Study on the levels of trace elements in mild and severe psoriasis.

BACKGROUND: Psoriasis is a chronic, recurrent skin disorder characterized histologically by cutaneous inflammation, increased epidermal proliferation, hyperkeratosis, angiogenesis, abnormal keratinization, shortened maturation time and parakeratosis. Data on the involvement of trace elements in the pathogenesis of psoriasis is limited. METHODS: The elements namely Na, K, Ca, P, S, Mg, Cu, Zn, and Fe were analyzed in the serum samples of mild and severe psoriasis patients with a control group using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). Patients were assessed as per standard clinical diagnostic criteria and classified into mild and severe psoriasis groups using Psoriasis Area Severity Index (PASI) score. RESULTS: In mild psoriasis, the levels of K, P, Cu, and Mg were increased significantly (p<0.001), while in severe psoriasis P, Mg, and Cu were increased significantly (p<0.001). The S and Fe concentrations were decreased significantly (p<0.001) in both mild and severe psoriasis types when compared to control. CONCLUSIONS: There is a disturbance in the under-study element contents and also element-element interdependency in psoriasis serum when compared to controls.

Endobronchial ultrasound-guided transbronchial needle aspiration in the diagnosis of lymphoma.

BACKGROUND: The diagnostic accuracy of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for the diagnosis of lymphoma in patients with mediastinal lymphadenopathy is not well defined. METHODS: A retrospective review was performed of all patients with mediastinal lymphadenopathy referred for EBUS-TBNA between August 2005 and December 2006 in whom lymphoma was suspected based on prior history or clinical presentation. Mediastinal biopsy specimens were taken using a linear array ultrasonic bronchoscope (Olympus XBF-UC 160F) and a 22-gauge cytology needle (NA-202C Olympus) with on-site cytopathological support. The EBUS-TBNA result was compared with a reference standard of pathological tissue diagnosis or a composite of > or =6 months of clinical follow-up with radiographic imaging. RESULTS: Of 236 patients who underwent EBUS-TBNA, 25 were eligible for inclusion. Indications for EBUS-TBNA were suspected mediastinal recurrence of lymphoma (n = 13) and mediastinal lymphadenopathy of unknown cause (n = 12). Adequate lymph node sampling was accomplished in 24/25 patients (96%); there were no complications. EBUS-TBNA identified lymphoma in 10 patients and benign disease in 14 patients. There was one false negative EBUS-TBNA for lymphoma (lymphoma prevalence 11/25 (44%)). Follow-up over a median of 10.5 months (range 1-19) confirmed stable or regressive lymphadenopathy in all 14 patients without a lymphoma diagnosis, consistent with a benign diagnosis. Overall, EBUS-TBNA had a sensitivity of 90.9%, specificity of 100%, positive predictive value of 100% and negative predictive value of 92.9% for the diagnosis of lymphoma. CONCLUSIONS: EBUS-TBNA is an accurate, safe and useful tool in the investigation of suspected lymphoma with isolated mediastinal adenopathy, and may diminish the need for more invasive procedures such as mediastinoscopy.

Determination of uranium in aqueous attenuating samples using gamma-ray spectrometry.

In the present work, a method for determination of uranium concentration in aqueous solution in standard geometry from attenuating samples has been developed based on modification of the empirical approach of Venkataraman and Croft [2003. Determination of plutonium mass using gamma-ray spectrometry. Nucl. Instrum. Methods Phys. Res. A 505, 527-530]. The method makes use of the multiple gamma (gamma)-rays emitted by 235U and depends on the empirical relation between apparent mass of the sample and gamma-ray energy. It was possible to determine uranium concentration in the range of 12-400mg/ml rapidly by this method without applying transmission corrections.

SDHB, SDHC, and SDHD mutation screen in sporadic and familial head and neck paragangliomas.

