Use of cool perches by broiler chickens.

Broilers under commercial conditions might experience relatively high temperatures during summer and leg disorders year round that may be partially alleviated by providing them with access to cooled perches. It is unknown, however, how perch temperature and factors such as height and position of the perch affect perch use. Furthermore, little is known regarding gender effects. Eight thousand 1-d-old, mixed-sex broilers were exposed to three perch treatments to determine preferences for water-cooled perches over ambient temperature perches and preferences for height, location, and temperature section of the perch. The experimental treatments were as follows: 1) three cool perches 15 cm above the floor (Cool 15), 2) three ambient perches 7.5 cm off the floor (Ambient 7.5), 3) three ambient perches 15 cm high (Ambient 15), and 4) control chambers with no perches. Total number of birds perching, their positions, and temperature section within the perch were recorded. The results indicate a strong preference for high perches as birds grow (P < 0.0001). The cooler sections of the perch were utilized more than warmer sections within the cool treatments (P < 0.05). Females showed a stronger tendency to perch than males, particularly within the cool treatment (P < 0.0001). The higher perch use could be one of the reasons for the higher eviscerated body weight found in females with access to cool perches (P < 0.05). Differences in mean body weight were not significant (P = 0.07). Potential beneficial effects of perch access in final body weights needs to be further investigated.

Peak exercise cardiac power output; a direct indicator of cardiac function strongly predictive of prognosis in chronic heart failure.

OBJECTIVES: This study assessed the prognostic value of peak cardiac power output, measured non-invasively during maximal cardiopulmonary exercise testing, against other exercise-derived haemodynamic variables in patients with chronic congestive heart failure. METHOD AND RESULTS: Two hundred and nineteen unselected, consecutive patients with congestive heart failure (166 men, mean (+/-SD) age of 56+/-13 years) who underwent maximal symptom limited cardiopulmonary treadmill exercise testing with non-invasive estimation of cardiac output using carbon dioxide re-breathing techniques, were followed-up for a mean period of 4.64 (4.47--4.82, 95% CI) years. Cardiac power output was calculated from the product of cardiac output and mean arterial blood pressure. All cause mortality was 12.3% (27 deaths). Peak and resting cardiac power output, peak mean arterial blood pressure, peak and resting cardiac output and peak VO(2)were all predictive of outcome on univariate analyses. Peak cardiac power output, either entered continuously or categorically with a cut-off value of 1.96 watts, was the only independent predictor of mortality (P=0.0004 for values < or >1.96 watts and P=0.001 for continuous values) using multivariate analysis. A relative risk ratio of 5.08 (1.94-13.3, 95% CI) was obtained for a cardiac power output <1.96 watts. CONCLUSION: Peak cardiac power output is an independent predictor of mortality that can be measured non-invasively using cardiopulmonary exercise testing. It can give further prognostic power to a peak VO(2)in the assessment of patients with congestive heart failure.

The reduced expression of surrogate light chains in B cell precursors from senescent BALB/c mice is associated with decreased E2A proteins.

Senescent mice exhibit decreased numbers of pre-B cells in the bone marrow. Herein, we show that the molecules, lambda5 and VpreB, which comprise the surrogate light chain component of the pre-B cell receptor, are reduced in pro-B/early pre-B cells derived in vitro from the bone marrow of 18-27 months old BALB/c mice after stimulation with IL-7. Both lambda5 and VpreB expression were decreased at the mRNA level as indicated by semi-quantitative RT-PCR; this suggests that the reduced surrogate light chains seen in senescent B cell precursors result from dysfunctional transcriptional regulation. The transcription of surrogate light chains is regulated, in part, by E2A (E47) gene products. Levels of E2A proteins, including E47, were decreased in senescent B cell precursors by up to 90%. Reduced E2A (E47) expression and subsequent reduced transcription of the surrogate light chain components lambda5 and VpreB may, in part, explain the diminished production of B lineage cells observed in senescence.

Transgenic human lambda 5 rescues the murine lambda 5 nullizygous phenotype.

