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.

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.

Plasma growth hormone concentration in corticosteroid-treated children.

Endogenous plasma growth hormone concentrations were measured in 23 children who were receiving daily corticosteroid therapy and in 10 control asthmatic children who had not received steroids for at least 8 months. The growth hormone concentrations were similar in the two groups of patients both during the fasting state and after insulin-induced hypoglycemia. 12 children, who were studied while receiving a large dose of prednisone and again 2 wk after steroid withdrawal, also showed no change in growth hormone concentration in relation to corticosteroid therapy. These findings suggest that deficiency of growth hormone is not the major mechanism responsible for the dwarfism of corticosteroid-treated children.

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.

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.

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.

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.

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.

Somatostatin in acute bleeding oesophageal varices. Clinical evidence.

Following the demonstration that somatostatin lowered portal pressure in cirrhotic patients with portal hypertension, 2 uncontrolled reports suggested that the hormone might be useful in the control of acute variceal haemorrhage. Subsequently, a number of randomised controlled trials have indicated that somatostatin may have an efficacy as good as or better than either vasopressin or combined vasopressin and nitroglycerin therapy and is associated with fewer side effects. Somatostatin has an efficacy comparable to balloon tamponade, histamine-2-receptor antagonists and injection sclerotherapy. One double-blind randomised controlled trial demonstrated a significant benefit of somatostatin over placebo in the control of variceal bleeding whereas a second did not show any significant difference between treatments. In all the controlled trials, the average control rate achieved with somatostatin administration was 69% and it was not associated with any major side effects. Somatostatin administration has also been shown in uncontrolled series to be very effective in controlling postinjection sclerotherapy bleeding from the varices per se, and from oesophageal ulcers and oesophagitis. Few data are available on the long acting analogue of somatostatin, octreotide, but preliminary data suggest that it may be as effective and safe as the native hormone in controlling the acute variceal bleeding and postinjection sclerotherapy haemorrhage. It is concluded that there may be a case for instituting somatostatin therapy as soon as the patient enters hospital to facilitate sclerotherapy, and for continuing treatment for 5 days after sclerotherapy when the risk of recurrent bleeding is highest.

Review article: the relative effectiveness of somatostatin and octreotide therapy in pancreatic disease.

Somatostatin and octreotide inhibit basal and stimulated pancreatic secretion, stimulate reticuloendothelial system activity, modulate the cytokine cascade and are cytoprotective with respect to the pancreas. These effects of somatostatin and octreotide suggest that both drugs may be useful either in the treatment of pancreatic disorders, or in preventing acute pancreatitis following procedures on the pancreas. In recent years it has become clear that somatostatin is a useful and effective therapy for severe acute pancreatitis and in preventing complications following endoscopic retrograde cholangiopancreatography (ERCP), whereas octreotide has no beneficial effect and may be deleterious in both these indications. The differences in the therapeutic efficacy of somatostatin and octreotide in acute pancreatitis and ERCP appears to be related to their differential effects on sphincter of Oddi motility--the native hormone relaxing, and the analogue increasing, its contractility. Consequently, any beneficial effects of octreotide in both acute pancreatitis and ERCP are offset by the increased contractility of the sphincter of Oddi, which results in retention of activated enzymes within the pancreas and further autodigestion of the gland. Somatostatin and octreotide are equally effective in promoting the closure of pancreatic fistulae. However, the time to closure after commencement of therapy is much more variable and longer in patients treated with subcutaneous octreotide than those receiving intravenous somatostatin, possibly as a result of fluctuations in pancreatic enzyme secretion between consecutive administrations of the hormone. Furthermore, the initial potent inhibitory effect of octreotide on pancreatic secretion is lost after 7 days of continuous subcutaneous administration. Therefore, in terms of cost-effectiveness, somatostatin would appear to be the treatment of choice for pancreatic fistulae. Octreotide markedly reduces the complication rates after elective pancreatic surgery. It remains to be established whether somatostatin is as effective as octreotide in this indication.

Assay of gastricsin and individual pepsins in human gastric juice.

AIMS: To develop and validate an analytical procedure for the quantitation of pepsins and gastricsin in human gastric juice and to assess its potential in a controlled gastric secretory study. METHODS: High performance ion-exchange chromatography was used to separate human pepsin 1, 3a, 3b, 3c and gastricsin from gastric juice. Computed chromatographic areas for each enzyme were quantified by relation to a known amount of a secondary standard porcine pepsin. The assay procedure was validated by recovery and analytical precision studies. Gastric secretions after pentagastrin and insulin stimulation from 10 patients with portal hypertension were used to assess the potential of the analytical procedure. RESULTS: The assay precision varied from 1.5 to 9.0% within batch and 7.5 to 18.1% between batch, with about 100% recoveries of porcine pepsin A from human gastric juice over the assay range 0.025-0.5 mg/ml. A fourfold increase in combined pepsin and gastricsin concentration was observed following pentagastrin and insulin stimulation. The mean percentage content of pepsins 3a, 3b, 3c, and 1 in non-stimulated gastric juice were 4%, 72%, 12% and 1.4%, respectively, and did not change significantly after gastric stimulation. An approximate doubling of the percentage of gastricsin (10% to 20%) relative to the pepsins was observed, however, after both insulin and pentagastrin stimulation. CONCLUSIONS: This procedure for quantifying individual human pepsins and gastricsin in gastric juice is simple and reliable. It may be of considerable importance in determining the mechanisms involved in the control and secretion of these digestive enzymes in man, including the effect of anti-ulcer drugs and our understanding of the pathophysiology of peptic ulcer disease.

