Weaning practices in phenylketonuria vary between health professionals in Europe.

BACKGROUND: In phenylketonuria (PKU), weaning is considered more challenging when compared to feeding healthy infants. The primary aim of weaning is to gradually replace natural protein from breast milk or standard infant formula with solids containing equivalent phenylalanine (Phe). In addition, a Phe-free second stage L-amino acid supplement is usually recommended from around 6 months to replace Phe-free infant formula. Our aim was to assess different weaning approaches used by health professionals across Europe. METHODS: A cross sectional questionnaire (survey monkey®) composed of 31 multiple and single choice questions was sent to European colleagues caring for inherited metabolic disorders (IMD). Centres were grouped into geographical regions for analysis. RESULTS: Weaning started at 17-26 weeks in 85% (n = 81/95) of centres, >26 weeks in 12% (n = 11/95) and < 17 weeks in 3% (n = 3/95). Infant's showing an interest in solid foods, and their age, were important determinant factors influencing weaning commencement. 51% (n = 48/95) of centres introduced Phe containing foods at 17-26 weeks and 48% (n = 46/95) at >26 weeks. First solids were mainly low Phe vegetables (59%, n = 56/95) and fruit (34%, n = 32/95).A Phe exchange system to allocate dietary Phe was used by 52% (n = 49/95) of centres predominantly from Northern and Southern Europe and 48% (n = 46/95) calculated most Phe containing food sources (all centres in Eastern Europe and the majority from Germany and Austria). Some centres used a combination of both methods.A second stage Phe-free L-amino acid supplement containing a higher protein equivalent was introduced by 41% (n = 39/95) of centres at infant age 26-36 weeks (mainly from Germany, Austria, Northern and Eastern Europe) and 37% (n = 35/95) at infant age > 1y mainly from Southern Europe. 53% (n = 50/95) of centres recommended a second stage Phe-free L-amino acid supplement in a spoonable or semi-solid form. CONCLUSIONS: Weaning strategies vary throughout European PKU centres. There is evidence to suggest that different infant weaning strategies may influence longer term adherence to the PKU diet or acceptance of Phe-free L-amino acid supplements; rendering prospective long-term studies important. It is essential to identify an effective weaning strategy that reduces caregiver burden but is associated with acceptable dietary adherence and optimal infant feeding development.

Early feeding practices in infants with phenylketonuria across Europe.

BACKGROUND: In infants with phenylketonuria (PKU), dietary management is based on lowering and titrating phenylalanine (Phe) intake from breast milk or standard infant formula in combination with a Phe-free infant formula in order to maintain blood Phe levels within target range. Professionals use different methods to feed infants with PKU and our survey aimed to document practices across Europe. METHODS: We sent a cross sectional, survey monkey® questionnaire to European health professionals working in IMD. It contained 31 open and multiple-choice questions. The results were analysed according to different geographical regions. RESULTS: Ninety-five centres from 21 countries responded. Over 60% of centres commenced diet in infants by age 10 days, with 58% of centres implementing newborn screening by day 3 post birth. At diagnosis, infant hospital admission occurred in 61% of metabolic centres, mainly in Eastern, Western and Southern Europe. Breastfeeding fell sharply following diagnosis with only 30% of women still breast feeding at 6 months.53% of centres gave pre-measured Phe-free infant formula before each breast feed and 23% alternated breast feeds with Phe-free infant formula. With standard infant formula feeds, measured amounts were followed by Phe-free infant formula to satiety in 37% of centres (n = 35/95), whereas 44% (n = 42/95) advised mixing both formulas together. Weaning commenced between 17 and 26 weeks in 85% centres, ≥26 weeks in 12% and < 17 weeks in 3%. DISCUSSION: This is the largest European survey completed on PKU infant feeding practices. It is evident that practices varied widely across Europe, and the practicalities of infant feeding in PKU received little focus in the PKU European Guidelines (2017). There are few reports comparing different feeding techniques with blood Phe control, Phe fluctuations and growth. Controlled prospective studies are necessary to assess how different infant feeding practices may influence longer term feeding development.

International practices in the dietary management of fructose 1-6 biphosphatase deficiency.