The human lambda 5 (hu lambda 5) gene is the structural homologue of the murine lambda 5 (m lambda 5) gene and is transcriptionally active in pro-B and pre-B lymphocytes. The lambda 5 and VpreB polypeptides together with the Ig mu H chain and the signal-transducing subunits, Ig alpha and Ig beta, comprise the pre-B cell receptor. To further investigate the pro-B/pre-B-specific transcription regulation of hu lambda 5 in an in vivo model, we generated mouse lines that contain a 28-kb genomic fragment encompassing the entire hu lambda 5 gene. High levels of expression of the transgenic hu lambda 5 gene were detected in bone marrow pro-B and pre-B cells at the mRNA and protein levels, suggesting that the 28-kb transgene fragment contains all the transcriptional elements necessary for the stage-specific B progenitor expression of hu lambda 5. Flow cytometric and immunoprecipitation analyses of bone marrow cells and Abelson murine leukemia virus-transformed pre-B cell lines revealed the hu lambda 5 polypeptide on the cell surface and in association with mouse Ig mu and mouse VpreB. Finally, we found that the hu lambda 5 transgene is able to rescue the pre-B lymphocyte block when bred onto the m lambda 5-/- background. Therefore, we conclude that the hu lambda 5 polypeptide can biochemically and functionally substitute for m lambda 5 in vivo in pre-B lymphocyte differentiation and proliferation. These studies on the mouse and human pre-B cell receptor provide a model system to investigate some of the molecular requirements necessary for B cell development.

Senescent BALB/c mice exhibit decreased expression of lambda5 surrogate light chains and reduced development within the pre-B cell compartment.

Although senescent BALB/c mice (approximately 2 years old) have reduced numbers of small pre-B cells, early pre-B cells (CD43+CD25+B220+) are present in comparable numbers within the bone marrow of both young (3-6-month-old) and senescent BALB/c mice. The transition of CD43+ pre-B cells to the CD43- pre-B cell compartments is dependent on proliferation and clonal maturation dictated by the pre-B cell receptor (mu/lambda5/VpreB). In vivo, senescent CD43+B220+ pro-B/early pre-B cells demonstrated reduction of lambda5 mRNA, by RT-PCR analysis, and of both surface and cytoplasmic lambda5 protein. Decreased lambda5 protein expression was also seen among pro-B/pre-B cells derived from senescent bone marrow after stimulation in vitro with IL-7. We propose that diminished expression of the lambda5 surrogate light chain results in decreased pre-B cell receptor formation and contributes to reduced recruitment of nascent CD43+ pre-B cells into the CD43- large and small pre-B cell compartments.

Autoantibodies to T-lineage cells in aged mice.

Aging is accompanied by a marked decline in protective immune function. This loss of effective immunity is largely due to alterations in the T-cell compartment. There are major impairments in both the production of new T-cells within the thymus and in the functions of mature T-cells in peripheral lymphoid organs. The mechanism(s) underlying this age-related decline in T-lineage cells is not clear. Here, we demonstrate that aging is accompanied by the appearance of appreciable titers of anti-T-lineage autoantibodies. The autoantibodies, which are exclusively of the IgM class, begin to appear at 1 year of life and are universally found in the sera of 2-year-old mice. Among thymocytes, all CD4/CD8 subsets reacted with the autoantibodies, with the CD4+8+ subset showing the greatest reactivity. The autoantibodies also bound to resting peripheral CD4+ and CD8+ cells. Following activation with either anti-CD3 or with TCR-independent stimulators, reactivity to peripheral T-cells was diminished, suggesting that the determinants recognized by the autoantibodies are downregulated in response to activation signals. Lastly, thymocytes freshly isolated from old, but not young, mice have IgM antibodies bound to their surfaces. Thus, circulating autoantibodies in old mice have access to the thymus and bind to thymocytes in situ. These results lead to the proposal that the presence of anti-T-lineage autoantibodies in vivo interferes with normal T-cell development and/or function in aged animals.

Graft-versus-host-disease-associated lymphoid hypoplasia and B cell dysfunction is dependent upon donor T cell-mediated Fas-ligand function, but not perforin function.

