Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation

The tumour suppressor gene PTEN , which maps to 10q23.3 and encodes a 403 amino acid dual specificity phosphatase (protein tyrosine phosphatase; PTPase), was shown recently to play a broad role in human malignancy. Somatic PTEN deletions and mutations were observed in sporadic breast, brain, prostate and kidney cancer cell lines and in several primary tumours such as endometrial carcinomas, malignant melanoma and thyroid tumours. In addition, PTEN was identified as the susceptibility gene for two hamartoma syndromes: Cowden disease (CD; MIM 158350) and Bannayan-Zonana (BZS) or Ruvalcaba-Riley-Smith syndrome (MIM 153480). Constitutive DNA from 37 CD families and seven BZS families was screened for germline PTEN mutations. PTEN mutations were identified in 30 of 37 (81%) CD families, including missense and nonsense point mutations, deletions, insertions, a deletion/insertion and splice site mutations. These mutations were scattered over the entire length of PTEN , with the exception of the first, fourth and last exons. A 'hot spot' for PTEN mutation in CD was identified in exon 5 that contains the PTPase core motif, with 13 of 30 (43%) CD mutations identified in this exon. Seven of 30 (23%) were within the core motif, the majority (five of seven) of which were missense mutations, possibly pointing to the functional significance of this region. Germline PTEN mutations were identified in four of seven (57%) BZS families studied. Interestingly, none of these mutations was observed in the PTPase core motif. It is also worthy of note that a single nonsense point mutation, R233X, was observed in the germline DNA from two unrelated CD families and one BZS family. Genotype-phenotype studies were not performed on this small group of BZS families. However, genotype-phenotype analysis inthe group of CD families revealed two possible associations worthy of follow-up in independent analyses. The first was an association noted in the group of CD families with breast disease. A correlation was observed between the presence/absence of a PTEN mutation and the type of breast involvement (unaffected versus benign versus malignant). Specifically and more directly, an association was also observed between the presence of a PTEN mutation and malignant breast disease. Secondly, there appeared to be an interdependent association between mutations upstream and within the PTPase core motif, the core motif containing the majority of missense mutations, and the involvement of all major organ systems (central nervous system, thyroid, breast, skin and gastrointestinal tract). However, these observations would need to be confirmed by studying a larger number of CD families.      

Blockade of Ca2+‐activated K+ channels in T cells: an option for the treatment of multiple sclerosis?

Voltage- and Ca(2+)-dependent K(+) channels in the membrane of both T and B lymphocytes are important for the cellular immune response. In the current issue of the European Journal of Immunology, Reich et al. demonstrate that selective blockade of the intermediate-conductance Ca(2+)-activated K(+) channel (the IK channel encoded by the KCNN4 gene) prevents cytokine production in the spinal chord and ameliorates the development of EAE caused by injection of myelin oligodendrocyte glycoprotein (MOG)(35-55) in mice. These data renew the focus on the IK channel as a potential target for the development of new immune-suppressant drugs for the treatment of autoimmune diseases.     

Lack of FasL expression in cultured human retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune privilege of the eye, but due to contradictory published results, it is unclear whether RPE cells express this molecule. The purpose of this study was to investigate the expression of FasL in RPE cells in vitro and in vivo. Cultured human fetal and adult RPE cells were examined by flow cytometry, Western blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from tissue sections, were positive for FasL, indicating a discrepancy between RPE cells in vitro and in vivo with regard to this molecule.     

The renin–angiotensin system in kidney development: role of COX‐2 and adrenal steroids

Recent data from studies in rodents with targeted gene disruption and pharmacological antagonists have shown that the renin–angiotensin–aldosterone system (RAAS) and cyclooxygenase type‐2 (COX‐2) are necessary for late stages of kidney development. The present review summarizes data on the developmental changes of RAAS and COX‐2 and the pathways by which they are activated; their possible interplay and the mechanisms by which they affect kidney development. Intrarenal and circulating renin and angiotensin II (ANG II) are stimulated at birth in most mammals. In rats, renin and ANG II stay significantly elevated in the suckling period while aldosterone stabilizes at an adult level. COX‐2 is stimulated in thick ascending limb of Henle's loop in the suckling period at a time when urine concentrating ability is not developed. Data suggest that this induction is mediated by combined low plasma glucocorticoid concentration and by a low NaCl intake. Studies with selective inhibitors of COX‐2 and COX‐2 null mice show that COX‐2 activity stimulates renin secretion from JG‐cells during postnatal kidney development and that lack of COX‐2 activity leads to pathological change in cortical architecture and eventually to renal failure. In the postnatal period, ANG II initiates and maintains pelvic and ureteric contractions necessary for urine flow. Lack of ANG II in the neonatal period is thought to cause injury by a chronic increase of renal pelvic pressure. Aldosterone is crucial for survival and growth in the neonatal period through its effects on sodium reabsorption and the intrarenal sensitivity to aldosterone is increased in the postnatal period. Final maturation of the kidney occurs through an intimate interplay between a low dietary sodium intake and a non‐responsive HPA‐axis which stimulates cortical COX‐2 activity. COX‐2 supports increased activity of the RAAS and may contribute to a low concentrating ability.     

