Neuralgia of the glossopharyngeal nerve in a patient with posttonsillectomy scarring: recovery after local infiltration of procaine-case report and pathophysiologic discussion

We describe a patient with a three-year history of severe progressive left-sided glossopharyngeal neuralgia (GPN) that failed to adequately respond to various drug therapies. The application of lidocaine spray to the posterior pharyngeal wall provided no more than short-term relief. Apart from a large hypertrophic tonsillectomy scar on the left side all clinical and radiologic findings were normal. In terms of therapeutic local anaesthesia, the hypertrophic tonsillectomy scar tissue was completely infiltrated with the local anaesthetic (LA) procaine 1%. The patient has been almost completely pain-free ever since, and the lidocaine spray is no longer needed. Six weeks after the first treatment a repeat infiltration of the tonsillectomy scar led to the complete resolution of all symptoms. The patient has become totally symptom-free without the need to take any medication now for two and a half years. This is the first report of a successful therapeutic infiltration of a tonsillectomy scar using an LA in a patient with GPN that has been refractory to medical treatment for several years. A possible explanation may be that the positive feedback loop maintaining neurogenic inflammation is disrupted and "sympathetically maintained pain" resolved by LA infiltration.

Is Parkinson's disease of early onset a separate disease entity?

Two groups of patients suffering from Parkinson's disease were studied. The first group consisted of 23 patients with an onset age before 40 years; in the second group of 21 patients the onset was after age 50. The clinical findings and the course of the disease were very similar in each group. In spite of a longer disease duration in the patients with early onset of the disease there was no difference in motor impairment; the younger patients did better in mental testing and they were taking less dopaminergic medication. These differences are thought to be due to the age difference rather than to the existence of different disease entities. In the early onset group more familial cases (mostly affecting siblings) were found than in the older ones. The points in favour of there being a hereditary subgroup of early onset Parkinson's disease or of environmental factors causing the disease are reviewed.

Autonomic disturbances and cardiovascular reflexes in idiopathic Parkinson's disease

A total of 22 patients suffering from idiopathic Parkinson's disease and 20 age-matched volunteers were questioned about autonomic disturbances and all underwent four non-invasive tests examining cardiovascular reflexes. Significantly more autonomic disturbances were reported by the patients than by the controls. Resting blood pressure was significantly decreased in patients taking dopamine agonists, whereas it was normal in those patients who only received levodopa and anticholinergics. Resting heart rate and resting beat-to-beat variation were normal in the patients, as were the blood pressure response to standing and the postural heart rate response. No pathological response to the Valsalva manoeuvre could be detected. On the other hand, the heart rate variation evoked by deep breathing as well as the blood pressure response and the heart rate response to sustained isometric exercise were significantly diminished in the patients with idiopathic Parkinson's disease. These findings indicate a central disturbance of cardiovascular reflex control, whereas the corresponding peripheral pathways seem to be normal.

Long-term results of therapeutic local anesthesia (neural therapy) in 280 referred refractory chronic pain patients

BACKGROUND: Can the application of local anesthetics (Neural Therapy, NT) alone durably improve pain symptoms in referred patients with chronic and refractory pain? If the application of local anesthetics does lead to an improvement that far exceeds the duration of action of local anesthetics, we will postulate that a vicious circle of pain in the reflex arcs has been disrupted (hypothesis). METHODS: Case series design. We exclusively used procaine or lidocaine. The inclusion criteria were severe pain and chronic duration of more than three months, pain unresponsive to conventional medical measures, written referral from physicians or doctors of chiropractic explicitly to NT. Patients with improvement of pain who started on additional therapy during the study period for a reason other than pain were excluded in order to avoid a potential bias. Treatment success was measured after one year follow-up using the outcome measures of pain and analgesics intake. RESULTS: 280 chronic pain patients were included; the most common reason for referral was back pain. The average number of consultations per patient was 9.2 in the first year (median 8.0). After one year, in 60 patients pain was unchanged, 52 patients reported a slight improvement, 126 were considerably better, and 41 pain-free. At the same time, 74.1 % of the patients who took analgesics before starting NT needed less or no more analgesics at all. No adverse effects or complications were observed. CONCLUSIONS: The good long-term results of the targeted therapeutic local anesthesia (NT) in the most problematic group of chronic pain patients (unresponsive to all evidence based conventional treatment options) indicate that a vicious circle has been broken. The specific contribution of the intervention to these results cannot be determined. The low costs of local anesthetics, the small number of consultations needed, the reduced intake of analgesics, and the lack of adverse effects also suggest the practicality and cost-effectiveness of this kind of treatment. Controlled trials to evaluate the true effect of NT are needed.

