Clinical and biochemical effects of the E139K missense mutation in the TIMP3 gene, associated with Sorsby fundus dystrophy.

PURPOSE: To determine the phenotypic and biochemical characteristics of the p.E139K missense variant in tissue inhibitor of metalloproteinase 3 (TIMP3) associated with Sorsby fundus dystrophy (SFD). METHODS: The coding regions and adjacent intronic sequence of TIMP3 were amplified by polymerase chain reaction and then analyzed by bidirectional sequencing. Allele-specific PCR was used to determine the minimum allele frequency of the mutant allele in ethnically matched controls. Clinical examination and imaging of affected individuals with color fundus photography, scanning laser ophthalmoscope (fundal autofluorescence), and optical coherence tomography was performed. A mutant construct of the TIMP3 protein was created and expressed in human retinal pigment epithelium (ARPE19) cells, which were then assayed for oligomerization and intrinsic matrix metalloproteinase (MMP) inhibitory activity. RESULTS: Three affected individuals from a family of Welsh origin each harbored one allele of the TIMP3 missense variant c.415 G>A, (p.E139K), which was not identified in 534 ethnically matched control chromosomes and thus presumed pathogenic. The mutant protein was shown to dimerize in culture cells and retain its MMP inhibitory activity. Retinal examination was variable between eyes of affected individuals and between family members. Drusen-like deposits were common to all three affected individuals and yellow subretinal deposits, exudative maculopathy, and geographic atrophy were also observed. Optical coherence tomography (OCT) images of affected individuals demonstrated hyperreflectivity of the RPE-photoreceptor-choroid complex. CONCLUSIONS: The TIMP3 p.E139K mutation is another cause of SFD. It is the second TIMP3 sequence variant reported that does not affect the number of cysteine residues in the mutant protein yet dimerizes in vitro. The clinical presentation of this family is in keeping with previous clinical reports of this disorder.

ABCA4 mutations and discordant ABCA4 alleles in patients and siblings with bull's-eye maculopathy.

AIM: To determine the frequency and nature of mutations in the gene ABCA4 in a cohort of patients with bull's-eye maculopathy (BEM). METHODS: A panel of 49 subjects (comprising 40 probands/families, 7 sibling pairs and a set of three sibs) with BEM, not attributable to toxic causes, was ascertained. Blood samples from each patient were used to extract genomic DNA, with subsequent mutation screening of the entire coding sequence of ABCA4, using single-strand conformational polymorphism (SSCP) analysis and direct sequencing. RESULTS: Fourteen probands (35%) were found to have a potentially disease-causing ABCA4 sequence variant on at least one allele. Three patients had a Gly1961Glu missense mutation, the most common variant in Stargardt disease (STGD), with 2 of these subjects having a macular dystrophy (MD) phenotype and a second ABCA4 variant previously associated with STGD. The second most common STGD mutation, Ala1038Val, was seen in one patient with cone-rod dystrophy (CORD). Five novel ABCA4 variants were detected. Two sibships were identified with a similar intra-familial phenotype but discordant ABCA4 variants. CONCLUSIONS: Variations in the ABCA4 gene are common in BEM. Two sibships showed discordant ABCA4 variants. One of these sibships illustrates that ABCA4 variants can be identified in families that have another molecular cause for their disease, due to the high prevalence of ABCA4 disease alleles in the population. The discordance evident in the second sibship may yet also be a chance finding in families with macular disease of another genetic cause, or it may represent a complex mode of inheritance determined/modified by the combination of ABCA4 alleles.

Administration of adrenocorticotropic hormone during chicken embryonic development prematurely induces pituitary growth hormone cells.

