Plasmid Delivery and Single-Cell Plasmid Expression Analysis for CRISPR/dCas9-Based Epigenetic Editing.

To fully exploit the potentials of reprogramming the epigenome through CRISPR/dCas9 systems for epigenetic editing, there is a growing need for improved transfection methods. With the utilization of constructs often with large sizes and the wide array of cell types used to read out the effect of epigenetic editing in different biological applications, it is evident that ongoing optimalization of transfection protocols tailored to each specific experimental setup is essential. Whether the goal is the production of viral particles using human embryonic kidney (HEK) cells or the direct examination of epigenomic modifications in the target cell type, continuous refinement of transfection methods is crucial. In the hereafter outlined protocol, we focus on optimization of transfection protocols by comparing different reagents and methods, creating a streamlined setup for transfection efficiency optimization in cultured mammalian cells. Our protocol provides a comprehensive overview of flow cytometry analysis following transfection not just to improve transfection efficiency but also to assess the expression level of the utilized construct. We showcase our transfection protocol optimization using HEK293T Lenti-X™ and breast cancer MCF-7 cell lines, using a single-guide RNA-containing plasmid. Specifically, we incorporate heat shock treatment for increased transfection efficiency of the MCF-7 cell line. Our detailed optimization protocol for efficient plasmid delivery and measurement of single-cell plasmid expression provides a comprehensive instruction for assessing both transient and sustained effects of epigenetic reprogramming.

Olfactory function in HIV-positive subjects.

The aim of this study was to reinvestigate previous reports of chemosensory dysfunction in HIV-positive subjects. Odor thresholds, odor discrimination and odor identification were assessed using the Sniffin' Sticks test battery. Seventy-four HIV-positive patients were tested. According to CDC criteria, 38 subjects were classified as stage A, 10 as stage B and 26 as stage C. None of the subjects exhibited severe cognitive impairment. Compared to normative data all subjects had normal odor identification and discrimination. However, odor thresholds were well below the median of a normal population. There were no significant differences between stage A, B or C subjects. This may be interpreted as indicating that olfactory dysfunction is among the primary deficits of HIV infection and occurs independently of disease stage. These results confirm previous work suggesting that odor thresholds are elevated early in HIV infection whereas a decline in identification and discrimination abilities is correlated with reduced cognitive abilities.

Chemotherapy-related hemolytic-uremic syndrome following treatment of a carcinoma of the nasopharynx.

OBJECTIVE: A cisplatin-containing regimen followed by radiation therapy is the recommended treatment for patients with advanced nasopharyngeal carcinoma. We report a case of a 58-year-old woman with hemolytic-uremic syndrome (HUS) who received induction chemotherapy for undifferentiated squamous cell carcinoma of the nasopharynx. PATIENTS AND METHODS: During the 2nd course of chemotherapy (consisting of bleomycin, cisplatin and epirubicin), the patient developed hemolytic anemia, thrombocytopenia, and acute renal failure. After HUS had been diagnosed, the patient was transferred to the intensive care unit. RESULTS: Twice daily therapeutic plasma exchange (TPE) with fresh-frozen plasma, hemodialysis and high-dose cortisone was performed. Two weeks after the start of plasma exchange, thrombocytes and renal function began to normalize. Low-dose cortisone was continued until the patient recovered from hemolytic anemia. Six weeks after the administration of the second course of chemotherapy, the patient had fully recovered from HUS, and radiation therapy was carried out as planned. The patient responded well to treatment, but died 9 months after the diagnosis due to liver metastases. CONCLUSION: We demonstrated that early TPE with fresh-frozen plasma and high-dose cortisone is a potentially successful treatment modality for the usually fatal, fulminant form of chemotherapy-induced HUS.

Ifosfamide and mitoxantrone in the treatment of recurrent and/or metastatic squamous cell carcinoma of the head and neck.

