Genetic and phenotypic overlap of specific obsessive-compulsive and attention-deficit/hyperactive subtypes with Tourette syndrome.

BACKGROUND: The unique phenotypic and genetic aspects of obsessive-compulsive (OCD) and attention-deficit/hyperactivity disorder (ADHD) among individuals with Tourette syndrome (TS) are not well characterized. Here, we examine symptom patterns and heritability of OCD and ADHD in TS families. METHOD: OCD and ADHD symptom patterns were examined in TS patients and their family members (N = 3494) using exploratory factor analyses (EFA) for OCD and ADHD symptoms separately, followed by latent class analyses (LCA) of the resulting OCD and ADHD factor sum scores jointly; heritability and clinical relevance of the resulting factors and classes were assessed. RESULTS: EFA yielded a 2-factor model for ADHD and an 8-factor model for OCD. Both ADHD factors (inattentive and hyperactive/impulsive symptoms) were genetically related to TS, ADHD, and OCD. The doubts, contamination, need for sameness, and superstitions factors were genetically related to OCD, but not ADHD or TS; symmetry/exactness and fear-of-harm were associated with TS and OCD while hoarding was associated with ADHD and OCD. In contrast, aggressive urges were genetically associated with TS, OCD, and ADHD. LCA revealed a three-class solution: few OCD/ADHD symptoms (LC1), OCD & ADHD symptoms (LC2), and symmetry/exactness, hoarding, and ADHD symptoms (LC3). LC2 had the highest psychiatric comorbidity rates (⩾50% for all disorders). CONCLUSIONS: Symmetry/exactness, aggressive urges, fear-of-harm, and hoarding show complex genetic relationships with TS, OCD, and ADHD, and, rather than being specific subtypes of OCD, transcend traditional diagnostic boundaries, perhaps representing an underlying vulnerability (e.g. failure of top-down cognitive control) common to all three disorders.

Parental mood during pregnancy and post-natally is associated with offspring risk of Tourette syndrome or chronic tics: prospective data from the Avon Longitudinal Study of Parents and Children (ALSPAC).

Little is known about risk factors for Tourette syndrome (TS) and chronic tic disorders (CT) but maternal psychological morbidity in pregnancy may be associated with TS/CT. We examined whether pre- and post-natal parental anxiety and/or depression are associated with risk of TS/CT in the Avon Longitudinal Study of Parents and Children. We compared self-reported anxiety and depression measures collected prospectively at four time points (18 and 32 weeks prenatally, and 8 weeks and 8 months post-natally) among parents of children who subsequently met criteria for TS/CT at 13 years of age as compared to other children from the cohort. We adjusted for various socioeconomic measures and tested both for time period-specific exposure and chronic exposure using multivariable logistic regression models. 122 children had TS/CT (50 TS, 72 CT) and 5968 children had no tics. In crude analyses, both pre- and post-natal maternal anxiety and depression, but only post-natal paternal depression at 8 months, showed associations with TS/CT. In the final, adjusted multivariable models, chronic maternal anxiety (odds ratio 2.17, 95% CI 1.23, 3.84, p = 0.007) and pre-natal maternal depression (odds ratio 1.86, 95% CI 1.02, 3.39, p = 0.04) showed associations with TS/CT though the latter was consistent with chance (p = 0.07) after adjustment for past maternal depression. We find associations between maternal psychological morbidity pre- and post-natally and risk of future TS/CT in offspring. These associations may reflect either shared genetic susceptibility or a pre-natal exposure. Further work is required to see if these findings can be replicated in larger datasets.

Genome-wide association study of obsessive-compulsive disorder.

