Cumulative life stressors and stress response to threatened preterm labour as birth date predictors.

PURPOSE: Preterm birth represents one of the main causes of neonatal morbimortality and a risk factor for neurodevelopmental disorders. Appropriate predictive methods for preterm birth outcome, which consequently would facilitate prevention programs, are needed. We aim to predict birth date in women with a threatened preterm labour (TPL) based on stress response to TPL diagnosis, cumulative life stressors, and relevant obstetric variables. METHODS: A prospective cohort of 157 pregnant women with TPL diagnosis between 24 and 31 weeks gestation formed the study sample. To estimate the stress response to TPL, maternal salivary cortisol, α-amylase levels, along with anxiety and depression symptoms were measured. To determine cumulative life stressors, previous traumas, social support, and family functioning were registered. Then, linear regression models were used to examine the effect of potential predictors of birth date. RESULTS: Lower family adaptation, higher Body Mass Index (BMI), higher cortisol levels and TPL diagnosis week were the main predictors of birth date. Gestational week at TPL diagnosis showed a non-linear interaction with cortisol levels: TPL women with middle- and high-cortisol levels before 29 weeks of gestation went into imminent labour. CONCLUSION: A combination of stress response to TPL diagnosis (salivary cortisol) and cumulative life stressors (family adaptation) together with obstetric factors (TPL gestational week and BMI) was the best birth date predictor. Therefore, a psychosocial therapeutic intervention program aimed to increase family adaptation and decrease cortisol levels at TPL diagnosis as well as losing weight, may prevent preterm birth in symptomatic women.

Analysis of the epitope structure of Plum pox virus coat protein.

Typing of the particular Plum pox virus (PPV) strain responsible in an outbreak has important practical implications and is frequently performed using strain-specific monoclonal antibodies (MAbs). Analysis in Western blots of the reactivity of 24 MAbs to a 112-amino-acid N-terminal fragment of the PPV coat protein (CP) expressed in Escherichia coli showed that 21 of the 24 MAbs recognized linear or denaturation-insensitive epitopes. A series of eight C-truncated CP fragments allowed the mapping of the epitopes recognized by the MAbs. In all, 14 of them reacted to the N-terminal hypervariable region, defining a minimum of six epitopes, while 7 reacted to the beginning of the core region, defining a minimum of three epitopes. Sequence comparisons allowed the more precise positioning of regions recognized by several MAbs, including those recognized by the 5B-IVIA universal MAb (amino acids 94 to 100) and by the 4DG5 and 4DG11 D serogroup-specific MAbs (amino acids 43 to 64). A similar approach coupled with infectious cDNA clone mutagenesis showed that a V74T mutation in the N-terminus of the CP abolished the binding of the M serogroup-specific AL MAb. Taken together, these results provide a detailed positioning of the epitopes recognized by the most widely used PPV detection and typing MAbs.

In utero exposure to mycophenolate mofetil: a characteristic phenotype?

Mycophenolate mofetil (MMF) is a widely prescribed immunosuppressive agent after solid organ transplantation. Potential teratogenic effects after in utero exposure to MMF in experimental studies and clinical observations in humans has been postulated in recent literature. However, a specific pattern of malformation has not been identified yet. We present a newborn patient, born to a recipient of renal transplantation, who became pregnant while taking MMF as immunosuppressive therapy. The newborn exhibited cleft lip and palate, bilateral microtia and atretic external auditory canals, chorioretinal coloboma, hypertelorism, and micrognathia. An extensive review of the literature documented six other cases with similar malformations after in utero exposure to MMF. A consistent pattern of malformations comprising cleft lip and palate, microtia and external auditory canals could be observed in five of the six cases. A different malformative pattern observed in one of the patients could be attributed to a different agent rather than MMF. The possible teratogenic effects of other immunosuppressive drugs, such as tacrolimus and prednisone, to which this patient was also exposed, are discussed herein. In addition, the differential diagnosis with other dysmorphic syndromes that can present with a similar phenotype, such as CHARGE syndrome, 18q deletion and hypertelorism-microtia-clefting (HMC) syndrome, is presented. We conclude that in utero exposure to MMF can cause a characteristic phenotype and propose the existence of a mycophenolate-associated embryopathy whose main features are: cleft lip and palate, microtia with atresia of external auditory canal, micrognathia and hypertelorism. Ocular anomalies, corpus callosum agenesis, heart defects, kidney malformations, and diaphragmatic hernia may be part of the phenotypic spectrum of MMF embryopathy. The human teratogenicity of MMF is reinforced by this report, and the current contraceptive recommendations about its use in fertile women are stressed.