Mutations within three genes, SDHB, SDHC, and SDHD, encoding distinct subunits of a hetero-oligomeric protein known as the mitochondrial complex II, a component of the mitochondrial electron transport chain and the Krebs cycle have been implicated in the pathogenesis of hereditary paraganglioma (PGL). This study describes a mutation screen of SDHB, SDHC, and SDHD in blood and tumor samples of 14 sporadic and three familial cases of head and neck PGL (HNP). Germline mutations in SDHB and SDHD were identified in two of the three affected individuals with familial HNP. The SDHB mutation was a novel 3 base pair, in-frame deletion of AGC at nucleotide 583-585 encoding serine (delS195). The SDHD mutation was a C to T transition within codon 81 causing substitution of proline with leucine (P81L). In contrast to familial cases, no germline or somatic mutations were identified in the 14 sporadic cases of HNP. The presence of mutations within SDHB and SDHD in two of the three samples of familial PGLs and absence of mutations in sporadic cases is consistent with the significant contribution of these genes to familial but not sporadic PGL. The etiology of sporadic PGL remains to be elucidated.

Mutation analysis of Connexin 31 (GJB3) in sporadic non-syndromic hearing impairment.

Mutations in GJB3, the gene encoding the gap junction protein Connexin 31 (CX31), have been pathogenically linked to erythrokeratodermia and non-syndromic autosomal dominant (DFNA2) or recessive hereditary hearing impairment (HHI). To determine the contribution of CX31 to sporadic deafness, we assessed 63 individuals with non-syndromic hearing impairment for CX31 mutations. Single coding exon of CX31 was amplified from genomic DNA and then sequenced. Single nucleotide sequence alteration was present in 15 out of 63 patients (24%), all of the positives being heterozygous for the four different single base pair changes that were detected: C94T, C201T, C357T and C798T. Of these, only C94T transition, identified in two patients, results in amino acid change, R32W, while the other three changes are single nucleotide polymorphisms (SNPs). The R32W substitution in CX31 has been previously documented and is speculated to manifest variable penetrance, similar to the polymorphic allele encoding CX26M34T. Over one-third of all samples were also screened with denaturing high-performance liquid chromatography (dHPLC). Seven out of 25 individuals screened were determined to be positive for CX31 sequence variation. Sequence analysis of the 25 individuals screened identified nucleotide alterations in all of the 7 'positives' and in none of the 16 'negatives' yielding a specificity and sensitivity of 100%. Thus, dHPLC represents a highly efficient CX31 screening technique. This study suggests that while sequence alterations are common, pathogenic mutations of CX31 are infrequent in sporadic non-syndromic hearing impairment.

Assessment of denaturing high-performance liquid chromatography (DHPLC) in screening for mutations in connexin 26 (GJB2).

Mutations in the gene GJB2 encoding connexin 26 (Cx26), a gap junction protein, have been shown to be responsible for a majority of recessive nonsyndromic hereditary hearing impairment in children. Over 60 different mutations in Cx26 have been reported. To obviate the need for direct sequencing of each specimen, a variety of screening techniques have been used to detect mutations in Cx26. However, each of these methods has significant shortcomings including expense, time consumption, and limited sensitivity. Denaturing high-performance liquid chromatography (DHPLC) has been recently introduced as a rapid and highly sensitive method of detecting sequence alterations. We have assessed the efficacy of DHPLC as a screening assay for detecting mutation in Cx26 coding region in 154 patients with hereditary hearing impairment. The GJB2 coding exon was amplified in one or two fragments, analyzed by DHPLC, and sequenced. Sequence analysis identified sequence variations in 34 patients concordant with abnormal DHPLC results. Three novel Cx26 mutations were identified: a single base pair substitution 511G>A, a 4 bp insertion 504insAACG, and a 3 bp deletion 358delAGG in three unrelated patients. In 120 patients with normal Cx26 sequence, DHPLC was normal. These results yield sensitivity and specificity of 100% for DHPLC-based detection of Cx26 mutations, and demonstrate that DHPLC is a highly sensitive and specific method of screening for sequence variations in Cx26 that is time and labor efficient. Further, our experience suggests that DHPLC screening alone followed by DNA sequencing only when DHPLC is abnormal may be adequate for identification of all sequence alterations in Cx26.

Cochlear gene delivery through an intact round window membrane in mouse.

Cochlear gene transfer studies in animal models have utilized mainly two delivery methods: direct injection through the round window membrane (RWM) or intracochlear infusion through a cochleostomy. However, the surgical trauma, inflammation, and hearing loss associated with these methods lead us to investigate a less invasive delivery method. Herein, we studied the feasibility of a vector transgene-soaked gelatin sponge, Gelfoam, for transgene delivery into the mouse cochlea through an intact RWM. The Gelfoam absorbed with liposomes and adenovirus, but not with adeno-associated virus (AAV), was successful in mediating transgene expression across an intact RWM in a variety of cochlear tissues. The Gelfoam technique proved to be an easy, atraumatic, and effective, but vector-dependent, method of delivering transgenes through an intact RWM. Compared with the more invasive gene delivery methods, this technique represents a safer and a more clinically viable route of cochlear gene delivery in humans.

