In vitro resistance development for RO-0335, a novel diphenylether nonnucleoside reverse transcriptase inhibitor.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of current combination therapies for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance and serious side effects can compromise the benefits of the first generation compounds in this class (efavirenz and nevirapine). To study potential pathways leading to resistance against the novel diphenylether NNRTI, RO-0335, sequential passage experiments at low multiplicity of infection (MOI) were performed to solicit a stepwise selection of resistance mutations. Two pathways to loss of susceptibility to RO-0335 were observed, containing patterns of amino acid changes at either V106I/A plus F227C (with additional contributions from A98G, V108I, E138K, M230L and P236L) or V106I/Y188L (with a potential contribution from L100I, E138K and Y181C). Characterization of the observed mutations by site-directed mutagenesis in the isogenic HXB2D background demonstrated that a minimum of two or more mutations were required for significant loss of susceptibility, with the exception of Y188L, which requires a two-nucleotide change. Patterns containing F227C or quadruple mutations selected by RO-0335 showed a low relative fitness value when compared to wild-type HXB2D.

Lymphatic malformations: a proposed management algorithm.

OBJECTIVE: The aim of this study was to develop a management algorithm for cervicofacial lymphatic malformations, based on the authors' experience in managing these lesions as well as current literature on the subject. STUDY DESIGN AND METHODS: A retrospective medical record review of all the patients treated for lymphatic malformations at our institution during a 10-year period (1998-2008) was performed. DATA COLLECTED: age at diagnosis, location and type of lesion, radiologic investigation performed, presenting symptoms, treatment modality used, complications and results achieved. RESULTS: 14 patients were identified. Eight (57%) male and six (43%) female. There was an equal distribution between the left and right sides. The majority (71%) of cases were diagnosed within the first year of life. The majority of lesions were located in the suprahyoid region. The predominant reason for referral was an asymptomatic mass in 7 cases (50%) followed by airway compromise (36%) and dysphagia (14%). Management options employed included: observation, OK-432 injection, surgical excision and laser therapy. In 5 cases (36%) a combination of these were used. CONCLUSION: Historically surgical excision has been the management option of choice for lymphatic malformations. However due to the morbidity and high complication rate associated this is increasingly being questioned. Recent advances in sclerotherapy e.g. OK-432 injection have also shown significant promise. Based on experience in managing these lesions as well as current literature the authors of this paper have developed an algorithm for the management of cervicofacial lymphatic malformations.

Is intralesional cidofovir worthwhile in juvenile recurrent respiratory papillomatosis?

OBJECTIVE: To investigate the efficacy of intralesional cidofovir in the treatment of recurrent respiratory papillomatosis (RRP) in children. METHODS: Prospective observational study of four consecutive children with RRP treated at an academic tertiary children's hospital. Laryngo-bronchoscopy was performed at three- to five-weekly intervals. Photodocumentation was obtained and disease severity assessed using an anatomical RRP severity score. Surgical debulking of large papillomas was then performed, and cidofovir (5 mg/ml) injected into any remaining papillomas as well as submucosally at the sites of resected papillomas. The efficacy of cidofovir was assessed by the change in papilloma severity score over the course of the treatment. RESULTS: Complete disease remission was obtained in one patient, with a partial response seen in two others. One patient showed no significant response. The greatest beneficial effect was seen after the fourth cidofovir injection; however, two patients demonstrated a deterioration in severity scores after treatment was withheld at this point. Both responded well to further cidofovir injections. However, a clear plateau in the response to cidofovir was seen in all patients by the eighth injection. CONCLUSION: Intralesional cidofovir may help control papilloma regrowth and reduce disease severity in many children with RRP. In most cases, cidofovir would appear to be less efficacious in causing disease eradication. There appears to be little evidence to support prolonged treatment regimes (i.e. more than eight treatments).

Complications in paediatric airway management.

Management of the paediatric airway can be difficult. This management in turn can be associated with complications which can be life threatening. We present three cases in which such complications occurred and then go on to discuss the different ways in which these can be prevented.

Type I laryngeal cleft: late presentation.

Laryngeal cleft anomalies are rare congenital defects of the posterior laryngotracheal wall that usually present with a history of recurrent respiratory tract infections during the early neonatal period. Presentation of type I clefts can be subtle and subsequently can result in late diagnosis or indeed failure to present. We report on the case of a type I laryngeal cleft presenting with a history of recurrent lower respiratory tract infections and severe gastro-oesophageal reflux disease, at 19 years of age. This is the oldest documented initial presentation of a congenital laryngeal cleft, and emphasizes the importance of maintaining a suspicion of the diagnosis into early adulthood. We further highlight the role of gastro-oesophageal reflux disease in the presentation of laryngeal clefts.