BACKGROUND: In fructose 1,6 bisphosphatase (FBPase) deficiency, management aims to prevent hypoglycaemia and lactic acidosis by avoiding prolonged fasting, particularly during febrile illness. Although the need for an emergency regimen to avoid metabolic decompensation is well established at times of illness, there is uncertainty about the need for other dietary management strategies such as sucrose or fructose restriction. We assessed international differences in the dietary management of FBPase deficiency. METHODS: A cross-sectional questionnaire (13 questions) was emailed to all members of the Society for the Study of Inborn Errors of Metabolism (SSIEM) and a wide database of inherited metabolic disorder dietitians. RESULTS: Thirty-six centres reported the dietary prescriptions of 126 patients with FBPase deficiency. Patients' age at questionnaire completion was: 1-10y, 46% (n = 58), 11-16y, 21% (n = 27), and >16y, 33% (n = 41). Diagnostic age was: <1y, 36% (n = 46); 1-10y, 59% (n = 74); 11-16y, 3% (n = 4); and >16y, 2% (n = 2). Seventy-five per cent of centres advocated dietary restrictions. This included restriction of: high sucrose foods only (n = 7 centres, 19%); fruit and sugary foods (n = 4, 11%); fruit, vegetables and sugary foods (n = 13, 36%). Twenty-five per cent of centres (n = 9), advised no dietary restrictions when patients were well. A higher percentage of patients aged >16y rather than ≤16y were prescribed dietary restrictions: patients aged 1-10y, 67% (n = 39/58), 11-16y, 63% (n = 17/27) and >16y, 85% (n = 35/41). Patients classified as having a normal fasting tolerance increased with age from 30% in 1-10y, to 36% in 11-16y, and 58% in >16y, but it was unclear if fasting tolerance was biochemically proven. Twenty centres (56%) routinely prescribed uncooked cornstarch (UCCS) to limit overnight fasting in 47 patients regardless of their actual fasting tolerance (37%). All centres advocated an emergency regimen mainly based on glucose polymer for illness management. CONCLUSIONS: Although all patients were prescribed an emergency regimen for illness, use of sucrose and fructose restricted diets with UCCS supplementation varied widely. Restrictions did not relax with age. International guidelines are necessary to help direct future dietary management of FBPase deficiency.

Analysis of combination anti-angiogenesis therapy on markers of coagulation, platelet activation and angiogenesis in patients with advanced cancer.

The effects of combination anti-angiogenesis therapy (marimastat, captopril and fragmin) on plasma levels of coagulation initiator tissue factor (TF), platelet marker soluble P-selectin and angiogenic vascular endothelial growth factor (VEGF) were tested in 25 patients with advanced cancer. They had higher soluble P-selectin (P<0.001) and TF (P<0.001), but not VEGF (P=0.066) than 25 age and sex-matched controls. VEGF and TF correlated significantly (r=0.8, P<0.001) in cancer patients. Soluble P-selectin, TF and VEGF did not change at 4- and 8-weeks whilst on treatment. We provide further evidence linking coagulation and angiogenesis but combination anti-angiogenesis therapy does not influence plasma soluble P-selectin, TF or VEGF.

Plasma vascular endothelial growth factor, angiopoietin-2, and soluble angiopoietin receptor tie-2 in diabetic retinopathy: effects of laser photocoagulation and angiotensin receptor blockade.