Detection of fungi in the nasal mucosa using polymerase chain reaction.

HYPOTHESIS: Fungi have been increasingly recognized as important pathogens in sinusitis. However, detection of fungus with conventional culture techniques is insensitive and unreliable. Polymerase chain reaction (PCR) is an exquisitely sensitive assay that can detect the DNA of 10 or less fungal elements. The aim of this study was to compare the sensitivity of conventional culture techniques using PCR analysis. METHODS: Nasal swabs and DNA samples were collected from the nasal cavities of control subjects and patients with chronic sinusitis. Fungal-specific PCR analysis and standard cultures were performed on every sample. chi2 analysis was used to test for statistical differences between groups. RESULTS: PCR analysis detected fungal DNA in 42% and 40% of control subjects and patients with chronic sinusitis while standard cultures were positive in 7% and 0%, respectively. There was no statistically significant difference in the prevalence of fungi in the normal volunteers and patients with chronic rhinosinusitis. CONCLUSION: PCR is significantly more sensitive than nasal swab cultures in detecting the presence of fungi in nasal mucosa. In addition, our study suggests that the presence of fungi alone is insufficient to implicate it as the pathogen in chronic sinusitis.

A surgical approach appropriate for targeted cochlear gene therapy in the mouse.

Therapeutic manipulations of the mammalian cochlea, including cochlear gene transfer, have been predominantly studied using the guinea pig as the experimental model. With the significant developments in mouse genomics and the availability of mutant strains of mice with well-characterized hearing loss, the mouse justifiably will be the preferred animal model for therapeutic manipulations. However, the potential advantages of the mouse model have not been fully realized due to the surgical difficulty of accessing its small cochlea. This study describes a ventral approach, instead of the routinely used postauricular approach in other rodents, for accessing the mouse middle and inner ear, and its application in cochlear gene transfer. This ventral approach enabled rapid and direct delivery of liposome-transgene complex to the mouse inner ear while avoiding blood loss, facial nerve morbidity, and mortality. Transgene expression at 3 days was detected in Reissner's membrane, spiral limbus, spiral ligament, and spiral ganglion cells, in a pattern similar to that previously described in the guinea pig. The successful access and delivery of material to the mouse cochlea and the replication of gene expression seen in the guinea pig demonstrated in this study should promote the use of the mouse in future studies investigating targeted cochlear therapy.

Cochlear microinjection and its effects upon auditory function in the guinea pig.

Microinjection through the round window membrane has been found to represent a method for vector delivery in intracochlear gene transfer in animal models but breaches the round window membrane, making it necessary to evaluate animals for possible postinjection hearing loss. In the present study healthy guinea pigs were evaluated for their baseline click auditory brainstem response (ABR) thresholds. Each animal was then injected with saline via the round window membrane. After 1 week auditory function was evaluated by click ABR. Animals with increased ABR thresholds were retested at 4 weeks. Animals with 1-week postoperative ABRs similar to baseline were not retested. Results showed that postoperative ABR thresholds in five animals (71%) remained unchanged from baseline, while two animals had increases of 20-25 dB in ABRs after 1 week but recovered baseline ABRs after 4 weeks. The mean baseline ABR threshold was 25.7 dB and was 27.9 dB after 1 week after injection. The difference between preoperative and 1-week postoperative averages was not significant (P = 0.707). In this preliminary study saline microinjection through the round window membrane did not cause permanent hearing loss in the guinea pigs tested, and any damage caused by microinjection appeared to be reversible.

Safety of adeno-associated virus as cochlear gene transfer vector: analysis of distant spread beyond injected cochleae.