Iron chelation improves endothelial function in patients with coronary artery disease.

BACKGROUND: Some epidemiological studies have shown that increased iron stores are associated with increased cardiovascular events. Redox-active iron may contribute to lipid peroxidation, endothelial cell activation, and generation of reactive oxygen species (especially hydroxyl radical, via Fenton chemistry). Increased oxidative stress is associated with impaired action of endothelium-derived nitric oxide in patients with atherosclerosis. METHODS AND RESULTS: To test the hypothesis that reducing vascular iron stores would reverse endothelial dysfunction, we examined the effects of the iron chelator deferoxamine (500 mg intra-arterially over 1 hour) on vasomotor function in forearm resistance vessels of patients with coronary artery disease by venous occlusion plethysmography. Patients with coronary artery disease had impaired endothelium-dependent vasodilation in response to methacholine compared with healthy control subjects (P<0.001). Deferoxamine infusion decreased serum iron levels (P<0.001). Deferoxamine improved the blood flow response to methacholine in patients with coronary artery disease (P<0.01 by 2-way repeated-measures ANOVA) but had no effect on the response to sodium nitroprusside. In normal volunteers, deferoxamine had no effect on the response to methacholine. The nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine abolished augmentation of the methacholine response associated with deferoxamine. The hydroxyl radical scavenger mannitol had no effect on the methacholine response. CONCLUSIONS: Deferoxamine improved nitric oxide-mediated, endothelium-dependent vasodilation in patients with coronary artery disease. These results suggest that iron availability contributes to impaired nitric oxide action in atherosclerosis.

Minor salivary glands: causing major problems.

Tumours arising in the parapharyngeal space (PPS) are rare and account for approximately 0.5% of all head and neck neoplasms. These neoplastic processes represent a wide variety of both benign (80%) and malignant lesions arising from the diverse range of structures within and surrounding the PPS. The PPS is typically conceptualized as a potential neck space in the shape of an inverted cone with its base at the skull base and apex at the greater cornu of the hyoid. Because of this unique structure, lesions must often grow to a considerable size before symptoms become apparent and clinical detection is possible. A rare case of mucoepidermoid tumour of the minor salivary glands arising in the prestyloid parapharyngeal space is described. The complex anatomical and pathological considerations within this region present a substantial challenge to the head and neck surgeon in the evaluation and management of these lesions.

Unusual cause of subglottic stenosis in an adult.

Subglottic foreign bodies presenting, as chronic subglottic stenosis is extremely rare in adults. A high index of suspicion and a careful history is of paramount importance in the diagnosis of a subglottic foreign body. Laser should not be used to excise granulation tissue to expose the foreign body because of the danger and potential of a fire particularly when the nature of the foreign body is not known. Rigid bronchoscopes are more beneficial than flexible ones in the removal of foreign bodies especially in long-standing cases.

Reduced hyaluronan in keloid tissue and cultured keloid fibroblasts.

Extracellular matrix hyaluronan is prominent during wound healing, appearing at elevated levels early in the repair process. It is prevalent throughout the course of fetal wound healing, which is scar-free, but decreases late in adult wound repair, that is often marked by scarring. To determine whether aberrant hyaluronan metabolism is associated with the excessive scarring that characterizes keloids, cultured fibroblasts derived from keloids and from the dermis of normal human skin and scar were compared. Levels of hyaluronan in 48 h conditioned media of keloid-derived cultures were significantly lower than in cultures of normal skin and scar fibroblasts. Profiles of hyaluronan polymer size were comparable in these two cell types, suggesting that excessive hyaluronan degradation was not involved. Hydrocortisone decreased hyaluronan levels approximately 70% in the conditioned media of both keloid and normal fibroblasts. Diminished hyaluronan accumulation in keloid-derived cells compared with normal fibroblasts was also observed in an in vitro wound healing model. Histolocalization of hyaluronan in keloids, normal skin, and scar samples confirmed the biochemical observations that the dermis of keloids, which comprises most of the scar tissue, contained markedly diminished levels of hyaluronan. Alterations in hyaluronan in the epidermis overlying keloids, however, were also observed. A modest increase in hyaluronan staining intensity was observed in the epidermis of keloids, as well as changes in the patterns of distribution within the epidermis, compared with that in normal skin and scar. Increased hyaluronan was present in the granular and spinous layers of the keloid epidermis Abnormalities are present apparently in both the overlying epidermis as well as in the dermis of keloids. Aberrations in signaling between keloid stroma and keloid epidermis may underlie abnormalities that contribute to the excessive fibrosis characteristic of these lesions.