Treatment of fetal rats and embryonic chickens with exogenous glucocorticoids induces premature GH cell differentiation. However, it is unknown whether the developing adrenal gland is capable of mounting this response autonomously. The present study determined whether stimulation of the adrenal gland in developing chicken embryos through administration of ACTH could induce a premature increase in GH cells. We found that plasma corticosterone and ACTH levels increased between embryonic day (e) 11 and e17, consistent with GH cell (somatotroph) ontogeny. Injection of ACTH into eggs on e9, e10, or e11 increased somatotrophs on e14. In contrast, thyroid-stimulating hormone, CRH, alpha-MSH, GHRH, and TRH were ineffective. Culture of e11 pituitary cells with ACTH failed to induce somatotrophs, suggesting an indirect action of ACTH on GH cells in vivo. Intravenous administration of ACTH dramatically increased plasma levels of corticosterone within 1 h and increased the percentage of pituitary somatotrophs within 24 h. Although ACTH administration increased the relative abundance of pituitary GH cells, there was no effect on plasma levels of GH, IGF-I, or IGF-II, or in hepatic expression of IGF-I or IGF-II mRNA. We conclude that ACTH administration can increase the population of GH cells in the embryonic pituitary. However, this treatment alone does not lead to downstream activation of hepatic IGF production. These findings indicate that the embryonic adrenal gland, and ultimately anterior pituitary corticotrophs, may function to regulate pituitary GH cell differentiation during embryonic development.

GABAergic neurons in the lateral superior olive of the hamster are distinguished by differential expression of gad isoforms during development.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that is synthesized by two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67. Using in situ hybridization and immunocytochemical techniques in hamsters, we investigated the postnatal development of GAD isoforms within the lateral superior olive (LSO) where GABAergic neurons form part of a descending efferent projection to the cochlea. In the neonatal hamster LSO, GAD67 immunoreactivity, GAD67 transcript labeling, and intense GABA immunostaining are at low levels. However, robust GAD65 mRNA expression is found throughout the LSO during the early postnatal period. The neonatal GABAergic expression patterns are in stark contrast to the adult where the LSO has robust GAD67 mRNA expression and weak GAD65 mRNA expression. Cells exhibiting intense GABA immunolabeling were also found in the same LSO locations as robust GAD67 mRNA expression and intense GAD67 immunoreactivity. Additionally, GAD67-positive cells in the LSO were retrogradely labeled from the cochlea confirming that these cells are a part of the lateral olivocochlear system. The late onset of GAD67 expression and intense GABA immunoreactivity in LSO neurons are consistent with the relatively late maturation of the lateral olivocochlear neurons inferred from previous studies. During development, these data lead us to conclude that the GABAergic portion of the lateral olivocochlear system is distinguished by preferential GAD67 expression, intense GABA immunoreactivity, and relatively late postnatal onset.

Clinical characterisation of a family with retinal dystrophy caused by mutation in the Mertk gene.

BACKGROUND/AIM: MERTK, a tyrosine kinase receptor protein expressed by the retinal pigment epithelium (RPE), is mutated in both rodent models and humans affected by retinal disease. This study reports a survey of families for Mertk mutations and describes the phenotype exhibited by one family. METHODS: 96 probands with retinal dystrophy, consistent with autosomal recessive segregation, were screened by direct sequencing. A family homozygous for a likely null allele was investigated clinically. RESULTS: A novel frame shifting deletion was identified in one of 96 probands. Other polymorphisms were detected. The deletion allele occurred on both chromosomes of four affected family members. Electrophysiology demonstrated early loss of scotopic and macular function with later loss of photopic function. Visual acuities and visual fields were preserved into the second decade. Perception of light vision was present in a patient in the fourth decade. A "bull's eye" appearance and a hyperautofluorescent lesion at the central macula were consistent clinical findings. CONCLUSIONS: Mutations in Mertk are a rare cause of ARRP in humans. The study extends the phenotypic characteristics of this retinal dystrophy and shows distinctive clinical signs that may improve its clinical identification. The moderate severity and presence of autofluorescence implies that outer segment phagocytosis is not entirely absent.

Does smoking influence the type of age related macular degeneration causing visual impairment?

AIMS: To assess the influence of smoking on the type of age related macular degeneration (AMD) lesion causing visual impairment in a large cohort of patients with AMD at a tertiary referral UK centre. METHODS: Prospective, observational, cross sectional study to analyse smoking data on 711 subjects, of western European origin, in relation to the type of AMD lesion present. Colour fundus photographs were graded according to a modified version of the international classification. Multiple logistic regression analysis was performed, adjusting for age and sex using the statistical package SPSS ver 9.0 for Windows. chi(2) tests were also used to assess pack year and ex-smoker data. RESULTS: 578 subjects were graded with neovascular AMD and 133 with non-neovascular AMD. There was no statistically significant association found between smoking status or increasing number of pack years and type of AMD lesion. The odds of "current smokers" compared to "non-smokers" developing neovascular rather than non-neovascular AMD when adjusted for age and sex was 1.88 (95% CI: 0.91 to 3.89; p = 0.09). CONCLUSIONS: Smoking is known to be a risk factor for AMD and this study suggests that smokers are at no more risk of developing neovascular than atrophic lesions.