A phase II study was performed to assess the safety and efficacy of ifosfamide and mitoxantrone in recurrent and/or metastatic squamous cell carcinomas of the head and neck. Treatment consisted of ifosfamide 1500 mg/m2 in 1000 ml saline, infused over 60 min and mesna 20% of the total dose of ifosfamide in three doses for 3 days combined with mitoxantrone 12 mg/m2 given as a short infusion on day 1. Treatment courses were repeated every 4 weeks until a total of six cycles. Twenty-two patients entered this trial, 13 of whom had received chemo- and radiation therapy, and nine patients who underwent radiation therapy with or without prior surgery. We observed no objective response, with the exception of two patients who experienced minor response (reduction of tumor size of 25%). The dose-limiting toxicity was myelosuppression with grade 3/4 leukocytopenia in seven patients (32%) and grade 3/4 neutropenia in 15 (68%). Severe organ toxicity except alopecia (91%) was not observed. Ifosfamide combined with mitoxantrone does not improve the therapeutic armentarium in recurrent squamous cell carcinoma of the head and neck.

Patterns of non-conductive olfactory disorders in eastern Austria: a study of 120 patients from the Department of Otorhinolaryngology at the University of Vienna.

BACKGROUND: About 1% of the population suffer from disorders of the chemosensory system. In the United States at least two million people have problems related to smell and taste. The sense of smell enables the individual to determine the flavour of food and beverages and is most important as a sophisticated warning system. For the present investigation, we collected data on the age pattern and causes of olfactory disorders in eastern Austria. METHODS: 120 patients with non-conductive olfactory disorders were examined over a 9-month period starting from July 1998 at the outpatient clinic of the Ear Nose and Throat Department of the University of Vienna. Data concerning the underlying population taken from the 1998 population census in Vienna were used for comparison, in order to gain a more representative estimation of the distribution of these disorders. The diagnosis was based on thorough history taking, physical examination, CT scan, and olfactory testing for sensitivity by means of so-called "sniffin' sticks". RESULTS: The patients' ages ranged from 16 to 86 years (mean, 54.5 years; 74 females, 46 males). Those older than 50 years seem to have a higher risk of developing olfactory disorders. Only 15 of the female patients were pre-menopausal. Olfactory disorders were most frequently caused by viral infections in the upper respiratory tract (n = 51). Fifteen patients reported head trauma as a cause of olfactory loss, and 45 causes were idiopathic. Most of those in whom the olfactory disorder had been in existence for less than 3 months were anosmic (84%), very few were hyposmic (16% of a total of 19 patients). In contrast, 38% (of a total of 29 patients) in whom the disease had been in existence for 3 and 6 months were hyposmic. Parosmia was reported in 16 cases. Most parosmias appeared after viral infection (56%). Eight of the 120 patients reported dysgeusia. CONCLUSIONS: The present study is a first step towards an assessment of olfactory disorders in Austria. We found similar causes of non-conductive olfactory disorders as have been reported in the literature for other countries, namely upper respiratory infection leading to postviral olfactory disorders, and head trauma. With increasing age women seem to suffer more often from chemosensory dysfunction than men, which may be related to hormonal factors.

Olfactory function in patients with olfactory groove meningioma.

OBJECTIVES: Olfactory meningiomas are rare benign tumours and represent about 12% of all basal meningiomas. Anosmia is thought to be among the first symptoms, even though patients often present with headaches or visual problems. However, so far no detailed psychophysical tests of olfactory function have been performed in a large number of those patients. METHODS: Twelve patients (five men, seven women; mean age 52 years) with olfactory meningiomas were examined. In all patients extensive preoperative and postoperative lateralised olfactory testing was performed using the "Sniffin' Sticks" test battery, a psychometric testing tool. In eight cases the meningioma was lateralised (five left, three right), in four patients a bilateral meningioma was found. In addition to a detailed ear, nose, and throat examination MRI was performed in all patients. RESULTS: In preoperative testing six patients were found to be anosmic on the side of the tumour, two were hyposmic. Four patients were normosmic. Postoperative investigations showed lateralised anosmia in four patients on the operated side, three were normosmic on the contralateral side and one hyposmic. The remaining eight patients were completely anosmic postoperatively. CONCLUSIONS: (1) Contrary to expectations, olfactory testing seems to be of little help in detecting olfactory meningiomas. (2) The likelihood of normal postoperative olfactory function contralateral to the tumour was high when the tumour was less than 3 cm in diameter and preoperative normosmia had been established. (3) Preservation of olfactory function ipsilateral to the tumour seems to be extremely difficult, irrespective of tumour size or surgical approach.