BACKGROUND: Proliferative diabetic retinopathy (PDR) may be a response to abnormal angiogenic growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), and the soluble angiopoietin receptor tie-2. The authors hypothesised the following: (a) there are differences in plasma levels of these growth factors in different grades of diabetic retinopathy; and (b) that the effects of intervention with panretinal laser photocoagulation (PRP) for PDR, and angiotensin receptor blockade (using eprosartan) for patients with other grades of diabetic retinopathy will be to reduce levels of the growth factors. METHODS: Cross sectional and interventional study (using PRP and eprosartan) in diabetic patients. VEGF, Ang-2, and tie-2 were measured by ELISA. RESULTS: VEGF (p<0.001) and Ang-2 levels (p<0.001) were significantly higher in 93 diabetic patients compared to 20 healthy controls, with the highest levels in grade 2 and grade 3 diabetic retinopathy (p<0.05). Tie-2 was lower in diabetics compared to controls (p = 0.008), with no significant differences between the diabetic subgroups. Overall, VEGF significantly correlated with Ang-2 (p<0.001) and tie-2 (p = 0.004) but the correlation between Ang-2 and tie-2 levels was not significant (p = 0.065). Among diabetic patients only, VEGF levels were significantly correlated with Ang-2 (p<0.001) and tie-2 (p<0.001); the correlation between Ang-2 and tie-2 levels was also significant (p<0.001). There were no statistically significant effects of laser photocoagulation on plasma VEGF, Ang-2, and tie-2 in the 19 patients with PDR, or any effects of eprosartan in the 28 patients with non-proliferative diabetic retinopathy. CONCLUSION: Increased plasma levels of VEGF and Ang-2, as well as lower soluble tie-2, were found in diabetic patients. The highest VEGF and Ang-2 levels were seen among patients with pre-proliferative and proliferative retinopathy, but there was no relation of tie-2 to the severity of retinopathy. As the majority of previous research into Ang-2 and tie-2 has been in relation to angiogenesis and malignancy, the present study would suggest that Ang-2 and tie-2 may be used as potential indices of angiogenesis in diabetes mellitus (in addition to VEGF) and may help elucidate the role of the angiopoietin/tie-2 system in this condition.

Combination antiangiogenesis therapy with marimastat, captopril and fragmin in patients with advanced cancer.

Marimastat, low molecular weight heparins and captopril have antiangiogenic activity in vitro and in animal models. We studied the safety and efficacy of the combination of these drugs in patients with advanced cancer. In all, 50 patients were enrolled. Captopril was given orally at a dose of 50 mg bd daily. Fragmin was administered as a daily subcutaneous injection of 200 units kg(-1) for the first 28 days and 5000 units thereafter. Marimastat was given at 10 mg bd orally. Serum, plasma and urinary angiogenic factors were measured at baseline and after 1 month of treatment. Inhibition of release of tumour necrosis factor alpha (TNF-alpha) from peripheral lymphocytes was used as a surrogate pharmacodynamic end point. There was one case of haemorrhagic stroke and one upper gastrointestinal haemorrhage. The commonest toxicity was myalgia. One of 10 patients with renal cancer had a partial response, and three patients had a prolonged period of stable disease. The treatment significantly inhibited phytohaemagglutinin (PHA)-stimulated TNF-alpha release from patient's lymphocytes. The combination of marimastat, fragmin and captopril is well tolerated and has in vivo activity. Inhibition of PHA-stimulated TNF-alpha release from lymphocytes is a surrogate pharmacodynamic marker of metalloprotease inhibition.

Angiopoietin/tie-2 as mediators of angiogenesis: a role in congestive heart failure?

Angiogenic factors, in particular vascular endothelial growth factor (VEGF) and the angiopoietins, Ang-1 and -2, have recently generated significant interest, especially in oncology. The process of angiogenesis is also thought to occur in response to ischaemic conditions, which lie at the core of cardiovascular disease states such as coronary artery disease and congestive heart failure. However, current data do not conclusively show evidence of angiogenesis per se in these conditions, despite (for example) the presence of high levels of VEGF and Ang-2. High levels of these angiogenic factors in heart disease also have not translated into clinically significant new vessel formation, as in accelerated cancer growth or proliferative retinopathy. Indeed, we would hypothesize that these angiogenic markers--especially the angiopoietins--do not necessarily translate into new vessel formation in congestive heart failure (CHF), but may well reflect disturbances of endothelial integrity in CHF.

Plasma angiopoietin-1, angiopoietin-2 and Tie-2 in breast and prostate cancer: a comparison with VEGF and Flt-1.