Abdominal Near-Infrared Spectroscopy Detects Low Mesenteric Perfusion Early in Preterm Infants with Hemodynamic Significant Ductus Arteriosus.

BACKGROUND: The therapeutic decision to close patent ductus arteriosus in preterm infants entails great uncertainty. Near-infrared spectroscopy is a noninvasive bedside technique used to monitor mixed regional oxygen saturation. We hypothesized that near-infrared spectroscopy could identify preterm infants at risk of mesenteric hypoperfusion associated with hemodynamically significant ductus arteriosus. METHODS: This is a prospective observational study including consecutively admitted preterm infants with a gestational age <32 weeks. Mesenteric regional oxygenation was blindly monitored using an INVOS 5100 device. The presence of a hemodynamically significant patent ductus arteriosus was routinely confirmed by echocardiography/Doppler. Statistical analysis including Bland-Altman plots was performed to assess near-infrared spectroscopy intraobserver repeatability. RESULTS: A total of 72 preterm infants were enrolled. The daily mean regional oxygen saturation for preterm infants was determined both in mesenteric and cerebral regions and plotted against time. We identified a differential temporary baseline. Hemodynamic significant ductus arteriosus was associated with lower blood pressures and lower regional splanchnic oxygenation. There was a significant relationship between reversal diastolic flow in the descending aorta and the regional oxygen saturation, which remained significant after controlling for ductal size and nil per os. CONCLUSIONS: The simultaneous monitoring of splanchnic near-infrared spectroscopy and echocardiography could identify low mesenteric perfusion in the presence of hemodynamic ductus arteriosus.

Targeting the extracellular domain of fibroblast growth factor receptor 3 with human single-chain Fv antibodies inhibits bladder carcinoma cell line proliferation.

PURPOSE: Previous gene expression studies have shown that fibroblast growth factor receptor 3 (FGFR3) is overexpressed in early stages of bladder cancer. To study the potential use of therapeutic antibodies against FGFR3, we have produced a collection of human single-chain Fv (scFv) antibody fragments by using phage display libraries. EXPERIMENTAL DESIGN: Two "naïve" semi-synthetic human scFv libraries were used to select antibodies against the extracellular domain of FGFR3alpha(IIIc). The reactivity of the selected scFvs with a recombinant FGFR3 was characterized by an enzyme immunoassay and surface plasmon resonance analysis and with RT112 bladder carcinoma cells by a fluorescence-activated cell sorter. The capacity of the selected scFvs to block RT112 cell proliferation was determined. RESULTS: We have isolated six human scFv antibody fragments directed against FGFR3. These human scFvs specifically bound FGFR3, but not the homologous molecule FGFR1. Biacore analysis was used to determine the affinity constants, which ranged from 12 to 40 nmol/L. Competition analysis showed that the FGF9 ligand was able to block the binding of two scFvs, 3C and 7D, to FGFR3, whereas FGF1 only blocked 7D. Immunoprecipitation and flow cytometric analysis confirmed the specificity of the antibodies to native membrane FGFR3. Two scFvs, 3C and 7D, gave an strong immunofluorescence staining of RT112 cells. Moreover, they recognized equally well wild-type and mutant FGFR3 containing the activating mutation S249C. Furthermore, they blocked proliferation of RT112 cells in a dose- and FGF-dependent manner. CONCLUSION: Our results suggest that these human anti-FGFR3 scFv antibodies may have potential applications as antitumoral agents in bladder cancer.

