Transplanted human pancreatic islets after long-term insulin independence.

Long-term insulin independence after islets of Langerhans transplantation is rarely achieved. The aims of this study were to identify the histological and immunological features of islets transplanted in a type 1 diabetic patient who died of a cerebral hemorrhage after >13 years insulin independence. Islets were pooled from two donors with respectively one and five HLA mismatches. Insulin-positive islets were found throughout the right and left liver, and absent in the pancreas. Two- and three-dimensional analysis showed that islets lost their initial rounded and compact morphology, had a mean diameter of 136 µm and were constituted of an unfolded epithelial band of 39.1 µm. Leukocyte phenotyping showed no evidence of a tolerogenic environment in the islet-containing portal spaces. Finally, HLA typing of microdissected islets showed HLA from the best matched donor in all 23 microdissection samples, compared to 1/23 for the least matched donor. This case report demonstrates that allogeneic islets can survive over 13 years while maintaining insulin independence. Allogeneic islets had unique morphologic features and implanted in the liver regardless of their size. Finally, our results suggest that, in this case, rejection had been prevalent over autoimmunity, although this hypothesis warrants further investigation.

Immune response to bacterial dextrans. II. T cell control of antibody isotypes.

The isotype distribution of Dextran B 512 (Dex)-specific plaque-forming cells (PFC) and serum antibodies was studied after in vivo immunization in C57BL/6 mice. Although IgG2b and IgG3 could also be detected in most individuals, the majority of non-IgM PFC were of the IgA isotype. All classes other than IgM were T cell-dependent, as shown by their complete absence in athymic "nude" mice. This unusual isotype pattern was further investigated by studying the antibody responses to the same Dex epitope coupled to a protein carrier, and to a different hapten coupled to the carrier Dex or to a protein. The results show that IgA responses are epitope-related and selectively associated with anti-Dex antibodies: no IgA PFC are detected against a hapten coupled to Dex or proteins, while the enhanced levels of helper cell reactivity provided by protein carrier to Dex result in the appearance of IgG1 antibodies in addition to IgA. These results indicate that T cells that modulate isotype patterns in these responses can discriminate between Dex- and DNP-specific B cells in the response to the same carrier. Since the same idiotype is detected on a large fraction of the IgM and IgA anti-Dex response and antiidiotypic helper cells have previously been detected in normal C57BL/6 mice, we suggest that idiotype-specific T cells control the production of IgA antibodies upon immunization with Dex.

Immunocompetent autoreactive B lymphocytes are activated cycling cells in normal mice.

Frequencies of B cell clonal precursors producing antibodies that react with mouse thyroglobulin, mouse erythrocytes, beef hemoglobin, KLH, and sheep erythrocytes were determined by limiting dilution analyses among small, resting lymphocytes, and among large activated cells from normal adult mice. While frequencies of clones reacting with external antigens were equally distributed in large and small B cells, most, if not all, autoreactive B lymphocytes were found in the large cell fraction. Analysis of antithyroglobulin hybridomas isolated from normal mice revealed dissociation constants ranging from 10(-6) to 5-6 X 10(-7). Treatment of normal donors with antimitotic drugs dramatically decreases the frequencies of autoreactive B cells, but not those of B lymphocytes reacting with external antigenic molecules. Taken together, these experiments show that immunocompetent, autoreactive B lymphocytes are activated and cycling cells in the peripheral lymphoid tissues of normal individuals.

Regulation of B lymphocyte development by the truncated immunoglobulin heavy chain protein Dmu.

The development of B lymphocytes from progenitor cells is dependent on the expression of a pre-B cell-specific receptor made up by a mu heavy chain associated with the surrogate light chains, immunoglobulin (Ig)alpha, and Igbeta. A variant pre-B cell receptor can be formed in which the mu heavy chain is exchanged for a truncated mu chain denoted Dmu. To investigate the role of this receptor in the development of B cells, we have generated transgenic mice that express the Dmu protein in cells of the B lineage. Analysis of these mice reveal that Dmu expression leads to a partial block in B cell development at the early pre-B cell stage, probably by inhibiting VH to DHJH rearrangement. Furthermore, we provide evidence that Dmu induces VL to JL rearrangements.

Cytotoxic T lymphocyte antigen 4 is induced in the thymus upon in vivo activation and its blockade prevents anti-CD3-mediated depletion of thymocytes.