Alteration in monocyte function in patients with breast cancer.

Migration, phagocytosis and lysozyme production of peripheral monocytes from 36 patients with breast carcinoma was compared with a group of 36 healthy controls. A significant reduction in monocyte random migration and migration towards a chemotactic agent (P less than 0.001) was observed in patients with breast cancer. Furthermore monocyte phagocytic activity was also significantly decreased (P less than 0.001) in breast cancer patients compared to the controls. In contrast lysozyme production by peripheral monocytes was significantly increased in patients with breast cancer compared to controls (P less than 0.001). The changes in monocyte function in patients with breast carcinoma were not due to abnormal blood biochemistry or to direct continuous serum inhibitors, suggesting that the defect may be intrinsic. This defect in monocyte function may play an important role in the control of malignancies by cellular immune processes.

The effect of colorectal malignancies on monocyte migration, lysozyme production, and phagocytosis.

Peripheral monocyte migration, phagocytosis and lysozyme production has been investigated in 46 patients with colorectal malignancies. The diagnosis of colorectal carcinoma was based on clinical, radiological, and endoscopic examinations and confirmed histopathologically after removal of the tumour. The results were compared with a group of 36 normal healthy controls. Monocytes from patients with colorectal malignancies displayed reduced migration towards the chemotactic agent (P less than 0.001), random migration (P less than 0.001), and phagocytic activity (P less than 0.01) compared to normal controls. Lysozyme production by monocytes from patients with colorectal malignancies was significantly increased (P less than 0.01) compared to control subjects. The impairment in monocyte function in patients with colorectal carcinoma was not due to direct serum factors or to abnormal blood biochemistry, suggesting that the defect is intrinsic to the monocytes. These results suggest that an impairment in monocyte function may play an important role in the control of malignancies by cellular immune processes.

The effect of portal venous flow on the washout of a regionally injected marker substance 99mTc-methylene diphosphonate after hepatic arterial blockade with degradable starch microspheres.

Patients with hepatic metastases derived from colorectal carcinoma have a poor prognosis. Regional chemotherapy, either alone, or combined with agents such as degradable starch microspheres (DSM) that reduce or abolish intrahepatic arterial flow and potentiate the delivery of cytotoxics to hepatic metastases, have not significantly improved survival. We have investigated one positive mechanism, namely the effect of portal venous washout of cytotoxics, for the poor efficacy of drugs administered either alone or in combination with DSM via the hepatic artery in the rat. Using a radiolabelled marker, 99mTc-methylene diphosphonate (MDP), to represent a cytotoxic drug, the initial studies indicated that with the hepatic artery and portal vein clamped, a volume of 0.05 ml of the marker administered via the hepatic artery resulted in the most uniform intrahepatic distribution with minimal washout into the systemic circulation (21 +/- 3.7%). When the hepatic artery was clamped, the washout of MDP was reduced from 100% (with clamps on the portal vein and hepatic artery) to 84.2 +/- 7.7%. DSM administered concomitantly with MDP, resulted in a greater reduction of the portal venous washout of the marker (63 +/- 2.4%). Administration of DSM and MDP via the hepatic artery and with the portal vein clamped further reduced the washout of the marker to (21 +/- 2.26), results similar to those observed with inflow vessel clamps. Following restoration of portal venous flow, there was a rapid washout of 53.7 +/- 7.6% of the marker into the systemic circulation. The results of this study suggest that portal venous washout of regionally delivered cytotoxics, either alone or in combination with DSM, offer an explanation for the poor efficacy of regional chemotherapy in improving the prognosis of patients with hepatic metastases.

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.

Fluctuations in the secretion of pancreatic enzymes between consecutive doses of octreotide: implications for the management of fistulae.

