In vitro selection of aptamers and their applications.

The introduction of the in-vitro evolution method known as SELEX (Systematic Evolution of Ligands by Exponential enrichment) more than 30 years ago led to the conception of versatile synthetic receptors known as aptamers. Offering many benefits such as low cost, high stability and flexibility, aptamers have sparked innovation in molecular diagnostics, enabled advances in synthetic biology and have facilitated new therapeutic approaches. The SELEX method itself is inherently adaptable and offers near limitless possibilities in yielding functional nucleic acid ligands. This Primer serves to provide guidance on experimental design and highlight new growth areas for this impactful technology.

pHAST (pH-Driven Aptamer Switch for Thrombin) Catch-and-Release of Target Protein.

A pH-driven DNA nanomachine based on the human α-thrombin binding aptamer was designed for the specific catch-and-release of human α-thrombin at neutral and acidic pH, respectively. In neutral conditions, the thrombin aptamer component of the nanomachine is exposed and exists in the G-quadruplex conformation required to bind to the target protein. At slightly acidic pH, the polyadenine tail of the nanomachine becomes partially protonated and A+(anti)•G(syn) mispairing results in a conformational change, causing the target protein to be released. Förster resonance energy transfer (FRET) was used to monitor conformational switching over multiple pH cycles. Electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy were used to show pH dependent protein binding and release by the nanomachine. This approach could be applied generally to existing G-rich aptamers to develop novel biosensors, theranostics, and nanoswitches.

Hypothyroid-associated sensorineuronal deafness.

A case of panhypopituitarism with hypothyroid-induced deafness in a man improving with hormone replacement is reported. A review of the medical literature reveals conflicting evidence regarding the underlying mechanism and prognosis of the defect in this context, but the association with hypothyroidism is more than spurious. Sensorineuronal hearing loss was initially evident, resolving both subjectively and on formal audiological evaluation after administering thyroxine. Central pathology affecting the eighth cranial nerve, as opposed to a conductive or mixed component is the likely culprit.

Long term effect of external pituitary irradiation on IGF1 levels in patients with acromegaly free of adjunctive treatment.

OBJECTIVE: It is established that external pituitary irradiation (EPI) effectively reduces serum GH levels in acromegaly. However, its effect in normalising serum IGF1 has been disputed. We looked at the number of our patients who achieved persistently normal IGF1 levels whilst free of adjunctive treatment for at least 1 year after EPI. PATIENTS AND DESIGN: We identified 63 acromegalic patients between 1964 and 2004 who received EPI. Six were excluded: three had surgery after EPI, two had no medical records available, and one had a pituitary Yttrium implant. MEASUREMENTS: Patients received 4500-5000 cGy in fractionated doses. IGF1 levels were correlated with their respective age-related reference ranges. RESULTS: After EPI, the number of patients with normal IGF1 and free of adjunctive medical treatment for at least 1 year were four patients by 3 years, nine patients by 5 years and seventeen by 10 years, with the current number of 25/57 (44%). Concordance between IGF1 levels and random GH dropped from 90% at the time of EPI to 65% at 3 years, 66% at 5 years and 71% at 10 years. CONCLUSIONS: We have demonstrated that, with time, EPI achieves a normal IGF1 in significant numbers of patients with acromegaly, thus obviating the need for life-long expensive medical therapy. For each patient this benefit has to be weighed against the possibility of new hypopituitarism as a result of the treatment. Any decision to use EPI is easier in the context of pre-existent hypopituitarism.

Poor responses to a test dose of subcutaneous octreotide predict the need for adjuvant therapy to achieve 'safe' growth hormone levels.

OBJECTIVE: Somatostatin analogues are an established treatment in acromegaly. This study was designed to evaluate whether the acute serum growth hormone (GH) response to a test dose of octreotide in acromegaly predicts longer-term response to the drug at 3 years. DESIGN AND METHODS: In 23 patients, GH responses across 8 h to a subcutaneous test dose (50 microg) of octreotide were compared with GH levels after 3 years of therapy. The majority had pituitary surgery as primary therapy and at 3 years were receiving at least 600 microg octreotide daily subcutaneously or 20 mg LAR monthly intramuscularly. RESULTS: Seven had a test day GH Nadir of 5 mU/l or less of whom 4 achieved GH < 5 mU/l at 3 years. Sixteen had a test day nadir GH of 10 mU/l or less and of these 8 achieved GH < 5 mU/l at 3 years. Seven of the 23 had a GH Nadir >10 mU/l and of these 3 had achieved GH <5 mU/l at 3 years. However all of these 3 had received external pituitary irradiation within 4 years of the 3 year assessment, as compared with 3 of the <5 mU/l nadir group and 5 of the <10 mU/l nadir group. CONCLUSIONS: In patients on optimal long-term doses of octreotide for acromegaly, absence of a nadir GH <10 mU/l in the 8 h after a test dose was associated with failure to achieve GH levels associated with a normal life expectancy (5 mU/l or less) unless adjunctive external pituitary irradiation was given. As well as testing tolerability a test dose of octreotide may help in determining which patients should be offered early external pituitary irradiation or therapy with a GH receptor antagonist if surgery has failed to achieve 'safe' GH levels.

