DLA-DRB1 polymorphisms in dogs defined by sequence-specific oligonucleotide probes (SSOP)

To date, DNA sequences for 29 dog DLA-DRB1 alleles have been reported. However, no data exists on the frequencies of these alleles within the general dog population, nor is there any indication of whether there is interbreed variation of allele distribution. We have addressed this by establishing a molecular based sequence-specific oligonucleotide probing (SSOP) method to identify all of the known broad DRB1 types and we have used this to type a random panel of dogs. A series of oligonucleotide probes were designed to detect known polymorphisms in the three DRB1 hypervariable regions, together with two distinctive motifs in other regions of exon 2. This set of probes enabled us to assign broad DRB1 types. Two hundred and eighteen dogs were SSOP typed for DRB1. All but 4 of the published DLA-DRB1 alleles were identified in these animals. Interbreed variation in both allele distributions and allele frequencies were observed, which may be useful in the study of genetic variation between breeds. This variation also has implications for the selection of control groups for studies aimed at identifying MHC associations with disease susceptibility in the dog.     

Use of a non-radioactive hybridisation assay for direct detection of gram-negative bacteria carrying TEM beta-lactamase genes in infected urine

DNA in infected urines from 81 patients with urinary tract infection was hybridised directly with a non-radioactive DNA probe specific for bacterial genes coding for TEM-type beta-lactamase. The results were assessed by means of a computerised image analysis system and compared with those obtained following isolation of the infecting organism, conventional sensitivity testing and isoelectric focusing (IEF) procedures for the detection of TEM-type beta-lactamase. Of the 27 ampicillin-resistant gram-negative organisms isolated in pure culture from the urines, 14 were shown by both hybridisation and IEF to carry a gene for TEM beta-lactamase production. Only four discordant results were obtained: three "false positive" direct hybridisation results, one due to urine pigmentation, and one, possibly, to a TEM beta-lactamase gene which was not being expressed, and one "false negative" result due to insufficient cell numbers in the urine. The system is capable of screening large numbers of samples and is applicable to any gene for which a suitable DNA probe is available.     

Immunohistochemical localization of apolipoproteins A-I and B in human carotid arteries

Decreased plasma levels of apolipoprotein A-I (apo A-I) and increased plasma levels of apolipoprotein B (apo B) have been shown to correlate with increased risk of atherosclerosis. While many studies have investigated the plasma levels of these apolipoproteins with regard to their value as predictors of cardiovascular disease, comparatively little is known about their precise tissue localization in atherosclerotic plaques. The purpose of this study was to determine the tissue localization of apo A-I and apo B in atherosclerotic segments of human carotid arteries through the use of immunohistochemical techniques. With tissue samples obtained from surgery and autopsy, apo A-I and apo B were found to be present in atherosclerotic plaques and absent in normal arterial tissue. In the plaques, both apo A-I and apo B were found extracellularly, primarily in the lipid core, but also in connective tissue. In addition, both apo A-I and apo B were found intracellularly in foam cells. This similar intracellular and extracellular distribution of apo A-I and apo B was unexpected, in view of their differing associations with atherosclerosis.     

Regulation of ST2L expression on T helper (Th) type 2 cells

T1/ST2L, an IL-1 receptor homologue, is selectively expressed on murine Th2 cells and specific anti-ST2L antibodies can profoundly modulate the Th1/Th2 balance in vivo. Naive CD4+ T cells do not express ST2L but do so on activation with specific antigen in the presence of IL-4 or when stimulated with low doses of antigen in the absence of exogenously added IL-4. Similarly enhanced ST2L expression occurred after stimulation of Th2 cells with antigen or the mitogen ConA in the presence of APC. Restimulation of Th2 cells in the presence of IFN-gamma led to a decreased expression of ST2L to below basal levels. Conversely, Th2 cells cultured with IL-4 led to increased ST2L expression. The reduced expression of ST2L in response to high doses of antigen is also reversed by the neutralization of IFN-gamma. Using an ST2L promoter/luciferase reporter gene construct, we show that the distal but not proximal ST2L promoter is responsible for specific gene expression in Th2 cells. IL-4 enhances, whereas IFN-gamma suppresses ST2L expression via direct modulation of the distal promoter of the ST2L gene. These data provide a mechanistic explanation for the selective expression of ST2L on Th2 cells.     

Prolactin-screening tumors and hypogonadism in 22 men.

We studied 22 men with prolactin-secreting pituitary tumors and hypogonadism. Twenty complained of impotence, nine had visual impairment, and three experienced galactorrhea. None of the 17 patients undergoing operation or radiotherapy, or both, were subsequently normoprolactinemic. In all 13 patients treated with bromocryptine major clinical improvement was associated with a decrease in serum prolactin levels and in nine with an increase in serum testosterone. Two patients receiving testosterone replacement therapy showed improved potency only after bromocryptine was administered. The results indicate that hyperprolactinemia frequently induces hypogonadism in men, that bromocryptine ameliorates symptoms of disease previously unchanged by operation or radiotherapy, and that the impotence observed may not be solely the result of hypogonadism.     

