Purpose: In breast cancer, the EGF receptors host an increasing number of therapeutic targets and the interactive mechanisms of actions of the receptors and their ligands justify investigation of the EGF family as an entity. Experimental design: Paired tissue samples of normal breast tissue and primary breast carcinomas were examined in a prospective study of 163 patients. A third sample was obtained from the paired ipsilateral metastatic lymph node from 58 of these patients. The mRNA expression of four EGF receptors (HER1 - HER4) and 11 activating ligands was quantified with real-time RT-PCR. Results: Expression of HER2, HER3, and HER4 mRNA was upregulated in primary carcinomas compared to normal breast tissue while HER1 was downregulated. The mRNA expression of HER3 and HER4 differed between primary breast carcinomas and lymph node metastases whereas there was no difference in the expression of HER1 and HER2. The combination of low HER3 and low HER4 expression in the primary carcinoma was significantly more frequent in lymph node-negative patients as compared to lymph node positive patients. Distinct correlation patterns of the receptors and their corresponding activating ligands appeared in both normal breast tissue and in carcinomas, notably for the HER3 and HER4 receptors and their 3 specific ligands: HB-EGF, NRG2, and NRG4. Conclusion: HER2, HER3, and HER4 showed increased mRNA expression in carcinomas and were positively correlated to each other and to specific activating ligands. Furthermore, low HER3 and HER4 expression in the carcinomas correlated to the absence of lymph node metastases.

Abstract Introduction: The Human Epidermal Growth Factor Receptor 4 (HER4) of the EGF receptor family has been characterized in both normal and malignant human breast tissue and HER4 overexpression has been shown to predict prolonged survival compared to HER4 receptor negative disease. In our study we investigated the HER4 expression in normal breast tissue, primary breast carcinoma and in ipsilateral metastatic axillary lymphnodes at the time of primary breast cancer surgery. Material and methods: Paired tissue samples from normal breast tissue and primary breast carcinomas were obtained from 169 patients. Out of these a third sample was obtained from 66 patients with metastatic lymphnodes. The primary tumour specimens were sampled uniformly at random. The mRNA expression of HER4 was quantified with real time RT-PCR and expressed relative to the householdgene (HMBS) in arbitrary units (arb.u.). Results: The mRNA expression of HER4 was significantly higher in breast carcinoma with a mean of 2.26 arb.u. [95% c.i.:1.87 to 2.65 arb.u.] than in the paired sample of normal breast tissue with a mean of 0.82 arb.u. [95% c.i.: 0.47 to 1.16 arb.u.] (p=0.0001). The mRNA expression of HER4 was also significantly higher in breast carcinoma than in the corresponding lymphnode mean 0.75 arb. u. [95% c.i.: 0.43 to 1.07 arb.u.] (p=0.015). There was no significant difference observed in mRNA expression of HER4 between the metastatic lymphnode and the corresponding normal breast tissue (P>0.05). When HER4 expression was compared between the primary tumours that had metastasised and those that had not, there was no significant difference (P>0.05). Conclusion: The HER4 expression was high in the primary tumour as compared to normal breast tissue and the corresponding lymphnode. In view of previously published relations between a low expression of HER4 and a poor prognosis our results warrant further studies in order to evaluate whether suppression of HER4 in tumour cells could be involved in lymphogenic metastatic spread. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-01-09.

Vitamin B12 deficiency is a major public health problem: however, there is no consensus on how to diagnose this deficiency. In the present review we attempt to give a balanced update. We include only issues related to diagnosing and treating vitamin B12 deficiency in the adult population and do not cover inherited diseases of vitamin B12 deficiency except for those related to classical pernicious anemia.

Once more—thanks to funding through Radiometer Medical A/S—an Astrup competition was arranged in connection with the XXIX Nordic Congress in Clinical Chemistry held in Malmö, Sweden, in April 2004. Clinical biochemists throughout the Nordic countries were invited to participate in the competition and among the 16 abstracts submitted for the 2004 Astrup competition 3 were selected for oral presentation at the opening ceremony of the Congress. The selection was made by a jury comprising Jørgen Hjelm Poulsen, Denmark; Ulf-Haakon Stenman, Finland; TorArne Hagve, Norway; Johan Stenflo, Sweden, and chaired by Ebba Nexø, Denmark. The final ranking of the three selected presentations was not an easy task. The jury evaluated the scientific content and gave due attention to the presentation of the work as well as the discussion that followed the presentation. The originality of the work and its potential for clinical medicine were also considered. Based on an evaluation of all these considerations, Anna Blom was selected as the winner of the 2004 Astrup competition. Jens Peter Gøtze was awarded the second prize and Boe Sandahl Sørensen the third prize. A common denominator for the 2004 Astrup lectures was the use of molecular biology to answer important research questions and to develop assays that could be used in daily clinical practice. Anna Blom constructs mutant proteins to explain how bacteria and viruses infect humans by paralysing the complement system and shows that viruses use a strategy involving the production of proteins homologous to the endogenous complement inhibitors. The exciting prospect of this work is the possibility of developing vaccines that could counteract the paralysis of the complement system induced by the viral proteins. The subject of Jens Gøtze’s paper is B-type natriuretic peptide (BNP), the peptide that recently has come into focus as a marker for heart failure. Gøtze addresses—and confirms—the hypothesis that the production of BNP mRNA is increased in the hypoxic myocardium, leading to an increased release of proBNP. The results support proBNP/BNP as a plasma marker of potential use for identifying patients at cardiac risk despite no overt cardiac dysfunction. Finally, Bo Sørensen demonstrates the use of real-time RT-PCR in the clinical setting. Using the amplification of the EGF receptor (HER2) as an example, he shows that RT-PCR may prove an important addition to identification of HER2 amplifications by immunohistochemistry and FISH. He also addresses the use of RT-PCR for measurement of mRNA expression and offers advice for those seeking to establish such assays. Together, these three papers illustrate the high level of research carried out in the Nordic countries within fields related to clinical biochemistry.