Fibroblast growth factor receptor 3 is overexpressed in urinary tract carcinomas and modulates the neoplastic cell growth.

PURPOSE: Fibroblast growth factor receptor 3 (FGFR3) mutations have been associated with achondroplastic syndromes and urinary bladder carcinomas. Here we describe changes in FGFR3 mRNA and protein expression in transitional carcinomas and determine the effect of monoclonal antibodies against FGFR3 in RT-112 cell line proliferation. EXPERIMENTAL DESIGN: We used microarray tools to evaluate FGFR3 mRNA expression in 22 urinary bladder carcinomas at different stages (noninvasive pTa, lamina propria invasive pT1, and muscular invasive pT2) and 7 nonneoplastic tissue controls. FGFR3 protein expression was evaluated by Western blotting in 15 different carcinomas and 3 nonneoplastic controls. Two hundred thirty-seven urinary bladder and renal pelvis carcinomas and 21 negative controls were tested on tissue microarrays by immunohistochemistry. The effect on cell proliferation in the RT-112 bladder cancer cell line of monoclonal antibodies against FGFR3 was also evaluated. RESULTS: Overexpression of FGFR3 mRNA was found in pTa and pT1 stage carcinomas (fold change >8) and in pT2 carcinomas (fold change >4). Nonneoplastic urinary bladder samples do not express FGFR3 protein. However, 83% of pTa, 100% of pT1, and 50% of pT2 carcinomas expressed FGFR3 as determined by Western blotting. By immunohistochemistry, FGFR3 was positive in 71.4% of pTa, 72% of pT1, and 49.2% of pT2 cases as well as 61.5% of upper urinary tract carcinomas. Proliferation of the RT-112 cell line was inhibited with monoclonal antibodies against FGFR3. CONCLUSIONS: FGFR3 seems to play an important role in transitional cell carcinoma development. Our results suggest that FGFR3 antagonists could be developed as possible therapeutics for treatment of urinary tract carcinoma.

The Real-World Routine Use of Caffeine Citrate in Preterm Infants: A European Postauthorization Safety Study.

BACKGROUND: Caffeine citrate is the treatment of choice for apnea of prematurity (AOP). Regulatory agencies have requested real-world data on drug utilization and safety, a postauthorization safety study, of a pharmaceutical-grade caffeine citrate, Peyona, to confirm its benefit for preterm infants. OBJECTIVES: To investigate the clinical use, outcomes, and safety profile of this pharmaceutical-grade caffeine citrate in the routine treatment of preterm infants with a gestational age (GA) <37 weeks. METHODS: We conducted a multicenter, noninterventional, prospective study in five European countries. Patients eligible for study enrollment were <37-week GA neonates who received treatment with the pharmaceutical-grade caffeine citrate and whose parents had given informed consent. RESULTS: A total of 506 preterm infants were enrolled from 21 institutions. The pharmaceutical-grade caffeine citrate doses were administered intravenously, orally, or via both routes. The main indication of use was AOP treatment (58%) followed by AOP prophylaxis (37%). Median treatment duration was 21 days. The primary cause of study termination was AOP resolution (n = 407; 80%). Hundred and six patients (21%) required supplemental oxygen on day 28; 48 patients (9.5%) had bronchopulmonary dysplasia at 36 weeks' postmenstrual age. Twenty-three adverse drug reactions were observed in 21 neonates (4.2%); the most frequent was tachycardia (2.3%) and only one (seizures) was considered serious. Thirty-one patients (8.1%) had hepatic or renal functional impairment; the side effects were manageable, and these patients also benefitted from treatment. CONCLUSIONS: The use of this caffeine citrate is safe for the management of AOP in a real-world setting.

Oxygen saturation after birth in preterm infants treated with continuous positive airway pressure and air: assessment of gender differences and comparison with a published nomogram.