Obsessive-compulsive disorder (OCD) is a common, debilitating neuropsychiatric illness with complex genetic etiology. The International OCD Foundation Genetics Collaborative (IOCDF-GC) is a multi-national collaboration established to discover the genetic variation predisposing to OCD. A set of individuals affected with DSM-IV OCD, a subset of their parents, and unselected controls, were genotyped with several different Illumina SNP microarrays. After extensive data cleaning, 1465 cases, 5557 ancestry-matched controls and 400 complete trios remained, with a common set of 469,410 autosomal and 9657 X-chromosome single nucleotide polymorphisms (SNPs). Ancestry-stratified case-control association analyses were conducted for three genetically-defined subpopulations and combined in two meta-analyses, with and without the trio-based analysis. In the case-control analysis, the lowest two P-values were located within DLGAP1 (P=2.49 × 10(-6) and P=3.44 × 10(-6)), a member of the neuronal postsynaptic density complex. In the trio analysis, rs6131295, near BTBD3, exceeded the genome-wide significance threshold with a P-value=3.84 × 10(-8). However, when trios were meta-analyzed with the case-control samples, the P-value for this variant was 3.62 × 10(-5), losing genome-wide significance. Although no SNPs were identified to be associated with OCD at a genome-wide significant level in the combined trio-case-control sample, a significant enrichment of methylation QTLs (P<0.001) and frontal lobe expression quantitative trait loci (eQTLs) (P=0.001) was observed within the top-ranked SNPs (P<0.01) from the trio-case-control analysis, suggesting these top signals may have a broad role in gene expression in the brain, and possibly in the etiology of OCD.

Genome-wide association study of Tourette's syndrome.

Tourette's syndrome (TS) is a developmental disorder that has one of the highest familial recurrence rates among neuropsychiatric diseases with complex inheritance. However, the identification of definitive TS susceptibility genes remains elusive. Here, we report the first genome-wide association study (GWAS) of TS in 1285 cases and 4964 ancestry-matched controls of European ancestry, including two European-derived population isolates, Ashkenazi Jews from North America and Israel and French Canadians from Quebec, Canada. In a primary meta-analysis of GWAS data from these European ancestry samples, no markers achieved a genome-wide threshold of significance (P<5 × 10(-8)); the top signal was found in rs7868992 on chromosome 9q32 within COL27A1 (P=1.85 × 10(-6)). A secondary analysis including an additional 211 cases and 285 controls from two closely related Latin American population isolates from the Central Valley of Costa Rica and Antioquia, Colombia also identified rs7868992 as the top signal (P=3.6 × 10(-7) for the combined sample of 1496 cases and 5249 controls following imputation with 1000 Genomes data). This study lays the groundwork for the eventual identification of common TS susceptibility variants in larger cohorts and helps to provide a more complete understanding of the full genetic architecture of this disorder.

Identification of a candidate modifying gene for spinal muscular atrophy by comparative genomics.

Spinal muscular atrophy (SMA) is a common recessive disorder characterized by the loss of lower motor neurons in the spinal cord. The disease has been classified into three types based on age of onset and severity. SMA I-III all map to chromosome 5q13 (refs 2,3), and nearly all patients display deletions or gene conversions of the survival motor neuron (SMN1) gene. Some correlation has been established between SMN protein levels and disease course; nevertheless, the genetic basis for SMA phenotypic variability remains unclear, and it has been postulated that the loss of an additional modifying factor contributes to the severity of type I SMA. Using comparative genomics to screen for such a factor among evolutionarily conserved sequences between mouse and human, we have identified a novel transcript, H4F5, which lies closer to SMN1 than any previously identified gene in the region. A multi-copy microsatellite marker that is deleted in more than 90% of type I SMA chromosomes is embedded in an intron of this gene, indicating that H4F5 is also highly deleted in type I SMA chromosomes, and thus is a candidate phenotypic modifier for SMA.

Meta-analysis of association between obsessive-compulsive disorder and the 3' region of neuronal glutamate transporter gene SLC1A1.

The neuronal glutamate transporter gene SLC1A1 is a candidate gene for obsessive-compulsive disorder (OCD) based on linkage studies and convergent evidence implicating glutamate in OCD etiology. The 3' end of SLC1A1 is the only genomic region with consistently demonstrated OCD association, especially when analyzing male-only probands. However, specific allele associations have not been consistently replicated, and recent OCD genome-wide association and meta-analysis studies have not incorporated all previously associated SLC1A1 SNPs. To clarify the nature of association between SLC1A1 and OCD, pooled analysis was performed on all available relevant raw study data, comprising a final sample of 815 trios, 306 cases and 634 controls. This revealed weak association between OCD and one of nine tested SLC1A1 polymorphisms (rs301443; uncorrected P = 0.046; non-significant corrected P). Secondary analyses of male-affecteds only (N = 358 trios and 133 cases) demonstrated modest association between OCD and a different SNP (rs12682807; uncorrected P = 0.012; non-significant corrected P). Findings of this meta-analysis are consistent with the trend of previous candidate gene studies in psychiatry and do not clarify the putative role of SLC1A1 in OCD pathophysiology. Nonetheless, it may be important to further examine the potential associations demonstrated in this amalgamated sample, especially since the SNPs with modest associations were not included in the more highly powered recent GWAS or in a past meta-analysis including five SLC1A1 polymorphisms. This study underscores the need for much larger sample sizes in future genetic association studies and suggests that next-generation sequencing may be beneficial in examining the potential role of rare variants in OCD.