Background: The trefoil factors (TFF1–3) are cysteine‐rich peptides expressed in the gastrointestinal tract where they play a critical role in mucosal protection and repair. The expression is up‐regulated at sites of ulceration in various chronic inflammatory diseases. Recently, we presented an ELISA method for measurement of TFF3. The aims of the present study were to develop and evaluate ELISAs for the other two known human trefoil peptides, TFF1 and TFF2, and to carry out a cross‐sectional study on serum TFF levels in patients with inflammatory bowel disease (IBD). Methods: The TFF1‐ELISA was based on two polyclonal rabbit antibodies and the TFF2‐ELISA on a monoclonal mouse antibody and a polyclonal rabbit antibody. RhTFF1 and 2 were employed to prepare the calibrators. TFF1–3 were assayed in serum from IBD patients (n=41) and controls (n=13). Results: The TFF1‐ (TFF2‐) ELISA had a detection limit of 3 pmol/L (6 pmol/L) and an analytical imprecision (CVA) of 7.0–8.8 for mean concentrations of 24–120 pmol/L (6.1–8.0 for mean concentrations of 17–77 pmol/L). The central reference intervals (n=300) were 140–1400 pmol/L (37–190 pmol/L). There was no variation with age and menstrual cycle. Food intake reduced concentrations of TFF1 by ∼15%, but did not influence concentrations of TFF2. TFF1 and TFF3 were increased in serum from IBD patients. Conclusions: We have developed assays for measuring TFF1 and TFF2. Finding increased TFF concentrations in serum from IBD patients suggests that measurements of trefoil peptides may be of clinical relevance in IBD.

Since plasma methylmalonic acid (ALMA) was introduced in Denmark in 1992 both MMA and P-cobalamins have been increasingly employed to diagnose vitamin B-12 deficiency, and the consumption of vitamin B-12 has been almost doubled during the same period. The controversy about diagnosing vitamin B-12 deficiency is reflected in a huge geographical variation both in requisition of NIMA and P-cobalamins and in the vitamin B-12 consumption. On the basis of studies performed so far we conclude that NMA is a reliable method with a good nosographic sensitivity. However, the nosographic specificity is sparsely examined, and the evidence to use MMA as a primary test for diagnosing vitamin B-12 deficiency is still insufficient. We recommend P-cobalamins as the first line test, and if P-cobalamins are in the grey area 125-250 pmol/l we recommend supplementation with NIMA. For patients who also have an MMA in the grey area (0.29-0.75 mumol/l) we recommend that the patient is reexamined after about a year.

BACKGROUND:Vitamin B(12) in plasma is complexed to the carrier proteins transcobalamin (TC) and haptocorrin. The TC-B(12) complex is filtered in the glomeruli and reabsorbed in the renal tubules by receptor-mediated endocytosis, providing a route for a significant renal accumulation of vitamin B(12). The present study investigates the role of the rodent kidney in B(12) homeostasis by examining the distribution of vitamin B(12) in rats during vitamin B(12) depletion or B(12) load, and compares kidney accumulation with the vitamin distribution in other tissues including brain, liver, testes, intestine, spleen and plasma.METHODS:Fifteen rats were fed on a diet containing different concentrations of B(12) supplemented with s.c. injections of B(12). Twenty four hours prior to sacrifice, all animals were injected with [(57)Co]B(12). The vitamin contents of kidneys, liver, spleen, brain, testis, intestine, skeletal muscle, serum and urine were analysed. Both total tissue vitamin B(12) accumulation and [(57)Co]B(12) were determined to compare steady-state B(12) and the distribution of an acutely injected dose. In the kidney, free and protein-bound B(12) was determined by gel filtration.RESULTS:The rat kidneys accumulated more B(12) during normal and loaded conditions than any other tissue. A 110-fold increase in vitamin content was observed from the deficient to the loaded conditions in the kidney compared with a 3.5-fold increase in the liver. In contrast to all other organs, significantly smaller amounts of acutely injected B(12) accumulated in the kidneys in the vitamin-deprived state compared with both the normal and the vitamin-loaded condition.CONCLUSIONS:The present study suggests a significant role for the rodent kidney in vitamin B(12) metabolism. We propose a model for rat tissue uptake consistent with the presence of two different TC-B(12) receptors and renal uptake following filtration of TC-B(12) in the glomeruli. The presented model allows for the reduced renal uptake and accumulation in vitamin-deprived conditions, thus reserving the vitamin for other tissues, including nerve tissue and bone marrow, which are more sensitive to vitamin B(12) deficiency.

