Calcitriol modulation of cardiac contractile performance via protein kinase C.

Vitamin D(3) deficiency enhances cardiac contraction in experimental studies, yet paradoxically this deficiency is linked to congestive heart failure in humans. Activated vitamin D(3) (1alpha,25-dihydroxyvitamin D(3)) or calcitriol, decreases peak force and activates protein kinase C (PKC) in isolated perfused hearts. However, the direct influence of this hormone on adult cardiac myocyte contractile function is not well understood. Our aim is to investigate whether 1alpha,25-dihydroxyvitamin D(3) acutely modulates contractile function via PKC activation in adult rat cardiac myocytes. Sarcomere shortening and re-lengthening were measured in electrically stimulated myocytes isolated from adult rat hearts, and the vitamin D(3) response (10(-10) to 10(-7) M) was compared to shortening observed under basal conditions. Maximum changes in sarcomere shortening and relaxation were observed with 10(-9) M 1alpha,25-dihydroxyvitamin D(3). This dose decreased peak shortening, and accelerated contraction and relaxation rates within 5 min of administration, and changes in the Ca(2+) transient contributed to the peak shortening and relaxation effects. The PKC inhibitor, bis-indolylmaleimide (500 nM) largely blocked the acute influence of the most potent dose (10(-9) M) on contractile function. While peak shortening and shortening rate returned to baseline within 30 min, there was a sustained acceleration of relaxation that continued over 60 min. Phosphorylation of the Ca(2+) regulatory proteins, phospholamban, and cardiac troponin I correlated with the accelerated relaxation observed in response to acute application of 1alpha,25-dihydroxyvitamin D(3). Accelerated relaxation continued to be observed after chronic addition of 1alpha,25-dihydroxyvitamin D(3) (e.g. 2 days), yet this sustained increase in relaxation was not associated with increased phosphorylation of phospholamban or troponin I. These results provide evidence that 1alpha,25-dihydroxyvitamin D(3) directly modulates adult myocyte contractile function, and protein kinase C plays an important signaling role in the acute response. Phosphorylation of key Ca(2+) regulatory proteins by this kinase contributes to the enhanced relaxation observed in response to acute, but not chronic calcitriol.

Do frozen sections help achieve adequate surgical margins in the resection of oral carcinoma?

This retrospective study examined the notes of 82 patients who underwent an attempted curative resection of oro-pharyngeal carcinoma, and who had frozen sections taken, over a four year period. Three hundred and fifty mucosal, 179 deep tissue, and 22 nerve frozen sections had been taken. Concordance between cryostat and paraffin sections was 99.5% although no false positives or negatives for invasive tumour were found. Nine (11%) patients who had frozen sections which were reported as dysplastic or positive for invasive tumour underwent further local resection; excision was then found to be complete in 8 (10%) of these patients with further frozen sections. In 15 patients the margins of the main resection specimen were judged to be close to the tumour (<5mm), 5 patients had squamous epithelial dysplasia and 12 patients had invasive tumour at a resection margin. Ten of the 12 patients with margins containing invasive tumour had negative cryostat sections intraoperatively, which demonstrated problems with sampling which is the major drawback. During follow up, 14 patients developed local recurrence. Only one of the patients who had frozen section guided further local resection, developed local recurrence. Frozen sections help achieve tumour clearance.

Minichromosome maintenance (Mcm) proteins, cyclin B1 and D1, phosphohistone H3 and in situ DNA replication for functional analysis of vulval intraepithelial neoplasia.