Combined ATRX/IDH1 immunohistochemistry predicts genotype of oligoastrocytomas

AIMS To assess whether in oligoastrocytomas ATRX deficiency - as a surrogate of the alternative lengthening of telomeres (ALT) pathway - has a role in predicting the presence or absence of loss of heterozygosity of 1p and 19q (LOH), the genetic signature of oligodendroglial differentiation and a favourable prognostic marker. METHODS AND RESULTS A series of 54 oligoastrocytomas were investigated by immunohistochemistry as well as microsatellite analysis for LOH 1p19q. Genetic findings were correlated with morphological assessment. CONCLUSIONS ATRX deficiency was mutually exclusive with LOH. Conversely, ATRX-proficient tumours immunoreactive for R132H-mutant isocitrate dehydrogenase 1 (IDH1) showed a high rate (85%) of LOH. A more oligodendroglioma-like morphology was associated with a higher rate of LOH even in the morphologically ambiguous group of oligoastrocytomas. Our findings support the concept that oligoastrocytomas represent a morphological grey zone rather than a group of truly "mixed" or "intermediate" tumours. More precise classification of diffuse gliomas may also improve grading of borderline cases. We propose an immunohistochemical algorithm for classification of morphologically ambiguous diffuse gliomas. This article is protected by copyright. All rights reserved.

Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.

Stabilisation of precipitates of pedogenic dissolved organic matter by multivalent cations

Purpose Precipitation of dissolved organic matter (DOM) by multivalent cations is important for biogeochemical cycling of organic carbon. We investigated to which extent cation bridges are involved in DOM precipitation and how cross-links by cations and water molecule bridges (WaMB) stabilise the matrix of precipitated DOM. Materials and methods DOM was precipitated from the aqueous extract of a forest floor layer adding solutions of Ca(NO3)2, Al(NO3)3 and Pb(NO3)2 with different initial metal cation/C (Me/C) ratios. Precipitates were investigated by differential scanning calorimetry before and after ageing to detect cation bridges, WaMB and restructuring of supramolecular structure. Results and discussion Twenty-five to sixty-seven per cent of the dissolved organic carbon was precipitated. The precipitation efficiency of cations increased in the order Ca < Al < Pb, while the cation content of precipitates increased in the order Pb < Ca < Al. The different order and the decrease in the WaMB transition temperature (T*) for Al/C > 3 is explained by additional formation of small AlOOH particles. Thermal analysis indicated WaMB and their disruption at T* of 53–65 °C. Like cation content, T* increased with increasing Me/C ratio and in the order Ca < Pb < Al for low Me/C. This supports the general assumption that cross-linking ability increases in the order Ca < Pb < Al. The low T* for high initial Me/C suggests less stable and less cross-linked precipitates than for low Me/C ratios. Conclusions Our results suggest a very similar thermal behaviour of OM bound in precipitates compared with soil organic matter and confirms the relevance of WaMB in stabilisation of the supramolecular structure of cation-DOM precipitates. Thus, stabilisation of the supramolecular structure of the DOM precipitates is subjected to dynamics in soils.

Self-reported nonadherence to antiretroviral therapy as a predictor of viral failure and mortality.

OBJECTIVE To determine the effect of nonadherence to antiretroviral therapy (ART) on virologic failure and mortality in naive individuals starting ART. DESIGN Prospective observational cohort study. METHODS Eligible individuals enrolled in the Swiss HIV Cohort Study, started ART between 2003 and 2012, and provided adherence data on at least one biannual clinical visit. Adherence was defined as missed doses (none, one, two, or more than two) and percentage adherence (>95, 90-95, and <90) in the previous 4 weeks. Inverse probability weighting of marginal structural models was used to estimate the effect of nonadherence on viral failure (HIV-1 viral load >500 copies/ml) and mortality. RESULTS Of 3150 individuals followed for a median 4.7 years, 480 (15.2%) experienced viral failure and 104 (3.3%) died, 1155 (36.6%) reported missing one dose, 414 (13.1%) two doses and, 333 (10.6%) more than two doses of ART. The risk of viral failure increased with each missed dose (one dose: hazard ratio [HR] 1.15, 95% confidence interval 0.79-1.67; two doses: 2.15, 1.31-3.53; more than two doses: 5.21, 2.96-9.18). The risk of death increased with more than two missed doses (HR 4.87, 2.21-10.73). Missing one to two doses of ART increased the risk of viral failure in those starting once-daily (HR 1.67, 1.11-2.50) compared with those starting twice-daily regimens (HR 0.99, 0.64-1.54, interaction P = 0.09). Consistent results were found for percentage adherence. CONCLUSION Self-report of two or more missed doses of ART is associated with an increased risk of both viral failure and death. A simple adherence question helps identify patients at risk for negative clinical outcomes and offers opportunities for intervention.

