oocytes (PI-1: three aspirations yielding six oocytes; PI-2: two aspirations yielding 14 oocytes; and PI-3: two aspirations yielding 12 oocytes). The oocyte recovery rate was better in negative control cattle, with 32 oocytes obtained from the two cattle in a single superstimulation and aspiration session. Oocytes were processed individually for SCNT, evaluated, and tested for BVDV. Nearly all (31/32) oocytes from the three PI donors were positive for BVDV by PCR, with detected viral RNA copy number ranging from 1 to 1.1 x 10(5). The proportion of oocytes acceptable for SCNT embryo production (based on oocyte quality and maturation status) was only 16 to 35% from PI donors, but was 81% from control donors. Therefore, routine testing of unacceptable (discarded) oocytes could be an effective approach to identify batches that might contain infected oocytes from PI donors. Identification and removal of high-risk batches of oocytes would minimize the risk of BVDV transmission through SCNT embryo production.

Experimental risk assessment of bovine viral diarrhea virus transmission via in vitro embryo production using somatic cell nucleus transfer.

The objective of this study was to perform a comprehensive risk assessment on infectious disease transmission in the system of in vitro embryo production via somatic cell nucleus transfer (SCNT) technology using bovine viral diarrhea virus (BVDV) as a model. The risks of BVDV transmission in each step of the SCNT embryo production procedure, from donor cells to preimplantation SCNT embryo culture, were carefully examined using a sensitive real-time polymerase chain reaction assay. The identified primary source of BVDV transmission in SCNT embryo production was donor cell infection, most likely caused by contaminated fetal bovine serum in the culture medium. The risk of disease transmission through contaminated oocytes from an abattoir was relatively low, and it can be greatly minimized by cumulus cell removal and adequate oocyte washing procedures. Of the 200 cumulus-oocyte complexes (COCs) and more than 1500 cumulus cell-free oocyte (CFO) samples collected from multiple sources over a course of 7 months, only 2.5% of the COCs were BVDV positive, and all of the CFOs (100%) were BVDV negative. To evaluate the risk of BVDV introduction during in vitro SCNT embryo culture, 324 SCNT embryos were produced from 18 different cell lines using oocytes from 26 different batches collected over a course of 9 months. The embryos were cultured in vitro for 7 days and then tested for BVDV. All of the 324 SCNT embryos (100%) were negative, indicating that the embryo culture system is virtually risk-free for BVDV transmission. Based on these results, a standard operational protocol (SOP) for SCNT embryo production was proposed to greatly minimize the risk of BVDV transmission through the SCNT embryo production system. This SOP could be a starting point to produce a SCNT system that is virtually risk-free for disease transmission in general.

Evaluation of tumor markers carcinoembryonic antigen, cytokeratin 19 fragment and cancer-associated antigen 72-4 in neoplastic and non-neoplastic canine effusions differentiation / Avaliação dos marcadores tumorais antígeno carcinoembrionário, fragmento de citoqueratina 19 e antígeno associado ao câncer 72-4 na diferenciação de efusões neoplásicas e não neoplásicas caninas

The concentration of tumor markers in body fluids can be used for diagnosis and prognosis of patients. This study aimed to investigate the performance of tumor markers cytokeratin 19 fragment (CYFRA 21-1), cancer-associated antigen 72-4 (CA 72-4) and carcinoembryonic antigen (CEA) in the neoplastic and non-neoplastic canine effusions. In thirty-two neoplastic (n=16) and non-neoplastic (n=16) samples of canine thoracic or abdominal effusions, tumor markers were measured. Significant statistical difference was found only for the CYFRA 21-1 marker. The levels were significantly higher for the neoplastic group. The lack of significance between groups for markers CA 72-4 and CEA can be explained by the presence of other diseases in the non-neoplastic group, causing elevated levels of these markers. This study concludes that CYFRA 21-1 performed well, showing good sensitivity, specificity and accuracy in the diagnosis of neoplastic effusions in dogs. However, further investigations are necessary in patients with malignancy as those with benign effusions...

Os níveis de marcadores tumorais em líquidos corporais podem ser usados para diagnóstico e prognóstico de pacientes. Este estudo objetiva investigar o desempenho dos marcadores tumorais fragmento de citoqueratina 19 (CYFRA 21-1), antígeno asociado ao câncer 72-4 (CA 72-4) e antígeno carcinoembrionário (CEA) em efusões caninas neoplásicas e não neoplásicas. Os marcadores tumorais foram mensurados em 32 amotras de efusões torácicas e abdominais de cães, 16 neoplásicas e 16 não neoplásicas. Foi encontrada diferença estatística somente para o marcador CYFRA 21-1, onde os níveis foram significativamente altos no grupo neoplásico. A falta de significância entre os grupos de marcadores CA 72-4 e CEA pode ser explicada pela presença de outras doenças no grupo não neoplásico, o que causou elevação dos níveis destes marcadores. Este estudo conclui que o marcador CYFRA 21-1 teve bom desempenho, pois mostrou boa sensibilidade, especificidade e acurácia no diagnóstico de efusões neoplásicas em cães. Entretanto, mais estudos são necessários tanto em pacientes portadores de efusões benignas quanto malignas...