Anti-HIV-1 activity of calanolides used in combination with other mechanistically diverse inhibitors of HIV-1 replication.

The natural product (+)-calanolide A, a unique non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 replication, is currently being evaluated in clinical trials in the USA. (+)-Calanolide A, the congeners costatolide and dihydrocostatolide, and (+)-12-oxo(+)-calanolide A, were evaluated in combination with a variety of mechanically diverse inhibitors of HIV replication to define the efficacy and cellular toxicity of potential clinical drug combinations. These assays should be useful in prioritizing the use of different combination drug strategies in a clinical setting. The calanolides exhibited synergistic antiviral interactions with other nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors. Additive interactions were also observed when the calanolides were used with representative compounds from each of these classes of inhibitors. No evidence of either combination toxicity or antagonistic antiviral activity was detected with any of the tested compounds. The combination antiviral efficacy of three-drug combinations involving the calanolides, and the efficacy of two- and three-drug combinations using a (+)-calanolide A-resistant challenge virus (bearing the T139I amino acid change in the reverse transcriptase), was also evaluated in vitro. These assays suggest that the best combination of agents based on in vitro anti-HIV assay results would include the calanolides in combination with lamivudine and nelfinavir, since this was the only three-drug combination exhibiting a significant level of synergy. Combination assays with the (+)-calanolide A-resistant strain yielded identical results as seen with the wild-type virus, although the concentration of the calanolides had to be increased.

Anti-human immunodeficiency virus type 1 (HIV-1) activity of 2'-fluoro-2',3'-dideoxyarabinosyladenine (F-ddA) used in combination with other mechanistically diverse inhibitors of HIV-1 replication.

2'-Fluoro-2'3'-dideoxyarabinosyladenine (F-ddA), a nucleoside reverse transcriptase inhibitor of human immunodeficiency virus (HIV) replication, is currently being evaluated in clinical trials. Future monotherapy for the treatment of HIV is unlikely owing to the rapid emergence of drug-resistant viruses, so F-ddA was evaluated in combination with a variety of mechanistically diverse inhibitors of HIV replication. Such in vitro studies provide insights into whether certain drug combinations yield synergistic antiviral activity or, more importantly, antagonistic antiviral activity or synergistic cytotoxicity. F-ddA exhibited synergistic antiviral interactions with representatives of each of the major classes of anti-HIV compounds, including other nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors. Greatest levels of synergistic interaction were detected when F-ddA was used in combination with the non-nucleoside compounds nevirapine and costatolide, the nucleoside analogues and costatolide, the nucleoside analogues AZT, ddC and 3TC and the protease inhibitors ritonavir and nelfinavir. No evidence of either combination toxicity or antagonistic antiviral activity was detected with any of the tested compounds.

Persistent otitis media with effusion: a new experimental model.

OBJECTIVES/HYPOTHESIS: Numerous mechanical animal models for the creation of otitis media with effusion (OME) have been described since the 1920s. However, there are many problems associated with these models, including high infection rates, unreliability, and high resolution rates. The aim of the current study was to create a suitable mechanical animal model that would produce a sterile and long-lasting effusion. STUDY DESIGN: A new technique using an external surgical approach on specific pathogen-free rats is described. METHOD: The eustachian tubes of 56 rats were obstructed in the mid portion along the skull base with gutta percha. RESULTS: All animals developed an effusion within 1 week of the procedure. The resolution rate was 8%, with 80% maintaining sterile effusions for up to 1 year. CONCLUSIONS: This new procedure for an OME model has proved consistently reliable in creating a persistent and long-lasting effusion. It has a low infection rate and should benefit future studies on the prolonged effects of OME on the tympanic membrane and middle ear.

Intensive nutritional counselling in bulimia nervosa: a role for supplementation with fluoxetine?