Vulval intraepithelial neoplasia (VIN) is defined histopathologically by distinctive abnormalities of cellular maturation and differentiation. To investigate the functional properties of VIN, the expression of several proteins involved in the regulation of the cell cycle as well as in situ DNA replication competence was analysed by immunohistochemistry. Snap-frozen vulval biopsies were graded as normal squamous epithelium (n=6), undifferentiated HPV positive VIN 1 (n=3), VIN 2 (n=8) and VIN 3 (n=20). Immunohistochemistry was performed using the following markers: cyclin D1 (expressed in middle/late G1), cyclin B1 (expressed in G2/early M), phosphorylated histone H3 (expressed during mitosis) and minichromosome maintenance (Mcm) proteins 2 and 5 (expressed during the cell cycle, but not in differentiated or quiescent cells). In situ DNA replication competence was used to identify S-phase cells. The percentage of positively stained nuclei in three representative microscopic fields was calculated per biopsy. In normal vulva, the expression of all markers was restricted to the proliferative compartment of the basal layer of the epithelium. In contrast in high-grade VIN, the majority of epithelial cells expressed the Mcm proteins from basal to superficial layer. The detection of cyclins B1 and D1, phospho-histone H3 and in situ DNA replication was also found through the full thickness of these lesions but by a lower proportion of the cells. This is consistent with these markers providing a series of 'snapshots' of the cell cycle status of individual cells. The low-grade VIN showed reduced expression of the cell cycle markers in relation to the level of dysplasia. The combination of these analyses establishes that the majority of VIN cells remain in a functional replicative or prereplicative state of the cell cycle. Clinical application of these analyses may provide a basis for improved diagnosis of VIN.

Rectal lymphoma in ulcerative colitis treated with azathioprine.

Primary malignant lymphoma of the bowel is a rare complication of inflammatory bowel disease. The association of gastrointestinal lymphoma, inflammatory bowel disease and prior immunosuppression remains unclear. We report the first case of azathioprine-treated ulcerative colitis developing rectal lymphoma.

Lethal invasive mucormycosis: case report and recommendations for treatment.

A case of lethal invasive mucormycosis (IM), a rare fungal infection which predominantly affects immunocompromised patients, is reported in a 73-year-old female patient who presented with a cervical abscess. The patient had asthma treated with steroids and had previously undiagnosed diabetes mellitus. Despite surgical treatment and parenteral antibiotic therapy, there was fatal progression of the condition. The pathogenesis, histological appearances and treatment of mucormycosis are discussed, particularly the importance of urgent histological examination of debrided tissue to distinguish this condition from necrotizing fasciitis (NF) earlier than microbiological culture alone would allow, thus permitting the early introduction of appropriate antifungal therapy.

A study of anal intraepithelial neoplasia in HIV positive homosexual men.

OBJECTIVES: To determine the prevalence of high grade anal intraepithelial neoplasia (HGAIN), the value of anal cytology in screening for HGAIN, and the characterisation of epidemiological factors and human papillomavirus (HPV) types. METHODS: Prospective cohort study of HIV positive homosexual men. Subjects were interviewed, underwent STD, anal cytological, and HPV screening at enrolment and at subsequent follow up visits with anoscopy and biopsy at the final visit. 57 enrolled, average CD4 count 273 x 10(6)/l (10-588); 41 completed the cytological surveillance over the follow up period (181 visits, average follow up 17 months), 38 of these had anoscopy and anal biopsy. RESULTS: Oncogenic HPV types were detected in 84% and high grade dyskaryosis in 10.5% (6/57) at enrollment. There was a 70% incidence of high grade dyskaryosis during follow up in patients with negative/warty or low grade dyskaryosis at enrollment. Anoscopy correlated with histology in high grade AIN lesions (sensitivity 91%, specificity 54%) and cytology was 78% sensitive (18/23) for HGAIN on biopsy. CONCLUSIONS: AIN and infection with multiple oncogenic HPV types are very common among immunosuppressed HIV positive homosexual men. Apparent progression from low to high grade cytological changes occurred over a short follow up period, with no cases of carcinoma. All 23 cases of HGAIN were predicted by cytology and/or anoscopy. Future studies focusing on the risk of progression to carcinoma are needed before applying anal cytology as a screening tool for AIN in this population.

A survey of general practitioners in north-east Scotland on practice-based counselling services.

