Urs Zumsteg

β cells are responsible for CXCR3-mediated T-cell infiltration in insulitis

T cell–mediated loss of insulin-secreting β cells in the islets of Langerhans is the hallmark of type 1 diabetes. The molecular basis for the directed migration of autoreactive T cells leading to insulitis is presently unknown. Here we demonstrate that in response to inflammation, β cells secrete the chemokines CXC ligand 10 and CXC ligand 9, which specifically attract T-effector cells via the CXC chemokine receptor 3. In mice deficient for this receptor, the onset of type 1 diabetes is substantially delayed. Thus, in the absence of known etiological agents, CXC receptor 3 represents a novel target for therapeutic interference early in type 1 diabetes.

A cross‐sectional international survey of continuous subcutaneous insulin infusion in 377 children and adolescents with type 1 diabetes mellitus from 10 countries

Objective:  To document current practices using continuous subcutaneous insulin infusion (CSII) by downloading electronically the 90‐d pump data held within the pump memory and relating that to clinical data from children and adolescents in different pediatric diabetes centers from Europe and Israel.

Randomized controlled comparison of two cognitive behavioral therapies for obese children : mother versus mother-child cognitive behavioral therapy

Background: Parent-child treatments have been shown to be superior to child-focused treatments of childhood obesity. Yet until now, the comparative effectiveness of parent-only and parent-child approaches has been little studied. Method: Fifty-six obese children and their families were randomly assigned to a 16-session cognitive behavioral therapy (CBT) for the parents only or for a combined treatment of parents and children. Children’s percent overweight, the body mass index of their mothers, and behavioral and psychological problems of children and mothers were assessed. Results: Both treatments reduced children’s percent overweight significantly and equally by 6-month follow-up. Also both treatments provided similar results in reducing general behavior problems (externalizing and internalizing behavior problems), global and social anxiety, and depression. Conclusions: Our results point to a comparable efficacy of the two treatments. Further, psychological well-being of both mothers and children can be improved in a CBT for obese children and their parents. Future studies should focus on finding ways to improve the adherence of families to long-term treatment of obesity in childhood.

Functional IL-18 Is produced by primary pancreatic mouse islets and NIT-1 beta cells and participates in the progression towards destructive insulitis.

Background: Preclinical stages of type 1 diabetes are characterized by infiltrating T cells and by the peri- and intra-islet accumulation of pro-inflammatory mediators, such as IFNγ. Methods/Results: Using quantitative PCR we demonstrated that mRNA specific for the IFNγ-inducing cytokine IL-18 is upregulated in NIT-1 beta cells and intact mouse islets upon exposure to IL-1β, IFNγ and TNFα. The biological activity of IL-18 was blocked using caspase inhibitors and anti-IL-18 antibodies. Increased IL-18 expression was also detected in islets during advanced stages of insulitis and correlated with elevated transcripts for IFNγ and for the IL-18 receptor. Conclusion: Thus, beta cells produce bioactive IL-18 in the course of insulitis and actively contribute to the exacerbation of inflammation leading to their own demise.

EXPRESSION OF RECEPTORS FOR MELANIN-CONCENTRATING HORMONE (MCH) IN DIFFERENT TISSUES AND CELL LINES

ABSTRACT Melanin-concentrating hormone (MCH) is a potent orexigenic neuropeptide and a physiological antagonist of α-melanocyte-stimulating hormone (α-MSH) in the brain as well as at peripheral sites, including the pigmentary systems of specific vertebrates. Two receptor subtypes for MCH, MCH-R1 and MCH-R2, have been cloned, but other receptor subtypes are likely to exist. Based on our own data and the current literature, we have compared the expression of different receptors for MCH in various mammalian cell lines and tissues. Summarizing all data currently available, we conclude that the two cloned MCH receptors, MCH-R1 and MCH-R2, exhibit differences in their expression pattern, although MCH-R1 is generally colocalized in all tissues where MCH-R2 expression is found. It appears that MCH-R1 is more abundant and has a wider distribution pattern than MCH-R2. Other hypothetical MCH-R subtypes may be expressed in specific tissues, e.g., in the pigment cell system.

Strict sun protection results in minimal skin changes in a patient with xeroderma pigmentosum and a novel c.2009delG mutation in XPD (ERCC2).

Abstract:  We examined the clinical, molecular and genetic features of a 16‐year‐old boy (XP2GO) with xeroderma pigmentosum (XP) and progressive neurological symptoms. The parents are not consanguineous. Increased sun sensitivity led to the diagnosis of XP at 2 years of age and a strict UV protection scheme was implemented. Besides recurrent conjunctivitis and bilateral pterygium, only mild freckling was present on his lips. He shows absent deep tendon reflexes, progressive sensorineural deafness and progressive mental retardation. MRI shows diffuse frontal cerebral atrophy and dilated ventricles. Symptoms of trichothiodystrophy (brittle hair with a tiger‐tail banding pattern on polarized microscopy) or Cockayne syndrome (cachectic dwarfism, cataracts, pigmentary retinopathy and spasticity) were absent. XP2GO fibroblasts showed reduced post‐UV cell survival (D37 = 3.8 J/m2), reduced nucleotide excision repair, reduced expression of XPD mRNA and an undetectable level of XPD protein. Mutational analysis of the XPD gene in XP2GO revealed two different mutations: a common p.Arg683Trp amino acid change (c.2047C>T) known to be associated with XP and a novel frameshift mutation c.2009delG (p.Gly670Alafs*39). The latter mutation potentially behaves as a null allele. While not preventing neurological degeneration, early diagnosis and rigorous sun protection can result in minimal skin disease without cancer in XP patients.

