Conformational differences of ovine and human corticotrophin releasing hormone A CD,IR, NMR and dynamic light scattering study

The differences in the conformational properties of ovine (o) and human (h) CRH in aqueous solution, structure-inducing TFE and in the presence of detergent micelles and lipid vesicles have been investigated by circular dichroism, Fourier transform infrared spectroscopy, NMR and dynamic light scattering. o-CRH was found to exist as a monomer with little regular structure in dilute aqueous solution. Association at concentrations higher than 10^?3 mol/L results predominantly in dimers. The induction of a substantial amount of intermolecular β-structure seems to be the result of interactions of the C-terminal hexapeptide and the N-terminal region 6-12 of o-CRH chains in antiparallel orientation. In contrast, h-CRH exhibits a high tendency of association which is highly sensitive to the pH. The formation of tetramers at millimolar peptide concentration is related to a helical content of ca. 50%. The potentially helical, highly hydrophobic region 6-20 enlarged by more hydrophobic residues in position 23 and 25 is proposed to stabilize the h-CRH associates. In the presence of structure inducing TFE (<40%v) both CRH peptides exist as monomers. o-CRH reveals about 72% helicity, in h-CRH the formation of about 85% helix is observed. The differences in helicity of the two CRH molecules are located in the C-terminal heptapeptide, as concluded on the basis of NMR studies. Both peptides bind to detergent micelles at pH 4 as well as 7.4 associated with an increase in the α-helical content. Interaction of the two peptides with DMPC vesicles was found exclusively at pH 4. Above the phase transition temperature of DMPC the α-helical content in h-CRH increases slightly; however, o-CRH reveals a substantial amount of β-type structure. The intramolecular type of β-structure is associated with a deeper insertion of the o-CRH region 6-12 into the hydrophobic region of the lipid bilayer, whereas the corresponding region of h-CRH is kept in the bilayer surface. The higher helicity of h-CRH might explain to some extent its higher affinity to the CRH receptor, CRH antibodies and the CRH binding protein. © Munksgaard 1996.     

Immune function during ageing in man: relation between serological abnormalities and cellular immune status

In a group of 176 apparently healthy aged people living at home (age range 62-86 years) the prevalences of monoclonal immunoglobulin and of autoantibodies did not differ from those found in other studies, but remarkably 38% of the people studied had cold lymphocytotoxic antibodies. The occurrence of serological abnormalities showed no age dependency. The participants were grouped according to serological abnormalities. The various groups showed no differences in peripheral blood mononuclear cell composition (concentration of B lymphocytes and T lymphocytes or of T lymphocyte subsets with OKT 4 and OKT 8 phenotype) or function (lymphocyte stimulation with phytohaemagglutinin, concanavalin A, pokeweed mitogen and antigen cocktail). Compared with a control group of young blood donors, the lymphocyte stimulation responses tended to be lower in the old age groups. It is concluded that, as measured in this study, humoral abnormalities during ageing are not associated with changes in B and T lymphocyte subsets or function.     

Alloantigen-specific T-cell anergy induced by human keratinocytes is abrogated upon loss of cell-cell contact

The activation of primary human T cells largely depends on the expression of both major histocompatibility complex (MHC) class II and B7 molecules on antigen-presenting cells (APC), whereas APC expressing HLA class II but not B7 antigens are expected to induce anergy. According to this concept, interferon-γ (IFN-γ)-activated keratinocytes (KC) expressing HLA class II but not B7 costimulatory antigens should be able to induce anergy. However, in terms of anergy versus activation contradicting data have been published on the outcome of interaction between T cells and human KC. In addition, it has been shown that human KC can express a B7-like molecule with unknown function, whereas MHC expression may be functionally impaired. To evaluate this item we transfected the human A431 KC cell line with B7-1 coding sequences and up-regulated HLA-DR by treatment with IFN-γ, yielding A431^DR,B7-1 cells. Irradiated A431^DR,B7-1 cells were found to be capable of inducing vigorous proliferative primary T-cell responses in resting allogeneic T cells, whereas A431^DR cells could induce proliferation only when interleukin-2 (IL-2) was added. These data indicate that KC can present alloantigens, and that lack of costimulatory molecules on KC is the main reason why these cells cannot induce primary T-cell responses. Surprisingly, however, no evidence could be obtained of stable anergy induction by A431^DR cells, as T cells contacted with A431^DR cells and then transferred to A431^DR,B7-1 cells clearly demonstrated alloresponsiveness. T-cell non-responsiveness was maintained only when T cells remained in contact with A431^DR cells. These data indicate that, despite expression of HLA class II in the absence of B7 costimulatory molecules, human KC cannot induce stable anergy but rather induce short-term anergy in primary resting T cells.     

