Lamina propria plasma cells in inflammatory bowel disease: intracellular detection of immunoglobulins using flow cytometry

This is the first application of flow cytometry for the detection of lamina propria plasma cells and their intracellular immunoglobulins in patients with inflammatory bowel disease compared to healthy controls. The study has been focused on the distribution of IgA, IgG, IgM and the four IgG subclasses. Plasma cells were detected as high CD38 positive cells. For fixation and permeabilisation a single step reagent, Ortho Permeafix®, was used.

By flow cytometry, in patients with inflammatory bowel disease compared to healthy controls, a higher percentage of IgG⁺ cells can be observed, in Crohn's disease also a higher percentage of IgM⁺ cells. Regarding the IgG subclass distribution, patients with Crohn's disease show an increase in IgG2⁺ cells, patients with ulcerative colitis an increase in IgG1⁺ and IgG3⁺ cells. These results do agree with and expand the results of earlier immunohistochemical and functional studies, which are favoured today. For the determination of lymphocyte subset proportions and the detection of intracellular antigens, flow cytometry provides a useful alternative to well-established immunohistochemical methods. By analysing a larger number of cells, this method is more reproducible and less prone to interobserver variations than immunohistochemistry, which needs the pre-selection of a mucosal area, the microscopic scoring of a limited number of cells and the circumvention of high background staining. The optimized flow cytometric protocol used in this study might be a promising tool for further investigations of various purposes.     

Diabetes insipidus renalis

Ohne Zusammenfassung     

Indolent systemic mastocytosis with elevated serum tryptase, absence of skin lesions, and recurrent severe anaphylactoid episodes

BACKGROUND: In contrast to aggressive mastocytosis, patients with indolent systemic mastocytosis (ISM) usually present with urticaria pigmentosa-like skin lesions. In those who lack skin lesions, mastocytosis is often overlooked or confused with endocrinologic, allergic, or other internal disorders. CASE REPORT AND RESULTS: We report on a 33-year-old male patient in whom severe hypotensive episodes occurred after contact with ants or yellow jackets. Since no specific IgE was detected, the serum tryptase concentration was measured and found to be clearly elevated (70 ng/ml). Consecutive staging and examination of the bone marrow revealed ISM. The patient was advised to circumvent insect contact, to take antihistamines on demand, and to carry an epinephrine self-injector for emergency events. In a retrospective analysis of 40 patients seen between 1988 and 2003, only 2 had a life-threatening mediator-related episode before ISM was diagnosed. CONCLUSIONS: Our report confirms the diagnostic value of tryptase in patients with suspected mastocytosis. In addition, the report suggests that the lack of typical skin lesions does not exclude an indolent form of mastocytosis even if the serum tryptase is clearly elevated. Finally, our case further shows that mastocytosis can be an important differential diagnosis to be considered in patients with unexplained anaphylactoid or other mediator-related symptoms.     

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Basic renal physiology for molecular biologists

Journal of Molecular Medicine -     

Significant association of strictures and internal fistula formation in Crohn’s disease

Intestinal inflammation in Crohn’s disease (CD) may be complicated by the occurrence of strictures and fistulae. The pathogenesis of fistula formation is unknown. We therefore wanted to determine whether mechanical factors might contribute to the development of fistulae. Furthermore, we tried to define the path of internal fistulae through the muscular layer. For this purpose, surgical resection specimens from 42 consecutive patients with CD were prospectively studied. In gross examination the whole bowel was cut into circumferential cross sections 0.3 cm thick. Abnormal areas were histologically examined. Strictures were found in 38 patients (90.5%), and fistulae were observed in 27 (64.3%) patients. In 11 (40.7%) specimens fistulae were found within a stricture, in 15 (55.6%) at the proximal end, and in 1 (3.7%) no stricture was found. In 7 (25.9%) cases with fistulae, herniated mucosa was found within the muscularis propria or the subserosa. In 7 (25.9%) cases a blood vessel was identified near a fistula traversing the muscularis propria. From these findings we conclude that that mechanical factors may contribute to fistula formation. This is further supported by the fact that fistulae appear to traverse the muscular layer along piercing vessels. Received: 21 January 2000 / Accepted: 7 March 2000     

Temperature rising elution fractionation of a random ethene/styrene copolymer

A random ethene/styrene copolymer containing 13.8 mol-% styrene was prepared with the Ziegler-Natta catalyst system Me₂Si(Me₄Cp)(N-t-butyl)TiCl₂/methylaluminoxane and characterized by means of preparative temperature rising elution fractionation (TREF) combined with size exclusion chromatography, NMR, differential scanning calorimetry and wide-angle X-ray scattering analyses of the copolymer fractions. Efforts are made to describe the distribution of the styrene content of the copolymers using the Stockmayer-Tacx distribution function. Both, comonomer distribution and molar mass distribution strongly support the presence of a single type of catalytically active center.     