A 45-year-old female with hypokalemic rhabdomyolysis due to VIP-producing composite pheochromocytoma.

The watery diarrhea, hypokalemia and achlorhydria (WHDA) syndrome due to vasoactive intestinal polypeptide (VIP)-producing extra-pancreatic tumors is rare. We report on a 45-year-old woman who suffered from persistent secretory diarrhea for six years and who was admitted to hospital with complaints of muscular weakness and myalgia. Biochemical testing revealed pronounced rhabdomyolysis due to severe hypokalemia. Gastrointestinal evaluation of long-standing diarrhea including endoscopy of the upper and lower gastrointestinal tract and the small intestine did not show any pathologies. An abdominal computed tomography scan revealed a mass of 4 × 5 cm in the left adrenal gland demonstrating a strong uptake in the 123I-labelled metaiodobenzylguanidine scintigraphy. Plasma levels of chromogranin A, calcitonin, parathormone, basal renin and most prominently VIP were increased in line with a increased 24 hour urinary secretion of noradrenaline, dopamine, normetanephrine and vanillymandelic acid. A WDHA (watery diarrhea, hypokalaemia, achlorhydria) syndrome with hypokalemic rhabdomyolysis due to a VIP-producing adrenal tumor was diagnosed that was removed surgically. The histological evaluation demonstrated a composite pheochromocytoma. Diarrhea stopped immediately after surgery together with a normalization of laboratory parameters. In conclusion, this case report focuses on the rare clinical presentation of secretory diarrhea and electrolyte disturbances in combination with hypokalemic rhabdomyolysis which was caused by a VIP-producing composite pheochromocytoma.

Characterization of direct and indirect cerebral revascularization for the treatment of European patients with moyamoya disease.

BACKGROUND: The best revascularization strategy for moyamoya disease (MMD) remains unknown. Our aim was to characterize angiographic revascularization effects of a bilateral standardized revascularization approach, consisting of superficial temporal artery (STA)-middle cerebral artery (MCA) bypass and encephalomyosynangiosis (EMS) on one hemisphere and single EMS on the contralateral hemisphere of each patient, and to compare the effects of both revascularization strategies on cerebral hemodynamics. METHODS: In 30 patients (18 females/12 males, age 8-63 years), standardized revascularization was performed. Digital subtraction angiography was performed preoperatively and at 7 days, 6 months and 12 months postoperatively. STA-MCA and EMS functions were graded I (poor), II (medium) or III (extensive) according to angiographic aspects. In 20 patients, cerebrovascular reserve capacity (CVRC) was assessed pre- and postoperatively (at 12 months) using xenon CT. RESULTS: After 12 months, STA-MCA/EMS function was grade 1 in 40/40%, grade 2 in 27/26%, and grade 3 in 27/10% of hemispheres, respectively. Twelve months after surgery, single EMS showed grade I in 37%, grade II in 27%, and grade III in 20% of hemispheres. Combined revascularization improved CVRC significantly compared to preoperative measurement (preoperative: 16.5 ± 34.6% vs. postoperative: 60.8 ± 64.22%; p < 0.05). Single EMS did not improve CVRC significantly (preoperative: 21.8 ± 35.9% vs. postoperative: 34.8 ± 63.0%; p < 0.05). CONCLUSIONS: Combined and indirect revascularization may be successfully applied in a bilateral standardized approach. STA-MCA/EMS is superior to single EMS in restoring CVRC in adult MMD patients.

Effect of mouth opening on bypass function after combined revascularization for Moyamoya disease.

Moyamoya disease represents a rare steno-occlusive disease of the internal carotid artery (ICA) with a reactive and pathological basal network of collateral vessels. It may lead to ischemic stroke or intracerebral hemorrhage. Treatment options are either direct or indirect revascularization procedures or a combination thereof. Specialized centers report sufficient revascularization in most patients and low complication rates.Between 2005 and 2008, direct extra-intracranial bypass surgery in combination with encephalomyosynangiosis (EMS) was performed in 71 Moyamoya patients at the Mannheim University Medical Center.Following one case of reversible neurological deficits associated with mouth opening, we prospectively evaluated the effect of mouth opening on bypass function in this patient and four further consecutive patients by digital subtraction angiography.Three out of five patients showed alterations in bypass patency upon mouth opening. The obstruction was located at the junction of the bypass and the temporal muscle. Two temporary occlusions and one case of decreased flow were observed. One patient demonstrated reversible hemiparesis and aphasia.