The adeno-associated virus (AAV), inoculated into the perilymph, has been shown to be an effective vector for mediating intracochlear transgene expression. The unexpected finding of transgene expression in the contralateral cochlea in previous work raised concern about dissemination of the virus from the target tissue. The current study was undertaken to assess the extent of AAV dissemination following its introduction into the inner ear. Adult male guinea pigs were injected with recombinant AAV into their left ears and sacrificed at 2 or 4 weeks. Various organs including the cochleae were harvested to characterize the presence and expression of the viral DNA. Virus DNA was detected via polymerase chain reaction in the infused and contralateral cochlea and in the cerebellum but not in any other organs, including cortex, heart, lung, liver, spleen, and kidney. Although the viral presence was established in the cerebellum, transgene expression in this organ was undetectable with either Western blot or immunohistochemistry. Transgene expression was demonstrated via immunohistochemistry in multinucleated giant cells in the bone marrow spaces adjacent to the infused and contralateral cochleae. Collectively, these results suggest potential routes for AAV dissemination from the infused cochlea via the cochlear aqueduct or by extension through the temporal bone marrow spaces. This study reinforces the need to investigate factors that mitigate viral leakage.

Human nonsyndromic hereditary deafness DFNA17 is due to a mutation in nonmuscle myosin MYH9.

The authors had previously mapped a new locus-DFNA17, for nonsyndromic hereditary hearing impairment-to chromosome 22q12.2-q13. 3. DFNA17 spans a 17- to 23-cM region, and MYH9, a nonmuscle-myosin heavy-chain gene, is located within the linked region. Because of the importance of myosins in hearing, MYH9 was tested as a candidate gene for DFNA17. Expression of MYH9 in the rat cochlea was confirmed using reverse transcriptase-PCR and immunohistochemistry. MYH9 was immunolocalized in the organ of Corti, the subcentral region of the spiral ligament, and the Reissner membrane. Sequence analysis of MYH9 in a family with DFNA17 identified, at nucleotide 2114, a G-->A transposition that cosegregated with the inherited autosomal dominant hearing impairment. This missense mutation changes codon 705 from an invariant arginine (R) to histidine (H), R705H, within a highly conserved SH1 linker region. Previous studies have shown that modification of amino acid residues within the SH1 helix causes dysfunction of the ATPase activity of the motor domain in myosin II. Both the precise role of MYH9 in the cochlea and the mechanism by which the R705H mutation leads to the DFNA17 phenotype (progressive hearing impairment and cochleosaccular degeneration) remain to be elucidated.

Molecular pathogenesis in sporadic head and neck paraganglioma.

HYPOTHESIS: Similar to familial tumors, sporadic head and neck paragangliomas are associated with chromosomal deletions at either 11q13 or 11q22-23. BACKGROUND: Familial paragangliomas are inherited in an autosomal dominant pattern with genomic imprinting of the maternal allele. Genetic studies of familial paragangliomas have localized the causative genetic defect to two separate loci: 11q13.1 and 11q22-23. The molecular pathogenesis of sporadic head and neck paragangliomas has not been studied. METHODS: Blood and tumor samples from patients with sporadic head and neck paragangliomas were screened for deletions on chromosome 11 using DNA microsatellite markers and polymerase chain reaction. Polymerase chain reaction-amplified alleles from tumor specimens were compared with those from the blood of eight patients. A greater than 50% reduction in band intensity (as determined by densitometric analysis) between blood and tumor sample was indicative of a chromosomal deletion. RESULTS: Three of the eight patients were found to have deletions at chromosome 11q: two at chromosome 11q22-23 and one at 11q13. CONCLUSIONS: Sporadic head and neck paragangliomas are associated with deletions at chromosome 11q13 and 11q22-23. It is thus likely that sporadic and familial paragangliomas share a similar molecular pathogenesis.

Identification of aquaporin 5 (AQP5) within the cochlea: cDNA cloning and in situ localization.

Dysfunction of fluid and electrolyte homeostasis is considered to cause variety of inner ear disorders. One group of candidate proteins that may play a critical role in the inner ear fluid homeostasis is the aquaporins, a family of proteins whose members have well defined roles in fluid transport in variety of organs. This study reports the identification of AQP5, a member of the aquaporin family, within the rat inner ear and its in situ localization. AQP5 was initially identified within rat cochlear RNA via RT-PCR and sequence analysis of the amplified fragments. Immunoblot of cochlear homogenate yielded a predominant AQP5-immunoreactive band of M(r) 35 kDa. The anti-AQP5 immunoreactivity, indicating expression of the AQP5 polypeptide, was localized within the cochlea in situ to the cell types that form the lateral wall of the cochlear duct-the external sulcus (ES) cells and the cells of the spiral prominence. Expression of AQP5 was observed in the apical turn but not the basal turn of the cochlea; nor was it observed in the vestibular neuroepithelia or its supporting cells. The restricted expression of AQP5 to the apical turns of the cochlea suggests its potential role in low frequency hearing.

Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector.

The utility of lentivirus as a gene delivery vector in the cochlea was evaluated in vitro and in vivo. Lentivirus transduction was assessed through expression analysis of a reporter gene, green fluorescent protein (GFP), integrated within the viral genome. In vitro characterization of lentivirus-GFP was assessed by infection of explants from cochleas of neonatal rat. The lentiviral vector transduced both spiral ganglion neurons (SGNs) and glial cells. In vivo characterization of lentivirus-GFP was assessed by directly infusing the vector into the guinea pig cochlea via an osmotic minipump. Sections of lentivirus-infused cochlea revealed a highly restricted fluorescence pattern limited to the periphery of the perilymphatic space. Transduction of SGNs and glial cells by lentivirus in vitro but not in vivo suggests limited dissemination of the viral vector from the perilymphatic space. The cellular and tissue architecture of the lentivirus-infused cochlea was intact and free of inflammation. Restricted transduction of cell types confined to the periphery of the perilymphatic space by the lentivirus is ideal for stable production of gene products secreted into the perilymph.

Cationic liposome mediated transgene expression in the guinea pig cochlea.

Sensorineural hearing loss affects nearly 10% of the American population that is refractory to conventional therapy. Gene therapy represents an intervention with potential therapeutic efficacy. We studied the feasibility of cationic liposome mediated gene transfer within the guinea pig cochlea in vivo following direct microinjection into the cochlea. Transgene expression was persistent up to 14 days in the neurosensory epithelia and surrounding tissue without toxicity and inflammation in the target organ. This study represents the first successful use of cationic liposomes for cochlear gene transfer thus providing a safe and rapid alternative to the use of recombinant viral vectors in gene therapy for inner ear disorders.

Point mutation in the MITF gene causing Waardenburg syndrome type II in a three-generation Indian family.

Waardenburg syndrome (WS) is an autosomal-dominant neural crest cell disorder phenotypically characterized by hearing impairment and disturbance of pigmentation. A presence of dystopia canthorum is indicative of WS type 1, caused by loss of function mutation in the PAX3 gene. In contrast, type 2 WS (WS2) is characterized by normally placed medial canthi and is genetically heterogeneous; mutations in MITF (microphthalmia associated transcription factor) associated with WS2 have been identified in some but not all affected families. Here, we report on a three-generation Indian family with a point mutation in the MITF gene causing WS2. This mutation, initially reported in a Northern European family, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking the HLH-Zip or Zip structure necessary for normal interaction with its target DNA motif. Comparison of the phenotype between the two families demonstrates a significant difference in pigmentary disturbance of the eye. This family, with the first documented case of two unrelated WS2 families harboring identical mutations, provides additional evidence for the importance of genetic background on the clinical phenotype.

The guinea pig cochlear AE2 anion exchanger: cDNA cloning and in situ localization within the cochlea.

This study has characterized the repertoire of the anion exchanger (AE) family members expressed within the guinea pig organ of Corti, the auditory neuroepithelia. Both AE2 and AE3 cDNAs were present, but AE1 cDNA was not detected. The more abundant AE2 was sequenced and its expression characterized in the cochlea. The 3888 base pairs (bp) AE2 sequence, compiled from multiple clones, includes 150 bp of upstream non-coding sequence and 3717 bp of open reading frame encoding a protein of 1238 amino acids. Immunoblot of cochlear homogenate revealed a single AE2-immunoreactive band of Mr 180 kDa. In situ hybridization and immunohistochemical analysis localized AE2 expression to several tissues and cell types within the guinea pig inner ear, including superior half of the spiral ligament and within the interdental cells lining the spiral limbus. However, AE2 was not clearly detected in the outer hair cells (OHC) of the organ of Corti by either immunohistochemistry or in situ hybridization. The results of these studies imply a physiologic role of AE2 in the cochlear homeostasis, but do not support its role as a potential 'motor protein' in mediating the in vitro-observed voltage-gated, ATP-independent OHC motility.