OBJECTIVE: The aims of the paper are to determine whether nutritional counselling is associated with an improvement in bulimic symptomatology, whether this improvement is maintained during post-treatment follow-up, and whether the addition of fluoxetine 3 x 20 mg/day confers additional benefit. METHOD: Psychological, pharmacological and combined psychopharmacological treatments of bulimia nervosa were reviewed briefly. Sixty-seven patients referred to specialist eating disorder services who fulfilled strict diagnostic criteria were treated with intensive nutritional counselling and randomly assigned to either fluoxetine 3 x 20 mg/day or placebo. After a 1-week 'wash-out', active treatment was given over 8 weeks, followed by post-treatment interviews at 12 and 20 weeks. RESULTS: Both groups of patients improved significantly during treatment. In some respects, the fluoxetine group did slightly better as demonstrated by the items 'restraint', 'weight concern' and 'shape concern' (p < 0.05 vs p < 0.0001) on the Eating Disorder Examination (EDE). Fluoxetine patients decreased their energy intake and lost a modest amount of weight. They went on to regain weight during the follow-up period, returning to levels higher than they were initially. These patients also appeared more likely to have a recurrence of symptoms, as shown by the fall in percentage of binge-free patients and by changes in the EDE. CONCLUSION: Nutritional counselling is an effective means of treating bulimia nervosa, with improvement maintained up to 3 months follow-up. The addition of fluoxetine may confer some benefit during active treatment, but its discontinuation may contribute to a higher rate of recurrence of symptoms post treatment. Of course, this study cannot be extrapolated to the efficacy of fluoxetine when used as the only form of treatment in patients for whom intensive nutritional counselling or other structured psychological programs are not available.

Highly potent oxathiin carboxanilide derivatives with efficacy against nonnucleoside reverse transcriptase inhibitor-resistant human immunodeficiency virus isolates.

The structure-activity relationships of a series of compounds related to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) oxathiin carboxanilide have been described (R. W. Buckheit, Jr., T. L. Kinjerski, V. Fliakas-Boltz, J. D. Russell, T. L. Stup, L. A. Pallansch, W. G. Brouwer, D. C. Dao, W. A. Harrison, R. J. Schultz, J. P. Bader, and S. S. Yang, Antimicrob. Agents Chemother. 39:2718-2727, 1996). From these studies, the furanyl-containing analog UC10 was identified as the most potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication and a promising candidate for further development. Three new UC analogs (UC040, UC82, and UC781) have been determined to inhibit laboratory-derived and low-passage-number, primary virus isolates at low nanomolar concentrations in both established and fresh human cells. Each of the compounds synergistically interacted with the nucleoside analogs zidovudine, dideoxyinosine, dideoxycytosine, and lamivudine to inhibit HIV-1 replication. As a group, the UC compounds were found to be less active against viruses with the L100I, K103N, and Y181C amino acid changes in the RT and, upon in vitro selection, yielded resistant virus with the Y181C mutation in the RT. The most potent of the three new compounds, UC781, contains a furanyl side chain, similar to UC10, but differs in having an extended ether side chain instead of an oxime chain. The broad therapeutic index of UC781 (>62,000) resulted in effective inhibition of NNRTI-resistant virus isolates at high nanomolar concentrations. Furthermore, UC781 and the NNRTI costatolide were able to synergistically inhibit HIV-1 replication when used in combination, suggesting that UC781 may interact with the RT differently than the other UC analogs. The favorable anti-HIV properties of the UC compounds suggest they should be considered for further clinical development.

Isolation of viruses from clinical specimens in microtitre plates with cells inoculated in suspension.

Virus isolation is essential for the provision of a full diagnostic virology service. Present methods are time consuming, expensive and relatively inflexible for routine use. Our objective was to audit our existing virus isolation system and to develop a sensitive, flexible virus isolation system which could be adapted for use in a busy routine laboratory which is required to provide a service for a wide range of clinical situations. We carried out a pilot study which compared conventional roller tube monolayer cultures to a microplate system using cells inoculated in suspension and showed that the microplate method using extra cell lines could provide a more sensitive system for virus isolation. This system was adapted for routine use using six cell lines inoculated in suspension and the results are presented for 2610 specimens for virus isolation and 972 for Clostridium difficile toxin (CDT) detection. There were 516 viruses isolated and 229 specimens positive for CDT using this system. Polioviruses (92), echoviruses (35), coxsackieviruses (15) and untyped enteroviruses (13) were isolated in RMK, E6-vero and RD cells. Adenoviruses (137) were isolated in HEp2 and E6-vero cells. Herpes simplex virus (HSV) was isolated from 149 specimens in E6-vero, FCL and HFF9 cells. Myxoviruses (38) and paramyxoviruses were isolated in RMK cells. HEp2 was the only cell line necessary to isolate the 33 respiratory syncytial viruses (RSV). Cytomegaloviruses (CMV) (2) and varicella zoster (1) virus (VZV) were isolated only in the human fibroblast cell line HFF9. Rubella virus was isolated from a baby with congenital rubella in RMK, E6-vero and additionally in BGM cells. In conclusion, the use of cells inoculated in suspension in microtitre plates for virus isolation was sensitive and convenient. It allowed the use of six cell lines for routine virus isolation without using additional laboratory staff time. It improved turnaround times. It was also safer microbiologically than conventional isolation in tube monolayers. The precise identification of virus isolates was simplified.