Peptides of the trefoil factor family (TFF1, TFF2 and TFF3) are co-secreted with mucus in most organ systems and are believed to interact with mucins to produce high-viscosity, stable gel complexes. We have previously demonstrated that cells in the GI tract possess binding sites to TFF2 and that injected TFF2 ends up in the mucus layer. In the present study, tissue binding and metabolism of parenterally administered human TFF1 and TFF3 in rats were described and compared to the immunohistochemical localization of the TFF peptides. 125I-TFF1 monomer and 125I-TFF3 mono- and dimer were given intravenously to female Wistar rats. The tissue distribution was assessed by gamma counting of organ samples and by autoradiography of histological sections. The degradation of 125I-TFF3 was studied by means of trichloracetic acid (TCA) precipitation and the saturability of the binding by administration of excess unlabelled peptide. The TFF peptides were localized in histologic sections from the GI tract by immunohistochemistry. Injected TFF3 dimer (12%) was taken up by the GI tract. At autoradiography, grains were localized to the same cells that were immunoreactive to TFF2. The binding could be displaced by excess TFF3. Similar binding was observed for the TFF1 and TFF3 monomers apart from binding in the stomach, where the uptake was only 15% in comparison to the dimer. There was no specific binding outside the GI tract and no binding to TFF1 or TFF3 immunoreactive cells. In conclusion, the TFF2-binding cells in the gastrointestinal tract seem to have basolateral, receptor-like activity to all three TFF peptides. The mucous neck cells of the stomach predominantly take up TFFs with two trefoil domains, indicating a different receptor-like activity in the stomach compared to the rest of the GI tract.

BACKGROUND:Vitamin B-12 deficiency is usually accompanied by elevated concentrations of serum total homocysteine (tHcy) and methylmalonic acid (MMA). Folate deficiency also results in elevated tHcy. Measurement of these metabolites can be used to screen for functional vitamin B-12 or folate deficiency. OBJECTIVE:We assessed the prevalence of vitamin B-12 and folate deficiency in a population-based study (n = 1562) of older persons living in Oxford City, United Kingdom. DESIGN:We postulated that, as vitamin B-12 or folate concentrations declined from adequate to impaired levels, tHcy (or MMA) concentrations would increase. Individuals were classified as being at high risk of vitamin B-12 deficiency if they had low vitamin B-12 (< 150 pmol/L) or borderline vitamin B-12 (150-200 pmol/L) accompanied by elevated MMA (> 0.35 micromol/L) or tHcy (> 15.0 micromol/L). Individuals were classified as being at high risk of folate deficiency if they had low folate (< 5 nmol/L) or borderline folate (5-7 nmol/L) accompanied by elevated tHcy (> 15 micromol/L). RESULTS:Cutoffs of 15.0 micro mol/L for tHcy and 0.35 micro mol/L for MMA identified persons with normal or elevated concentrations. Among persons aged 65-74 and >or= 75 y, respectively, approximately 10% and 20% were at high risk of vitamin B-12 deficiency. About 10% and 20%, respectively, were also at high risk of folate deficiency. About 10% of persons with vitamin B-12 deficiency also had folate deficiency. CONCLUSION:Use of tHcy or MMA among older persons with borderline vitamin concentrations may identify those at high risk of vitamin B-12 deficiency who should be considered for treatment.

This study investigates the renal and urinary levels of epidermal growth factor (EGF) in rats under long-term treatment with alpha- or beta-adrenergic agonists. Urine samples were obtained on days 7, 14 and 21, and renal tissue samples on day 21. EGF was quantified by ELISA and tissue sections were used for immunohistochemistry and in situ hybridization. Fractional kidney weight was increased in the alpha-adrenergic agonist-treated group by 35% when compared with controls. Histological examination of the kidney revealed well-defined wedge-shaped areas of tubular dilatations and luminal amorphous material in the distal tubules. Concomitantly, reduced levels of EGF and EGF mRNA were observed, and also the urinary levels of EGF were reduced. Together, these observations indicate alpha-adrenergic treatment to affect the distal tubules. Treatment with the beta-adrenergic agonist did not change fractional kidney weight, but initially the urinary excretion of EGF was reduced. The data add further evidence to the suggestion that activity of the sympathetic nervous system influences renal homeostasis of EGF, either directly or indirectly through renal histopathological changes.

Fetuses of diabetic mothers who were exposed to excessive glucose show delayed maturation. Under these conditions, altered growth factor expression or signaling may have important regulatory influences. We examined the role of epidermal growth factor (EGF) in lung development and maternal diabetes in the rat. In order to evaluate the possible role of glucose for the expression of EGF and the growth of lung tissue, we performed in vitro studies with organotypic cultures of fetal alveolar cells obtained from control rats.