Biomedical subjects
R B Pearce
Publications and source records attributed to R B Pearce.
Fungal volatilization of arsenic and antimony and the sudden infant death syndrome.
Fungal volatilization of antimony and other group Vb elements has been proposed to have a causal role in the sudden infant death syndrome (SIDS; cot death). The ability of fungi to produce volatile arsenic and antimony compounds in pure culture was examined using Scopulariopsis brevicaulis, reported as an inhabitant of PVC cot mattress covers, and Phaeolus schweinitzii, a wood decay fungus known to be a good volatilizer of arsenic. Volatile arsenic compounds were detected from all cultures grown on arsenic-supplemented media, but antimony volatilization was not reliably detected. Although antimony levels above the baseline sensitivity of the analytical technique were detected in four (out of 24) of the samples analyzed, the concentrations recorded were too low to be reliably interpreted as evidence for volatilization. Our results are discussed in relation to hypotheses regarding the causes of SIDS.
Fine-mapping of the mouse T lymphocyte fraction (Tlf) locus on chromosome 9: association with autoimmune diabetes.
Tlf (T lymphocyte fraction) defines a locus that governs the unusually high fraction of circulating T lymphocytes in the nonobese diabetic (NOD) mouse. We previously mapped Tlf to proximal Chromosome 9 in BC1 mice. Here, Tlf was tine-mapped on Chromosome 9 using 8 markers covering the 43 cM interval from D9Mit90 at 9 cM to D9Mit35 at 52 cM. Markers for diabetic genes on Chromosomes 3, 4, 5, 6, and 17 were also examined for effects on the Tlf phenotype. By both parametric and nonparametric tests. Tlf associated with two areas on Chromosome 9, one with the segment bounded by D9Mit66 (15 cM) and D9Mit2 (17 cM) and a second region near D9Mit71 (29 cM). This linkage pattern was observed both in BC1 and F2 populations. Thus, the Tlf phenotype is possibly governed by two genes on Chromosome 9. An influence by sex on the penetrance of Tlf was evident in that linkage was strongest for female F2 mice and male BC1 mice. One locus controlling the T lymphocyte fraction may be Idd2 since historically a subline of NOD mice with a low T cell fraction showed a low incidence of diabetes. Candidate genes for Tlf are Ets1 and Fli1, proximally and Igif distally.
Clarification of the Ins2 gene sequence: relevance to glucose intolerance in NON/Lt mice.
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Acetaldehyde-serum protein adducts inhibit interleukin-2 secretion in concanavalin A-stimulated murine splenocytes: a potential common pathway for ethanol-induced immunomodulation.
Variable immunobiological changes occur with alcohol consumption. Previous studies have shown that acetaldehyde forms stable adducts with serum proteins, including albumin. These adducts are elevated in persons and animals consuming ethanol. We examined the effect of serum protein-acetaldehyde adducts formed with fetal bovine serum (FBS) on concanavalin A-stimulated murine splenocytes. Interleukin-2 (IL-2) secretion and IL-2 receptor (IL-2R) expression were determined as a function of the effect of the acetaldehyde-protein adduct(s). FBS was incubated with acetaldehyde (500, 100, 50, 25, 10, and 0 microM) for 1 hr at 37 degrees C. Excess acetaldehyde was removed by ultrafiltration using a 500 molecular weight cut-off membrane in 3 volumes. Free as well as bound acetaldehyde was quantified using fluorigenic HPLC before and after incubation. Recovered acetaldehyde correlated with the amount added (r2 = 0.996). Splenocytes were cultured for 48 hr in complete medium containing 5% acetaldehyde-treated and 5% untreated FBS with 4 micrograms/ml concanavalin A. Although cell viability was unchanged, acetaldehyde-treated FBS mixed with native FBS decreased IL-2 secretion in a dose-dependent manner. The percentage of cells expressing IL-2R was reduced only at the highest acetaldehyde-FBS dose. Therefore, immunological effects ascribed to ethanol may result in part from the toxic properties of acetaldehyde-protein adducts on IL-2 secretion.