Functional characterisation and serial imaging of abnormal fundus autofluorescence in patients with retinitis pigmentosa and normal visual acuity.

AIM: To characterise and monitor abnormal fundus autofluorescence (AF) in patients with retinitis pigmentosa (RP) who have good visual acuity. METHODS: 21 patients with a clinical diagnosis of RP were examined. All had rod-cone dystrophy (ISCEV standard electroretinograms (ERGs)), visual acuity of 6/9 or better, and manifested a parafoveal ring of high density fundus AF. Repeat AF imaging was performed after periods of between 2 years and 5 years in 12 patients. Pattern ERG (PERG) and multifocal ERG (mfERG) were performed in 20 cases. Visual fields (VF), photopic and scotopic fine matrix mapping and small field PERGs were performed in representative cases. RESULTS: The rings of high density AF varied in size between patients (from 4 degrees -16 degrees diameter). MfERGs showed relative preservation over the central macular area, correlating with the size of AF ring and with PERG and psychophysical data. Progressive constriction of the AF ring was demonstrated at follow up in three patients. Serial PERG, mfERG, and VFs, performed in one of these cases, showed evidence of deterioration concordant with ring constriction. CONCLUSIONS: High density rings of AF, seen in some patients with RP with good visual acuity, demarcate areas of preserved central photopic function. MfERGs correlate with the area encircled by high density AF and the PERG data. The size of the ring of AF can show progressive constriction accompanied by increasing macular dysfunction.

Fluorescence in-situ hybridisation on biopsies from clam ileocystoplasties and on a clam cancer.

The incidence of carcinoma following an enterocystoplasty increases with time and is a major concern after such procedures. The aim of this study was to investigate genetic instability (in the form of numerical chromosomal aberrations) at the enterovesical anastomosis in patients who had undergone a clam ileocystoplasty using fluorescent in-situ hybridisation (FISH). Fluorescent in-situ hybridisation was performed on touch preparation samples prepared from fresh endoscopic biopsies obtained from the enterovesical anastomosis and native bladder remnant (control specimens) of 15 patients who had undergone a clam ileocystoplasty. Fluorescent in-situ hybridisation was also performed on one squamous cell cancer specimen. Significant aneusomic changes were found at the enterovesical anastomosis in all 15 patients. Alterations in chromosome 18 copy number were the most frequent abnormal finding (trisomy 18, n=8; monosomy 18, n=7). Nine patients were monosomic for chromosome 9. Isolated monosomy 8 and trisomy 8 were each found in one patient. The control specimens were all normal. An unusually high incidence of polysomic cells was found in the clam tumour specimen, reflecting the aggressive nature of this cancer. Chromosomal numerical abnormalities occur at the enterovesical anastomosis following a clam ileocystoplasty and chromosome 18 appears to be a particularly good marker of genetic instability. The results of this study indicate that morphologically normal tissue obtained from the enterovesical anastomosis displays evidence of chromosomal instability that may predispose to tumour formation. However, further prospective, blinded, longitudinal studies are required to establish whether predetermined FISH signal patterns in enterocystoplasty cells in urine or obtained by biopsy predict the presence or absence of tumour.

Hepatocyte growth factor/scatter factor and prostate cancer: a review.

Men who die from prostate cancer do so from uncontrolled metastatic disease. A better understanding of the mechanisms involved in the progression and metastasis of prostate cancer may lead to novel therapeutic approaches to prevent its natural progression. Hepatocyte Growth Factor / Scatter factor (HGF/SF) has been demonstrated to elicit a number of key functions in numerous tissues that are important in the progression, invasion and metastasis of cancer. Studies have demonstrated that the activity of HGF/SF and its receptor c-Met are linked to disease progression in numerous cancers. However, research into these functions, which include activities as a mitogen, a motogen and an anti-apoptotic and angiogenic factor in prostate cancer are limited. This article reviews the published evidence of the roles HGF/SF plays in prostate cancer progression and highlights the clinical and therapeutic potential of research into this pleiomorphic cytokine.

