Construction and analysis of a Vibrio cholerae delta-aminolevulinic acid auxotroph which confers protective immunity in a rabbit model.

Vibrio cholerae CVD101 is a very effective live vaccine. Although this strain does not produce active cholera toxin because of a mutation in the gene for the cholera toxin A subunit, it still shows residual pathogenicity. To attenuate CVD101 further, we set out to isolate derivatives of CVD101 which were limited in their ability to proliferate in vivo. Two delta-aminolevulinic acid auxotrophs of CVD101, designated V286 and V287, were isolated by transposon mutagenesis and penicillin enrichment. Southern blotting revealed that the mutants differed with respect to the location of the transposon insertion. Under aerobic conditions, in the absence of delta-aminolevulinic acid, both mutants showed diminished growth compared with CVD101. The growth of V286 was most severely affected. Microaerophilic growth of both mutants was less affected. Competition experiments with a rabbit model showed that strain V286 was found in numbers 10(3)- to 10(4)-fold lower than its parental strain. This observation indicates that strain V286 is impaired in its ability to colonize the rabbit intestine. It also supports an important role for aerobic growth in the colonization of the intestine by V. cholerae. Vaccination of rabbits with a single dose of strain V286 resulted in full protection against challenge with a virulent strain. Strain V286 was not shed from rabbits in a cultivatable form. Our results suggest that delta-aminolevulinic acid auxotrophy can attenuate V. cholerae by limiting its ability to colonize without affecting its capacity to induce protective immunity. Furthermore, this type of mutation may prevent the spread of V. cholerae vaccine strains in the environment.     

Sequencing-based typing reveals new insight in HLA-DPA1 polymorphism

Abstract: An HLA-DPA1 sequencing-based typing (SBT) system has been developed to identify DPA1 alleles. Up to now eight DPA1 alleles have been defined. Six can be discriminated based upon exon 2 polymorphism. The three subtypes of DPA1*01: DPA1*0101, DPA1*0102 and DPA1*0103, have identical exon 2 sequences but show differences in exon 4. Exon 4 sequences were known for only the three DPA1*01 subtypes and for DPA1*0201. We now present additional sequence information for exon 4 and the unknown segments at the 3' end of exon 2. Additionally with the use of this sequencing technique it is also possible to identify previously unidentified polymorphism. We have studied the exon 2 and exon 4 polymorphism of DPA1 in 40 samples which include all known DPA1 alleles. A new allele, DPA1*01 new, was identified which differs by one nucleotide in exon 2 from DPA1*0103, resulting in an aspartic acid at codon 28. The DPA1*01 subtypes DPA1*0101 and DPA1*0102 could not be confirmed in samples which previously were used to define these subtypes, and consequently they do not exist. The exon 4 sequence of DPA1*0201 is corrected based on sequence data of DAUDI, the cell line in which DPA1*0202 was originally defined. The exon 4 regions of the remaining four alleles were resolved: the exon 4 regions of the alleles DPA1*02021 and DPA1*02022 were found to be identical to the — corrected — DPA1*0201 whereas the exon 4 region of DPA1*0301 differs by one nucleotide compared to DPA1*0103. The DPA1*0401 exon 4 region differs by one nucleotide compared to the corrected DPA1*0201. As is found in other class II genes, all exons of DPA1 show some polymorphism, but the polymorphism in exon 2 is sufficient to identify the different alleles.     

Heterogeneity revealed by integrated genomic analysis uncovers a molecular switch in malignant uveal melanoma

