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.     

A rapid radiometric assay for measuring phagocytosis of Saccharomyces cerevisiae in macrophage cultures

A new assay was developed to measure yeast phagocytosis in cultures of murine resident peritoneal macrophages. Saccharomyces cerevisiae was radiolabeled during exponential growth in nutrient broth supplemented with [3H]glucose. Following ingestion of the radiolabeled heat-killed yeast particles for 15 min, phagocytic capacities were measured in harvested macrophage lysates by liquid scintillation spectrometry. The new procedure compares favorably with light microscopic techniques and appears to be a more sensitive method for quantitating phagocytic function. Dose-response studies indicate, that over a wide range of dexamethasone concentrations, the radiometric procedure consistently measures greater inhibitory effects for the steroid induced suppression of phagocytosis.     

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.     

Monoclonal antibody definition of the DRB3 allele, HLA-Dw25

Monoclonal antibodies are powerful tools for analyzing HLA antigen polymorphism. We have investigated the serological and biochemical nature of the DRw52-related antigen defined by the monoclonal antibody NDS10. A detailed analysis of the population distribution of NDS10 reactivity revealed that the epitope was present on a subpopulation of DRw52 positive cells. A distinct pattern of reactivity was found within DR3 individuals: all of the B18,DR3 cells were NDS10 positive, whereas the A1,B8,DR3 cells were negative. All of the DR5(w11) cells and two of three DRw12 cells reacted with NDS10. NDS10 reactivity with DRw6 was not restricted to either of the serologically defined subtypes; three of 17 DRw13 and nine of 10 DRw14 cells were NDS10 positive. NDS10 was unreactive with all of the DRw8 cells tested. Two-dimensional gel analyses revealed that the NDS10 molecule precipitated from DR3, DR5(w11) and DRw6(w14) cell lines had an identical beta chain profile. These data indicate that NDS10 recognises the Dw25 allele of the DRw52 complex.     

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.     

Biosynthesis of two stage-specific membrane proteins during transformation of Plasmodium gallinaceum zygotes into ookinetes

We have studied the synthesis and expression of surface proteins in zygotes of Plasmodium gallinaceum during their transformation to mature ookinetes. The cells were biosynthetically labelled in vitro using [35S]methionine and proteins were immunoprecipitated with rabbit anti-ookinete serum or monoclonal antibodies. Early zygotes (approx. 2 h post-gametogenesis and fertilization) synthesized and expressed on their surface a protein of Mr 26 000 as observed under reducing conditions on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (31 000 under non-reducing conditions) and continued to do so for 8-10 h; thereafter synthesis of the Mr 26 000 protein declined and little or none was synthesized in the mature ookinetes (greater than 20 h post-gametogenesis). Between 3-5 h post-gametogenesis, zygotes also began to synthesize a protein of Mr 28 000 (34 000 under non-reducing conditions). Synthesis and expression of this surface protein continued throughout development; and the Mr 28 000 protein was the predominant surface protein synthesized by the mature ookinete. Mr 26 000 and Mr 28 000 proteins have been designated earlier as PgO-1 and PgO-2 respectively (Carter and Kaushal, Mol. Biochem. Parasitol. (1984) 13, 235-241). Neither protein was synthesized in the gametocytes prior to gametogenesis. Both proteins could be labelled with [3H]glucosamine or [3H]mannose. When zygotes were incubated with [3H]palmitic acid both PgO-1 and PgO-2 bound fatty acids in covalent linkage. The two proteins do not otherwise appear to be structurally related. They were differentially immunoprecipitated by different monoclonal antibodies and gave rise to distinct patterns of peptides following digestion with proteases such as Staphylococcus aureus V-8, trypsin and chymotrypsin.     

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.