Allogeneic bone marrow transplant recipients often exhibit a graft-versus-host-disease (GVHD)-associated immune deficiency that can be prolonged and lead to life-threatening infections. We have examined the role of donor T cell-mediated cytotoxic function in the development of GVHD-associated immune deficiency. A major histocompatibility complex-matched model of allogeneic bone marrow transplantation was employed in which lethally irradiated C3H.SW mice received a nonlethal dose of T cells from either perforin-deficient (B6-perforin 0/0), Fas-ligand (FasL)-defective (B6-gld), or normal (B6) allogeneic donor mice. T cell-depleted marrow from B6-Ly-5.1 congenic donor mice was transplanted along with the donor T cell populations to determine the effects of donor T cell-mediated cytotoxicity on engraftment. Our results demonstrate that recipients of perforin-deficient or normal allogeneic T cells exhibit profound lymphoid hypoplasia and severely reduced splenic proliferative responses to lipopolysaccharide in vitro. In contrast, GVHD-associated lymphoid hypoplasia is dramatically reduced and in vitro B cell function is intact in recipients of FasL-defective allogeneic T cells. Engraftment of myeloid and erythroid lineage cells occurs irrespective of donor T cell cytotoxic function. Although recipients of perforin-deficient or normal allogeneic T cells exhibited hematopoietic engraftment exclusively of donor origin, recipients of FasL-defective donor T cells exhibited significant mixed chimerism (Ly-5.1/Ly-5.2). Because only marrow of donor origin was transplanted, this finding suggests that Fas-mediated antirecipient cytotoxicity is required for clearance of residual hematopoietic stem cells of host origin that persist following lethal irradiation.

Autoantibodies inhibit interleukin-7-mediated proliferation and are associated with the age-dependent loss of pre-B cells in autoimmune New Zealand Black Mice.

Surface IgM+B220+ B cell precursors can be categorized as either leukosialin (CD43/S7) negative (late stage pre-B cells) or positive (pro-B/early pre-B cells). In autoimmune New Zealand Black (NZB) mice, bone marrow small pre-B cells (IgM-CD43-B220+) and pro-B/early pre-B cells (IgM-CD43+B220+) declined significantly with age. In particular, subpopulations of pro-B/early pre-B cells expressing the heat stable antigen (HSA) were found in lower proportions with age. Significant decreases in interleukin-7 (IL-7) colony forming units (CFU) were also seen in NZB mice by 6 to 8 months of age and accompanied alterations in the numbers of pro-B and pre-B cells in bone marrow. Concomitant with reduced numbers of B lineage precursor cells and IL-7 CFU in vivo, NZB mice produced serum IgM antibodies that strongly inhibited IL-7 CFU responses in vitro. Two monoclonal IgM antibodies (5G9, 2F5) derived from LPS stimulated 10-month-old NZB splenocytes recognized pre-B cell surface antigens on both pre-B cell lines and on IL-7 stimulated bone marrow pro-B/pre-B cells. However, these monoclonal antibodies (MoAb) failed to significantly stain ex vivo bone marrow cells. The 5G9 and 2F5 MoAbs also partially inhibited IL-7 CFU in vitro. These results suggest that NZB bone marrow becomes increasingly deficient in B cell precursors and especially in IL-7 responsive pre-B cells with age. IgM serum antibodies and monoclonal IgM antibodies derived from older NZB mice inhibit pre-B cell growth to IL-7. The production of such autoantibodies may interfere with B cell development in aging NZB mice by preventing IL-7-mediated proliferation.

IL-10 inhibits IL-7-mediated murine pre-B cell growth in vitro.

B lymphopoiesis in the bone marrow is mediated by both positive and negative regulatory cytokines. In this report, we demonstrate that interleukin-10 (IL-10) may function to inhibit murine IL-7-dependent pre-B cell growth. Recombinant IL-10 (rmIL-10) inhibited BALB/c bone marrow IL-7 colony-forming unit (CFU) in a concentration-dependent manner, and growth was restored when IL-10 was neutralized with the monoclonal anti-IL-10 antibody, SXC-1. Enriched populations of B220+ bone marrow B lineage cells were also inhibited in their responses to IL-7 by exposure to rmIL-10, suggesting that pre-B cells were directly susceptible to rmIL-10 inhibition. Heterogeneity in the capacity of IL-7 CFU to be inhibited by IL-10 was evident. Although 60% of IL-7 CFU were inhibited by rmIL-10 at 5 U/mL, approximately 20% of IL-7 CFU were not inhibited by rmIL-10 concentrations up to 50 U/mL. Prior incubation of bone marrow cells for 24 hours with IL-7 prevented rmIL-10-mediated growth inhibition, suggesting that prior rIL-7 stimulation of pre-B cells abrogates the inhibitory effects of rmIL-10. These experiments indicate that IL-10, at these concentrations, may function as a potent negative growth regulator for a significant fraction of IL-7-responsive pre-B cells.

