T-cell specificity in murine autoimmune haemolytic anaemia induced by rat red blood cells

Autoimmune haemolytic anaemia (AIHA) can be induced in mice by repeated injections with rat red blood cells (RBC). Here we describe the identification of rat and murine RBC antigens recognized by T-cells from mice with this disease. Splenic T-cells from mice with AIHA proliferated in response to multiple murine RBC membrane components, each of which is recognized by rat RBC induced autoantibodies. Thus, there were responses to murine autoantigen fractions that correspond in apparent molecular mass with the anion channel Band 3, with spectrin from the membrane skeleton and with the high and low molecular mass glycophorins, and the equivalent fractions from rat RBC also stimulated proliferation by T-cells. It was confirmed that purified Band 3 from murine and rat RBC also elicited responses. In contrast with the results in AIHA, T-cells from healthy control mice failed to respond to the antigens from either species, with the exception of proliferation induced by murine spectrin in one experiment and weak responses elicited by rat Band 3. It is suggested that T-cells activated by multiple cross-reactions between rat and murine RBC proteins, and by epitope spreading, are necessary to drive autoantibody production in this model of AIHA.     

The presence of immunosuppressive ''p15E-like'' factors in the serum and urine of patients suffering from malign and benign breast tumours.

Autoantibodies in sera from patients with systemic lupus erythematosus (SLE) and onchocerciasis recognize calreticulin (CaR), a calcium-binding protein, as antigen. In this study we present the immunological properties of two synthetic peptides prepared to correspond to the 1-24 and 7-24 amino acid sequence of CaR. In contrast to information previously reported for the recombinant protein, the CaR-peptide analogues appeared immunoreactive to anti-Ro/SSA autoimmune sera. Human sera from patients with SLE, Sjögren's syndrome (SS), rheumatoid arthritis (RA), as well as mixed connective tissue disease (MCTD), demonstrated a positive autoimmune response (binding of antibodies), to the CaR-peptide analogues. These findings suggest that anti-calreticulin autoantibodies are not restricted to any disease specificity.     

Preferential activation of peripheral blood V gamma 9+ gamma/delta T cells by group A, B and C but not group D or F streptococci.

Previous studies have established that inactivated mycobacteria are potent and selective activators of V gamma 9+/V delta 2+ human gamma/delta T cells. Here we have analysed the proliferative response of human gamma/delta T cells to five serologically distinct groups of streptococci. While heat-inactivated streptococci of all five serogroups tested (A, B, C, D and F) induced a strong proliferative response in peripheral blood mononuclear cells (PBMC), only groups A, B and C elicited a selective activation of V gamma 9+ gamma/delta T cells in 10 (serogroup B) or 11 (serogroups A and C) of 11 tested healthy individuals. In striking contrast, groups D and F streptococci failed to activate gamma/delta T cells in nine of 11 donors and induced only a weak gamma/delta T cell response in two additional individuals. Depletion of V gamma 9+ T cells before culture completely eliminated all gamma/delta T cell responses to streptococci. These data indicate that groups A, B and C (but not D or F) streptococci can be included in the growing list of selective ligands for V gamma 9+/V delta 2+ human gamma/delta T cells.     