[D-(p-Benzoylphenylalanine)13, tyrosine19]-melanin-concentrating hormone, a potent analogue for MCH receptor crosslinking

A photoreactive analogue of human melanin‐concentrating hormone was designed, [d‐Bpa13,Tyr19]‐MCH, containing the d‐enantiomer of photolabile p‐benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous‐flow solid‐phase methodology using Fmoc‐strategy and PEG‐PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [d‐Bpa13,Tyr19]‐MCH at its Tyr19 residue was carried out enzymatically using solid‐phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed‐phase mini‐column and HPLC. Saturation binding analysis of [125I]‐[d‐Bpa13,Tyr19]‐MCH with G4F‐7 mouse melanoma cells gave a KD of 2.2±0.2×10−10 mol/l and a Bmax of 1047±50 receptors/cell. Competition binding analysis showed that MCH and rANF(1–28) displace [125I]‐[d‐Bpa13,Tyr19]‐MCH from the MCH binding sites on G4F‐7 cells whereas α‐MSH has no effect. Receptor crosslinking by UV‐irradiation of G4F‐7 cells in the presence of [125I]‐[d‐Bpa13,Tyr19]‐MCH followed by SDS‐polyacrylamide gel electrophoresis and autoradiography yielded a band of 45–50 kDa. Identical crosslinked bands were also detected in B16‐F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS‐7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [125I]‐[d‐Bpa13,Tyr19]‐MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein‐coupled receptor, most likely with a varying degree of glycosylation. Copyright

INTERACTION OF MELANIN-CONCENTRATING HORMONE (MCH), NEUROPEPTIDE E-I (NEI), NEUROPEPTIDE G-E (NGE), AND α-MSH WITH MELANOCORTIN AND MCH RECEPTORS ON MOUSE B16 MELANOMA CELLS

Melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between α-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-Rpc) and the different melanocortin (MC) receptors. Radioreceptor assays using [125I]MCH, [125I]α-MSH and [125I]NEI as radioligands and bioassays were performed with MC1-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC50s of α-MSH and NEI or NGE for [125I]MCH displacement from mouse MCH-Rpc were 80-fold and, respectively, > 300-fold higher than that of MCH, and the IC50s for MCH and NEI or NGE for [125I]α-MSH displacement from mouse MC1-R were 50,000-fold and > 200,000-fold higher than that of α-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [125I]α-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 μM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.

Antagonist and agonist activities of the mouse agouti protein fragment (91-131) at the melanocortin-1 receptor.

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91–131) (AP91–131) at the melanocortin type-1 receptor (MC1-R) were assessed using B16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP91–131 was about 3-fold less potent than the natural agonist α-melanocyte-stimulating hormone (α-MSH) in displacing the radioligand [125I]-[Nle4, D-Phe7]-α-MSH (Ki 6.5±0.8 nmol/l). α-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP91–131; the IC50 values for AP91–131 in the two assay systems were 91±22 nM and 95±15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP91–131 with IC50 values of 9.6±1.8 nM and 5.0±2.4 nM, respectively. This indicates inverse agonist activity of AP91–131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP91–131 in the adenylate cyclase and melanin assays. On the other hand, AP91–131 inhibited cell growth similar to α-MSH (IC50 11.0±2.1 nM; maximal inhibition 1.8-fold higher than that of α-MSH). Furthermore, MC1-R was down-regulated by AP91–131 with about the same potency and time-course as with α-MSH. These results demonstrate that AP91–131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different α-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.

Panhypopituitarism Presenting as Life-Threatening Heart Failure Caused by an Inherited Microdeletion in 1q25 Including LHX4

Clinical presentation of hypopituitarism in the neonate may be variable, ranging from absent to severe nonspecific symptoms and may be life-threatening in patients with adrenocorticotropic hormone deficiency. The LIM homeobox gene 4 (LHX4) transcription factor regulates early embryonic development of the anterior pituitary gland. Autosomal dominant mutations in LHX4 cause congenital hypopituitarism with variable combined pituitary hormone deficiency (CPHD). We report on a neonate with unexplained heart failure and minor physical anomalies, suggesting a midline defect. She was diagnosed with complete CPHD. Cardiac function was rescued by replacement with hydrocortisone and thyroxine; hypoglycaemia stopped under growth hormone therapy. Magnetic resonance imaging revealed a dysgenetic pituitary gland suggesting an early developmental defect. Array comparative genomic hybridization showed a maternally inherited 1.5-megabase microdeletion in 1q25.2q25.3, including the LHX4 gene. Haploinsufficiency of LHX4 likely explains the predominant pituitary phenotype in the proposita and we suggest variable intrafamilial penetrance of the inherited microdeletion. To the best of our knowledge, we are the first to report on heart failure as a rare nonspecific symptom of treatable CPHD in the newborn. Variably penetrant pituitary insufficiency, including this severe and atypical presentation, can be correlated with LHX4 insufficiency and highlights the role of LHX4 for pituitary development.