B220- bone marrow progenitor cells from New Zealand black autoimmune mice exhibit an age-associated decline in Pre-B and B-cell generation.

New Zealand Black (NZB) autoimmune mice exhibit progressive, age-dependent reduction in bone marrow pre-B cells. To ascertain the capacity of NZB bone marrow B220- cells to generate pre-B cells in a supportive environment, B-lineage (B220+) cell-depleted and T-cell-depleted bone marrow cells from NZB mice at 1 to 3, 6, and 10 to 11 months of age were adoptively transferred into irradiated (200R) C.B17 severe combined immunodeficient (SCID) mice. Bone marrow pre-B cells (sIgM- CD43[S7]- B220+) were assessed 3 and 10 weeks posttransfer. Pre-B cells and B cells were reconstituted in SCID recipients of older NZB progenitor cells by 10 weeks posttransplant, in contrast to the very low numbers of pre-B cells present in the donor bone marrow. However, B220- bone marrow progenitor cells from greater than 10-month-old NZB donors were deficient in the reconstitution of both pre-B and B cells in SCID recipients at 3 weeks post-transfer. This reflected a slower kinetics of repopulation, because older NZB-->SCID recipients had numbers of both pre-B and B cells similar to recipients of young NZB progenitor cells by 10 weeks posttransplant. Adoptive transfer of equal mixtures of BALB/c and older NZB bone marrow B220- progenitor cells into irradiated C.B17 SCID recipients failed to demonstrate active suppression. These results suggest that, with age, NZB bone marrow has reduced numbers and/or function of early B220- B-lineage progenitors. Consistent with this hypothesis, B220- bone marrow cells from older NZB mice were deficient in progenitors capable of yielding interleukin-7 (IL-7) responsive pre-B cells in vitro on stimulation with the pre-B-cell potentiating factor, insulin-like growth factor 1 (IGF-1).

IFN-gamma abrogates IL-7-dependent proliferation in pre-B cells, coinciding with onset of apoptosis.

These studies investigated the mechanism by which interferon-gamma (IFN-gamma) inhibits the interleukin-7 (IL-7)-dependent proliferation of BALB/c bone marrow B-cell precursors in vitro. Low concentrations (1 U/ml) of recombinant murine IFN-gamma (rmIFN-gamma) caused a approximately 80% suppression of IL-7 colony-forming units (CFU) formation in semi-solid media, in part through a direct affect on isolated B220+ pre-B cells. IFN-gamma did not induce apoptosis in small resting pre-B cells in BALB/c bone marrow. There was no difference in the proportion of apoptotic B220+ pre-B cells in IFN-gamma-treated cultures compared to cultures treated with IL-7 alone. However, IL-7-responsive pre-B cells generated from bone marrow had a 30-50% loss in cells in S+G2/M phases of the cell cycle and an increase of up to twice as many in apoptotic cells within 48 hr of exposure to IFN-gamma. Notably, expression of the tyrosine phosphatase B220 was increased in the IFN-gamma-treated pre-B cells. Interestingly, although there was no substantial change in IL-7 receptor mRNA expression upon IFN-gamma treatment, a small decrease in binding of biotinylated IL-7 to IFN-gamma-treated pre-B cells was observed. These results suggest that IFN-gamma inhibits IL-7 responsiveness in pre-B cells, resulting in a subtle down-regulation of IL-7 binding, inhibition of proliferation and, ultimately, apoptosis.

Suppression of B-cell development as a result of selective expansion of donor T cells during the minor H antigen graft-versus-host reaction.

