Bisphenol-A-glycidyldimethacrylate (BisGMA) is used in many resin-based dental materials. It was shown in vitro that BisGMA was released into the adjacent biophase from such materials during the first days after placement. In this study, the uptake, distribution, and excretion of [14C]BisGMA applied via gastric and intravenous administration (at dose levels well above those encountered in dental care) were examined in vivo in guinea pigs to test the hypothesis that BisGMA reaches cytotoxic levels in mammalian tissues. [14C]BisGMA was taken up rapidly from the stomach and intestine after gastric administration and was widely distributed in the body following administration by each route. Most [14C] was excreted within one day as 14CO2. The peak equivalent BisGMA levels in guinea pig tissues examined were at least 1000-fold less than known toxic levels. The peak urine level in guinea pigs that received well in excess of the body-weightadjusted dose expected in humans was also below known toxic levels. The study therefore did not support the hypothesis.

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1) pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1) residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

Objectives: To study the clinical outcome, treatment

response, T-cell subsets and functional consequences of a

novel tumour necrosis factor (TNF) receptor type 1

(TNFRSF1A) mutation affecting the receptor

cleavage site.

Methods: Patients with symptoms suggestive of tumour

necrosis factor receptor-associated periodic syndrome

(TRAPS) and 22 healthy controls (HC) were screened for

mutations in the TNFRSF1A gene. Soluble TNFRSF1A and

inflammatory cytokines were measured by ELISAs.

TNFRSF1A shedding was examined by stimulation of

peripheral blood mononuclear cells (PBMCs) with phorbol

12-myristate 13-acetate followed by flow cytometric

analysis (FACS). Apoptosis of PBMCs was studied by

stimulation with TNFa in the presence of cycloheximide

and annexin V staining. T cell phenotypes were monitored

by FACS.

Results: TNFRSF1A sequencing disclosed a novel V173D/

p.Val202Asp substitution encoded by exon 6 in one

family, the c.194–14G.A splice variant in another and

the R92Q/p.Arg121Gln substitution in two families.

Cardiovascular complications (lethal heart attack and

peripheral arterial thrombosis) developed in two V173D

patients. Subsequent etanercept treatment of the V173D

carriers was highly effective over an 18-month follow-up

period. Serum TNFRSF1A levels did not differ between

TRAPS patients and HC, while TNFRSF1A cleavage from

monocytes was significantly reduced in V173D and R92Q

patients. TNFa-induced apoptosis of PBMCs and T-cell

senescence were comparable between V173D patients

and HC.

Conclusions: The TNFRSF1A V173D cleavage site

mutation may be associated with an increased risk for

cardiovascular complications and shows a strong

response to etanercept. T-cell senescence does not seem

to have a pathogenetic role in affected patients.

Objective: Liver regeneration is mainly based on cellular

self-renewal including progenitor cells. Efforts have been

made to harness this potential for cell transplantation, but

shortage of hepatocytes and premature differentiated

progenitor cells from extra-hepatic organs are limiting

factors. Histological studies implied that resident cells in

adult liver can proliferate, have bipotential character and

may be a suitable source for cell transplantation.

Methods: Particular cell populations were isolated after

adequate tissue dissociation. Single cell suspensions were

purified by Thy-1 positivity selection, characterised in vitro

and transplanted in immunodeficient Pfp/Rag2 mice.

Results: Thy-1+ cells that are mainly found in the portal

tract and the surrounding parenchyma, were isolated from

surgical liver tissue with high yields from specimens with

histological signs of regeneration. Thy-1+ cell populations

were positive for progenitor (CD34, c-kit, CK14, M2PK,

OV6), biliary (CK19) and hepatic (HepPar1) markers

revealing their progenitor as well as hepatic and biliary

nature. The potential of Thy-1+ cells for differentiation in

vitro was demonstrated by increased mRNA and protein

expression for hepatic (CK18, HepPar1) and biliary (CK7)

markers during culture while progenitor markers CK14,

chromogranin A and nestin were reduced. After

transplantation of Thy-1+ cells into livers of immunodeficient

mice, engraftment was predominantly seen in the

periportal portion of the liver lobule. Analysis of in situ

material revealed that transplanted cells express human

hepatic markers HepPar1 and albumin, indicating functional

engraftment.

Conclusion: Bipotential progenitor cells from human

adult livers can be isolated using Thy-1 and might be a

potential candidate for cell treatment in liver diseases.

Systemic mastocytoses represent neoplastic proliferations

of mast cells. In about 20% of cases systemic

mastocytoses are accompanied by clonal haematopoietic

non-mast cell-lineage disorders, most commonly myeloid

neoplasms. A case of systemic mastocytosis carrying the

characteristic mutation at codon 816 (D816V) in the KIT

gene of mast cells, with two concurrent accompanying

clonal haematopoietic non-mast cell-lineage disorders,

chronic myeloproliferative disease, unclassifiable and

precursor B lymphoblastic leukaemia is documented. Both

accompanying clonal haematopoietic non-mast cell-lineage

disorders carried the wild-type KIT gene, but had a

novel t(13;13)(q12;q22) involving the FLT3 locus at

13q12. The chronic myeloproliferative disease, unclassifiable

and the precursor B lymphoblastic leukaemia were

cured by syngenous stem cell transplantation, but the

systemic mastocytosis persisted for more than 10 years.