Structure and mechanism of an active lipid-linked oligosaccharide flippase

The flipping of membrane-embedded lipids containing large, polar head groups is slow and energetically unfavourable, and is therefore catalysed by flippases, the mechanisms of which are unknown. A prominent example of a flipping reaction is the translocation of lipid-linked oligosaccharides that serve as donors in N-linked protein glycosylation. In Campylobacter jejuni, this process is catalysed by the ABC transporter PglK. Here we present a mechanism of PglK-catalysed lipid-linked oligosaccharide flipping based on crystal structures in distinct states, a newly devised in vitro flipping assay, and in vivo studies. PglK can adopt inward- and outward-facing conformations in vitro, but only outward-facing states are required for flipping. While the pyrophosphate-oligosaccharide head group of lipid-linked oligosaccharides enters the translocation cavity and interacts with positively charged side chains, the lipidic polyprenyl tail binds and activates the transporter but remains exposed to the lipid bilayer during the reaction. The proposed mechanism is distinct from the classical alternating-access model applied to other transporters.

The Vacuolar H+-ATPase B1 Subunit Polymorphism p.E161K Associates with Impaired Urinary Acidification in Recurrent Stone Formers

Mutations in the vacuolar–type H+-ATPase B1 subunit gene ATP6V1B1 cause autosomal–recessive distal renal tubular acidosis (dRTA). We previously identified a single-nucleotide polymorphism (SNP) in the human B1 subunit (c.481G.A; p.E161K) that causes greatly diminished pump function in vitro. To investigate the effect of this SNP on urinary acidification, we conducted a genotype-phenotype analysis of recurrent stone formers in theDallas and Bern kidney stone registries. Of 555 patients examined, 32 (5.8%) were heterozygous for the p.E161K SNP, and the remaining 523 (94.2%) carried two wild–type alleles. After adjustment for sex, age, body mass index, and dietary acid and alkali intake, p.E161K SNP carriers had a nonsignificant tendency to higher urinary pH on a random diet (6.31 versus 6.09; P=0.09). Under an instructed low–Ca and low–Na diet, urinary pH was higher in p.E161K SNP carriers (6.56 versus 6.01; P,0.01). Kidney stones of p.E161K carriers were more likely to contain calcium phosphate than stones of wild-type patients. In acute NH4Cl loading, p.E161K carriers displayed a higher trough urinary pH (5.34 versus 4.89; P=0.01) than wild-type patients. Overall, 14.6% of wild-type patients and 52.4% of p.E161K carriers were unable to acidify their urine below pH 5.3 and thus, can be considered to have incomplete dRTA. In summary, our data indicate that recurrent stone formers with the vacuolar H+-ATPase B1 subunit p.E161K SNP exhibit a urinary acidification deficit with an increased prevalence of calcium phosphate– containing kidney stones. The burden of E161K heterozygosity may be a forme fruste of dRTA.

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein-Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA). RESULTS The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Blocking the epithelial-to-mesenchymal transition pathway abrogates resistance to anti-folate chemotherapy in lung cancer.

Anticancer therapies currently used in the clinic often can neither eradicate the tumor nor prevent disease recurrence due to tumor resistance. In this study, we showed that chemoresistance to pemetrexed, a multi-target anti-folate (MTA) chemotherapeutic agent for non-small cell lung cancer (NSCLC), is associated with a stem cell-like phenotype characterized by an enriched stem cell gene signature, augmented aldehyde dehydrogenase activity and greater clonogenic potential. Mechanistically, chemoresistance to MTA requires activation of epithelial-to-mesenchymal transition (EMT) pathway in that an experimentally induced EMT per se promotes chemoresistance in NSCLC and inhibition of EMT signaling by kaempferol renders the otherwise chemoresistant cancer cells susceptible to MTA. Relevant to the clinical setting, human primary NSCLC cells with an elevated EMT signaling feature a significantly enhanced potential to resist MTA, whereas concomitant administration of kaempferol abrogates MTA chemoresistance, regardless of whether it is due to an intrinsic or induced activation of the EMT pathway. Collectively, our findings reveal that a bona fide activation of EMT pathway is required and sufficient for chemoresistance to MTA and that kaempferol potently regresses this chemotherapy refractory phenotype, highlighting the potential of EMT pathway inhibition to enhance chemotherapeutic response of lung cancer.