IL-4 and IL-10 modulate autoimmune haemolytic anaemia in NZB mice

New Zealand Black (NZB) mice spontaneously develop autoimmune haemolytic anaemia (AIHA). Here the effect of injecting NZB mice with plasmids encoding IL-4 (pIL-4) or IL-10 (pIL-10) on NZB disease was tested. Both constructs delayed the development of anaemia as judged by increased haematocrit values as compared with controls, but neither altered the IgG1 to IgG2 red blood cell (RBC) bound autoantibody levels. The increased haematocrit value was associated temporally with increased RBC bound IgG in NZB mice treated with pIL-10, but not pIL-4. By contrast, up-regulation of splenic macrophage FcgammaRIIb2 mRNA was associated temporally with increased haematocrit values in NZB mice given pIL-4. However, no such increase occurred in NZB mice that inhaled a peptide containing a dominant T-cell epitope, although this treatment is known to bias the autoimmune response towards Th2 and to reduce the severity of anaemia. It is considered that IL-4 treatment, in part, ameliorates NZB anaemia by increasing the expression of the inhibitory FcgammaRIIb2 and thereby reducing the capacity of splenic macrophages to phagocytose autoantibody coated RBC, but that this mechanism does not explain the beneficial effects of the inhaled peptide.     

Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes

Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.     

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.     

Regulation of human tonsillar T-cell proliferation by the active metabolite of vitamin D3.

We have examined the effects of 1,25(OH)2D3 on T-cell populations isolated by buoyant density and E rosetting from human tonsils. Cell proliferation was assessed by measuring the incorporation of 125iododeoxyuridine; interleukin-2 (IL-2) production was measured using an IL-2-dependent cell line, and the number of 1,25(OH)2D3 receptors was measured by whole-cell nuclear association assay. At a concentration of 10(-7) M, 1,25(OH)2D3 inhibited mitogen-induced T-cell proliferation in all E+ T-cell populations. This effect was more pronounced in the cells from the intermediate and high density layers and was reflected both in cell proliferative responses and in relative IL-2 synthesis. By adding the 1,25(OH)2D3 during the course of the mitogen assay, we demonstrated that activation of the T cell precedes the 1,25(OH)2D3-mediated inhibition. Cells that had been preincubated with mitogen in the presence of the 1,25(OH)2D3 were refractory to further stimulation by mitogens. Receptors for 1,25(OH)2D3 could not be detected in unstimulated T cells. However, activation led to the expression of high-affinity receptors for 1,25(OH)2D3. Co-incubation of the cells with mitogen and 1,25(OH)2D3 increased the number of receptors compared with mitogen alone. The effects provide further evidence for the hypothesis that 1,25(OH)2D3 is an important potential modulator of the immune system through its action on T cells. Taking our observations in conjunction with the known capacity of monocytes to hydroxylate the precursor metabolite (and thus synthesize the active form of cholecalciferol), the results support the suggestion that 1,25(OH)2D3 plays a role as a local mediator of mononuclear phagocyte-T cell interaction in human lymphomedullary tissues.     

The partnership between breast cancer advocates and scientists

The National Breast Cancer Coalition (NBCC) is a grassroots organization that represents breast cancer advocates and is committed to eradicating breast cancer. NBCC defines a breast cancer advocate as someone who has been personally affected by the disease (e.g., a breast cancer survivor, family member, or friend), represents a constituency, and is motivated to join the fight against the disease. One of the organization's goals is to ensure that breast cancer advocates have a seat at the table when decisions are made about breast cancer research and policy. To accomplish this goal, NBCC educates advocates so that they can participate in and make meaningful contributions to legislative, scientific, and regulatory decision-making bodies. In addition to creating educational opportunities for advocates, NBCC has spearheaded several initiatives designed to directly increase the quality and quantity of breast cancer research. NBCC has also played a major role in making funding available to breast cancer researchers. Two of NBCC's most notable programs include Project LEAD, an intensive science-training course for breast cancer advocates, and the Environmental Initiative, a collection of activities designed to improve research into the relationship between breast cancer and the environment. Breast cancer advocates trained by NBCC have partnered with the scientific community and individual scientists to improve the peer review, design, and implementation of breast cancer research.     

Quantification of the morphological features of a full microvascular network

Investigations into the changes that occur in microvasculature following the surgical procedure called delay have brought about the need for a computer system capable of quantifying the morphological features of a full microvascular network in terms of average vessel length, diameter, and tortuosity. Both the formulaic conventions that have been developed to measure these quantities as well as their implementation in the form of a HP-9000/UNIX based computer software system that we developed specifically for this purpose are discussed. Reliability studies performed using the final system to measure the microcirculatory network of a mouse latissmus dorsi muscle (LDM) showed 95% confidence intervals within 5% of means and coefficients of variability within 7% of means for all quantities measured in large (150–300 μm), medium (50–150 μm), and small (<50 μm) diameter vessels. These variations were significantly smaller than the changes that were observed in a preliminary study comparing these microvascular network parameters before and after delay in the hairless mouse LDM, showing the proposed quantification methods to be well suited to the study of the microvascular changes following delay. It is hoped that the formulaic conventions, implementation process and reliability data will provide a useful comparison for other researchers interested in measuring similar features of microcirculatory networks.     