AIMS: The goal of the study was to compare preductal SpO2 in the first 10 min after birth in preterm infants treated with non-invasive continuous positive airway pressure (CPAP) and air with a published nomogram of preductal SpO2 in preterm infants who received no medical intervention, and to examine gender differences. DESIGN: Prospective observational study. PATIENTS AND METHODS: We enrolled infants of ≤32 weeks gestation who were spontaneously breathing with heart rate >100 bpm, and treated with face mask CPAP and air during postnatal stabilisation. SpO2 limits were targeted at ≥75% at 5 min and ≥85% at 10 min and heart rate at >100 bpm. FIO2 was titrated against SpO2. Preductal SpO2, airway pressure and FIO2 were recorded with a data acquisition system from birth until stabilisation. Babies receiving supplemental oxygen (>21%), positive pressure ventilation, were intubated and/or received chest compressions or drugs were excluded. RESULTS: Measurements were obtained in 102 babies with median gestational age of 29 (range: 24-31) weeks. Median SpO2 was significantly higher in the observational group than in the reference range at 3 min (82% (CI 71% to 85%) vs 76% (CI 67% to 83%); p<0.05), at 4 min (87% (CI 81% to 90%) vs 81% (CI 72% to 88%); p<0.05), at 5 min (92% (CI 88% to 95%) vs 86% (CI 80% to 92%); p<0.05), at 6 min (94% (CI 90% to 97%) vs 90% (CI 81% to 95%); p<0.05), at 7 min (95% (CI 92% to 97%) vs 92% (CI 85% to 95%); p<0.05), at 8 min (96% (CI 93% to 98%) vs 92% (CI 87% to 96%); p<0.05) and at 9 min (97% (CI 92% to 99%) vs 93% (CI 87% to 96%); p<0.05). Female babies achieved targeted SpO2 significantly earlier than male babies. CONCLUSIONS: Preterm babies receiving CPAP and air and especially female subjects achieve reference oxygen saturation more rapidly than spontaneously breathing preterm babies without respiratory aid.

Situación actual de la leucemia mieloide crónica en Costa Rica / (Current situation of chronic myeloid leukemia in Costa Rica)

Resumen Justificación y objetivo: la leucemia mieloide crónica constituye un paradigma de reversión de neoplasia con un tratamiento específico basado en los inhibidores de tirosina quinasa. Aunque la situación epidemiológica ha sido estudiada en países primermundistas, los estudios en Latinoamérica son escasos. Con el fin de actualizar la situación real de la LMC en la región centroamericana, el estudio pretende describir la epidemiología de la leucemia mieloide crónica en Costa Rica. Métodos: se evaluaron 133 pacientes con la enfermedad, mediante monitoreo hematológico y molecular. Se analizó la respuesta de estos casos a tratamiento conforme a las siguientes variables: respuesta hematológica, respuesta molecular y supervivencia global, libre de evento, progresión, así como la prevalencia de mutaciones que confieren resistencia al tratamiento. Resultados: la respuesta hematológica completa fue del 97,7%, y la molecular mayor, a los 12 meses, fue del 43,4%. El seguimiento recomendado por la guía European LeukemiaNet se alcanzó solo en un 68,4% de los pacientes en el primer año, bajando al 57,7%, posteriormente. Un total de 92 pacientes alcanzó respuesta molecular mayor en algún momento; de ellos, el 87,0% conservó respuesta. La supervivencia libre de evento a 3 años fue del 65,7%, libre de progresión del 92,2% y global del 89,2%. La mutación más frecuente encontrada en el gen ABL fue la T315I. Conclusión: el tratamiento de la leucemia mieloide crónica en Costa Rica presenta una eficacia comparable a lo reportado en otros países, con una respuesta molecular mayor inferior a lo esperado, debido a dificultades de acceso al medicamento y monitoreo de la enfermedad.