Vascular endothelial growth factor (VEGF) and microvessel density in squamous cell carcinomas of the larynx: an immunohistochemical study.

The distribution of vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, and microvessel density (MVD) were assessed in laryngeal carcinomas by means of immunohistochemistry. Correlation of VEGF with MVD and clinical parameters (T stage, N stage, histological grading, survival, recurrence-free interval) was also examined. VEGF expression was evaluated semi-quantitatively and was observed in varying intensity (i) in tumour cells, (ii) in the stromal department as diffuse, sometimes strong reactivity, especially in close proximity to tumour masses and (iii) in macrophages and endothelial cells. Normal epithelium presented no VEGF reactivity except in the immediate vicinity of tumour transformation. Forty percent of our specimens exhibited substantial VEGF reactivity, whereas 20% showed no staining in tumour cells and stroma. These results could be positively correlated with MVD. Moreover, high-graded carcinomas revealed higher VEGF expression, but there was no association of tumour stage or lymph node status with VEGF or MVD. There was a trend in the survival and recurrence analysis towards a higher risk of disease relapse and shorter survival time for patients with enhanced VEGF expression. Apart from tumour cells, macrophages seem to be a substantial source of VEGF in carcinomas. This observation supports the concept of a pivotal role of these cells in tumour defence--in our case, promoting tumour formation by contributing to neovascularization. VEGF was also found in the connective tissue, where it seems to be bound on collagens and probably builds a reservoir for rapid enzymatic mobilization.

Topical ephedrine administration and nasal chemosensory function in healthy human subjects.

OBJECTIVE: To investigate dose-related effects of ephedrine on olfactory function in healthy subjects. DESIGN: Placebo-controlled, randomized, double-blind study. METHODS: Drug effects were assessed using olfactory and trigeminal psychophysical measures (intensity ratings, odor discrimination, butanol and formic acid thresholds); nasal patency was assessed by means of anterior rhinoresistometry. The investigation was performed in 24 healthy volunteers; subjects were assigned to treatments A, B, or C (3 groups with 8 subjects each; 4 women and 4 men per group). All subjects received either placebo or ephedrine in both nostrils; group A subjects received placebo, and group B and C subjects received ephedrine in dosages of 0.12 and 0.24 mg, respectively. RESULTS: Treatment with ephedrine produced a tendency toward an increase of nasal airflow. However, during the time of observation there was no significant difference between effects produced by the 2 dosages. Ephedrine had no systematic effect on measures of olfactory function. The only significant correlation to the nasal airflow was found for perceived intensity of the trigeminal stimuli, which increased with increasing flow. CONCLUSIONS: Ephedrine appeared to have neither negative nor major positive effects on intranasal chemosensory function in healthy subjects. This indicates that ephedrine may be used as a decongestant in studies on olfaction.

Angiogenic factors in laryngeal carcinomas: do they have prognostic relevance?

Angiogenesis is a cascade-like mechanism essential for tumour growth and metastasis. Recently developed inhibitors of angiogenesis have already proven their benefit in anticancer treatment. Therefore the existence of angiogenic molecules in individual tumours is of major interest. The factors most frequently contributing to angiogenesis are basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and some matrix metalloproteinases (MMPs). In order to evaluate the prognostic significance of these molecules we investigated the distribution pattern of VEGF, bFGF, MMP-2 and MMP-9 in 41 laryngeal carcinomas using immunohistochemical methods. The results were correlated with clinicopathological parameters, i.e. T and N stage, histological grading, freedom from disease, overall survival and microvessel density (MVD). Only VEGF revealed a correlation with MVD (p = 0.01) and a weak association with histological grading (p = 0.06). Even though there is controversy about published data on the prognostic relevance of angiogenic factors, our results suggest that the factors examined in this study are not suitable for prognosis in larynx cancer patients.

Localization of endothelin-1 and endothelin-3 in the cochlea.