Gene expression profiles as well as genomic imbalances are correlated with disease progression in uveal melanoma (UM). We integrated expression and genomic profiles to obtain insight into the oncogenic mechanisms in development and progression of UM. We used tumor tissue from 64 enucleated eyes of UM patients for profiling. Mutations and genomic imbalances were quantified with digital PCR to study tumor heterogeneity and molecular pathogenesis. Gene expression analysis divided the UM panel into three classes. Class I presented tumors with a good prognosis and a distinct genomic make up that is characterized by 6p gain. The UM with a bad prognosis were subdivided into class IIa and class IIb. These classes presented similar survival risks but could be distinguished by tumor heterogeneity. Class IIa presented homogeneous tumors while class IIb tumors, on average, contained 30% of non-mutant cells. Tumor heterogeneity coincided with expression of a set of immune genes revealing an extensive immune infiltrate in class IIb tumors. Molecularly, class IIa and IIb presented the same genomic configuration and could only be distinguished by 8q copy number. Moreover, UM establish in the void of the immune privileged eye indicating that in IIb tumors the infiltrate is attracted by the UM. Combined our data show that chromosome 8q contains the locus that causes the immune phentotype of UM. UM thereby provides an unique opportunity to study immune attraction by tumors.     

Visualization of Human Inner Ear Anatomy with High-Resolution MR Imaging at 7T: Initial Clinical Assessment

BACKGROUND AND PURPOSE:In many centers, MR imaging of the inner ear and auditory pathway performed on 1.5T or 3T systems is part of the preoperative work-up of cochlear implants. We investigated the applicability of clinical inner ear MR imaging at 7T and compared the visibility of inner ear structures and nerves within the internal auditory canal with images acquired at 3T.MATERIALS AND METHODS:Thirteen patients with sensorineural hearing loss eligible for cochlear implantation underwent examinations on 3T and 7T scanners. Two experienced head and neck radiologists evaluated the 52 inner ear datasets. Twenty-four anatomic structures of the inner ear and 1 overall score for image quality were assessed by using a 4-point grading scale for the degree of visibility.RESULTS:The visibility of 11 of the 24 anatomic structures was rated higher on the 7T images. There was no significant difference in the visibility of 13 anatomic structures and the overall quality rating. A higher incidence of artifacts was observed in the 7T images.CONCLUSIONS:The gain in SNR at 7T yielded a more detailed visualization of many anatomic structures, especially delicate ones, despite the challenges accompanying MR imaging at a high magnetic field.

Patients with sensorineural hearing loss (SNHL) have malfunction of the inner ear, cochlear nerve, or central auditory pathway. Treatment consists of amplification of sound or, in case of severe-to-profound SNHL, direct electrical stimulation of the cochlear nerve by a cochlear implant. MR imaging of patients with SNHL focuses on the integrity of the auditory pathways from the cochlea to the auditory cortex in the brain. In particular, the fluid-filled spaces of the labyrinth and internal auditory canal and the cerebellopontine angle are of interest because the most commonly identified pathologies that cause SNHL are found in these regions.^1–5One clear trend in the development of MR imaging systems has been the drive to higher magnetic field strengths.⁶ For clinical inner ear scanning, MR imaging scanners with a magnetic field strength of 1.5T or 3T are routinely used. The relatively recent introduction of commercial 7T scanners potentially enables an increased SNR, resulting in more detailed imaging of anatomic structures. Concerning inner ear imaging, the visualization of delicate and small-sized inner ear structures might benefit from such high-resolution imaging. This may result in new opportunities for obtaining normative measurements and evaluating pathologic alterations within the inner ear or associated nerves. Such detailed anatomic depiction has caused particular interest for assessment of candidates for cochlear implants because it gives decisive information on implantation feasibility, possible surgical risks, and choice of implant device. As such, it would aid in patient-specific preoperative planning of cochlear implantation and could provide valuable information for individualized assessment of insertion.Transition from a conventional 3T scanner to a stronger 7T scanner is challenging, however, due to technical complexities accompanying the higher magnetic field strength.⁶ One of these technical complexities is the increased inhomogeneity of the static (B₀) and radiofrequency (B₁) fields, typically featuring areas of low B₁ close to the temporal lobes. The B₀ inhomogeneities are caused primarily by the susceptibility difference between inner ear fluids and the surrounding bone, and the B₁ inhomogeneities, by the elliptic shape of the head.⁷ Both of these effects can result in loss of signal in the inner ear region as previously described by Takahara et al⁸ and van Egmond et al.⁹ Additionally, the specific absorption rate, for which regulatory safety limits are defined, scales approximately quadratic with field strength, ultimately limiting the imaging speed at high fields in vivo. Recently, we introduced geometrically tailored dielectric pads to locally tailor the B₁ distribution. These improved contrast homogeneity and transmit efficiency in the region of the inner ear without increasing the specific absorption rate, which contributed to the development of a high-resolution imaging protocol at 7T.¹⁰The aim of this study was the following: 1) to investigate the feasibility of clinical inner ear imaging at 7T MR imaging, and 2) to compare the visibility of inner ear structures and nerves within the internal auditory canal with images acquired at 3T.     