A murine model of bone marrow (BM) transplantation in which donor (B10.D2) and recipient (BALB/c) mice were major histocompatibility complex (MHC) (H-2d) and Mls-1 identical, but incompatible at multiple non-MHC minor histocompatibility (H) antigens, and at Mls-2,3 was used to examine regeneration of B-cell development during the minor H antigen graft-versus-host reaction (GVHR). Mice that received T-cell-depleted allogeneic BM regained significant pre-B cells (sIg- 14.8+) in their BM. Mice undergoing GVHR after transplantation with allogeneic BM + T cells had less than 2% pre-B cells in their BM at day 7 and only 12% to 14% pre-B cells at days 21 and 28 compared with greater than 20% pre-B cells in the allogeneic controls. After partial recovery, the pre-B cells in the BM of GVH mice again decreased to less than 3% by day 42. This abnormal pattern of pre-B cell development in mice undergoing GVHR was associated with a reduced response to interleukin-7 (IL-7) in vitro. The delay in B-lineage cell reconstitution in mice with GVHR correlated with the expansion of donor V beta 3+ T cells in both the spleen and BM. BM T cells from mice with GVHR as well as isolated V beta 3+ T cells inhibited IL-7 colony-forming units from normal BM in co-culture assays. This inhibition could be reversed with anti-interferon gamma (IFN gamma) antibody. These data suggest that the delay in appearance and the reduction in proportion and number of pre-B cells observed early during the GVH reaction in this model is caused, in part, by the inhibitory actions of IFN gamma derived from donor V beta 3+ T cells on B-lineage cell development.

B cell precursors are decreased in senescent BALB/c mice, but retain normal mitotic activity in vivo and in vitro.

The numbers of phenotypic (sIg- Ly5[220]+) and functional B cell precursors were significantly reduced in the bone marrow of senescent (22-24 months old) BALB/c mice when compared to their young (2-4 months old) cohorts. Little alteration in the numbers of B cell precursors occurred during the first 12 months of life in this strain. In contrast, an accelerated loss of B cell precursors between 15 and 18 months of age was observed. In particular, the levels of small Ly5(220)+ B cell precursors were decreased with advanced age, although a decline in numbers of large sIg- Ly5(220)+ B cell precursors was also evident. The percentages of large sIg- Ly5(220)+ B cell precursors in (S + G2/M) stages of cell cycle were similar (e.g., 60-80%) in aged and young BALB/c mice. Importantly, Ly5(220)+ pre-B cells from both young and aged BALB/c mice, either present in vivo or derived from Ly5(220)- cells in vitro, were capable of proliferation in response to rIL-7. These observations suggest that the aging process results in a progressive decline in the numbers of pre-B cells; however, this apparently is not due to failure of B lineage precursor cells to respond to growth mediators either in vivo or in vitro.

B cells in autoimmune (NZB x NZW)F1 mice show altered IgG isotype switching upon T cell-dependent antigenic stimulation in vitro.

Humoral immune responses of (NZB x NZW)F1 (BWF1) autoimmune mice to T cell-dependent antigens often exhibit a predominance of IgG2 antibodies, while normal mice produce IgG1 antibodies. In order to determine whether this results from differences in properties of the B cells or the T cells involved, the responses of both primary and secondary BWF1 B cells to the antigen DNP-hemocyanin (Hy) were measured in limiting dilution splenic fragment cultures in the presence of normal T cell help. Furthermore, the capacity of Hy-primed lymph node T cells from BWF1 mice to provide help to BALB/c nu/nu B cells was determined in modified splenic fragment cultures. These experiments indicated that (a) stimulation of primary BWF1 B cells with DNP-Hy and normal T cell help failed to yield significant numbers of clones which produced any of the IgG isotypes; (b) antigenic stimulation of BWF1 secondary B cell clones also demonstrated a paucity of IgG1, but elevated production of IgG2 isotypes; and (c) Hy-primed BWF1 lymph node T cells were comparable to those derived from BALB/c mice in their capacity to provide both help for nu/nu B cell responses and modulation of IgG isotype switching. BWF1 B cells apparently differ from normal murine B cells in their capacity to produce IgG antibodies upon T cell-dependent antigenic stimulation.