The distribution of endothelin-1 (ET-1) and endothelin-3 (ET-3) was studied by indirect immunostaining of decalcified guinea pig and rat cochleae. No species differences were observed. Perikarya and processes of spiral ganglion cells were highly reactive for both ET-1 and ET-3. The epithelial lining of the cochlear duct stained for ET-1 and ET-3, but reactivity for ET-1 was higher in the lining cells of the inner sulcus, Claudius', and Hensen's cells, while the tympanic covering layer of the basilar membrane stained stronger for ET-3 compared to ET-1. In the stria vascularis, all cell types stained for ET-3, while marginal cells were more reactive for ET-1. Spiral ligament fibroblasts were reactive for ET-1, but not for ET-3. Connective tissue cells of the spiral limbus stained for both endothelins. The region of synapses on outer hair cells reacted for ET-1 and ET-3 but sensory cells remained unstained. Endothelins are discussed to act as modulatory peptides, possibly interfering with nitric oxide, prostaglandins, and atrial natriuretic peptide in the lateral cochlear wall (lateral cochlear wall, i.e. stria vascularis and spiral ligament). The occurrence of endothelins in cochlear neurons suggest their potential role as neurotransmitters.

Localization of nitric oxide synthase I and III in the cochlea.

Nitric oxide synthase (NOS) isoforms I and III were localized in the guinea pig cochlea by indirect immunohistochemistry using frozen sections and paraffin sections. NOS I staining was observed in the cytoplasm of outer hair cells, in nerve cell somata and fibers of the spiral ganglion, and in axonal profiles of the spiral lamina next to the base of inner hair cells. In addition, lining cells of the inner sulcus and limbus, and cells of the spiral ligament stained for NOS I but vascular walls remained unstained. NOS III reactivity was seen in the cytoplasm of outer and inner hair cell, in lining cells of the limbus, and on the endolymphatic surface of marginal cells. Staining for NOS III of spiral ganglion perikarya showed varying intensity. Endothelial cells of cochlear glomeruli reacted for NOS III. NOS III in vascular endothelial cells implies regulatory effects of nitric oxide (NO) on vascular wall tonus and cochlear blood supply. NOS I in cochlear neurons indicates these cells as possible sources for NO during neuronal activity. Activated neurons may provide NO that adjusts cochlear perfusion to neuronal activity. Finally, NO that is liberated from hair cells or afferent synaptic terminals may act as an inhibitor on N-methyl-D-aspartate (NMDA) receptors (negative feed-back inhibition).

[Non-neoplastic changes in the salivary glands]. / Nichtneoplastische Speicheldrüsenveränderungen.

Non-neoplastic disorders of the salivary glands are divided into the following groups: malformations, salivary gland cysts, sialadenosis, sialolithiasis, sialadenitis, HIV-associated salivary gland disease, oncocytosis and necrotizing sialometaplasia (salivary gland infarction). Clinically, an etiological classification of sialadenitis is mandatory. Sialadenosis is distinguishable from sialadenitis by its clinical, radiological, and morphological characteristics. Non-neoplastic cysts make up about 6% of diseases of the salivary glands. Mucoceles represent the majority of these cysts (75%). HIV-associated salivary gland disease includes lymphoepithelial lesions and cysts involving the salivary gland tissue and/or intraglandular lymph nodes, and Sjögren's syndrome-like conditions, diffuse interstitial lymphocytosis syndrome, and other reported lesions of the major salivary glands. The diagnosis, differential diagnosis, symptoms and treatment of different non-neoplastic salivary gland disorders are discussed.

[Unique forms of the postthrombotic syndrome]. / Forme particulare de sindrom posttrombotic.

A prospective study was made with a view to analyse and to identify two particular forms of the post-thrombotic syndrome. The post-thrombotic syndrome of the shank can be recognized on the basis of the clinical and of the phlebographic examination. It has a serious prognosis and can be treated either conservatively or by surgery according to case. Its frequency is probably higher than it was presumed on the basis of the first observations. The post-thrombotic venous obstruction may be a cause of arterial ischemia during effort but this occurs only in very few cases. This particular syndrome is manifested as intermittent claudication which occurs in the clinical picture of chronic orthostatic venous failure. The probable mechanism is the direct effect of increased pressure in the arterial and venous circulation in vessels with normal walls, as a result of an important obstruction in the return circulation. Lumbal sympathectomy appears to improve durable claudication and hyperhidrosis.