Multiple post-translational modifications of mouse insulin-like growth factor binding protein-6 expressed in epithelial Madin-Darby canine kidney cells.

Insulin-like growth factors (IGFs), IGF receptors and IGF binding proteins (IGFBPs) participate in the regulation of proliferation and differentiation of epithelial cells. Expression of the growth-inhibitory murine IGFBP-6 in epithelial Madin-Darby canine kidney (MDCK) cells followed by 2D analysis revealed the presence of multiple isoforms. Metabolic labelling experiments showed that several IGFBP-6 isoforms are modified by phosphate and sulfate groups. Expression analysis of mutant IGFBP-6 further demonstrated that serine residue 143 is O-glycosylated. Substitution of serine 143 by alanine did slightly reduce the preferential sorting of mIGFBP-6 to the apical site in MDCK cells grown on semipermeable filters. Both the presence of multiple and heterogeneously modified isoforms of murine IGFBP-6 in MDCK cells, and the preferential secretion of non-glycosylated IGFBP-6 mutants to the apical side suggest that the major apical sorting signal is the protein moiety.

[Molecular principles of alternative treatment approaches for hormone-refractory prostate cancer]. / Molekulare Grundlagen alternativer Therapieansätze für das hormonrefraktäre Prostatakarzinom.

Prostate cancer is more frequently diagnosed in men from Western countries than from Asian societies. Therefore, nutritional factors such as phyto-oestrogens from soya are considered to cause this prostate cancer prevention effect. As there is no curative therapy for hormone-refractory prostate cancer, new strategies are in demand which might include phyto-oestrogens or inhibitors of histone deacetylases. Both approaches have in common the potential to reduce the aberrant androgen receptor and IGF receptor signalling. Furthermore, invasiveness and acquired survival strategies of tumours can be diminished. Reduced tumour cell proliferation and PSA secretion coincide with altered gene expression in the aforementioned processes. In addition, selective knock-down of genes by RNA interference afforded functional analyses regarding impact and succession of expression events involved in the beneficial effects caused by phyto-oestrogens and histone deacetylase inhibitors.

Left ventricular volume during supine exercise: importance of myocardial scar in patients with coronary heart disease.

Existing studies suggest that exercise-induced ischemia produces an increase in left ventricular end-diastolic volume; however, all of these studies have included patients with previous myocardial infarction. To test whether the end-diastolic volume response to exercise is related to the extent of myocardial scar, the results of gated radionuclide supine exercise tests performed on 130 subjects were reviewed. The patient group comprised 130 subjects were reviewed. The patient group comprised 130 men aged 35 to 65 years (mean +/- SD 52 +/- 5) with documented coronary heart disease. The extent of myocardial ischemia and scar formation was assessed by stress electrocardiography and thallium-201 scintigraphy. Patients were classified into three groups on the basis of left ventricular end-diastolic volume response at peak exercise: group 1 (n = 72) had an increase of end-diastolic volume greater than 10%, group 2 (n = 41) had a change in end-diastolic volume less than 10% and group 3 (n = 17) had a decrease in end-diastolic volume greater than 10% (n = 17). At rest there was no significant difference among groups in heart rate, systolic blood pressure, end-diastolic (EDVrest) or end-systolic volumes or ejection fraction (p greater than 0.05); however, at peak exercise the end-systolic volume response was significantly greater for group 1 (p less than 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of insulin-like growth factor-I and insulin-like growth factor binding proteins during thioacetamide-induced liver cirrhosis in rats.