Levels of Tap-1 and Tap-2 mRNA and expression of Kd and Db on splenic lymphocytes are normal in NOD mice.
It has been reported that the level of Tap-1 (transporter associated with antigen processing) mRNA and the expression of class I on splenocytes are low in NOD mice. Class I expression at 37 degrees C depends on an adequate supply of peptides, so a decrease in Tap could lead to lower class I levels. Since hypoexpression of class I correlated uniformly with the development of diabetes, it has also been suggested that Tap-1nod is diabetogenic. However, others report normal Tap-1 and class I levels in NOD mice. We examined Tap-1 and Tap-2 mRNA levels in NOD/Smrf mice using a reverse transcriptase-polymerase chain reaction method that detects > or = 25% changes in mRNA. We also assessed class I expression with three monoclonal antibodies. No difference in Tap-1 or Tap-2 mRNA levels for females of different ages or between diabetic and nondiabetic animals was observed. Tap-1 mRNA levels were identical between NOD/Smrf and BALB/cJ mice. Kd expression was significantly lower on NOD lymphocytes than in BALB/cJ cells, but the difference was due to the smaller size of the NOD splenic lymphocyte. When cells of the same size were analyzed, no difference in class I levels was observed. Class I levels were also identical in diabetic and age-matched nondiabetic NOD and BALB/c females. Both NOD Tap-1 mRNA and class I were increased by interferon-gamma. We find no evidence for impaired NOD Tap gene activity or class I expression, as previously reported for this strain.
Association of an androgen-responsive T cell phenotype with murine diabetes and Idd2.
T cells are involved in the induction and suppression of autoimmune diabetes in nonobese diabetic (NOD) mice. Because the incidence of diabetes is 13-fold greater in NOD/Smrf females, we searched for T cell phenotypes that showed sexual dimorphism and associated with diabetes in backcross segregants. The percentage of CD4+PBL was higher in NOD/Smrf females than males, was intermediate in [NOD X NON] F1 mice and approximated a 1:1 distribution in F1 mice backcrossed to either NOD or NON parental strains, suggesting primary control of the phenotype by an incompletely dominant gene, but not excluding additional effects by other genes. We term this primary gene Tlf(T lymphocyte frequency) because it also influenced the percentage of CD8+ T cells, although to lesser extent and independently from the MHC previously shown to lower the CD8+ T cell fraction in NON mice. Tlf segregated with diabetes in BC1 females, suggesting linkage with at least one diabetic locus. Genotyping of markers for Idd1, Idd2, and Idd3/10 revealed that Tlf mapped with Idd2 on chromosome 9. Dihydrotestosterone simultaneously lowered CD4+ PBL levels and prevented diabetes in NOD females while, in vitro, it had a differential effect on Con A elicited cytokines, increasing IL-2 22% and decreasing IL-4 39% (p < 0.0001). Thus the Tlf phenotype in NOD females, like diabetes, can be modulated by androgens.
Polymorphism in the mouse Tap-1 gene. Association with abnormal CD8+ T cell development in the nonobese nondiabetic mouse.
Tap-1 and Tap-2 genes code for a heterodimeric peptide transporter required for the normal maturation and surface expression of class I molecules. Polymorphic variants of these MHC encoded genes occur in rats and humans. After failing to amplify a 3' polymerase chain reaction (PCR) product from thymic and splenic cDNA of the nonobese nondiabetic (NON) strain, we considered it possible that Tap-1 polymorphism was present, since cDNA from CBA/J, C57BL/6, BALB/c, and NOD (nonobese diabetic) mice all yielded Tap-1 3' products. Overlapping PCR fragments spanning the highly conserved ATP-binding cassette (ABC) were generated for purposes of restriction endonuclease analysis, studies of IFN-gamma regulation, and sequencing. To avoid amplifying other members of the transporter family, we used a gel-purified 1670-bp Tap-1 PCR "long product" as template for nested PCR. Sequencing revealed three polymorphic alleles. The most divergent was for the NON strain and involved two non-conserved amino acid substitutions (Arg-->Cys397 and Leu-->Arg491) and three silent mutations. NON mice show an abnormal pattern of class I (Kb) expression and a sizeable reduction in the percentage of CD8+ cells in the blood and thymus. In F2 segregants, the low CD8 phenotype mapped to the MHC. Tap-1 genes of NON and C57BL/6 mice were equally sensitive to up-regulation by IFN-gamma. We conclude that the mouse Tap-1 transporter gene, like the Tap-2 of the rat and the Tap-1 and Tap-2 of the human, is polymorphic. The extensive variation and specific codon changes of Tap-1 in the NON mouse raise the possibility that this gene is the MHC locus responsible for altering the intrathymic development of CD8+ T cells.