The development of a normal T cell repertoire in the thymus is dependent on the interplay between signals mediating cell survival (positive selection) and cell death (negative selection or death by neglect). Although the CD28 costimulatory molecule has been implicated in this process, it has been difficult to establish a role for the other major costimulatory molecule, cytotoxic T lymphocyte antigen (CTLA)-4. Here we report that in vivo stimulation through the T cell receptor (TCR)-CD3 complex induces expression of CTLA-4 in thymocytes and leads to the association of CTLA-4 with the SH2 domain-containing phosphatase (SHP)-2 tyrosine phosphatase. Moreover, intrathymic CTLA-4 blockade dramatically inhibits anti-CD3-mediated depletion of CD4+CD8+ double positive immature thymocytes. Similarly, anti-CD3-mediated depletion of CD4+CD8+ double positive cells in fetal thymic organ cultures could also be inhibited by anti-CTLA-4 antibodies. Thus, our data provide evidence for a role of CTLA-4 in thymic selection and suggest a novel mechanism contributing to the regulation of TCR-mediated selection of T cell repertoires.

Imaging the pancreas: from ex vivo to non-invasive technology.

While many recently published reviews have covered non-invasive nuclear imaging techniques, the aim of this review is to focus on current developments in optical imaging technologies for investigating the pancreas. Several of these modalities are being developed into non-invasive, real-time monitoring routines for pancreatic diseases. However, they also provide pre-clinical ex vivo and/or intravital tools for three-dimensional quantitative assessments of cellular and molecular events, with levels of specificity and resolution difficult to achieve with other currently available modalities.

Cryohypophysectomy used in the treatment of a case of feline acromegaly.

A 10-year-old female spayed cat was diagnosed with acromegaly secondary to a pituitary tumour. At the time of diagnosis, the cat had insulin-resistant diabetes mellitus and its insulin-like growth factor-I levels were elevated. Clinical signs included polyuria, polydipsia and weight gain. Persistent hyperglycaemia and glucosuria were identified, and fructosamine levels remained elevated. Magnetic resonance imaging of the brain showed a pituitary tumour. Transsphenoidal cryohypophysectomy was used to treat the pituitary tumour. Postoperatively, the serum insulin-like growth factor-I levels decreased and the diabetes mellitus was controlled with routine levels of insulin. To the authors' knowledge, this is the second reported case of acromegaly treated with cryohypophysectomy, and the first that reports a favourable long-term outcome. Cryohypophysectomy may be a safe and effective treatment for cats with a pituitary mass resulting in acromegaly.

The biomechanical properties of the feline femur.

The purpose of this study was to determine the biomechanical properties of feline long bone by testing cadaver bone from mature cats in compression, three-point bending, notch sensitivity and screw pull-out strength. The determination of these properties is of clinical relevance with regard to the forces resulting in long bone fractures in cats as well as the behaviour and failure mode of surgical implants utilized for fracture stabilization and repair in the cat. Cadaveric cat femurs were tested in compression, three-point bending and in three-point bending after the addition of a 2.0 mm screw hole. Cortical screws, 2.7 mm in diameter, were inserted in cadaveric cat femur samples for screw pull-out testing. The mean maximum load to failure of mid diaphyseal feline femurs tested in compression was 4201+/-1218 N. Statistical analysis of the parameter of maximum load tested in compression revealed a statistical difference between sides (p=0.02), but not location (p=0.07), or location by side (p=0.12). The maximum strength of mid diaphyseal feline femurs tested in compression was 110.6+/-26.6 MPa. The modulus of elasticity of mid-diaphyseal cat femurs tested in compression was determined to be 5.004+/-0.970 GPa. The mean maximum load to failure of feline femurs tested in three-point bending was 443+/-98 N. The mean maximum load to failure of feline femurs tested in three-point bending after a 2.0 mm diameter hole was drilled in the mid-diaphyseal region of each sample through both cortices was 471+/-52 N. The mean maximum load required for screw pull-out of 2.7 mm cortical screws placed in feline femurs tested in tension was 886+/-221 N. This data should be suitable for investigating fracture biomechanics and the testing of orthopaedic constructs commonly used for fracture stabilization in the feline patient.

Evaluation of a short glass fibre-reinforced tube as a model for cat femur for biomechanical testing of orthopaedic implants.

The biomechanical testing of tubes made of third generation short glass fibre-reinforced (SGFR) material approximating cat femurs was performed in order to determine their suitability as cat femur surrogates for the biomechanical testing of orthopaedic implants. The tubes were tested in compression, three-point bending, notch testing, and screw pullout. Thin walled (B1-tubes) had a 13% lower maximum load to failure, a 19% higher maximum strength and a 13% lower elastic modulus compared to cat femurs tested in compression. B1-tubes maximum load to failure in three-point bending and screw pullout strength were considerably lower compared to cat femurs (29% and 63%, respectively). Notch testing was not performed on B1-tubes due to low bending strength. Thicker walled (B2-tubes) had a 23% higher maximum load to failure, a 10% higher maximum strength and a 21% lower elastic modulus compared to cat femurs tested in compression. The comparison of B2-tubes and cat femurs in three-point bending revealed a 7% increase in maximum load to failure for the B2-tubes. Drilled B2-tubes (notch testing) were weaker with a 30% lower load to failure compared to cat femurs. A screw pullout comparison of B2-tubes and cat femurs revealed a 2% increase in maximum load to failure for the B2-tubes. These tubes were intended to provide a model as a suitable surrogate for cat femurs for testing the bending strength of various orthopaedic constructs involving plates and screws. Testing revealed that third generation SGFR tubes were not suitable for these purposes and emphasizes the need to carefully evaluate the suitability of any model.

