Therapy with m-TOR inhibitors decreases the response to the pandemic influenza A H1N1 vaccine in solid organ transplant recipients.

Concern has been raised regarding the response to vaccination in solid organ transplant recipients (SOTR) undergoing immunosuppressant regimens and the possibility of rejection related to the immune response associated with pandemic influenza H1N1-2009 vaccination. The goal of this study was to assess the immunogenicity, efficacy and safety of the pandemic vaccine in SOTR. We performed a multicenter prospective study in SOTR receiving the pandemic vaccine. Immunological response was determined in serum 5 weeks after vaccination by microneutralization assays, and immunoglobulins were measured by ELISA. Three hundred and forty-six SOTR were included. Preexisting seroprotection was detected in 13.6% of cases and rates of seroconversion and seroprotection after vaccination were 73.1% and 82.9%, respectively. Patients with baseline antibody titers had better geometric mean titers (GMT)-post after pandemic vaccination (339.4 vs. 121.4, p < 0.001). Younger age, liver disease and m-TOR inhibitor therapy were independently associated with lower seroprotection and GMT-post. There were no major adverse effects or rejection episodes. Pandemic vaccine was safe in SOTR and elicited an adequate response, although lower than in healthy individuals. This is the first study describing a decreased response after vaccination in patients receiving mTOR inhibitors who presented lower seroprotection rates and lower GMT-post.

Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p.

Eukaryotic cells respond to DNA damage by arresting the cell cycle and modulating gene expression to ensure efficient DNA repair. The human ATR kinase and its homolog in yeast, MEC1, play central roles in transducing the damage signal. To characterize the role of the Mec1 pathway in modulating the cellular response to DNA damage, we used DNA microarrays to observe genomic expression in Saccharomyces cerevisiae responding to two different DNA-damaging agents. We compared the genome-wide expression patterns of wild-type cells and mutants defective in Mec1 signaling, including mec1, dun1, and crt1 mutants, under normal growth conditions and in response to the methylating-agent methylmethane sulfonate (MMS) and ionizing radiation. Here, we present a comparative analysis of wild-type and mutant cells responding to these DNA-damaging agents, and identify specific features of the gene expression responses that are dependent on the Mec1 pathway. Among the hundreds of genes whose expression was affected by Mec1p, one set of genes appears to represent an MEC1-dependent expression signature of DNA damage. Other aspects of the genomic responses were independent of Mec1p, and likely independent of DNA damage, suggesting the pleiotropic effects of MMS and ionizing radiation. The complete data set as well as supplemental materials is available at http://www-genome.stanford.edu/mec1.

Genomic expression programs in the response of yeast cells to environmental changes.

We explored genomic expression patterns in the yeast Saccharomyces cerevisiae responding to diverse environmental transitions. DNA microarrays were used to measure changes in transcript levels over time for almost every yeast gene, as cells responded to temperature shocks, hydrogen peroxide, the superoxide-generating drug menadione, the sulfhydryl-oxidizing agent diamide, the disulfide-reducing agent dithiothreitol, hyper- and hypo-osmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. A large set of genes (approximately 900) showed a similar drastic response to almost all of these environmental changes. Additional features of the genomic responses were specialized for specific conditions. Promoter analysis and subsequent characterization of the responses of mutant strains implicated the transcription factors Yap1p, as well as Msn2p and Msn4p, in mediating specific features of the transcriptional response, while the identification of novel sequence elements provided clues to novel regulators. Physiological themes in the genomic responses to specific environmental stresses provided insights into the effects of those stresses on the cell.

Update on the risk stratification of acute symptomatic pulmonary thromboembolism. / Actualización en la estratificación de riesgo del tromboembolismo pulmonar agudo sintomático.

Early mortality in patients with pulmonary thromboembolism (PTE) varies from 2% in normotensive patients to 30% in patients with cardiogenic shock. The current risk stratification for symptomatic PTE includes 4 patient groups, and the recommended therapeutic strategies are based on this stratification. Patients who have haemodynamic instability are considered at high risk. Fibrinolytic treatment is recommended for these patients. In normotensive patients, risk stratification helps differentiate between those of low risk, intermediate-low risk and intermediate-high risk. There is currently insufficient evidence on the benefit of intensive monitoring and fibrinolytic treatment in patients with intermediate-high risk. For low-risk patients, standard anticoagulation is indicated. Early discharge with outpatient management may be considered, although its benefit has still not been firmly established.

Postoperative sympathetic ophthalmia.