Can subjects with a positive allergen skin test be selected by a short questionnaire?

The objective was to evaluate a postal questionnaire screening procedure for selection of subjects with positive reactions to skin prick tests with common allergens. The project consisted of a screening, with subsequent skin prick test of two selected groups. The setting was the Glostrup Population Studies institute in Copenhagen, Denmark. Participants in the screening included 8000 subjects, aged 15–69 years. The subjects were randomly selected from the population of western Copenhagen County, Denmark. From the 6998 respondents (87.5%), 793 subjects were randomly selected (Random Group), and 788 subjects were chosen on the basis of their answers to the questionnaire (Symptom Group). Both groups were invited to take skin prick tests. Attendance rates were 75.5% (Random Group) and 80.6% (Symptom Group).
The main outcome measures were responses (yes or no) to the specific questions and the subjects' skin reaction (positive or negative). The association between symptoms and skin reactivity, adjusted for the effects of sex and age, was summarized by odds ratios. Symptoms on exposure to allergens were highly associated with positive skin reactivity. In the Symptom Group the percentage of subjects with at least one positive skin reaction was 57.7%, which was twice as much (28.4%) as in the Random Group. The results show that it was possible to select a group with high skin reactivity on the basis of the symptoms reported in the screening. Questions about exposure to allergens were the most appropriate for selection of this group.     

Arrested neuronal proliferation and impaired hippocampal function following fractionated brain irradiation in the adult rat

The generation of new neurons in the adult mammalian brain has been documented in numerous recent reports. Studies undertaken so far indicate that adult hippocampal neurogenesis is related in a number of ways to hippocampal function.Here, we report that subjecting adult rats to fractionated brain irradiation blocked the formation of new neurons in the dentate gyrus of the hippocampus. At different time points after the termination of the irradiation procedure, the animals were tested in two tests of short-term memory that differ with respect to their dependence on hippocampal function. Eight and 21 days after irradiation, the animals with blocked neurogenesis performed poorer than controls in a hippocampus-dependent place-recognition task, indicating that the presence of newly generated neurons may be necessary for the normal function of this brain area. The animals were never impaired in a hippocampus-independent object-recognition task. These results are in line with other reports documenting the functional significance of newly generated neurons in this region. As our irradiation procedure models prophylactic cranial irradiation used in the treatment of different cancers, we suggest that blocked neurogenesis contributes to the reported deleterious side effects of this treatment, consisting of memory impairment, dysphoria and lethargy.     

Intracellular mechanisms governing the acute phase of β-endorphin secretion from the corticotrope in vitro

It is not certain which protein kinase (A, C or both) is involved in the acute phase of β-endorphin (β-EP) release stimulated in the corticotrope by vasopressin (VP) and corticotropin-releasing factor (CRF). We have employed an isolated ovine anterior pituitary cell superfusion system to determine the dynamic effects of forskolin, a protein kinase A (PKA) stimulator, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator. Both secretagogues stimulated β-EP release within 5 min and therefore both PKA and PKC are potential mediators of the acute phase of hormonal stimulation of the corticotrope. Pretreatment with PMA specifically desensitized the pituitary cell columns to subsequent PMA exposure while not significantly altering sensitivity to forskolin or 50 mM KCl.     

An enzyme-based in situ hybridisation method for the identification of Streptococcus suis

A method for enzyme-based in situ hybridisation of Streptococcus suis was developed. It enables the light microscopic localization of bacterial ribosomal RNA (rRNA) in formalin-fixed paraffin-embedded tissues. A unique sequence in the 16S rRNA of S. suis was targeted. Different pretreatment protocols were applied to facilitate probe penetration and multiple detection systems were tested. The results were compared to those obtained by immunohistochemistry. Pretreatment was necessary to obtain a signal by in situ hybridisation. The use of proteinase-K pretreatment was optimal regarding sensitivity and preservation of tissue morphology. A strong specific in situ hybridisation signal was achieved in tissue sections containing S. suis in microcolonies and the microanatomy of the surrounding tissue was easily assessed. However, the signal distribution differed from that found immunohistochemically and low-grade infection could not be detected by in situ hybridisation. These findings were interpreted as reflecting the physiological state of the bacteria. Thus, this method could prove useful in future studies of the infection pathogenesis.