Effects of low-dose oral hydrocortisone replacement versus short-term reproduction of physiological serum cortisol concentrations on insulin action in adult-onset hypopituitarism.

OBJECTIVE: Hypercortisolism is associated with impaired glucose tolerance and insulin resistance. For many years hydrocortisone 30 mg was the standard total daily replacement dose in adult hypopituitarism. The use of this conventional dose has now been shown to result in mild biochemical hypercortisolism and might contribute to the increased cardiovascular risk reported in hypopituitarism. The use of lower doses of hydrocortisone replacement therapy might prevent some of the adverse metabolic effects seen with conventional doses. PATIENTS: In a randomized crossover study we assessed peripheral and hepatic insulin action in 15 ACTH-deficient patients with normal glucose tolerance on two occasions while receiving either a low-dose oral hydrocortisone replacement (LOR) therapy (15 mg at 0800, 5 mg at 1700) or a physiological hydrocortisone infusion (PHI), which achieved physiological serum cortisol concentrations. RESULTS: Exogenous glucose infusion rates required to maintain euglycaemia were similar for the LOR and the PHI protocols (26.2 +/- 0.4 vs. 23.8 +/- 0.6 micromol/kg/min, respectively). Endogenous glucose production was also similar (12.0 +/- 2.5 vs. 11.6 +/- 2.4 micromol/kg/min, respectively) and in the post-absorptive state suppressed to a similar extent following insulin (4.5 +/- 2.0 vs. 5.1 +/- 3.1 micromol/kg/min). CONCLUSION: Hydrocortisone replacement therapy at a dose of 15 mg with breakfast, 5 mg with evening meal does not increase peripheral or hepatic insulin resistance when compared to a hydrocortisone infusion designed to simulate physiological serum cortisol concentrations.

The effects on insulin action in adult hypopituitarism of recombinant human GH therapy individually titrated for six months.

There is controversy about the effect of replacement GH on insulin action in adult hypopituitary patients. GH replacement calculated from weight leads to unacceptable side effects in some patients. Recent studies suggest it should be individually titrated in adults using serum IGF-I levels. We have assessed the effect of titrated GH replacement on peripheral and hepatic insulin action in 13 adult-onset hypopituitary patients (8 males and 5 females; ages 47 +/- 10 yr, mean duration of hypopituitarism 6 yr) with confirmed GH deficiency (GHD; maximum GH <5 mU/liter during insulin induced hypoglycemia), ACTH deficiency, and normal glucose tolerance. All patients were on stable hydrocortisone replacement (15 mg with breakfast, 5 mg with evening meal) for at least 2 months before the trial. Insulin action was assessed by the euglycemic hyperinsulinemic glucose clamp technique (1 mU/kg x min) before and after 6 months of GH therapy. GH was started at 0.8 IU sc daily and titrated monthly until the serum IGF-I increased to within 1-2 SD of the mean of normal age-matched controls. Body mass index did not change significantly during the 6 months of GH therapy. Fasting plasma glucose and HbA1c increased significantly after 6 months (5.2 +/- 0.0 vs. 5.5 +/- 0.0 mmol/liter, P < 0.0001, and 4.5 +/- 0.1 vs. 4.7 +/- 0.1%, P < 0.0005, respectively). There was no increase in fasting serum insulin (51.6 +/- 10.2 vs. 60.0 +/- 10.2 pmol/liter, P = 0.12). Exogenous glucose infusion rates required to maintain euglycemia were similar after GH (23.0 +/- 0.4 vs. 21.1 +/- 0.3 micromol/kg x min, P = 0.6). Endogenous glucose production in the fasting state was also unchanged following GH (11.8 +/- 0.7 vs.12.3 +/- 0.9 micromol/kg x min, P = 0.5) and suppressed to a similar extent following insulin (4.4 +/- 0.8 vs. 5.5 +/- 0.8 micromol/kg x min, P = 0.3). In summary, GH therapy for 6 months, with serum IGF-I maintained in the upper physiological range, increased fasting plasma glucose and HbA1c. There was no effect on peripheral or hepatic insulin sensitivity. Patients receiving GH therapy require long-term monitoring of glucose tolerance.

Prevalence of diabetes and impaired glucose tolerance in adult hypopituitarism on low dose oral hydrocortisone replacement therapy.