BACKGROUND: Angiogenesis is essential for tumour growth and metastasis, and is coordinated by several classes of growth factors mediating their effect through receptors linked, in turn, to tyrosine kinase. These growth factors include angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF), which act through receptors Flt-1 and Tie-2. MATERIALS AND METHODS: In order to further determine abnormalities in levels of Ang-1, Ang-2, Tie-2, sFlt-1 and VEGF in human cancer (and their interrelationships), these molecules were measured in plasma from 30 patients with breast cancer, 30 patients with prostate cancer and 12 healthy controls per cancer group. RESULTS: In breast cancer, levels of Ang-1 (P=0.0005), Ang-2 (P=0.0173), Tie-2 (P=0.0001), and VEGF (P=0.0001) were all significantly raised, and plasma levels of sFlt-1 (P=0.045) were significantly reduced compared with controls. However, in prostate cancer, only levels of VEGF and Tie-2 were significantly higher (both P=0.001). There were no significant differences between levels of any molecule between the two groups of cancer. The only difference between the healthy control groups was lower Ang-1 in the women compared with men. Significant correlations were found between levels of Ang-1 and Tie-2 both in breast (r=0.498, P=0.005) and prostate cancer (r=0.643, P=<0.001). Angiopoietin-1 was also positively correlated with Ang-2 in both breast (r=0.422, P=0.02) and prostate cancer (r=0.543, P=0.002). CONCLUSIONS: Abnormal levels of Ang-1, Ang-2 and their receptor, Tie-2, are present in breast and prostate cancer, and their interrelationships may be important in the pathophysiology of these conditions.

Removal of neuroblastoma cells from bone marrow with monoclonal antibodies conjugated to magnetic microspheres.

Monoclonal antibodies bound to polystyrene microspheres containing magnetite have been used to remove tumour cells from bone marrow destined for autologous transplantation. The small magnetic beads can be targeted to the surface of tumour cells to render these magnetic. A flow system using permanent samarium cobalt magnets effects the rapid and efficient removal of "magnetic" tumour cells from bone marrow. The system is designed to be used with any type of tumour cell, so that by changing panels of monoclonal antibody it can be used for "cleaning" the bone marrow in many different malignancies.

Changes in cell surface antigen expression during hemopoietic differentiation.

Human bone marrow cells were separated on a fluorescence activated cell sorter (FACS) according to their binding of a series of monoclonal antibodies; the positive and negative fractions were cloned for erythroid burst and colony-forming units (BFU-E and CFU-E) and myeloid colony-forming units (CFU-GM), and cytocentrifuge slides were prepared for microscopy of maturing precursors. The pattern of antigen expression on hemopoietic progenitor and precursor populations has been established using antibodies defining blood group (A, I/i), HLA-associated (*A, B, C, DR, DC1), lineage specific, and transferrin receptor antigens. Like monomorphic HLA-DR, the antigen defined by monoclonal antibody OKT10 is expressed on the earliest progenitors and lost during differentiation, suggesting a role in interactions regulating the differentiation of these cells. The HLA-linked DC1 determinant, in contrast to HLA-DR, is not expressed at a detectable level on progenitor cells. Although a lineage-specific early antigen has not been identified, the transferrin receptor is expressed on the majority of erythroid progenitors, but only weakly on myeloid progenitors, and may provide an approach to isolating erythroid progenitors. These and earlier studies with monoclonal antibodies against HLA-DR and glycophorin now provide a detailed "map" of antigen expression during hemopoietic differentiation.

Mechanisms of uptake and the fate of serum proteins and horseradish peroxidase in cultured human glioma cells. A light- and electron-immunocytochemical study.

Previous studies have shown that serum proteins are taken up from extracellular oedema fluid by reactive astrocytes and by tumour astrocytes. The present investigation was designed to define the mechanism of this protein uptake. Two or 3-week-old explant cultures from 26 astrocytic gliomas, one anaplastic ependymoma, and five non-glial intracranial tumours were treated with either human IgG (12 mg/ml), human serum albumin, (44 mg/ml) or horseradish peroxidase (0.1--4.0 mg/ml) for 4--24 h. Human IgG and albumin were subsequently detected in cultured cells by the indirect peroxidase-antiperoxidase (PAP) method for light microscopy or by direct peroxidase conjugate technique for electron microscopy. Horseradish peroxidase activity was localised by treatment with diaminobenzidine and hydrogen peroxide. Results of the study show that human serum proteins and horseradish peroxidase are taken up by tumour astrocytes and ependymal cells, and by macrophages, but not by non-glial tumour cells nor by mesenchymal elements in the glioma cultures. Electron immunocytochemistry suggests that the serum proteins are taken up by smooth walled micropinocytic vesicles (approximately 80 nm in diameter) which fuse to form larger endocytic vesicles (200--300 nm); these vacuoles in turn fuse with secondary lysosomes to form cytoplasmic bodies 1.2--3 mum in diameter.