Mechanisms of neutrophil damage to human alveolar extracellular matrix: the role of serine and metalloproteases.

Many syndromes of lung injury are associated with accumulation of neutrophils within the pulmonary parenchyma. These neutrophils have the capacity to produce lung injury by products including proteases and reactive oxygen species (ROS). We examined the ability of activated neutrophils to solubilize human alveolar extracellular matrix (ECM), and by use of scavengers and inhibitors, evaluated the role of ROS and proteases in this process. Supernatants of phorbol myristate acetate-activated neutrophils routinely solubilized 10.2% +/- 0.8% (n = 30) of collagen in human alveolar ECM, as measured by hydroxyproline release. Scavengers of ROS had no significant effect on ECM solubilization. Inhibitors of metalloproteases partially inhibited ECM solubilization (38.5% +/- 4.6% inhibition by ethylenediaminetetraacetic acid [n = 6], and 37.0% +/- 14.7% by 1,10-phenanthroline [n = 6]; p less than 0.05). Inhibitors of the neutrophil serine proteases, elastase and cathepsin G, markedly inhibited ECM solubilization (100.9% +/- 3.7% by alpha 1-protease inhibitor [alpha 1-PI] [n = 6] and 81.9% +/- 0.1% by soybean trypsin inhibitor [n = 6]; p less than 0.01). Since alpha 1-PI completely inhibited solubilization, metalloprotease activity appeared to be related to serine protease activity. This finding was confirmed by the observation that addition of a metalloenzyme activator, p-aminophenylmercuric acetate, in the presence of alpha 1-PI, restored solubilization to the same level as that inhibited by metal chelators. We conclude that human neutrophil metalloproteases and serine proteases directly solubilize human alveolar ECM. Furthermore, neutrophil serine proteases activate latent metalloproteases. However, ROS were not demonstrated to play a major role in ECM solubilization in our system.     

CD19 signal transduction in normal human B cells: linkage to downstream pathways requires phosphatidylinositol 3-kinase, protein kinase C and Ca2+

CD19 is required for normal antibody responses in mice. We have shown that CD19 enhances the activation of extracellular signal-regulated kinase (ERK) 2 by membrane (m) IgM but otherwise little is known of CD19 signaling in primary human cells. We now ask which pathways link CD19 with ERK2 in human tonsillar B cells. In analyses of signaling intermediates, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin partially suppressed the release of Ca2+ induced by coligation of CD19 and mIgM but the selective PKC inhibitor bisindolylmaleimide I (BIM-I) did not. The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, BIM-I and wortmannin each had only a small effect on ERK2 activation induced by surface IgM alone but blocked the enhancement of that activation by CD1 9/mIgM coligation. To analyze the mitogen-activated protein kinase (MAPK) cascade, we measured activation of Raf, MAPK- or ERK kinase (MEK) 1 and ERK2. CD19 consistently enhanced activation of ERK2 and MEK1. However, synergistic activation of Raf was variably observed. In subpopulation analyses, synergistic activation of Raf1 was consistently observed in the IgDlow but not in the IgDhigh cells. Thus, in normal human B cells, PI3K is upstream of the Ca2+ response while PI3K, Ca2+ release and protein kinase C are all required for ERK2 activation, and CD19 enhances the MAPK cascade at multiple levels, depending on the state of differentiation.     

Inability of dendritic cells to prevent the blood transfusion effect in a mouse cardiac allograft model

The beneficial effect of blood transfusions before clinical renal transplantation is well established, but this can result in sensitization of some potential first graft recipients. Dendritic cells (DC), present in human blood, are potent stimulators of immune responses in vitro and in vivo, and in different systems they can overcome immune unresponsiveness. We therefore investigated whether DC could prevent the transfusion effect in a murine cardiac allograft model. C57BL/10 (C57) hearts were normally rejected by untreated DBA/2 recipients with a median survival time (MST) of 17 days. Long-term survival (MST greater than 100 days) was induced when the recipients were transfused with C57 blood 14 days before transplantation (d-14). Similar survival times were obtained when up to 1.5 x 10(5) splenic DC were added to the transfused blood. This number of DC or as few as 10(3), in "unsorted" preparations that were 70-85% pure, similarly enhanced when transfused alone at d-14. This enhancement was probably due to the contaminating cells rather than the DC since 10(3) lymphocytes prolonged survival, but an equivalent dose of "sorted" DC (ca. 93% pure, most contaminating cells being removed by sorting on an Ortho Cytofluorograf) did not. Transfusion of unsorted preparations containing 1.5 x 10(5) DC at d-3 led to accelerated graft rejection (MST 4 days). This sensitization was most likely due to the DC because equivalent numbers of lymphocytes were ineffective. Nevertheless, if a blood transfusion was given at d-14 followed by a normally sensitizing dose of DC at d-3, graft survival was still prolonged. Thus in no case were DC able to prevent the blood transfusion effect in this strain combination. Furthermore, DC of donor origin given to DBA/2 recipients with long-surviving C57 hearts, produced by prior blood transfusion, did not trigger rejection of the hearts.