How relevant are house dust mite-fungal interactions in laboratory culture to the natural dust system?
Both house dust and house dust mites Dermatophagoides pteronyssinus contained a wider range of fungi than laboratory mite cultures. In total, nine species of fungi were isolated from D. pteronyssinus in house dust, and these included three xerophilic species (Eurotium amstelodami, Aspergillus penicillioides and Wallemia sebi) commonly found in laboratory cultures of D. pteronyssinus. It is concluded that mites do interact with a similar range of fungi in natural dust and in laboratory culture, but that the diversity of fungal species in the laboratory is reduced and the density of individual fungal species in culture exceeds that of house dust. In a second experiment, dust samples were incubated at room temperature with 75% relative humidity. The diversity of fungi invariably declined from up to 13 genera to the few species recorded in laboratory culture. This suggests that the dominance of xerophilic fungi in laboratory mite rearings is mediated primarily by low relative humidity, and the exclusion of air-borne spores.
Studies of concanavalin A in nonobese diabetic mice. II. Lymphocyte tracking and phenotype responses.
We studied the tracking of 51chromium-labeled (resting) and Con A-induced (activated) tritiated thymidine-labeled syngeneic lymphocytes to adrenal, blood, brain, heart, liver, lymph nodes, pancreas, spleen, testis/ovary, thymus and thyroid in prediabetic, nonobese diabetic (NOD) mice and in age- and sex-matched C57BL/6 mice. 51Chromium-labeled cells showed no significant difference between strains in tracking to any tissue except lymph nodes (decreased in NOD vs. C57, P less than .05). Con A incubation resulted in no difference between strains in lymphocyte tracking to lymph nodes, but NOD mice had increased pancreatic tracking with Con A-incubated cells compared to C57 mice (P less than .05). Female NOD mice had reduced thymic tracking (P = .001) compared to C57 controls. Positive selection experiments showed the Con A-responsive cell to be a T cell. Both Lyt2+ (CD8+) and L3T4+ (CD4+) enriched T cell populations showed a labeling response to Con A. After 48 h of Con A incubation, the L3T4+/Lyt2+ ratio increased in splenocytes from NOD mice (P less than .05), whereas it decreased in C57 controls (P less than .01). Over the course of 6 days in culture, NOD splenocytes exposed to Con A characteristically exhibited a delayed expansion of the Lyt2+ population. We conclude that Con A incubation of NOD splenocytes results in T cells that, when reinfused, avoid the thymus and track preferentially to the pancreas. Con A immunomodulation as potential treatment for autoimmune disease warrants further study in this murine model.
Studies of concanavalin A in nonobese diabetic mice. I. Prevention of insulin-dependent diabetes.
Concanavalin A (Con A) administered in vivo inhibited the development of insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic mice. By experimental day 310 only one of 10 Con A-treated animals had developed IDDM compared to six of 11 cohort controls receiving buffered saline and 65% of colony controls. Treatment consisted of an initial 70-day course of Con A at a dose of 10 micrograms/g b. wt., i.p. in sterile saline every other day. After two deaths occurred in the Con A group, treatment was stopped at day 70. The remaining animals were given two 1-week booster doses beginning on experimental days 118 and 189. The single animal in the Con A group that eventually developed IDDM at day 293 had not received an injection for 107 days. Histological examination revealed peri-islet lymphocytosis but no islet infiltration in the Con A group. Surviving control animals showed both peri-islet and islet infiltrates. One-week Con A treatment significantly suppressed in vitro responses of spleen cells to Con A and allogeneic lymphocytes. The treatment increased the frequency of "blast-sized" cells in vivo and decreased the CD4+/CD8+ ratio among resting splenocytes. It is concluded that polyclonal T cell activation by Con A provides protection against autoimmune diabetes in nonobese diabetic mice concomitant with phenotypic and morphologic lymphocyte changes.