OBJECTIVE: Practice-based counselors have become very numerous in primary care and we sought to elicit the views of general practitioners in north-east Scotland about the present situation and possible developments in practice-based counselling services. DESIGN AND SUBJECTS: A questionnaire was mailed to the 352 general practitioners working in the area covered by Grampian Health Board. RESULTS: There was a 74% response rate: 46% of respondents had access to a practice-based counsellor and of those who did not, two-thirds wanted to acquire one. Larger practices and fundholding practices were more likely to have practice-based counsellors. There was a lack of knowledge about the medico-legal situation when referrals to counsellors were made. Seventy-nine per cent of respondents thought that the need/demand for counselling would increase during the next decade, and a majority considered provision of counselling through mental health teams, with local co-ordination of accreditation, training and supervision, to be desirable. CONCLUSION: Since the efficacy of counselling for mental health problems in primary care has not been established, and provision of counselling in primary care settings continues to increase, there is a need to mount coherent and appropriate research studies. There also appears to be a need for greater integration between primary care and mental health services to generate rational provision of practice-based counselling.

Visceral leishmaniasis (Kala-azar) in two patients with HIV-1 infection: atypical features and response to therapy.

Visceral leishmaniasis (VL) is a well recognized opportunistic infection in patients with HIV-1 infection, which may occasionally present with atypical features. We describe two patients with advanced HIV-1 infection (CD4<100/ mm3) in whom visceral leishmaniasis presented with atypical features, and their response to therapy. Atypical features of visceral leishmaniasis in the two infected patients include absence of fever, dissemination to the duodenal mucosa and to the skin as xanthoma-like lesions. Therapy and secondary prophylaxis remain unsatisfactory, and studies to evaluate combinations of amphotericin B and immunotherapy are needed.

Primary non-Hodgkin's lymphoma of the larynx in an AIDS patient.

A case of primary non-Hodgkin's lymphoma of the larynx in an AIDS patient is presented with a review of the literature. Non-Hodgkin's lymphomas in AIDS patients are common but the primary laryngeal presentation is very rare. The symptoms usually include dysphonia and progressive airway obstruction requiring tracheostomy. As with laryngeal non-Hodgkin's laryngeal lymphomas in non-HIV positive patients the majority are of B cell lineage and respond well to radiotherapy. Our patient had a high grade lymphoma of B cell lineage which showed a good response to radiotherapy. The role of chemotherapy and surgery is not yet established. We suggest that the diagnosis of AIDS should not influence the management of these patients unless the individual is in the terminal disease stage.

Tracheal microaspiration in adult cystic fibrosis.

Gastro-oesophageal reflux (GOR) has been implicated in the aetiology of lung disease. Cystic fibrosis (CF) patients have a high incidence of GOR symptoms with demonstrable episodes of oesophageal acidification. We studied 24-hour ambulatory tracheal and oesophageal pH in 11 CF patients with GOR symptoms to identify any episodes of tracheal acidification and define their temporal relation to oesophageal reflux and respiratory symptoms. 8 patients had evidence of significant GOR (DeMeester score mean 58; range 17-107) and in 6 it was gross (DeMeester score > 30). 4 patients had tracheal acidification (defined as tracheal pH < 5.5): all had greatly raised DeMeester scores. Two patterns of lowered tracheal pH were seen: a gradual drift downwards of tracheal pH to < 5.5 which recovered slowly, and an acute fall in tracheal pH to < 5.5 with rapid recovery. Only one patient had a fall in peak expiratory flow in conjunction with a decline in tracheal pH, and no association was found between the presence of tracheal microaspiration and pulmonary function. We conclude that tracheal acidification occurs in adult CF patients with GOR.

Structure and dynamics of pentaglycyl bridges in the cell walls of Staphylococcus aureus by 13C-15N REDOR NMR.