Outcome of laparoscopic cholecystectomy conversion: is the surgeon’s selection needed?

Background

Risk factors for conversion in cholecystectomy may be of clinical value. This study aimed to investigate whether a set of risk factors, including the surgeon’s specialization, can be used for the development of a preoperative strategy to optimize conversion outcome.

Methods

The data for all patients who underwent laparoscopic cholecystectomy at a single institution between January 2004 and December 2008 were retrospectively reviewed. Factors predictive for conversion were identified, and a preoperative strategy model was deduced.

Results

Of the 1,126 patients analyzed, 106 (9%) underwent laparoscopic cholecystectomy in an emergency setting. Delayed surgery was performed for 63 (46%) of 138 patients (12%) with acute cholecystitis. Preoperative endoscopic retrograde cholangiography was achieved for 161 of the patients (14%). Risk factors predictive of conversion (for 65 patients) were male gender [odds ratio (OR), 2.3; 95% confidence interval (CI), 1.3–3.9; p = 0.004], age older than 65 years (OR, 2.6; 95% CI, 1.4–4.8; p = 0.002), body mass index (BMI) exceeding 25 kg/m² (OR, 3.4; 95% CI, 1.7–7.1; p < 0.001), history of complicated biliary disease (HCBD) (OR, 5.6; 95% CI, 3.2–9.8; p = < 0.001), and surgery by a non-gastrointestinal (non-GI) surgeon (OR, 4.9; 95% CI, 2.2–10.6; p < 0.001). The conversion rate for patients with a history of no complications who had two or more risk factors (gender, age, BMI > 25) and for patients with a HCBD who had one or more risk factors was significantly higher if the surgery was performed by non-GI rather than GI surgeons.

Conclusion

Male gender, age older than 65 years, BMI exceeding 25 kg/m², HCBD, and surgery by a non-GI surgeon are predictive for conversion. A preoperative triage for surgeon selection based on risk factors and a HCBD is proposed to optimize conversion outcome.     

Transplant for TP53-mutated MDS and AML: because we can or because we should?

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Oculocerebrorenal syndrome of Lowe protein controls cytoskeletal reorganisation during human platelet spreading

Lowe syndrome (LS) is a rare, X-linked disorder characterised by numerous symptoms affecting the brain, the eyes, and the kidneys. It is caused by mutations in the oculocerebrorenal syndrome of Lowe (OCRL) protein, a 5-phosphatase localised in different cellular compartments that dephosphorylates phosphatidylinositol-4,5-bisphosphate into phosphatidylinositol-4-monophosphate. Some patients with LS also have bleeding disorders, with normal to low platelet (PLT) count and impaired PLT function. However, the mechanism of PLT dysfunction in patients with LS is not completely understood. The main function of PLTs is to activate upon vessel wall injury and stop the bleeding by clot formation. PLT activation is accompanied by a shape change that is a result of massive cytoskeletal rearrangements. Here, we show that OCRL-inhibited human PLTs do not fully spread, form mostly filopodia, and accumulate actin nodules. These nodules co-localise with ARP2/3 subunit p34, vinculin, and sorting nexin 9. Furthermore, OCRL-inhibited PLTs have a retained microtubular coil with high levels of acetylated tubulin. Also, myosin light chain phosphorylation is decreased upon OCRL inhibition, without impaired degranulation or integrin activation. Taken together, these results suggest that OCRL contributes to cytoskeletal rearrangements during PLT activation that could explain mild bleeding problems in patients with LS.