Study of electrophoretic mobility of cellular membranes isolated from rat liver.

Plasma membranes as well as mitochondrial and microsomal subfractions were subjected to zone electrophoresis. Treatment with neuraminidase, phospholipase A or C does not influence the movement of plasma membranes and smooth microsomes. Trypsin increases mobility of plasma membranes and smooth by about 20%, and further treatment with phospholipase C decreases mobility of plasma membranes, total smooth and smooth I microsomes, which, however, is not the case with smooth II microsomes. Low concentrations of trypsin also solubilize enzyme proteins of smooth microsomes from phenobarbital-treated rat liver, but electrophoretic mobility is not increased, indicating structural differences in induced membranes. The mobility of the outer and inner mitochondrial membranes is significantly higher than that of submitochondrial particles. For microsomes the negative surface charge density occurs in the decreasing order of: ribosomes--rough--smooth I--smooth II. A 10 mM CsCl gradient decreases the mobility of rough microsomes by 40% and of ribosomes by 20% but has no effect on total smooth micromes. On the other hand, 5mM MgCl2 decreased the mobility of all three fractions. EDTA-treated rough and EDTA-treated smooth microsomes have the same electrophoretic mobilities. However, the mobilities of non-treated rough and smooth microsomes differ significantly from each other.

Immunoglobulin VH gene replacements in a T-cell lymphoblastic lymphoma.

We have analysed the rearrangement status of the immunoglobulin heavy (IgH) chain locus during progression of a T-cell lymphoblastic lymphoma displaying multiple IgH rearrangements as demonstrated by variable heavy (VH) gene family specific polymerase chain reaction (PCR) analysis. The tumor was found to undergo diversification at the IgH locus between diagnosis and relapse through a mechanism of VH to VHDJH replacement. In subsets of the tumor at relapse, two separate VH gene segments were found to have replaced the VH gene utilized by a VHDJH rearrangement identified at diagnosis. The observed VH gene replacement events appear to have been mediated by a heptamer sequence homologous to the heptamer of the recombination signal sequence (RSS) located internally in the VH gene segment. These results support the notion that VH replacements contribute to the diversification of immunoglobulin genes.

Establishment and characterization of a mouse strain (TLL) that spontaneously develops T-cell lymphomas/leukemia.

In this study, a mouse strain (TLL) that spontaneously develops T-cell lymphomas/leukemia with an early onset and high incidence was established and characterized. All tumors analyzed were found to express the alpha,beta T-cell receptor, and the majority of them had a mature, CD3+CD4+CD8- immunophenotype. In a few cases, tumors with a more immature CD3+CD4+CD8+ phenotype were isolated. Expanded phenotyping using a broad panel of lymphocyte differentiation markers confirmed the mature T-cell phenotype of the tumors. Histologic and cell cycle analysis of the tumors revealed an aggressive lymphoblastic malignancy with a very high proliferation rate and widespread engagement of bone marrow and lymphoid as well as nonlymphoid organs. Thus, the TLL mouse strain represents a unique model for the analysis of the oncogenesis and progression of mature T-cell tumors and for the development of therapeutic measures to combat such tumors.

Transgenic Cre recombinase expression in germ cells and early embryogenesis directs homogeneous and ubiquitous deletion of loxP-flanked gene segments.

We report on the establishment of a transgenic mouse line expressing Cre recombinase under control of the c-kit promoter. Expression of Cre recombinase was only observed in late spermatogenesis and oogenesis, however, Cre-mediated deletion of floxed gene segments occurred at this stage as well as in early embryogenesis. As a consequence of this, a chimeric distribution of loxed alleles was found in a large fraction of these mice. The chimerism was very homogeneous in different organs and tissues of the same individual but varied between different individual offspring. The potential uses for this mouse line are discussed.

Functional expression of Cre recombinase in sub-regions of mouse CNS and retina.

We describe a strategy for generating CNS and retina sub-region-specific mutations using the Cre/loxP system. Transgenic mice expressing Cre recombinase under the control of the c-kit promoter were established. Functional Cre expression was predominantly found to be restricted to the CA1, CA2 and CA3 regions of the hippocampus, the anterior region of the dentate gyrus, and to the ganglion cell layer of the retina.