Although uncommon, SO is a fearful postoperative complication because of its potential to blind both eyes. It can result not only from penetrating ocular surgery but also from nonpenetrating ocular procedures. Thus, it is important to consider in any patient who has undergone ocular surgery and develops bilateral uveitis, particularly because prompt, sufficient treatment is required to maximize visual outcome. It is also important to note that the disease may present with a spectrum of clinical findings, none of which is pathognomonic. Thus, suspicion is important for making the diagnosis. Treatment should address the T-cell-mediated nature of the disease. With appropriate treatment, visual acuity of no less than 20/60 is likely. However, before the start of treatment, which consists of immunosuppressants, infection must be ruled out and potential side effects of treatments must be considered. Furthermore, any patient with a history of SO needs ample immunosuppressant coverage for ocular procedures. Better understanding of the pathogenesis of the disease may lead to safer treatments that result in improved visual outcome and a cure. Meanwhile, because of its relapsing nature, SO requires continual, close surveillance, even after many years of quiescence.

[Leydig cell tumor, gynecomastia, and inferior vena cava thrombosis]. / Tumor de células de Leydig, ginecomastia y trombosis de vena cava inferior.

Leydig cell tumor is a testicular tumor with a low incidence characterized by a high estrogens secretion from the tumoral cells. Its more frequent clinical presentation is a testicular nodule with or without other endocrine manifestations due to estrogenic hypersecretion. We're reporting a case of a Leydig cell tumor with high plasmatic levels of estradiol, gynecomastia and inferior cava vein thrombosis, which hasn't been described among its clinical features up to now. Vascular thrombotic phenomenons have already been reported in other clinical situations with hiperestrogesism and they could also be associated with these tumors. Patients with Leydig cell tumors could be at a higher risk of developing thromboembolic phenomenons because of tumoral hyperestrogenism and could present thrombotic complications among the clinical findings.

A simple prognostic score for risk assessment in patients with acute pancreatitis.

BACKGROUND: Acute pancreatitis (AP) is a common disease that poses potential serious problems. Its clinical course is often unpredictable. Identification of high risk patients enables early appropriate treatment. METHODS: We conducted a prospective study to develop a new prognostic method that can objectively and easily grade the severity of AP within the first 72 h of admission. The prediction rule was based on clinical and analytical parameters in 308 patients admitted in a community-based hospital. We validated the score in 193 additional patients in the same hospital. RESULTS: Independent prognostic factors related to poor prognosis were age >65 years, leucocytes >13,000/mm(3), albumin <2.5 mg/dL, calcium <8.5 mg/dL and reactive C protein >150 mg/dL. We assigned points to each of the independent factors for complicated AP in proportion to the regression coefficients. We defined three different risk groups according to the points obtained in the prediction rule. Low risk, 0 points (18% patients, 0% risk), moderate, 1-3 points (56% patients, 19% risk) and high, 4-6 points (26% patients, 73% risk). The sensitivity of this formula was 90% with specificity of 63%. The positive and negative predictive values were 50% and 94% respectively. CONCLUSIONS: Our simple prediction rule is an additional tool that may help physicians stratifying the severity of AP. Patients with high risk for complicated AP should be kept under close surveillance whereas low risk patients would not need special monitoring.

Rapid detection of Listeria monocytogenes by PCR-ELISA.

A rapid detection system specific for Listeria monocytogenes based upon the polymerase chain reaction was developed. The specificity of the primers and the probe annealing to the coding region of the mpl gene proved positive with the DNA from a total of 103 L. monocytogenes strains, while DNA from another 73 Listeria and non-Listeria strains tested negative. To facilitate detection with large numbers of samples, a microtitre plate assay was established with biotinylated probes. Use of a standard DNA prevented false-negative results when used as an internal amplification control in the PCR-ELISA. As the described method required approximately 5-6 h to be completed it may prove useful in the detection of L. monocytogenes in food.

The DIMINUTO gene of Arabidopsis is involved in regulating cell elongation.

We have isolated a recessive mutation named diminuto (dim) from T-DNA transformed lines of Arabidopsis thaliana. Under normal growth conditions, the dim mutant has very short hypocotyls, petioles, stems, and roots because of the reduced size of cells along the longitudinal axes of these organs. In addition, dim results in the development of open cotyledons and primary leaves in dark-grown seedlings. The gene for DIM was cloned by T-DNA tagging. DIM encodes a novel protein of 561 amino acids that possesses bipartite sequence domains characteristic of nuclear localization signals. Molecular and physiological studies indicate that the loss-of-function mutant allele does not abolish the response of seedlings to light or phytohormones, although the inhibitory effect of light on hypocotyl elongation is greater in the mutant than in wild type. Moreover, the dim mutation affects the expression of a beta-tubulin gene, TUB1, which is thought to be important for plant cell growth. Our results suggest that the DIM gene product plays a critical role in the general process of plant cell elongation.

Fine mapping of human PI 3-kinase associated p85 alpha transcripts in the YAC contig surrounding the spinal muscular atrophy gene.