Heterosexual transmission of AIDS.
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Chlorantine fast green BLL as a stain for callose in oak phloem.
Sections of oak bark were stained with chlorantine fast green BLL, used as a 0.25% aqueous solution. All tissues were unstained, except for local deposits of material associated with phloem cell walls, which stained deep green. This green-staining material also stained specifically with resorcinol blue and with the aniline blue fluorescence technique, the usual histochemical tests for callose. The chlorantine fast green-staining material was removed from sections by treatment with a beta-1,3-glucan hydrolase. It is concluded that chlorantine fast green BLL stains callose in plant sections and is a useful additional stain for the histochemical detection of this polymer.
AIDS and parasitism.
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Intestinal protozoal infections in AIDS.
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Changes in corticotropic responsiveness and mitochondrial ultrastructure of adrenocortical cells from the inner zone of the duck (Anas platyrhynchos) adrenal gland: the effects of cycloheximide, puromycin, and chloramphenicol.
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Functional significance of interrenal cell zonation in the adrenal gland of the duck (Anas platyrhynchos).
Slices of whole adrenal gland tissue, incubated in vitro in the presence of ACTH for 1 h and 2 h produced corticosterone and aldosterone in constant ratio (16:1). Tangential slices taken from the region immediately below the connective tissue capsule and slices taken from deeper regions of the gland consisted primarily of cells conforming to the distinct structural characteristics of the subcapsular zone (SCZ) and inner zone (IZ) tissues respectively. When samples were incubated in the presence of ACTH for 1 h and 2 h, the interrenal cells of the SCZ produced relatively more aldosterone than cells taken from the IZ of the gland. The corticosterone: aldosterone ratio for the IZ after 1 h (68:1) and after 2 h (102:1) were ten times greater than the ratios for the SCZ after 1 h (7:1) and after 2 h (10:1). The SCZ slices were not more than 60 cells thick and consisted of cells arranged in cords. These cells contained irregular nuclei, mitochondria with shelf-like cristae and a moderate abundance of smooth endoplasmic reticulum. In contrast, the production of large amounts of corticosterone by the cells of the IZ was associated with tissue containing more vascular space than the SCZ and the cells contained large round nuclei surrounded by an abundance of smooth endoplasmic reticulum and the mitochondria had tubular rather than shelf-like cristae.
Structural changes occurring in internal tissue of the duck (Anas platyrhynchos) following adenohypophysectomy and treatment in vivo and in vitro with corticotropin.
Adrenal glands from ACTH-treated intact ducks and chronically adenohypophysectomized ducks showed clear zonation into a subcapsular zone (SCZ) and an inner zone (IZ). Adenohypophysectomy caused ultrastructural changes in the IZ but not in the SCZ cells. These included increases in lipid droplets, changes in mitochondrial cristae from tubular to shelf-like, and changes in the shape of the nuclei from spherical to crenated. These changes were reversed by treatment with ACTH. Also, cells of the IZ, but not the SCZ, of adrenals from intact birds given ACTH showed more SER, more dense bodies, fewer lipid droplets and more prominent Golgi complexes. IZ cells incubated in buffer containing no ACTH developed mitochondria with shelf-like cristae and numerous opaque granules in the matrix. Exposure to buffer containing ACTH caused the mitochondrial cristae to become tubular and the matrix granules either decreased in number or disappeared. The granules could be extracted by incubating sections with chelating agents. The mitochondria in SCZ cells did not respond structurally to the presence of ACTH in the incubation medium but the matrix granules, like those in IZ cells, responded to the presence of chelating agents.