The additional impact of molecular techniques on the

correct diagnosis in haematological malignancies is

highlighted, and evidence is provided that, apart from

internal tandem duplications and mutations, FLT3 can be

activated by translocations.

Acute unilateral peripheral and central vestibular lesions

can cause similar signs and symptoms, but they require

different diagnostics and management. We therefore

correlated clinical signs to differentiate vestibular neuritis

(40 patients) from central ‘‘vestibular pseudoneuritis’’ (43

patients) in the acute situation with the final diagnosis

assessed by neuroimaging. Skew deviation was the only

specific but non-sensitive (40%) sign for pseudoneuritis.

None of the other isolated signs (head thrust test,

saccadic pursuit, gaze evoked nystagmus, subjective

visual vertical) were reliable; however, multivariate

logistic regression increased their sensitivity and specificity

to 92%.

Background: Immunodeficiency, centromeric instability

and facial dysmorphism (ICF syndrome) is a rare

autosomal recessive disease characterised by facial

dysmorphism, immunoglobulin deficiency and branching

of chromosomes 1, 9 and 16 after PHA stimulation of

lymphocytes. Hypomethylation of DNA of a small fraction

of the genome is an unusual feature of ICF patients which

is explained by mutations in the DNA methyltransferase

gene DNMT3B in some, but not all, ICF patients.

Objective: To obtain a comprehensive description of the

clinical features of this syndrome as well as genotype–

phenotype correlations in ICF patients.

Methods: Data on ICF patients were obtained by

literature search and additional information by means of

questionnaires to corresponding authors.

Results and conclusions: 45 patients all with proven

centromeric instability were included in this study. Facial

dysmorphism was found to be a common characteristic

(n=41/42), especially epicanthic folds, hypertelorism,

flat nasal bridge and low set ears. Hypo- or agammaglobulinaemia

was demonstrated in nearly all patients

(n=39/44). Opportunistic infections were seen in several

patients, pointing to a T cell dysfunction. Haematological

malignancy was documented in two patients. Life

expectancy of ICF patients is poor, especially those with

severe infections in infancy or chronic gastrointestinal

problems and failure to thrive. Early diagnosis of ICF is

important since early introduction of immunoglobulin

supplementation can improve the course of the disease.

Allogeneic stem cell transplantation should be considered

as a therapeutic option in patients with severe infections

or failure to thrive. Only 19 of 34 patients showed

mutations in DNMT3B, suggesting genetic heterogeneity.

No genotype–phenotype correlation was found between

patients with and without DNMT3B mutations.

Background: DLG5 p.R30Q has been reported to be

associated with Crohn disease (CD), but this association

has not been replicated in most studies. A recent analysis

of gender-stratified data from two case–control studies

and two population cohorts found an association of DLG5

30Q with increased risk of CD in men but not in women

and found differences between 30Q population frequencies

for males and females. Male–female differences in

population allele frequencies and male-specific risk could

explain the difficulty in replicating the association with CD.

Methods: DLG5 R30Q genotype data were collected for

patients with CD and controls from 11 studies that did not

include gender-stratified allele counts in their published

reports and tested for male–female frequency differences

in controls and for case–control frequency differences in

men and in women.

Results: The data showed no male–female allele

frequency differences in controls. An exact conditional

test gave marginal evidence that 30Q is associated with

decreased risk of CD in women (p=0.049, OR=0.87,

95% CI 0.77 to 1.00). There was also a trend towards

reduced 30Q frequencies in male patients with CD

compared with male controls, but this was not significant

at the 0.05 level (p=0.058, OR=0.87, 95% CI 0.74 to

1.01). When data from this study were combined with

previously published, gender-stratified data, the 30Q allele

was found to be associated with decreased risk of CD in

women (p=0.010, OR=0.86, 95% CI 0.76 to 0.97), but

not in men.

Conclusion: DLG5 30Q is associated with a small

reduction in risk of CD in women.

Introduction: A lack of studies on the effects of continuous blood pressure biofeedback led to the development of a screening and feedback software allowing for a `beat-to-beat' representation of arterial blood pressure. Through this method patients find that they can exert some control of their blood pressure. Patients and Methods: For evaluation purposes a pilot study was conducted with 84 hypertensive patients at the Klinik Hohenried, Germany. Two experimental groups which received the normal treatment program of the hospital and an additional 2 (20 patients) or 3-6 (22 patients) biofeedback sessions, respectively, were compared with one control group treated additionally with pseudo biofeedback (20 patients) and another control group (22 patients) which underwent the clinic program without biofeedback. Results: Both experimental groups yielded significantly more markedly blood pressure reductions (MAP) than the controls (p = 0.001), but patients who received 3-6 feedback sessions did not perform significantly better than patients who received only 2 sessions (p = 0.517). Conclusions: 2 sessions of feedback treatment can already have clinically relevant effects. This suggests a major influence of cognitive processes such as self-effectiveness and self-control.