Abstract Background and aim: Chronic myeloid leukemia is a paradigm of reversion of neoplasia with a specific treatment based on tyrosine kinase inhibitors. Although the epidemiological situation has been studied in first world countries, studies in Latin American countries are scarce. In order to update the real situation of the chronic myeloid leukemia in our Central American region, this study aims to describe the epidemiology of chronic myeloid leukemia in Costa Rica. Methods: 133 patients with the disease were evaluated through hematological and molecular monitoring. The response of these cases to treatment was analyzed by the following variables: haematological response, molecular response and overall survival, event-free, progression, as well as the prevalence of mutations that confer resistance to treatment. Results: The complete haematological response was 97.7% and the molecular response greater than 12 months was 43.4%. The follow-up recommended by the European LeukemiaNet guideline was reached in only 68.4% of the patients in the first year, decreasing to 57.7% later on. A total of 92 patients achieved a higher molecular response at some point, of which 87.0% retained a response. The 3-year event-free survival was 65.7%, progression free of 92.2% and overall of 89.2%. The most frequent mutation found in the ABL gene was T315I. Conclusion: The treatment of chronic myeloid leukemia in Costa Rica presents an efficacy comparable to that reported in other countries, with a lower molecular response than expected due to difficulties in accessing medication and monitoring the disease.

Virus variants with differences in the P1 protein coexist in a Plum pox virus population and display particular host-dependent pathogenicity features.

Subisolates segregated from an M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the helper component proteinase (HCPro) protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in certain subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings suggested the existence of uncharacterized attenuating factors in these subisolates. In this study, we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site-directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV-PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host.

A survey of intravenous sodium bicarbonate in neonatal asphyxia among European neonatologists: gaps between scientific evidence and clinical practice.

BACKGROUND: Birth asphyxia is characterized by intermittent periods of hypoxia/ischemia leading to metabolic acidosis. The use of intravenous sodium bicarbonate (IVSB) is still a matter of controversy in clinical practice. OBJECTIVE: To assess the use of IVSB in birth asphyxia by attending neonatologists in European hospitals. DESIGN/METHODS: Survey using a questionnaire administered to neonatologists in Europe whose electronic addresses were provided by the national councils of neonatology of each participating country. The questionnaire consisted of a brief theoretical introduction followed by demographic questions related to the respondent's professional qualification. This was followed by a clinical case of an asphyxiated term neonate with severe combined metabolic and respiratory acidosis and case-specific questions related to the perceived indication for administration of IVSB and use of additional therapies to address the clinical situation. Descriptive statistics and χ² analysis were performed. RESULTS: The questionnaire was answered by 259 neonatologists from 17 countries. IVSB was believed to be indicated by 109 (42.2%) of the respondents in this clinical scenario. Results differed significantly between countries, with IVSB use varying from as high as 68% in Portugal and <5% in Scandinavian countries. However, there were no differences in the choice of other therapeutic measures. CONCLUSIONS: Although scientific evidence suggests that IVSB is not effective in asphyxiated newborns and current guidelines do not recommend its use, 42.2% of the consulted neonatologists in Europe would use it, with significant differences between but not within countries. There were no differences regarding additional measures to overcome asphyxia. Strategies to implement use of internationally accepted guidelines are needed.

Human milk enhances antioxidant defenses against hydroxyl radical aggression in preterm infants.