The molecular basis for inherited bullous diseases

In the past 5 years enormous progress have been made in our understanding of the molecular basis for a number of inherited skin diseases characterized by easy blistering of the skin and the mucous membranes after minor physical trauma. This increased fragility of the skin or its appendages is due to molecular defects in genes coding for different intra- and extracellular structural proteins which are responsible for mechanical strength at their sites of expression. These diseases encompass the group of epidermolysis bullosa and disorders of cornification such as bullous forms of ichthyosis, palmoplantar keratoderma, and pachyonychia congenita. On the basis of clinical, morphological, and ultrastructural observations the epidermolysis bullosa group has been divided into three major categories. In epidermolysis bullosa simplex blister formation appears within the basal cell layer of the epidermis, and many mutations have been found in the genes of keratin 5 and 14 which are both expressed in basal keratinocytes. Epidermolytic hyperkeratosis leads to an epidermal separation in the suprabasal cell layers. In these patients numerous point mutations have now been described in the suprabasally expressed genes of keratin 1 and 10. In ichthyosis bullosa of Siemens blisters occur in the more upper suprabasal epidermis coincidental with the expression of keratin 2e, and mutations have been detected in the corresponding gene. In epidermolytic palmoplantar hyperkeratosis the suprabasal epidermal splitting is restricted to palms and soles of the patient. In keratin 9, which reveals such an exclusive expression pattern, molecular defects have indeed been recognized. Most recently in two different clinical subtypes of pachyonychia congenita, which is characterized by defective nails and focal palmoplantar hyperkeratosis, point mutations have been found in the genes coding for keratins 6, 16, and 17. In junctional epidermolysis bullosa the separation takes place within the dermal-epidermal basement membrane at the level of the lamina lucida, and mutations have been found in three genes coding for different laminin chains, in the ₄ gene of ₆₄ integrin, and in the gene of collagen XVII. In dystrophic epidermolysis bullosa the tissue separation occurs beneath the basement membrane within the papillary dermis at the level of the anchoring fibrils, and several mutations have been identified in the collagen VII gene. The rapid unraveling of molecular defects in these disabling or even lethal inherited skin diseases makes possible a more precise and earlier prenatal diagnosis, creates new options for suitable therapeutic regimens, and even offers the hope of curing these diseases by means of somatic cell gene therapy.Abbreviations BM Basement membrane - BPAg Bullous pemphigoid antigen - DEB Dystrophic epidermolysis bullosa - EB Epidermolysis bullosa - EBS Epidermolysis bullosa simplex - EHK Epidermolytic hyperkeratosis - EPPK Epidermolytic palmoplantar keratoderma - IBS Ichthyosis bullosa of Siemens - JEB Junctional epidermolysis bullosa - KIF Keratin intermediate filaments - NC Noncollagenous domain - NEPPK Nonepidermolytic palmoplantar keratoderma - PC Pachyonychia congenita     

Biochemical and morphological characterization of sympathetic neurons grown in a chemically-defined medium

Previous studies have demonstrated that individual neurons from neonatal rat superior cervical ganglion express a mixed adrenergic-cholinergic phenotype when grown under certain tissue culture conditions.^{9,14,15,29,30} The expression of this phenotype is critically influenced by a number of undefined components present in the culture medium.^18,23,33 In the present study, we have examined whether superior cervical ganglion neurons grown on a chemically defined serum-free medium similarly develop dual transmitter expression, or if under these conditions, neurons express only those properties characteristic of their adrenergic heritage. To address this issue, we established that superior cervical ganglion neurons could be maintained in culture for extended periods on the defined medium described by Bottenstein & Sato⁴ in the absence of supporting cells. We then studied the biochemical, immunocytochemical and ultrastructural characteristics of these neurons. We found that in defined medium, superior cervical ganglion neurons continued to express, in a modified form, certain of their expected adrenergic properties, including the development of tyrosine hydroxylase and dopamine-β-hydroxylase activities, stores of endogenous norepinephrine, synaptic vesicles with dense cores and tyrosine hydroxy lase-immunoreactive staining properties. Superior cervical ganglion neurons grown on a defined medium did not, however, acquire cholinergic traits in culture. In this paper we show that choline acetyltransferase activity did not reach detectable levels; the companion paper¹³ documents that cholinergic synapses were not formed.We conclude that superior cervical ganglion neurons, grown under serum-free culture conditions, develop certain properties characteristic of adrenergic neurons and do not express a mixed adrenergic cholinergic phenotype. A companion paper¹³ describes the electrophysiological properties of these neurons and demonstrates the frequent occurrence of electrotonic synapses in these cultures.     