OBJECTIVE: To determine whether variations in pancreatic enzyme secretion between consecutive subcutaneous administrations of octreotide explain why octreotide takes longer than somatostatin to facilitate the closure of gastrointestinal fistulae. METHODS: Pancreatic enzyme secretion was studied over a 3-day period in a patient with a catheter left in the pancreatic duct postoperatively. On days 1 and 3 the patient did not receive octreotide (control days) but on day 2 he received subcutaneous octreotide 100 micrograms every 8 h. Pancreatic juice was collected at 2-h intervals over the 3-day period. RESULTS: On the day of octreotide treatment, the patient's pancreatic secretory volume and protein output were significantly reduced (P < 0.001, Mann-Whitney U-test) compared with the 2 control days. The pancreatic secretory volume decreased markedly after the first injection of octreotide and remained low for the duration of the treatment period. The enzyme concentration of the pancreatic juice was also markedly reduced after the first injection of octreotide. However, approximately 4h after each octreotide injection the protein concentration of the pancreatic juice began to rise progressively, peaking approximately 6h after each administration of the analogue. CONCLUSION: Subcutaneous administration of octreotide produces a sustained decrease in the volume of pancreatic juice secreted, but enzyme secretion rises progressively between consecutive administrations of the analogue. The net effect is therefore the production of low volumes of pancreatic juice with a high enzyme concentration between consecutive injections of octreotide, which may delay the healing of the fistula tract. This may explain why longer treatment periods are required to achieve fistula closure with octreotide than with somatostatin, particularly in the case of pancreatic fistulae.

Effect of octreotide infusion on hepatic and tumour blood flow in two experimental models of liver metastases.

OBJECTIVE: To investigate the effects of octreotide infusion on hepatic and tumour blood flow in rats with experimentally induced liver tumours. DESIGN: Blood flow was determined in tumour-bearing rats using a dual reference microsphere technique before and after intravenous infusion of octreotide. METHODS: Tumours were induced in syngeneic rats by intraportal injection of K12-Tr and WB2054-M adenocarcinoma cells. Hepatic arterial and portal venous inflow, tumour blood flow and systemic arterial pressure were determined before and after octreotide infusion (0.05 microgram/min). RESULTS: In rats with K12-Tr tumours there was no change in tumour blood flow, hepatic arterial flow or portal venous inflow after octreotide infusion. In contrast, in rats with WB2054-M tumours, octreotide infusion resulted in a significant reduction in the blood flow to the hepatic tumour (from 0.37 to 0.135 ml/min/g) but had no effect on hepatic artery or portal venous inflow. CONCLUSION: The reduction in blood flow to tumours derived from WB2054-M cells could, at least in part, explain the inhibitory effect of octreotide on the growth and development of these tumours. However, octreotide had no effect on blood flow to tumours derived from K12-Tr cells, suggesting that the analogue must inhibit tumour growth by other mechanisms.

Induced hepatic arterial blockade by degradable starch microspheres in the rat.

Degradable starch microspheres (DSM, Spherex) have been shown to cause intermittent blockage of hepatic arterial flow and to increase the concentration of regionally injected cytotoxics. The Spherex monitoring system has been developed by Pharmacia, Sweden to establish the correct dose of DSM to optimize hepatic arterial blockade. Groups of normal rats received varying dosages of DSM and co-injected methylene diphosphonate (MDP) in order to reproduce the effect of reduction of passing fraction and marker flow rate as determined by the Spherex monitoring system. A flow reduction and significant decrease in passing fraction was achieved on injection of 4 mg of DSM via the hepatic artery.

A model of the hepatic perfusion index in the rat.

The hepatic perfusion index (HPI) is an indicator of the relative hepatic arterial to total liver blood flow as measured by dynamic flow scintigraphy. Hitherto, accurate assessment of the HPI in small animals has not been possible because of methodological difficulties. A reproducible method for measuring the HPI by dynamic scintigraphy in rats is described using a rapid intraventricular bolus administration of 0.04 ml 99Tcm sulphur colloid. There was no significant difference between the HPI determined by dynamic scintigraphy and and that calculated from absolute measurements of hepatic arterial and total liver blood flow. These results indicate that the HPI derived by dynamic scintigraphy in the rat is a true estimate of the ratio of the hepatic arterial to total liver blood flow.

Changes in the hepatic perfusion index during the growth and development of experimental hepatic micrometastases.

Micrometastases were induced in Fisher rats using an intraportal inoculation of 0.2 ml of 8 x 10(7) Walker carcinosarcoma cells. A control group received normal saline. The hepatic perfusion index (HPI) was measured during the growth and development of micrometastases. The HPI at 4 days (0.51 +/- 0.008) and at 6 days (0.65 +/- 0.16) was significantly raised when compared to controls (0.31 +/- 0.07) and at 2 days after inoculation (0.31 +/- 0.06). Hepatic artery flow did not change throughout the study period. However, portal venous inflow was decreased significantly at 4 and 6 days (0.57 +/- 0.16 and 0.55 +/- 0.11) when compared to controls (0.96 +/- 0.34). These results indicate that the change in the hepatic perfusion index is related to a decrease in portal venous inflow. The decrease in portal venous inflow could be a mechanical effect of the micrometastases on intrahepatic blood flow or to increased arteriovenous shunting.