BACKGROUND: Preterm infants endowed with an immature antioxidant defense system are prone to oxidative stress. Hydroxyl radicals are very aggressive reactive oxygen species that lack specific antioxidants. These radicals cannot be measured directly, but oxidation byproducts of DNA or phenylalanine in urine are reliable markers of their activity. Human milk has a higher antioxidant capacity than formula. OBJECTIVE: We hypothesized that oxidative stress associated with prematurity could be diminished by feeding human milk. DESIGN: We recruited a cohort of stable preterm infants who lacked perinatal conditions associated with oxidative stress; were not receiving prooxidant or antioxidant drugs, vitamins, or minerals before recruitment; and were fed exclusively human milk (HM group) or preterm formula (PTF group). Collected urine was analyzed for oxidative bases of DNA [8-hydroxy-2'-deoxyguanosine (8-oxodG)/2'-deoxyguanosine (2dG) ratio] and oxidative derivatives of phenylalanine [ortho-tyrosine (o-Tyr)/Phe ratio] by HPLC coupled to tandem mass spectrometry. Healthy term newborn infants served as control subjects. RESULTS: Both preterm groups eliminated greater amounts of metabolites than did the control group. However, the PTF group eliminated significantly (P < 0.02) higher amounts of 8-oxodG (8-oxodG/2dG ratio: 10.46 +/- 3.26) than did the HM group (8-oxodG/2dG ratio: 9.05 +/- 2.19) and significantly (P < 0.01) higher amounts of o-Tyr (o-Tyr/Phe ratio: 14.90 +/- 3.75) than did the HM group (o-Tyr/Phe ratio: 12.53 +/- 3.49). When data were lumped together independently of the type of feeding received, a significant correlation was established between the 8-oxodG/2dG and o-Tyr/Phe ratios in urine, dependent on gestational age and birth weight. CONCLUSION: Prematurity is associated with protracted oxidative stress, and human milk is partially protective.

Antenatal steroids and antioxidant enzyme activity in preterm infants: influence of gender and timing.

Antenatal steroids have improved the survival of preterm infants; however, the mechanism of action is not fully understood. We aimed to establish an association between antenatal steroids and antioxidant activity and postnatal oxidative stress. In a prospective cohort study, extremely preterm neonates receiving antenatal steroids (CORT) or not (NOCORT) were enrolled. An association between antenatal steroids and activities of antioxidant enzymes and glutathione cycle enzymes in cord blood was found. In addition, reduced oxidative stress (GSH/GSSG ratio, CORT vs. NOCORT, 35.68 + or - 12.20 vs. 28.38 + or - 9.92; p < 0.01) and, decreased oxidation of proteins (ortho-tyrosine/phenylalanine ratio, CORT vs. NOCORT, 8.66 + or - 2.45 vs. 12.55 + or - 4.41; p < 0.01) and DNA (8oxodG/2dG ratio, CORT vs. NOCORT, 6.73 + or - 2.18 vs. 9.53 + or - 3.83; p < 0.01) also was found. Antenatal steroids were associated with reduced oxygen supplementation, mechanical ventilation, and conditions such as bronchopulmonary dysplasia, intra-periventricular hemorrhage, or retinopathy of prematurity. The maximal effectiveness was when steroids were administered 2-4 days before delivery. Female preterm infants had less oxidative stress and increased antioxidant activity and better clinical outcomes than did male infants, independent of receiving or not antenatal steroids. Antenatal steroids are accompanied by a reduction in postnatal oxidative-stress-derived conditions and increased antioxidant enzyme activity. Both these effects seem to be influenced by specific timing and female gender.

Achievement of targeted saturation values in extremely low gestational age neonates resuscitated with low or high oxygen concentrations: a prospective, randomized trial.

OBJECTIVE: Extremely low gestational age neonates have very low oxygen saturation in utero and an immature antioxidant defense system. Abrupt increases in oxygen saturation after birth may cause oxidative stress. We compared achievement of a targeted oxygen saturation of 85% at 10 minutes of life when resuscitation was initiated with low or high fractions of inspired oxygen and levels were adjusted according to preductal pulse oxygen saturation values. METHODS: A prospective, randomized, clinical trial was performed in 2 level III neonatal referral units. Patients of < or = 28 weeks of gestation who required active resuscitation were randomly assigned to the low-oxygen group (fraction of inspired oxygen: 30%) or the high-oxygen group (fraction of inspired oxygen: 90%). Every 60 to 90 seconds, the fraction of inspired oxygen was increased in 10% steps if bradycardia occurred (< 100 beats per minute) or was decreased in similar steps if pulse oxygen saturation reached values of > 85%. Preductal pulse oxygen saturation was continuously monitored. RESULTS: The fraction of inspired oxygen in the low-oxygen group was increased stepwise to 45% and that in the high-oxygen group was reduced to 45% to reach a stable pulse oxygen saturation of approximately 85% at 5 to 7 minutes in both groups. No differences in oxygen saturation in minute-to-minute registers were found independent of the initial fraction of inspired oxygen used 4 minutes after cord clamping. No differences in mortality rates in the early neonatal period were detected. CONCLUSIONS: Resuscitation can be safely initiated for extremely low gestational age neonates with a low fraction of inspired oxygen (approximately 30%), which then should be adjusted to the infant's needs, reducing the oxygen load to the neonate.