Release of Cytokines and Soluble Cell Surface Molecules by PBMC after Activation with the Bispecific Antibody CD3 × CD19

Bispecific antibodies (BsAb) consist of two different heavy and light chains and may bind to two different antigens present on different cell types. With their dual specificity BsAb may recognize effector cells (e.g. T cells) on one hand and tumour cells (e.g. malignant B cells) on the other hand. The authors analysed whether T cell activation and subsequent killing of malignant B cells mediated by the bispecific antibody CD3 × CD19 was reflected by the release of cytokines. In addition, the authors investigated whether the in vitro cytokine release was similar to that observed in vivo in the patients treated with BsAb. The in vitro release of cytokines into the supernatant of cell cultures of peripheral blood mononuclear cells (PBMC) and malignant B cells was measured after incubation with either the bispecific antibody CD3 × CD19 or the monospecific anti-CD3 (aCD3) antibody in the presence or absence of interleukin (IL)-2. Release of tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-6, IL-8, IL-10, soluble (s) CD4, sCD8 and sCD25 by PBMC was equal under both conditions and could be used as an indicator for T cell activation. However, the cytokine pattern and level did not correlate with the cytotoxic capacity, which was 4 logs higher with BsAb + IL-2 compared to aCD3 + IL-2. The in vitro pattern of cytokine release was similar to that observed in vivo in the serum of patients treated with BsAb and IL-2, indicating the possibility of predicting cytokine release in future patients with other therapeutic regimens.     

Insulin aggregation in solution

The process of insulin aggregation in neutral solutions was studied by dynamic light scattering. Solutions of different concentrations were subjected to thermal and mechanical stress (37°, rotation) for a period of 4 weeks. The starting solutions contained exclusively one particle distribution of insulin in the association equilibrium with hexamers as the largest structures. After a lag period of about 8 days the solutions showed continuously increasing scattering intensities but did not evolve perceptible turbidity within the experimental period. A more rapid increase in scattering intensity was observed in diluted than in concentrated solutions. The analysis of scattering data unexpectedly revealed that insulin species did not grow continuously. After the lag period one additional relatively restricted size distribution with particles of a mean radius of about 100nm was found, the amount of which increased continuously with time. The occurrence of these particles seems to be related to adsorption phenomena of insulin to the solid interface. We assume the 100nm-class of aggregates to be a transient state in the physical destabilization process of insulin solutions.     

Immune Response in Asymptomatic Paraproteinemia

In a group of 20 patients with asymptomatic paraproteinemia, as judged after at least 3 years of follow-up, the primary and secondary antibody response to Helix pomatia hemocyanin (HPH) was defective as compared with the response in controls. The class of antibody was assessed by mercaptoethanol (ME) treatment of serum. A lowered response was found not only in the total but also in the ME-resistant (mainly 7S, IgG) antibody titer. Low anti-HPH antibody titers were preferentially found in the patients with high serum paraprotein levels, whereas in half of the patients with low serum paraprotein levels a completely normal antibody response was found. No differences in the total or 7S anti-HPH antibody response were found between patients with IgG, IgM, or IgA paraproteinemia. The polyclonal serum Ig levels were not predictive for the measured anti-HPH response. The anamnestic diphtheria and tetanus antibody response was not different from that of controls.     

Visualization of alveolar recruitment in a porcine model of unilateral lung lavage using 3He-MRI

Background: In the acute respiratory distress syndrome potentially recruitable lung volume is currently discussed. ³He‐magnetic resonance imaging (³He‐MRI) offers the possibility to visualize alveolar recruitment directly. Methods: With the approval of the state animal care committee, unilateral lung damage was induced in seven anesthetized pigs by saline lavage of the right lungs. The left lung served as an intraindividual control (healthy lung). Unilateral lung damage was confirmed by conventional proton MRI and spiral‐CT scanning. The total aerated lung volume was determined both at a positive end‐expiratory pressure (PEEP) of 0 and 10 mbar from three‐dimensionally reconstructed ³He images, both for healthy and damaged lungs. The fractional increase of aerated volume in damaged and healthy lungs, followed by a PEEP increase from 0 to 10 mbar, was compared. Results: Aerated gas space was visualized with a high spatial resolution in the three‐dimensionally reconstructed ³He‐MR images, and aeration defects in the lavaged lung matched the regional distribution of atelectasis in proton MRI. After recruitment and PEEP increase, the aerated volume increased significantly both in healthy lungs from 415 ml [270–445] (median [min–max]) to 481 ml [347–523] and in lavaged lungs from 264 ml [71–424] to 424 ml [129–520]. The fractional increase in lavaged lungs was significantly larger than that in healthy lungs (healthy: 17% [11–38] vs. lavage: 42% [14–90] (P=0.031). Conclusion: The ³He‐MRI signal might offer an experimental approach to discriminate atelectatic vs. poor aerated lung areas in a lung damage animal model. Our results confirm the presence of potential recruitable lung volume by either alveolar collapse or alveolar flooding, in accordance with previous reports by computed tomography.     

VALIDATION OF THE MOTIVATION FOR EATING SCALE

For items used in the Likert-type Motivation for Eating Scale (MFES), content domain was clearly specified and a panel of experts assessed the relevance of each item. Based on responses from 298 participants in the western United States, the MFES was evaluated for internal consistency and reliability using factor analysis and correlation techniques. The factor solution isolated four factors that replicated scale construction, including: environmental eating, emotional eating, physical eating, and social eating with alpha coefficients ranging from .75 to .95. Retesting after two weeks (N=88) yielded correlation coefficients that ranged between .55 and .77. Theorized relationships between subscale scores and certain demographic variables add support for concurrent validity. MFES subscales also correlated predictably with select subscales from the Emotional Eating Scale and the Three Factor Eating Questionnaire suggesting convergent validity (N=103). Findings provide tentative support for use of the MFES in community and college settings.