Whole cells and cell-wall fractions of Staphylococcusaureus have been labeled by various combinations of [1-13C]glycine, [15N]glycine, L--6-13C-lysine, L--6-15N-lysine, D--1-13C-alanine, and D--15N-alanine. The resulting materials have been examined using 13C and 15N solid-state, magic-angle spinning NMR techniques including cross-polarization, double cross-polarization, and rotational-echo double resonance. The results of these measurements indicate that the peptidoglycan glycyl bridges are complete (five units long) and form cross-links between three-quarters of all peptide stems. The pentaglycyl bridges are immobilized in lyophilized cell-wall fractions in a compact conformation with inter-residue spacings comparable to those of an alpha helix. The bridges have a similar compact conformation in intact whole cells, regardless of whether the cells have been lyophilized or were hydrated and frozen at -10 degrees C. The bridges are also in a time-averaged compact conformation in whole cells at 0 degrees C but with sizable structural fluctuations associated with local mobility. A small fraction of bridges are in extended-chain conformations.

Cross-Polarized Magic-Angle Spinning (sup13)C Nuclear Magnetic Resonance Spectroscopic Characterization of Soil Organic Matter Relative to Culturable Bacterial Species Composition and Sustained Biological Control of Pythium Root Rot.

We report the use of a model system that examines the dynamics of biological energy availability in organic matter in a sphagnum peat potting mix critical to sustenance of microorganism-mediated biological control of pythium root rot, a soilborne plant disease caused by Pythium ultimum. The concentration of readily degradable carbohydrate in the peat, mostly present as cellulose, was characterized by cross-polarized magic-angle spinning (sup13)C nuclear magnetic resonance spectroscopy. A decrease in the carbohydrate concentration in the mix was observed during the initial 10 weeks after potting as the rate of hydrolysis of fluorescein diacetate declined below a critical threshold level required for biological control of pythium root rot. Throughout this period, total microbial biomass and activity, based on rates of [(sup14)C]acetate incorporation into phospholipids, did not change but shifts in culturable bacterial species composition occurred. Species capable of inducing biocontrol were succeeded by pleomorphic gram-positive genera and putative oligotrophs not or less effective in control. We conclude that sustained efficacy of naturally occurring biocontrol agents was limited by energy availability to this microflora within the organic matter contained in the potting mix. We propose that this critical role of organic matter may be a key factor explaining the variability in efficacy typically encountered in the control of pythium root rot with biocontrol agents.

Structural constraints on the complex of elongation factor Tu with magnesium guanosine diphosphate from rotational-echo double-resonance NMR.

Rotational-echo, double-resonance (REDOR) NMR measurements of 31P-15N dipolar couplings have been made on a complex of Mg guanosine diphosphate (MgGDP) with uniformly 15N-labeled elongation factor Tu. The complex was embedded in a lyophilized buffer glass. The observed 15N REDOR dephasing by 31P was accounted for quantitatively by distances from 15N of Gly23 and Lys24 to P alpha and P beta of MgGDP as determined by X-ray crystallography of MgGDP complex formed using an elongation factor Tu that is missing a 15 residue loop in the vicinity of the binding site.

Sternocostoclavicular hyperostosis presenting with thoracic sinus formation.

Sternocostoclavicular hyperostosis (SCCH) is a condition which is well described in the Japanese literature but is rare in Western Europe. It is characterised by pain and swelling in the upper anterior part of the chest, which tends to be progressive. A patient is described with bilateral chronic discharging sinuses over the anterior ends of the clavicles in whom the diagnosis appeared to be one of SCCH.

A simple solid-state NMR method for determining peptidoglycan crosslinking in Bacillus subtilis.

The crosslink index of the peptidoglycan of intact cell walls of Bacillus subtilis grown in media containing L-[15N]aspartic acid has been determined by a combination of cross-polarization magic-angle spinning 15N NMR and gas chromatography/mass spectrometry. The crosslink index of 62% decreased to 52% when the bacteria were exposed to the antibiotic cephalothin.

Treatment of large airway obstruction in lung cancer using expandable metal stents inserted under direct vision via the fibreoptic bronchoscope.