Ontogeny of the hypothalamo-pituitary-adrenocortical axis in the chicken embryo: a review.

The embryo of the domestic fowl (Gallus domesticus) tenders one distinctive advantage over general mammalian models for investigating the development of the hypothalamo-pituitary-adrenocortical (HPA) axis. This is the relative simplicity with which the embryonic endocrine environment can be influenced without confounding maternal influences. The ease of direct manipulation of the embryonic endocrine system has facilitated analysis of the development and function of the HPA axis in the chick embryo. As the chick embryo develops, functional activation of the adrenal gland is regulated at three different levels: the adrenal gland itself, the anterior pituitary, and the hypothalamus. The adrenal gland appears capable of independent secretion of glucocorticoids from day 8 until shortly after day 14 of embryonic development, at which point the pituitary influences adrenocortical activity. Around the same age, the hypothalamic level of control also begins. The information covered in this review will describe the major steps in the development of the HPA axis in the chicken embryo and show that the chicken has an emblematic HPA neuroendocrine axis.

An atypical phenotype of macular and peripapillary retinal atrophy caused by a mutation in the RP2 gene.

AIMS: To determine the molecular basis and describe the phenotype of an atypical retinal dystrophy in a family presenting with bilateral, progressive central visual loss. METHODS: Family members were examined. Investigations included Goldman perimetry, electrophysiology, and autofluorescence imaging. Candidate gene screening was performed using SSCP and sequence analysis. The proband's lymphoblastoid cells were examined for protein expression. RESULTS: Fundal examination of the proband, his mother, and brother revealed peripapillary and macular atrophy. Autosomal dominant retinal dystrophy was suspected, but less severe disease in the mother led to screening for mutations in X linked genes. A 4 bp microdeletion in exon 3 of the RP2 gene, segregating with disease, was identified. No RP2 protein expression was detected. CONCLUSION: The distinct phenotype in this family, caused by this frameshifting mutation in RP2, broadens the phenotypic spectrum of X linked retinitis pigmentosa. The absence of RP2 protein suggests that loss of protein function and not novel gain of function could account for the atypical phenotype. A definitive diagnosis of X linked retinitis pigmentosa permits appropriate genetic counselling with important implications for other family members. Clinicians should have a low threshold for screening RP2 in families with retinal dystrophy, including posterior retinal disease, not immediately suggestive of X linked inheritance.

Somatostatin analogs in oncology: a look to the future.

In the past 15 years considerable advances have been made in our understanding of the molecular pharmacology of the mechanisms whereby somatostatin and its analogs mediate their direct and indirect antineoplastic effects. However, some important issues remain to be resolved, in particular the functional roles of the individual somatostatin receptors (SSTR-1-5) in tumor tissue and up- or downregulation of the hSSTRs with prolonged administration of somatostatin analogs. Answers to these questions are essential before we can maximize the therapeutic efficacy of somatostatin analogs in cancer. For example, is continuous administration more or less effective than intermittent therapy? The role of somatostatin analogs in the management of acromegaly and to a lesser extent neuroendocrine tumors is firmly established. The development of depot preparations of all 3 somatostatin analogs currently available for clinical use will undoubtedly improve both patient compliance and quality of life in patients with these conditions. There are only likely to be minor differences in the therapeutic efficacy of octreotide, lanreotide and vapreotide since all three analogs exert the majority of their antineoplastic effects via hSSTR-2 and hSSTR-5 and at the end of the day, price may well dictate which of these drugs oncologists use to provide symptomatic palliation of acromegaly and neuroendocrine tumors. Apart from some notable exceptions, somatostatin analog therapy has proven to be very disappointing in the management of advanced malignancy. Improvements in the management of solid tumors are likely to come only from combination therapy of somatostatin analogs with cytotoxic agents or other hormones in both advanced malignancy and in the adjuvant setting. Clinical trials with clear-cut objective outcome measures and health-related quality of life assessment are needed to evaluate the therapeutic efficacy of combination treatment in advanced malignancy and as an adjuvant to surgery. Particular attention needs to be paid to possible adverse effects of somatostatin analog therapy on the immune response to cancer. Further studies are required to establish whether the adverse effects of somatostatin analog therapy alone or in combination with cytotoxics or other hormones can be reversed with appropriate immunomodulatory treatment. Targeted somatostatin analog radiotherapy and chemotherapy are currently being investigated and the results of these studies are awaited with interest. Novel approaches using combinations of somatostatin analogs with antiangiogenic drugs or gene therapy are of particular interest and may provide important advances in the management of cancer in the not too distant future.