PCR-based diagnosis of acute and chronic cutaneous leishmaniasis caused by Leishmania (Viannia)

We evaluated PCR methods for diagnosis of acute and chronic cutaneous leishmaniasis (CL) in an area of Colombia where Leishmania (Viannia) is endemic. The PCR method specifically amplified whole linearized minicircle kinetoplast DNA (kDNA) of the Leishmania subgenus Viannia from biopsy lysates. PCR products were detected in agarose gels. For 255 acute cases, this PCR method had greater sensitivity (75.7%) than each conventional method, i.e., microscopic examination of Giemsa-stained lesion scraping (46.7%), biopsy culture (55.3%), aspirate culture (46.3%), and the conventional methods combined (70.2%). Among 44 cases of chronic CL, amplification of biopsy DNA was more sensitive (45.5%) than the individual (4.5 to 27.7%) and combined (27.3%) conventional methods. The detection of kDNA in biopsies from chronic lesions was enhanced by a chemiluminescent dot blot hybridization, which produced a sensitivity of 65.8% when alone and 90.9% when in combination with DNA extraction of biopsy lysates (P < 0.001). Three biopsies from 84 skin lesions of other etiologies were falsely positive by PCR (specificity, 96.4%). PCR detected kDNA more frequently in biopsies (detection level, 83.9%) than in aspirates (74.7%) from 103 cases of acute CL. Among aspirates from 53 chronic cases of CL, the alternative methods, DNA extraction and hybridization, increased sensitivity from 41.5 to 56.6% (P > 0.05). This enhanced PCR method in chronic biopsies was so much more sensitive than conventional methods that it should be considered the preferred diagnostic method for chronic CL. These findings support the appropriate incorporation of PCR into diagnostic strategies for cutaneous leishmaniasis.     

How clients with religious or spiritual beliefs experience psychological help‐seeking and therapy: A qualitative study

This qualitative study explored the process of help‐seeking and therapy among clients with religious or spiritual beliefs. Ten clients who were currently in, or had recently finished, therapy were interviewed. Participants reported using their religious or spiritual beliefs to cope with their psychological problems before and during therapy. Prior to therapy, they were worried that secular‐based help might weaken their faith. However, the experience of having psychological distress and the process of receiving therapy were both perceived as strengthening to faith and ultimately part of a spiritual journey. Contrary to expectations, a match between the spirituality or religious affiliation of the therapist and client was not considered important. This implies that the ‘religiosity gap’ between secular therapists and clients with religious/spiritual beliefs is bridgeable. Copyright © 2007 John Wiley & Sons, Ltd.     

Characterization of the receptor for IgM present on human B lymphocytes.

The presence of a receptor for the Fc of IgM (muFcR) was demonstrated on the pathological B cells of all of sixteen patients with hairy-cell leukaemia and most, but not all, of twenty-four cases of chronic lymphocytic leukaemia, by a rosette method employing ox erythrocytes sensitized with purified IgM (EAm). This muFcR was also demonstrated on a small population of normal human mononuclear cells from peripheral blood. Pathological B cells with this receptor (Bm) simultaneously expressed a different and distinct receptor for the Fc of IgG, and were detectable without preincubation in medium containing foetal calf serum (FCS). The muFcR on B cells was blocked by Fc5mu and IgM, but not by F(ab')2mu fragments, or by IgG, whether monomeric or aggregated. Monomeric IgM and IgM bound to its antigen blocked much more effectively than pentameric IgM. B cells also possessed surface immunoglobulin and the Ia-like P29, 34 antigen, and an antiserum to this antigen blocked the muFcR. The muFcR on B cells differs in a number of ways from the muFcR reported on T cells, and these differential characteristics are discussed in some detail. The muFcR was rapidly shed and resynthesized when washed Bm cells were maintained in medium not containing FCS and the general importance of this phenomenon in any study of muFcR is considered. It is suggested that Bm cells are memory cells and that the muFcR plays a part in the immune response.     

Langerhans' cell expression of the selectin ligand, sialyl Lewis x.

Cellular adhesion molecules play a central role in leucocyte migration through peripheral blood and tissues. A crucial stage in these events in selectin-mediated adhesion involving E-selectin expressed on activated endothelium interacting with a range of carbohydrate ligands expressed by specific subpopulations of leucocytes. As such mechanisms may be relevant to bone marrow-derived dendritic epidermal Langerhans' cell (LC) migration, expression of these carbohydrate ligands was assessed immunocytochemically in whole skin biopsies and in epidermal cell suspensions obtained from adult humans. Double-labelling experiments revealed that sialyl Lewis x, recognized by the monoclonal antibody CSLEX1, was expressed on epidermal LC (n = 9). Furthermore, expression was enhanced at 24 hr following epicutaneous application of antigen and in the inflammatory disorder psoriasis (n = 10). E-selectin was concomitantly strongly expressed on dermal endothelium in psoriasis and allergic contact dermatitis. Intradermal injection of the T-cell-derived cytokine interferon-gamma (IFN-gamma) led to increased LC expression of sialyl Lewis x. In epidermal cell suspensions, in contrast to keratinocytes, CD1a+ cells expressed sialyl Lewis x, intensity of which was enhanced after 4 days in culture. CSLEX1 staining could be abolished and CD15 (non-sialated Lewis x) expression induced by saponification and treatment with neuraminidase. Expression of other selectin ligands was also examined. While the cutaneous lymphocyte antigen defined by the monoclonal antibody HECA-452 reacted with a small minority of LC, sialyl Lewis a and sulphatide were not expressed under any experimental conditions. These studies indicate that E-selectin-sialyl Lewis x interactions are potentially important in LC migration, both into and out of skin.