OBJECTIVE: The liver plays a central role in insulin-like growth factor (IGF) homeostasis providing the majority of circulating IGF-I and some of its binding proteins (IGFBPs). In liver cirrhosis the IGF axis is severely disturbed, and these alterations are associated with reduced IGF-I, IGFBP-3 but elevated IGFBP-1 serum levels. METHODS: By Northern blotting and in situ hybridization (ISH), hepatic expression of IGF-I and of IGFBP was studied in a rat model of liver cirrhosis induced by thioacetamide. RESULTS: ISH revealed a homogeneous distribution of IGFBP-1, IGFBP-4 and IGF-I mRNA over hepatic parenchyma in normal and cirrhotic liver. Fibrous septa of cirrhotic liver were IGFBP-1 mRNA negative, whereas IGFBP-4 and IGF-I transcripts were detected in single cells. In normal liver, IGFBP-3 mRNA was distributed within nonparenchymal cells of the hepatic lobule and in the wall of the portal vein. In cirrhotic liver, IGFBP-3 transcripts were abundant in mesenchymal cells of fibrous tissue. IGFBP-3 mRNA expression was also prominent in cells at the septal-nodular interface most likely representing monocyte infiltration. IGFBP-3 mRNA expression was reduced in nonparenchymal liver cells located more distantly from the septal-nodular interface in the cirrhotic nodule that correlated with reduced IGFBP-3 mRNA expression observed in Kupffer cells (KC) and sinusoidal endothelial cells (SEC) isolated from macronodular cirrhotic livers. CONCLUSION: Cirrhosis is accompanied by an altered spatial expression of IGFBP-3 in liver tissue, which is characterized by decreased levels of IGFBP-3 mRNA in KC and SEC, but elevated IGFBP-3 expression in myofibroblast-like cells and inflammatory infiltrate.

Thyrohyoid approach to cidofovir injection: a case study.

Various techniques for injection into the larynx have been well described, including transoral injection with indirect laryngoscopy or direct laryngoscopy and transcutaneous injection through the thyroid cartilage or cricothyroid membrane. We describe a case not amenable to these techniques, in which entry through the thyrohyoid membrane for injection of cidofovir was successfully performed with a thyrohyoid approach. This technique is described, and comparisons are made with currently available techniques.

De novo aneurysm of the anterior communicating artery presenting with subarachnoid hemorrhage 7 years after initial cryptogenic subarachnoid hemorrhage: a case report and review of the literature.

Spontaneous subarachnoid hemorrhage (SAH) is usually caused by a ruptured cerebral aneurysm. Despite the use of initial four-vessel cerebral digital subtraction angiography (DSA), 15 % of all cases remain idiopathic. According to the initial computed tomographic scan, the spontaneous SAH can be divided into a perimesencephalic group associated with a benign nature and a nonperimesencephalic group with a similar clinical course as aneurysmal SAH. We present a case of a 49-year-old man with a de novo aneurysm formation of the anterior communicating artery with SAH 7 years after initial cryptogenic nonperimesencephalic SAH. This observation suggests that in some cases, long-term angiographic studies might be justified.

Glyceraldehyde 3-phosphate dehydrogenase activity in rat and human lenses and the fate of enzyme molecules in the aging lens.

The specific activity of the enzyme glyceraldehyde 3-phosphate dehydrogenase declines as a function of age. Immunotitration with monospecific antibodies demonstrated that with age there is an increase of catalytically defective, but antigenically reactive enzyme molecules in the lens. Antiserum, produced against denatured enzyme, removed the inactive molecules from lens homogenates, without effects on the levels of enzyme activity. These studies suggest that inactive enzyme molecules in aging lenses are totally devoid of catalytic activity, and are at least partially denatured.

CD4-binding of gp130 micelles isolated from SIVagmTYO-7.

The binding and binding inhibition of the SIVagmTYO-7 external glycoprotein gp130 in micellar form to the CD4 molecule on human Molt-4 clone 8 cells was investigated. The best binding of gp130 to Molt-4 clone 8 cells occurred at pH 5.5 to 6.5 at 37 degrees C after 4 h or at room temperature after 10 h. The dissociation constant of this reaction was 0.2-0.4 nM, with both soluble CD4 or CD4 on Molt-4 clone 8 cells. This value is close to 0.15 nM determined for the antihuman CD4 monoclonal antibody 30F16H5. After partial deglycosylation of gp130, a 90 kD product arose which still bound to CD4. Fully deglycosylated gp130 of 60 kD was still immunoprecipitable, but had lost the CD4 binding activity. Lens culinaris agglutinin was able to inhibit the gp130-CD4 interaction very efficiently, while the agglutinin of Phaseolus vulgaris was half as efficient and Canavalia ensiformis was inefficient. CD4 binding of gp130 micelles was also inhibited with several anti CD4 monoclonal antibodies directed against the OKT4a epitope as well as with soluble recombinant CD4.