Awake intubation made easy and acceptable.

This study examined the efficacy of a technique of administration of lignocaine 2% (with 1:200,000 adrenaline) to the nose, pharynx and larynx. A simple device constructed from a 22 gauge Optiva cannula attached to a medical oxygen supply via green "bubble" tubing and the barrel of a 3 ml syringe, similar to that described by Mackenzie, was used to administer aerosolized lignocaine initially via the nose and subsequently via a nasopharyngeal airway. Ten unsedated, unpremedicated volunteers were intubated. The mean time from the start of the administration of lignocaine to confirmation of placement of the endotracheal tube was 12 minutes (range 8 to 19 minutes). Intubating conditions were good and the procedure was well tolerated in all subjects. The mean dose of lignocaine was 4.8 mg.kg-1 (range 2.7 to 6.9 mg.kg-1) and plasma concentrations were well below toxic levels (highest concentration 3.12 mg.l-1). There was good haemodynamic stability and no episode of oxyhaemoglobin desaturation.

Drug therapy for non-variceal upper gastrointestinal bleeding. Assessment of options.

The efficacy of somatostatin and octreotide have been widely studied in the control of bleeding from oesophageal varices. It has also been suggested that these drugs may be useful for the control of non-variceal upper gastrointestinal (UGI) bleeding, including that from peptic ulcers. In approximately 80% of patients presenting with non-variceal UGI bleeding, haemorrhage ceases spontaneously and does not recur. However, the remaining 20% of patients require active treatment. Results from recent studies have indicated that somatostatin is an effective treatment for the control of non-variceal UGI bleeding in high-risk patients, i.e. those in whom haemorrhage does not cease spontaneously or is likely to recur. In contrast there is no good evidence available at present to support a role for octreotide, histamine (H(2) antagonists) or proton pump inhibitors in this indication. The efficacy of somatostatin in controlling bleeding in patients with non-variceal UGI bleeding at high risk of mortality upon admission, or rebleeding following endoscopy, coupled with an excellent safety and tolerability profile, suggests it may be a valuable therapeutic option in the management of non-variceal bleeding.

Do alterations in the rate of gastric emptying after injection sclerotherapy for oesophageal varices play any role in the development of portal hypertensive gastropathy?

Bleeding from portal hypertensive gastropathy (PHG) has been estimated to account for up to 30% of all upper gastrointestinal haemorrhage in patients with cirrhosis and portal hypertension. Although portal hypertension seems to be an essential prerequisite, the precise mechanisms responsible for the development of PHG are unknown. The aim of this study was to examine the role of injection sclerotherapy of oesophageal varices in the development of PHG. Gastric emptying was studied using a radionuclide test meal with the emptying characteristics of a slow liquid in 57 patients with cirrhosis and/or portal hypertension (median age 53 yrs), of whom 34 had received injection sclerotherapy for their oesophageal varices and 20 normal healthy volunteers (median age 42 yrs). As vagal damage is associated with more rapid emptying of liquids, despite hold up of solids, this technique might be expected to demonstrate such damage if gastric emptying was accelerated. The results indicated that there was no difference in the rate of gastric emptying between normal healthy volunteers and portal hypertensive patients. However, patients who had received injection sclerotherapy emptied their stomachs faster than those who had not (p < 0.05). Furthermore, the speed of gastric emptying correlated directly with the number of injections (r = 0.41; p = 0.02) and the volume of sclerosant injected (r = 0.39; p = 0.03). These observations suggest that injection sclerotherapy for oesophageal varices results in disturbances of gastric emptying that may contribute to the pathogenesis of portal hypertensive gastropathy.