The anti-apoptotic factor Bcl-2 can functionally substitute for the B cell survival but not for the marginal zone B cell differentiation activity of BAFF

The TNF family ligand B cell-activating factor (BAFF, BLyS, TALL-1) is an essential factor for B cell development. BAFF binds to three receptors, BAFF-R, transmembrane activator and CAML interactor (TACI), and B cell maturation antigen (BCMA), but only BAFF-R is required for successful survival and maturation of splenic B cells. To test whether the effect of BAFF is due to the up-regulation of anti-apoptotic factors, TACI-Ig-transgenic mice, in which BAFF function is inhibited, were crossed with transgenic mice expressing FLICE-inhibitory protein (FLIP) or Bcl-2 in the B cell compartment. FLIP expression did not rescue B cells, while enforced Bcl-2 expression restored peripheral B cells and the ability to mount T-dependent antibody responses. However, many B cells retained immaturity markers and failed to express normal amounts of CD21. Marginal zone B cells were not restored and the T-independent IgG3, but not IgM, response was impaired in the TACI-IgxBcl-2 mice. These results suggest that BAFF is required not only to inhibit apoptosis of maturating B cells, but also to promote differentiation events, in particular those leading to the generation of marginal zone B cells.     

No influence of atopic diseases on antibody titres following tetanus,diphtheria and hepatitis B immunisation among adults

Several studies have reported associations between reduced humoral immune response to vaccine antigens and diseases with modified reactions of the immune system. We have investigated the influence of atopic diseases on specific IgG levels to tetanus, diphtheria and hepatitis B (HB), following immunisation, in a general adult population. From the Study of Health in Pomerania, a total number of 3,920 subjects aged 20 to 79 years were included in the analyses. Information on immunisation history, as well as behavioural and socio-demographic characteristics were collected. Anti-tetanus IgG, anti-diphtheria IgG and anti-HBs IgG were measured by indirect enzyme-linked immunosorbent assay (ELISA). Odds ratios and 95% confidence intervals were calculated using logistic regression. Atopic diseases were reported by 14% of participants. Proportions of 67%, 34% and 10% had been vaccinated against tetanus, diphtheria and hepatitis B within the past ten years, respectively. Multi-variable analyses revealed no associations between the presence of atopic diseases and all of the three vaccine-specific antibody titres. We conclude that there is no reduced immune response related to antibody production following immunisations against tetanus, diphtheria and hepatitis B in adults with atopic diseases.     

Mechanismus der photopolymerisation des styrols, 4. Zur rolle der photo-(4π + 2π)-addukte als kettenüberträger und zur kinetik der photopolymerisation in gegenwart überlagerter,thermischer prozesse

The photopolymerization of styrene was investigated at 25°C, by following the variations in the rate of polymerization and the degree of polymerization with the light intensity, J_M, at different wavelengths of the exciting light (λ_e = 313 nm, λ_e = 335 nm). The corresponding data were analyzed in terms of the formalism between the rate R_P and the viscosity average degree of polymerization P _η. The kinetic evaluation shows that at least at small fractions of the absorbed light and at short duration of the irradiation the stabilization of the growing chain radicals will occur predominantly by bimolecular termination processes and not by chain transfer. This must be attributed to the fact that among the four theoretically possible isomeric photo-(4π + 2π)-intermediates (I) 1-phenyl-1,2,3,8a-tetrahydronaphthalene (diastereomers 1a and 1b ) and 2-phenyl-1,2,3,8a-tetrahydronaphthalene (diastereomers 1 ′ a and 1 ′ b ) the photostationary concentration [ 1a ]_∞ of the most reactive chain transfer agent 1a is—as a result of its very small quantum yield of formation ( φ_1a ? φ_1b + φ_1′a + φ_1′b )—extremely low and contributes only a few percent to the total photo-steady-state concentration of I. At higher temperatures (T > 25°C) and at small fractions of the absorbed light J_M/J_o (λ_e = 335 nm), the rate of photopolymerization R_P is controlled by the contributions of two independent routes of initiation: (1) the rate R, which accounts for the photo-free radical formation of the photo-intermediates I, and 2 the rate R, which is derived from the photoinduced free radical generation of the thermally produced Diels-Alder adducts 1a and 1b . As a further aspect of this analysis the results give strong evidence that the endo adduct 1a , when electronically excited, enters into a very efficient radical forming process, contrary to the other isomers 1b , 1 ′ b , and 1 ′ a , which seem to be mainly deactivated by their individual retro-Diels-Alder decompositions and will not enter into free radical formation to any noticeable extent: φ_S ^1a ? (φ_S ^1b + φ_S ^{1 ′ b} + φ_S ^{1 ′ a} ) An explanation of the very different stereodynamic behaviour is given.