Host-specific effect of P1 exchange between two potyviruses.

The potyviruses Plum pox virus (PPV) and Tobacco vein mottling virus (TVMV) have distinct host ranges and induce different symptoms in their common herbaceous hosts. To test the relevance of the P1 protein in host compatibility and pathogenicity, hybrid viruses were constructed in which the P1 coding sequence of PPV was completely or partially replaced by the corresponding sequences from TVMV. Infections induced by these chimeric viruses revealed that the TVMV P1 and a PPV/TVMV hybrid P1 proteins are functionally equivalent in herbaceous plants to the P1 protein of a PPV isolate adapted to these hosts, in spite of having high sequence divergence. Moreover, the presence of TVMV P1 sequences enhanced the competence of a low-infectivity PPV-D-derived chimera in Nicotiana clevelandii. Conversely, all PPV/TVMV hybrids were unable to infect Prunus persicae, a specific host for PPV, suggesting that TVMV P1 is not functionally competent in this plant. Together, these data highlight the importance of the P1 protein in defining the virus host range.

Plexin B1 is downregulated in renal cell carcinomas and modulates cell growth.

Plexins are a family of transmembrane receptors that interact with the repulsive axon guidance molecules (Semaphorins) in neural tissues. In extraneural tissues, plexins are involved in other cellular functions often altered in neoplastic cells, such as adhesion, migration, and apoptosis. Plexin B1 has been implicated in the regulation of Akt, which is an activated pathway in renal cell neoplasms, and only 1 report has emphasized its role as an oncogenic factor. Furthermore, plexin B1 is located in 3p21, which is a chromosomal region deleted frequently in renal cell carcinomas. In accordance with a hypothetical oncogenic role for plexin B1, we have shown by reverse transcription-polymerase chain reaction that plexin B1 is expressed in nonneoplastic renal tissue, and it is severely downregulated in clear cell renal carcinomas. We have also demonstrated by immunohistochemistry on tissue microarrays that plexin B1 protein is absent in more than 80% of renal cell carcinomas (169 in 209 carcinomas examined). Otherwise, all kinds of renal tubules showed strong membrane reactivity. Moreover, when we have induced plexin B1 expression with an expression vector in the renal adenocarcinoma cell line ACHN, a marked reduction in proliferation rate was produced. Altogether, this evidence suggests a possible role for plexin B1 in renal oncogenesis.

Transcriptional profiling of hematologic malignancies with a low-density DNA microarray.

BACKGROUND: High-density microarrays are powerful tools for expression analysis of thousands of genes simultaneously; however, experience with low-density microarrays in gene expression studies has been limited. METHODS: We developed an optimized procedure for gene expression analysis based on a microarray containing 538 oligonucleotides and used this procedure to analyze neoplastic cell lines and whole-blood samples from healthy individuals and patients with different hematologic neoplasias. Hierarchical clustering and the Welch t-test with adjusted P values were used for data analysis. RESULTS: This procedure detects 0.2 fmol of mRNA and generates a linear response of 2 orders of magnitude, with CV values of <20% for hybridization and label replicates. We found statistically significant differences between Jurkat and U937 cell lines, between blood samples from 15 healthy donors and 59 chronic lymphocytic leukemia (CLL) samples, and between 6 acute myeloid leukemia patients and 4 myelodysplastic syndrome patients. A classification system constructed from the expression data predicted healthy or CLL status from a whole-blood sample with a 97% success rate. CONCLUSION: Transcriptional profiling of whole-blood samples was carried out without any cellular or sample manipulation before RNA extraction. This gene expression analysis procedure uncovered statistically significant differences associated with different hematologic neoplasias and made possible the construction of a classification system that predicts the healthy or CLL status from a whole-blood sample.