Pharmacokinetics of octreotide in patients with cirrhosis and portal hypertension; relationship between the plasma levels of the analogue and the magnitude and duration of the reduction in corrected wedged hepatic venous pressure.

In healthy subjects octreotide is largely metabolised by the liver suggesting that the plasma half-life of the somatostatin analogue may be prolonged in patients with hepatic dysfunction. The aim of this study was therefore (a) to determine the pharmacokinetics of octreotide following its subcutaneous injection in 6 patients with cirrhosis and portal hypertension and (b) compare the magnitude and duration of the effects of intravenous administration of 250 micrograms somatostatin and 50 micrograms octreotide on corrected wedged hepatic venous pressure (WHVP) and to relate the findings to the plasma levels of the analogue 1 h after administration in 13 patients with cirrhosis and portal hypertension. Following subcutaneous administration of 50 micrograms octreotide the circulating half life (range 2.4 to 4.79 h) was prolonged whereas the clearance (range 2.101 to 4.775 L/h) was decreased compared to healthy controls. Intravenous bolus administration of 250 micrograms somatostatin or 50 micrograms octreotide resulted in a reduction in WHVP of approximately the same magnitude and duration despite appreciable quantities of the analogue in the blood 1 h after administration (1944 +/- 226 pg/ml). These results indicate that the circulating half-life of octreotide is prolonged in cirrhotics suggesting that the dosage regimens should be modified in such patients to avoid accumulation of the analogue in the blood which may result in undesirable side-effects or toxicity. Furthermore, since the magnitude and duration of the reduction in WHVP elicited by IV octreotide is similar to that observed with somatostatin, the analogue, like the native hormone, must be administered by continuous IV infusion to produce a sustained response and hence a therapeutic effect in the management of acute variceal bleeding.

Grading of benign prostatic obstruction can predict the outcome of transurethral prostatectomy.

PURPOSE: We investigate whether urodynamic grading of benign prostatic obstruction and detrusor contractility predicts the outcome of transurethral prostatectomy. MATERIALS AND METHODS: A total of 53 patients who were suitable candidates for transurethral prostatectomy completed an assessment protocol before and 3 months after surgery, which included International Prostate Symptom Score, uroflowmetry, ultrasonography (prostatic size and residual urine volume) and standard pressure flow study. The results of the pressure flow study were analyzed to grade obstruction (unequivocal, equivocal or no obstruction) and detrusor contractility (weak or normal) using our simplified pressure flow nomogram. RESULTS: Analysis of the pressure flow study data demonstrated that the efficiency of detrusor contraction was weak in 6 of 27 men with unequivocal, 11 of 23 with equivocal and 2 of 3 with no obstruction. Treatment outcome was significantly better in patients with unequivocal obstruction and normal detrusor contractility. Treatment failure occurred in 80% of patients with equivocal obstruction and impaired detrusor contractility, and 100% of the unobstructed group. Urodynamic grading of obstruction and detrusor contractility predicted treatment outcome with a sensitivity of 87%, specificity 93% and positive predictive value 95%. CONCLUSIONS: Urodynamic grading of benign prostatic obstruction and detrusor contractility can reliably predict treatment outcome and, therefore, enable the urologist to identify a subgroup of patients who would not benefit from surgery.

Somatostatin in the treatment of non-variceal upper gastrointestinal bleeding.

The efficacies of somatostatin and octreotide have been widely studied in the control of bleeding from oesophageal varices. It has also been suggested that these drugs may be useful for the control of non-variceal upper gastrointestinal (UGI) bleeding, including that from peptic ulcers. In approximately 80% of patients presenting with non-variceal UGI bleeding, haemorrhage ceases spontaneously and does not recur. However, the remaining 20% of patients require active treatment. Results from recent studies have indicated that somatostatin is an effective treatment for the control of non-variceal UGI bleeding in high-risk patients, i.e. those in whom haemorrhage does not cease spontaneously or is likely to recur. In contrast there is no good evidence available at present to support a role for octreotide in this indication. The efficacy of somatostatin in controlling bleeding in patients with non-variceal UGI bleeding at high risk of mortality upon admission, or rebleeding following endoscopy, coupled with an excellent safety and tolerability profile, suggests that it may be a valuable therapeutic option in the management of non-variceal bleeding.