During a search for transcribed sequences within the gene region for autosomal recessive spinal muscular atrophy (SMA), two cDNA clones were isolated from a human fetal brain and an adult spinal cord cDNA library, respectively, by use of the cosmid LA96B (LAS96). The clones sized 950 bp and 1733 bp detect a 7.7-kb transcript in all tested human tissues. An additional transcript of 6.6 kb is detectable in brain and kidney, and faintly in skeletal muscle and liver. Using comparative human Northern blot analysis, the isolated LA96B cDNA clones could be identified as parts of the 3' untranslated region from the phosphatidylinositol 3 (PI3)-kinase associated p85 alpha transcripts; these were unknown up to now. The 5' end of the gene was mapped to YAC-EFTA:A, whereas the 3' end was localized within the distal overlapping YAC 85 flanking the SMA candidate gene region.

The expression of beta 1 and beta 3 tubulin genes of Drosophila melanogaster is spatially regulated during embryogenesis.

In Drosophila beta tubulins are encoded by a small gene family and the four members of this family are differentially expressed. mRNAs transcribed from two of these genes, namely the beta 1 and beta 3 tubulin genes, are abundant during embryogenesis. While the beta 1 tubulin gene is constitutively expressed during development, beta 3 mRNA is restricted to two distinct phases: mid embryogenesis and metamorphosis. The transcription initiation sites are identical in both these stages and comparison of presumptive promoter regions reveals no extensive homologies between the genes. In situ localization shows beta 1 tubulin mRNA to be maternally expressed in the nurse cells of the egg chambers and evenly distributed during early embryogenesis. In contrast, during later stages of embryogenesis beta 1 tubulin transcripts are predominantly expressed in neural derivatives. The beta 3 tubulin gene expression is also spatially regulated, beta 3 mRNA being restricted to the mesoderm.

A 14 bp promoter element directs the testis specificity of the Drosophila beta 2 tubulin gene.

To analyze the regulation of gene expression during male germ cell development, we investigated the testis-specific expression of the Drosophila beta 2 tubulin gene. Germ line transformation experiments with the upstream region of the D.melanogaster beta 2 tubulin gene fused to the Escherichia coli lacZ gene resulted in the correct tissue specific expression of the reporter gene. Furthermore, we showed that the upstream sequences of the beta 2 tubulin gene of the distantly related species D.hydei can drive the expression of the lacZ gene testis specifically in D.melanogaster flies. A detailed deletion analysis showed that 53 bp of upstream and 23 bp (D.melanogaster) or 29 bp (D.hydei) of leader sequences are sufficient to confer tissue specificity. The short promoter regions contain a 14 bp motif at identical positions in both species, which acts as a position-dependent promoter element. In vitro mutagenesis and subsequent germline transformation experiments revealed that this sequence is the only element necessary for the testis-specific transcription of the beta 2 tubulin gene in Drosophila.

Determinants of glaucoma awareness in a general eye clinic.

PURPOSE: Heightened public awareness about glaucoma may increase the chance of identifying undetected cases. To ascertain determinants of glaucoma awareness, we surveyed a population visiting a general eye clinic. DESIGN: Cross-sectional study. PARTICIPANTS: 1197 general eye clinic patients and their companions. METHODS: We designed and administered a questionnaire about glaucoma to general eye clinic patients and their companions. We created multivariate logistic regression models to ascertain the effect of demographic and clinical features on the likelihood of being unaware of glaucoma. MAIN OUTCOME MEASURES: Adjusted odds ratio (OR) with 95% confidence intervals of survey attributes associated with self-perceived unfamiliarity with glaucoma. RESULTS: Glaucoma awareness overall (72%) approached that found in the subgroup self-reporting a diagnosis of glaucoma (80%). Survey attributes associated with an increased likelihood of being unaware of glaucoma were African American race (OR = 1.69 [1.28-2.20], Hispanic ethnicity (OR = 2.13 [1.46-3.02]), and less than a college education (OR = 1.67 [1.37-2.05]). Age was also a determinant of glaucoma awareness (for ages 50-64 years, OR = 0.60 [0.44-0.80] and for ages 65-79 years, OR = 0.56 [0.41-0.75] compared with ages less than 35 years). A self-report of glaucoma was not a determinant of glaucoma awareness (OR = 0.63 [0.33-1.17]), although there was a trend toward enhanced glaucoma awareness in this subgroup. Finally, respondents with a history of employment in the health field (OR = 0.63 [0.49-0.82]) myopia (OR = 0.68 [0.56-0.82]), glaucoma in a first-degree relative (OR = 0.68 [0.53-0.87]), and respondents who reported having a dilated eye examination (OR = 0.53 [0.42-0.66]) were less likely to be unaware of glaucoma than those who did not have these attributes. CONCLUSIONS: Although glaucoma awareness in this population was high, Hispanics, African Americans, and those with less than a college education were more likely to be unfamiliar with the disease. Interestingly, a self-report of having glaucoma was not a statistically significant determinant of glaucoma awareness.