Tri-iodothyronine increases insulin-like growth factor binding protein-4 expression in rat hepatocytes.

Previous in vivo studies demonstrated significant variations in insulin-like growth factor binding protein-1 (IGFBP-1), IGFBP-2 and IGFBP-4 hepatic mRNAs and/or serum levels depending on the rat thyroid status. In this study we employed cultured hepatocytes from adult rats to demonstrate a possible direct regulation of these genes by tri-iodothyronine (T3). Northern blot analysis revealed that IGFBP-1 and -4 messages were clearly expressed, whereas IGFBP-2 signal was barely detectable. No significant effects on IGFBP-1 mRNA level or on peptide secretion were detected in T3-cultured hepatocytes. In contrast, significant increases in IGFBP-4 mRNA steady-state levels as well as in IGFBP-4 secretion were observed in hepatocytes cultured for 12-24 h in the presence of T3. The T3 effect on IGFBP-4 transcript levels appears to consist of enhanced gene transcription and is independent of ongoing protein synthesis. The T3-increased IGFBP-4 expression in cultured hepatocytes is consistent with our in vivo experiments demonstrating an increase in hepatic IGFBP-4 mRNA and serum IGFBP-4 levels in T3-treated rats. Furthermore, significant decreases in hepatic IGFBP-4 message and serum IGFBP-4 levels were observed in hypothyroid rats compared with euthyroid controls. Our data establish an important direct role for thyroid hormone in regulating IGFBP-4 expression and consequently IGF activity.

Improved target volume definition for precision radiotherapy planning of meningiomas by correlation of CT and dynamic, Gd-DTPA-enhanced FLASH MR imaging.

In this methodological paper the authors report a fast, T1-weighted gradient-echo sequence (FLASH) for dynamic, Gd-DTPA-enhanced magnetic resonance (MR) imaging of meningiomas and its application in precision radiotherapy planning. Indications for radiotherapy included unresected tumors, tumor remaining after surgery, and recurrences. The patient's head was fixed in a stereotactic localization system which is usable at the CT, MR and the linear accelerator installations. By phantom measurements different materials (steel, aluminum, titanium, plastic, wood, ceramics) used for the stereotactic system were tested for mechanical stability and geometric MR image distortion. All metallic stereotactic rings (closed rings made of massive metal) led to a more or less dramatic geometric distortion and signal cancellation in the MR images. The best properties--nearly no distortion and high mechanic stability--are provided by a ceramic ring. If necessary, the remaining geometric MR image distortion can be 'corrected' (reducing displacements to the size of a pixel) by calculations based on modeling the distortion as a fourth order two-dimensional polynomial. The target volume was defined in dynamic, T1-weighted FLASH MR images, which were measured before, during, and after the controlled intravenous infusion of 0.1 mmol/kg body weight Gd-DTPA. The stereotactic localization technique allows the precise transfer of the target volume information from MR onto CT data to provide a map of the radiation attenuation coefficient for dose calculation. In genera, the superior soft tissue contrast of MR showed an excellent tumor delineation, especially in regions, such as the base of the skull, where the target often was obscured in CT images.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse insulin-like growth factor binding protein-6: expression, purification, characterization and histochemical localization.

The mitogenic and metabolic activities of insulin-like growth factors (IGF) are modulated by a family of six high affinity IGF binding proteins (IGFBPs). This study describes the expression of the mouse IGFBP-6 which is unique among IGFBPs in its preferential binding of IGF II, in insect cells using the baculovirus system. The purified, O-glycosylated IGFBP-6 was functional as shown by IGF binding and by inhibition of IGF II-stimulated DNA synthesis in human fibroblasts. Specific antibodies generated in chicken against the recombinant IGFBP-6 were used for Western blotting analysis and immunohistochemistry. Strong immunoreactivity was found in ossifying bones of the cranial base, in cell clusters of the pancreas anlage, in the trigeminal ganglion, on myoblasts, on motoneurons of the spinal cord of embryonic mice. In tissues of adult mouse, strong IGFBP-6 immunostaining was present in epidermal and peridermal layers of the skin, in meningeal layers, in long-striated skeletal muscle, and in the Langerhans' islets of the pancreas. No immunopositive staining was observed in lung and liver indicating that sites of synthesis and IGFBP action are different.