BACKGROUND: Self-expanding metal stents have been used successfully to overcome large airway obstruction due to malignant pulmonary disease. The technique has been modified to place stents under direct vision using the fibreoptic bronchoscope. The effect of this procedure on lung function and patient well being was investigated in a large series of patients. METHODS: Fifty six patients with malignant tracheobronchial tumours were treated for symptoms of life threatening airways obstruction or collapse of a lung by the insertion of an expandable metal stent(s) under local anaesthetic using a fibreoptic bronchoscope. All had inoperable cancer and 33 had relapsed after or failed to respond to radiotherapy, chemotherapy, or surgery. Forty seven had primary bronchial carcinomas and nine had metastases from other tumour sites. All but two patients had the stents inserted at one sitting. Measurements were performed in most of the patients before and after stenting and included objective measures (pulmonary function tests, arterial blood gas tensions) and non-objective measures (patient well being, performance status). RESULTS: Overall, 77% of patients showed symptomatic improvement. In those patients in whom measurements were performed two thirds showed improvement in forced expiratory volume in one second (FEV1), forced vital capacity (FVC), peak expiratory flow rate (PEFR) and arterial oxygen tension (PaO2). Medical Research Council dyspnoea score (MRC), Karnofsky, and visual analogue scores (for both breathing and well being) improved in 81% of patients. There were no perioperative deaths. Fifty one patients have died since the procedure with a mean survival of 77 days (range 1-477), and five patients are still alive after a mean of 207 days (range 135-274). CONCLUSIONS: In suitable patients with either extraluminal or intraluminal tumour, or both, the insertion of expandable metal stents using a fibreoptic bronchoscope and local anaesthetic is a valuable addition to other palliative therapies in the treatment of lung cancer.

Cross-links in cell walls of Bacillus subtilis by rotational-echo double-resonance 15N NMR.

The cross-link index of peptidoglycan of intact cell walls of Bacillus subtilis grown in media containing L-[2-13C,15N]aspartic acid has been determined by rotational-echo double-resonance 15N NMR. A cross-link index of 72% decreased to 47% when the bacteria were exposed to the antibiotic cephalosporin C. The fraction of 15N label routed from aspartic acid in the media to glutamic acid in the peptidoglycan peptide stems more than doubled for the cephalosporin-exposed cells, indicating interference with general cell wall nitrogen metabolism by the antibiotic.

Desmoplastic malignant mesothelioma: a review of 17 cases.

AIMS: To identify the histological features of desmoplastic mesothelioma, and to determine its incidence and prognosis. METHODS: Two hundred and fifty five cases of malignant mesothelioma were examined over seven years (1982-9) to identify the desmoplastic variant. Sections were cut at 5 microns and stained with haemotoxylin and eosin and with CAM 5.2 (Dakopatts). Asbestos fibre counts were carried out by light microscopy in 14 cases using the potash digestion method. RESULTS: Seventeen cases were identified as desmoplastic mesothelioma giving an incidence of 6.6%. In 11 cases the cell type in more cellular areas was sarcomatous and in six others it was biphasic. The mean survival time from onset of symptoms to death was 5.8 months for the sarcomatous variant and 6.8 months for the biphasic variant. Twelve of 16 patients had had previous occupational exposure to asbestos, ranging from five months to 43 years. The diagnosis of desmoplastic mesothelioma was only accepted if acellular connective tissue comprised 50% or more of the tumour bulk. Also seen was collagen necrosis, anastomosing bands of often hyalinised collagen with a prominent storiform pattern, and where cellular detail was present there were hyperchromatic nuclei. CONCLUSIONS: Desmoplastic mesothelioma is a rare variant of malignant mesothelioma with a storiform collagen pattern, collagen necrosis, bland acellular collagen and focal cytological features of malignancy. Though rare, it is important to recognise this variant and distinguish it from a pleural plaque, nonspecific reactive pleural fibrosis, pleurisy, rheumatoid disease, or, rarely, spindle cell sarcomas.