Evidence that the cis preference of the Tn5 transposase is caused by nonproductive multimerization.

The transposase (Tnp) of the bacterial transposon Tn5 acts 50- to 100-fold more efficiently on elements located cis to the site of its synthesis compared with those located in trans. In an effort to understand the basis for this cis preference, we have screened for Tnp mutants that exhibit increased transposition activity in a trans assay. Two mutations in the carboxyl terminus were isolated repeatedly. The EK345 mutation characterized previously increases Tnp activity eightfold both in cis and in trans. The novel LP372 mutation, however, increases Tnp activity 10-fold specifically in trans. Combining both mutations increases Tnp activity 80-fold. Interestingly, the LP372 mutation maps to a region shown previously to be critical for interaction with Inh, an inhibitor of Tn5 transposition, and results in reduced inhibition activity by both Tnp and Inh. Tnp also inhibits Tn5 transposition in trans, and this has been suggested to occur by the formation of inactive Tnp multimers. Because Inh and (presumably) Tnp inhibit Tn5 transposition by forming defective multimers with Tnp, the inhibition defect of the trans-active LP372 mutant suggests that the cis preference of Tnp may also be attributable to nonproductive Tnp-Tnp multimerization. In addition, we show that increasing the synthesis of EK345/LP372 Tnp, but not wild-type Tnp, leads to very high levels of transposition, presumably because this altered Tnp is defective in the inhibitory activity of the wild type protein.

Intron and upstream sequences regulate expression of the Drosophila beta 3-tubulin gene in the visceral and somatic musculature, respectively.

The morphogenetic programs involved in the differentiation of internal organs, such as the muscle system, during Drosophila embryogenesis have remained largely obscure. beta 3-tubulin has proved to be a good marker for mesoderm development as this tubulin isotype is detectable soon after mesoderm formation and during the process of mesoderm differentiation. The beta 3-tubulin gene is expressed in the somatic and pharyngeal musculature, the dorsal vessel, and the visceral musculature. To learn more about the programs underlying mesodermal differentiation, we have started to dissect the regulatory elements of the beta 3-tubulin gene by means of P-element-mediated transformation experiments. We show that expression of the beta 3-tubulin gene in the somatic muscles, the pharyngeal muscles, and the dorsal vessel is mediated by far upstream sequences. We also demonstrate that the first intron of the beta 3-tubulin gene bears a tissue-specific enhancer element that is required for expression in the visceral muscles and that also functions efficiently when cloned downstream of an indicator gene. The separability of elements driving beta 3-tubulin expression in the somatic and visceral mesoderm facilitates the investigation of the different programs involved in regulating the early differentiation of this germ layer.

Mapping of a human rRNA gene in the YAC contig surrounding the SMA candidate gene.

Using the yeast artificial chromosome (YAC) 116 flanking the autosomal recessive spinal muscular atrophy (SMA) gene region, we have screened a human fetal brain cDNA library and isolated the cDNA clone 14-3/9 with an insert size of 2.5 kb. The cDNA clone could be identified as part of the human rRNA gene coding for 28S rRNA with a total size of 5025 bp. The human 28S rRNA is involved in the organization of the 60S ribosomal subparticle and is arranged in a 13-kb pre-rRNA transcription unit that occurs in tandem repeat clusters. Multiple copies of the rRNA gene have been mapped by pulsed field blot hybridization in the YAC contig between YAC 66 and YAC 116, which encompasses the SMA candidate gene, and additionally in the distally localized YAC 153.

Beta 3 tubulin expression characterizes the differentiating mesodermal germ layer during Drosophila embryogenesis.

During embryogenesis, the beta 3 tubulin gene of Drosophila is transcribed predominantly in the mesoderm. We have raised antibodies specific to the C-terminal domain of the beta 3 tubulin and analysed by immunostaining the distribution of this tubulin isotype during Drosophila embryogenesis. The protein is first detectable in the cephalic mesoderm at maximal germband extension. Shortly afterwards, beta 3 tubulin is expressed in single cells at identical positions of the thoracic and abdominal segments. We suggest that these cells represent muscle pioneer cells of Drosophila. During later embryonic development the somatic musclature, visceral musculature, dorsal vessel and macrophages contain beta 3 tubulin. In dorsalizing mutants dorsal, snail and twist, which do not form a ventral furrow during gastrulation, beta 3 expression is greatly reduced but not completely abolished. Our analysis shows that beta 3 tubulin immunostaining characterizes the differentiation of mesodermal derivatives during embryogenesis.