OBJECTIVE: The conventional dosage of hydrocortisone, used for many years in the management of hypopituitarism (30 mg per day), has now been shown to be more than is physiologically necessary. On this conventional corticosteroid therapy studies have demonstrated an increased prevalence of diabetes and impaired glucose tolerance, which may contribute to the increased vascular morbidity and mortality reported in the condition. In these studies no information is available on oral glucose tolerance test (OGTT) timing in relation to administration of oral steroid and variable hydrocortisone doses were employed. PATIENTS: In order to assess glucose tolerance in patients treated with lower, more physiological doses, we performed a 75-g OGTT at least 1 month after hydrocortisone therapy was adjusted to 15 mg at 0800 h and 5 mg at 1700 h in 45 adult onset hypopituitary patients (30 M, 15 F). Mean (+/- SD) duration of hypopituitarism was 12 +/- 10 years, mean age 52 +/- 14 years and BMI 29.3 +/- 5.1 kg/m2. All were on hydrocortisone, 43 on thyroxine, 31 on sex steroids, 9 on desmopressin and 33 had documented growth hormone deficiency. Hydrocortisone 15 mg was taken at 0800 and the OGTT commenced at 0900. RESULTS: Using standard WHO criteria 36 patients (80%) had normal glucose tolerance, 1 (2%) had newly diagnosed diabetes and 8 (18%) had impaired glucose tolerance. Using the recently announced American Diabetes Association criteria for diagnosis 96% had normal glucose tolerance, 2% had diabetes and 2% impaired fasting glucose. CONCLUSION: The markedly reduced prevalence of diabetes and impaired glucose tolerance on lower hydrocortisone replacement doses in our series of patients with hypopituitarism, not previously known to be diabetic, is of great interest. This lower prevalence may eventually result in reduced vascular complication rates.

Achieving optimal diabetic control in adolescence: the continuing enigma.

The transition from childhood through adolescence to adulthood is a difficult stage, particularly for patients with type 1 diabetes. The yearning for autonomy and independence, as well as the hormonal changes around the time of puberty, can manifest in poor glycaemic control. The focus on diet and weight increases the prevalence of eating disorders, compounding the difficulties in supervising diabetes patients. This can be exacerbated by the realisation that hyperglycaemia induces weight loss and the use of this knowledge to further manipulate diabetes control to gain a desired body image. The management of adolescents with type 1 diabetes is therefore challenging and requires close collaboration between psychological medicine and diabetes teams. This review describes the difficulties frequently encountered, with a description of four cases illustrating these points. Case 1 demonstrates the problem of needle phobia in a newly diagnosed patient with type 1 diabetes leading to persistent hyperglycaemia, the recognition of weight loss associated with this and the development of bulimia. The patient's overall management was further complicated by risk-taking behaviour. By the age of 24 years, she has developed diabetic retinopathy and autonomic neuropathy and continues to partake in risk-taking behaviour. Case 2 illustrates how the lack of parental support shortly after the development of type 1 diabetes led to poor glycaemic control and how teenagers often omit insulin to accommodate lifestyle and risk-taking behaviour. Case 3 further exemplifies the difficulty in managing patients with needle phobia and the fear of hypoglycaemia. Case 4 adds further weight to the need for parental support and the impact of deleterious life events on glycaemic control by manipulation of insulin dosage.

Malignancy arising in cystic ovarian teratomas. A report of 24 cases.

Twenty-four cases with a malignant change involving one of the elements of an ovarian dermoid cyst are reported. The patients were older than those with benign ovarian dermoid cysts. Squamous carcinoma was most frequent with adenocarcinoma, undifferentiated carcinoma and a fibrosarcoma also occurring. Survival time after diagnosis was less than 1 year in 75% of the cases.

The histopathology of the epididymis in a group of cases of azoospermia with normal testicular function.

The histopathology of the head of the epididymis in cases of azoospermia associated with normal testicular function is described. In 2 specimens congenital absence of the vas deferens was associated with stuffing of all of the tubules with normal spermatozoa but no other abnormality. In 30 specimens from cases with idiopathic epididymal obstruction there were abnormalities in the vasa efferentia. These included both extreme narrowing of the lumen due to thickening of the wall by fibrosis and also distension of the lumen by sperm and degenerate debris. Small sperm granulomata were present in the tubular wall in 10 specimens and foreign body granulomata were present in 20 specimens. It is suggested that either persistent low grade infection, congenital abnormality or acute obstruction due to ischaemic or traumatic damage may be the initial lesion. If the infection or the obstruction is not speedily resolved, the condition may become self-perpetuating.

Histological grade and other prognostic factors in relation to survival of patients with breast cancer.

Records of 3085 patients registered with breast cancer at the Mersey Regional Cancer Registry have been analysed to evaluate the relative importance of possible prognostic factors. In a subgroup of 1759 patients, clinical stage and histological grade are shown to be strongly related to survival after treatment. In addition histological grade is related to the distribution of times to death after treatment. The results of this and 3 other studies have implications for the design and analysis of clinical trials in the primary treatment of breast cancer.

Pathological findings in three cases of fungal endocarditis complicating open-heart surgery.

Three cases of fungal endocarditis are described, each following a homograft valve replacement for aortic stenosis. The causative organism was C. albicans. The characteristic findings included soft, easily detached vegetations consisting of fungal colonies, aneurysms, embolization of large vessels, and lack of response to current therapeutic measures. The source of the infection is obscure.