3D MRI-Based Multicomponent FSI Models for Atherosclerotic Plaques

A three-dimensional (3D) MRI-based computational model with multicomponent plaque structure and fluid-structure interactions (FSI) is introduced to perform mechanical analysis for human atherosclerotic plaques and identify critical flow and stress/strain conditions which may be related to plaque rupture. Three-dimensional geometry of a human carotid plaque was reconstructed from 3D MR images and computational mesh was generated using Visualization Toolkit. Both the artery wall and the plaque components were assumed to be hyperelastic, isotropic, incompressible, and homogeneous. The flow was assumed to be laminar, Newtonian, viscous, and incompressible. The fully coupled fluid and structure models were solved by ADINA, a well-tested finite element package. Results from two-dimensional (2D) and 3D models, based on ex vivo MRI and histological images (HI), with different component sizes and plaque cap thickness, under different pressure and axial stretch conditions, were obtained and compared. Our results indicate that large lipid pools and thin plaque caps are associated with both extreme maximum (stretch) and minimum (compression when negative) stress/strain levels. Large cyclic stress/strain variations in the plaque under pulsating pressure were observed which may lead to artery fatigue and possible plaque rupture. Large-scale patient studies are needed to validate the computational findings for possible plaque vulnerability assessment and rupture predictions.     

Subtyping of Mycoplasma pneumoniae isolates based on extended genome sequencing and on expression profiles

Mycoplasma pneumoniae isolates from patients, collected over a period of 12 years in Germany, were characterized by various methods (parameters) including multilocus sequence typing, restriction fragment length polymorphisms, Western blotting with mono-specific antibodies directed against selected proteins or with polyspecific antibodies directed against the Triton X-114-soluble protein fraction, and two-dimensional gel electrophoresis. The results for 91 isolates from Germany, which were complemented with 14 isolates from the USA and 10 isolates from France, clearly showed that M. pneumoniae is a highly uniform species and that most of the isolates could be assigned to one of the two subtypes 1 and 2. The members of one subtype differ from the other with respect to the sequence of the P1 gene, the ORF6 gene, the P65 gene, and by a typical DNA restriction fragment pattern. We observed four isolates (variants), which seemed identical by the above mentioned criteria, but did not belong to either one of the two subtypes. They showed most of the subtype 2-specific features, but differed in the sequence of the P1 gene and showed a variation in the restriction fragment pattern. The appearance of subtype 1 or 2 over the last 12 years in Germany showed a dominance of subtype 1 between 1989 and 1996 and a dominance of subtype 2 between 1997 and 1998. The variant (neither subtype 1 nor subtype 2) was only detected in 1991 and 1995 but it had no epidemiological consequences.     

A subset of gamma delta lymphocytes is increased during HIV-1 infection.

The gamma delta T cell receptor (TcR) lymphocytes constitute 3-10% of human peripheral blood lymphocytes. Only a very small fraction of these cells is recognized by the delta TCS1 monoclonal antibody, directed against the V delta 1 chain of the receptor. We describe the immunological, virological and clinical data of a small group of seropositive subjects having high levels of gamma delta TcR T cells in the peripheral blood. Our flow cytometric studies show that most of these cells belong to the delta TCS1+ (V delta 1+), CD8 +/- (dim staining) subset. Patients with high gamma delta TcR T cell numbers were not characterized by the presence of an acute (IgM positive) or reactivated (as defined by high IgG titres against early antigen or IgA titres against viral capsidic antigen) Epstein-Barr virus infection. Cytomegalovirus infection was excluded by serological assays, and other herpes viral infections were not found after clinical examination. HIV p24 antigenaemia was present in two out of 11 subjects. AIDS patients had very high percentages of gamma delta TcR T cells. Altogether these data show that the selective expansion of delta TCS1+ cells in HIV1 seropositive subjects is not related to some exogenous antigen stimulation, but may be related to peculiar pathologic processes involving the immune system.     

Separate and variably shaped chromosome arm domains are disclosed by chromosome arm painting in human cell nuclei

Fluorescence in situ hybridization (FISH) with microdissection probes from human chromosomes 3 and 6 was applied to visualize arm and subregional band domains in human amniotic fluid cell nuclei. Confocal laser scanning microscopy and quantitative three-dimensional image analysis showed a pronounced variability of p- and q-arm domain arrangements and shapes. Apparent intermingling of neighbouring arm domains was limited to the domain surface. Three-dimensional distance measurements with pter and qter probes supported a high variability of chromosome territory folding.