Identification of a susceptibility locus for migraine with and without aura on 6p12.2-p21.1.

Migraine is the most common type of chronic episodic headache. To find novel susceptibility genes for familial migraine with and without aura, a genomewide screen was performed in a large family from northern Sweden. Evidence of linkage was obtained on chromosome 6p12.2-p21.1, with a maximum two-point lod score of 5.41 for marker D6S452. The patients with migraine shared a common haplotype of 10 Mb between markers D6S1650 and D6S1960.

A rapid screening technique for monoclonal antibodies with specificity for protein antigens.

A rapid technique for screening crude supernatants from antibody-producing hybridomas against protein antigen is described. The technique involves blotting of crude hybridoma supernatants to nitrocellulose filters and revealing specific binding using radiolabeled antibodies or antigen.

Idiotypic characterization of antibody-induced antibody responses.

Anti-idiotypic antisera were produced in syngeneic (C57BL/6) mice against a monoclonal anti-Dextran B512 (Dex) antibody (38-13). In radioimmunoassays, anti-idiotypic antibodies were shown to react with the homologous idiotype, while failing to recognize another monoclonal anti-Dex antibody, independently derived from C57BL/6 mice (D-16). Plaque inhibition tests confirmed the specificity of the anti-idiotypic antibodies and revealed that the 38-13 idiotype is expressed by about half of all anti-Dex antibodies produced in C57BL/6, but not in CBA mice. Injection of normal (but not athymic) C57BL/6 mice with low doses of 38-13 monoclonal antibodies, contained culture supernatants or ascitic fluids, resulted in a 10-20 fold increase in the numbers of anti-Dex PFC detected in the spleen 5 days later, the majority of which carried the 38-13 idiotype.

Analysis of VH gene utilisation in the non-obese diabetic mouse.

The immunoglobulin (Ig) heavy chain variable (VH) gene complexity and the VH gene utilisation pattern of the non-obese diabetic (NOD) mouse were investigated. We found that the NOD mouse displays a VH gene complexity which appears to be identical to that of the C57BL/6 mouse. Thus, Southern hybridisation using probes specific for 9 of the murine VH gene families revealed identical restriction fragment length polymorphism (RFLP) patterns in both mouse strains. As indicated by immunofluorescence analysis using allotype specific monoclonal antibodies the NOD mice were also found to carry the IgCH-1b allele. Collectively, these data suggest that the NOD mice carry an IgVH locus identical to that carried by C57BL/6. In contrast to the apparent identity at the level of germline VH gene repertoires, the pattern of VH gene utilisation differed considerably between these two mouse strains. Thus, in NOD mice the neonatal preference of D-proximal VH genes was found to be more pronounced than in C57BL/6 mice. Moreover, in contrast to adult C57BL/6 mice a D-proximal bias was evident also in adult NOD mice. On the basis of these findings we discuss the possibility that the distorted development of B cell repertoires in the NOD mouse could be directly or indirectly related to the T cell mediated, autoimmune process in the NOD mouse.

Activities and well-being in older age: effects of self-concept and educational attainment.

The positive effect of activities on well-being is proposed to be mediated by self-conceptualizations and facilitated by socioeconomic status. The hypothesized processes were estimated with LISREL VIII using data from a large cross-sectional survey with a sample of 679 adults aged 65 and older who were representative of older adults living in the Detroit area. Findings indicate that the frequency of performing both leisure and productive activities yields an effect on physical health and depression and that these effects are mediated in part by a sense of self as agentic, but less clearly by a sense of self as social. Furthermore, socioeconomic status, operationalized as formal educational attainment, facilitates the effect of leisure to a greater extent than that of productive activities.

Distraction osteogenesis in the irradiated canine mandible.

The potential use of distraction osteogenesis in mandibular reconstruction has been limited by its questionable efficacy in previously radiated bone. We studied five mongrel dogs that had a hemimandible rendered edentulous and underwent a full course of external beam radiation therapy (50 Gy/20 fractions). Six months after completion of radiotherapy, a 2-cm critical-size segmental mandibular defect was created and stabilized with a stainless steel plate. A proximal mandibular transport segment was then formed and an external lengthening apparatus (Orthofix) applied. By means of bifocal distraction osteogenesis, the defects were filled with new bone in a period of 30 days in four of five dogs. Histologic analysis and fluorochrome microscopy confirmed the formation of new cortical bone. Our results suggest that distraction osteogenesis is successful in previously radiated bone and that it may be a simple method of mandibular reconstruction following ablative head and neck surgery.