Salicylic acid-mediated and RNA-silencing defense mechanisms cooperate in the restriction of systemic spread of plum pox virus in tobacco.

Plum pox virus (PPV) is able to replicate in inoculated leaves of Nicotiana tabacum, but is defective in systemic movement in this host. However, PPV produces a systemic infection in transgenic tobacco expressing the silencing suppressor P1/HC-Pro from tobacco etch virus (TEV). In this work we show that PPV is able to move to upper non-inoculated leaves of tobacco plants expressing bacterial salicylate hydroxylase (NahG) that degrades salicylic acid (SA). Replication and accumulation of PPV is higher in the locally infected leaves of plants deficient in SA or expressing TEV P1/HC-Pro silencing suppressor. Accumulation of viral derived small RNAs was reduced in the NahG transgenic plants, suggesting that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco. Besides, expression of SA-mediated defense transcripts, such as those of pathogenesis-related (PR) proteins PR-1 and PR-2 or alternative oxidase-1, as well as that of the putative RNA-dependent RNA polymerase NtRDR1, is induced in response to PPV infection, and the expression patterns of these defense transcripts are altered in the TEV P1/HC-Pro transgenic plants. Long-distance movement of PPV is highly enhanced in NahG x P1/HC-Pro double-transgenic plants and systemic symptoms in these plants reveal that the expression of an RNA-silencing suppressor and the lack of SA produce additive but distinct effects. Our results suggest that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco, and that silencing suppressors, such as P1/HC-Pro, also alter the SA-mediated defense. Both an RNA-silencing and an SA-mediated defense mechanism could act together to limit PPV infection.

Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants.

RNA silencing has a well-established function as an antiviral defence mechanism in plants and insects. Using an Agrobacterium-mediated transient assay, we report here that NS1 protein from human influenza A virus suppresses RNA silencing in plants in a manner similar to P1/HC-Pro protein of Tobacco etch potyvirus, a well-characterized plant virus silencing suppressor. Moreover, we have shown that NS1 protein expression strongly enhances the symptoms of Potato virus X in three different plant hosts, suggesting that NS1 protein could be inhibiting defence mechanisms activated in the plant on infection. These data provide further evidence that an RNA silencing pathway could also be activated as a defence response in mammals.

Host-specific involvement of the HC protein in the long-distance movement of potyviruses.

Plum pox virus (PPV) is a member of the Potyvirus genus that, in nature, infects trees of the Prunus genus. Although PPV infects systemically several species of the Nicotiana genus, such as N. clevelandii and N. benthamiana, and replicates in the inoculated leaves of N. tabacum, it is unable to infect systemically the last host. The long-distance movement defect of PPV was corrected in transgenic tobacco plants expressing the 5"-terminal region of the genome of tobacco etch virus (TEV), a potyvirus that infects systemically tobacco. The fact that PPV was unable to move to upper noninoculated leaves in tobacco plants transformed with the same TEV transgene, but with a mutation in the HC protein (HC-Pro)-coding sequences, identifies the multifunctional HC-Pro as the complementing factor, and strongly suggests that a defect in an HC-Pro activity is responsible for the long-distance movement defect of PPV in tobacco. Whereas PPV HC-Pro strongly intensifies the symptoms caused by potato virus X (PVX) in the PPV systemic hosts N. clevelandii and N. benthamiana, it has no apparent effect on PVX pathogenicity in tobacco, supporting the hypothesis that long-distance movement and pathogenicity enhancement are related activities of the potyviral HC proteins. The movement defect of PPV in tobacco could also be complemented by cucumber mosaic virus in a mixed infection, demonstrating that at least some components of the long-distance machinery of the potyviruses are not strictly virus specific. A general conclusion of this work is that the HC-Pro might be a relevant factor for controlling the host range of the potyviruses.