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T K Huard

Publications and source records attributed to T K Huard.

13 recordsLinked to original sources
PubMed2006

Increasing prevalence of HIV-1 protease inhibitor-associated mutations correlates with long-term non-suppressive protease inhibitor treatment.

Treatment of human immunodeficiency virus type 1 with protease inhibitors (PIs) is associated with the emergence of resistance-associated mutations. Treatment-characterized datasets have been used to identify novel treatment-associated protease mutations. In this study, we utilized two large reference laboratory databases (>115,000 viral sequences) to identify non-established resistance-associated protease mutations. We found 20 non-established protease mutations occurring in 82% of viruses with a PI resistance score of 4-7, 62% of viruses with a resistance score of 1-3, and 35% of viruses with no predicted PI resistance. We correlated mutational prevalence to treatment duration in a treatment-characterized dataset of 2161 patients undergoing non-suppressive PI therapy. In the non-suppressed dataset, 24 mutations became more prevalent and three mutations became less prevalent after more than 48 months of non-suppressive PI-therapy. Longer durations of non-suppressive treatment correlated with higher PI resistance scores. Mutations at eight non-established positions that were more common in viruses with the longest duration of non-suppressive therapy were also more common in viruses with the highest PI resistance score. Covariation analysis of 3036 protease amino acid substitutions identified 75 positive and nine negative correlations between resistance associated positions. Our findings support the utility of reference laboratory datasets for surveillance of mutation prevalence and covariation.

Amino Acid Sequence↗
PubMed1999

A human prostatic stromal myofibroblast cell line WPMY-1: a model for stromal-epithelial interactions in prostatic neoplasia.

Here we report the characterization of an SV40 large-T antigen-immortalized stromal cell line, WPMY-1, derived from the same prostate as our previously described epithelial cell lines. The WPMY-1 cells were determined to be myofibroblasts on the basis of co-expression of smooth muscle alpha-actin and vimentin. They also show positive staining for androgen receptor, large-T antigen, and positive but heterogeneous staining for p53 and pRb. Their growth is stimulated by the synthetic androgen mibolerone to 145% of control (100%). Platelet-derived growth factor BB, epidermal growth factor and basic fibroblast growth factor, at 10 ng/ml, stimulated growth to 138, 143 and 146% of control, respectively. Transforming growth factor-beta, at 10 ng/ml, inhibited serum-induced growth to 65% of control in the presence of 1% serum, and bFGF-induced growth to 30% of control. A serum-free medium was developed for optimal growth of WPMY-1 cells. They show anchorage-independent growth in soft agar. Studies on paracrine interactions show that myofibroblast-conditioned medium causes a marked inhibition of growth in WPE1-10 cells, while conditioned medium from WPE1-10 prostatic epithelial cells caused only a small increase in the growth of WPMY-1 cells. WPMY-1 cells secrete very low levels of MMP-9 but high levels of MMP-2, markedly higher than the epithelial cells. These epithelial and myofibroblast cell lines, derived from the same prostate, provide novel and useful models for studies on paracrine stromal-epithelial interactions in carcinogenesis, tumor progression, prevention and treatment of prostate cancer and benign prostatic hyperplasia.

Actins↗
PubMed1992

alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins.

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.

Animals↗
PubMed1989

Effect of serum concentration on hybridoma cell growth and monoclonal antibody production at various initial cell densities.

Hybridoma cells (S3H5/gamma 2bA2) were cultivated using RPMI 1640 media with different serum concentrations at various initial cell densities. It was found that the serum requirement for cell growth was population-dependent. At an initial cell density of 1.10(4) cells/ml, the minimum serum concentration for cell growth was 5% (v/v) and reduced to 1% at the initial cell density of 1.10(5) cells/ml. When the initial density was increased to 1.10(6) cells/ml, serum was not needed for cell growth. The maximum cell density and the integral of viable cells over cultivation time increased as serum concentration or initial cell density was increased. However, specific antibody production rate was almost constant at 0.16 pg/cell.hr irrespective of serum concentration or initial cell density.

Animals↗
PubMed1986

Lectin binding studies on murine peritoneal cells: physicochemical characterization of the binding of lectins from Datura stramonium, Evonymus europaea, and Griffonia simplicifolia to murine peritoneal cells.

Purified 125I-labeled lectins from Datura stramonium, Evonymus europaea, and Griffonia simplicifolia (I-B4 isolectin) were used to analyze changes in the expression of carbohydrates on the surface of resident (PC) and thioglycollate-stimulated murine (C57B/6J) peritoneal exudate cells (PEC). The lectins from D. stramonium, E. europaea, and G. simplicifolia I-B4 bind specifically to PEC with relatively high affinity (Kd = 5.65 +/- 1.08 X 10(-7) M, 1.08 +/- 0.12 X 10(-8) M, and 1.33 +/- 0.15 X 10(-7) M, respectively). Assuming a single lectin molecule binds to each cell surface saccharide, the number of receptor sites per cell ranged for different cell samples from 22.3 to 50.0 X 10(6), from 3.8 to 4.8 X 10(6), and from 2.0 to 16.8 X 10(6) for D. stramonium, E. europaea, and G. simplicifolia I-B4 lectins, respectively. There were approximately 3- to 7-fold, 16- to 20-fold, and 2- to 20-fold increases in binding capacity for D. stramonium, E. europaea and G. simplicifolia I-B4, respectively, compared to the binding to resident, peritoneal cells. Scatchard plots of the binding of all three lectins to PEC were linear, suggesting that the receptor sites for these lectins are homogeneous and noninteracting. The binding capacity of these lectins to PEC was unchanged after trypsin digestion of cells. The expression of carbohydrates on the surface of PEC was also monitored by an agglutination assay. PEC were agglutinated by all three lectins whereas PC either were not agglutinated or were agglutinated only at high lectin concentrations. On the basis of our knowledge of the carbohydrate binding specificity of the D. stramonium and G. simplicifolia I-B4 lectins, we postulate that, parallel with thioglycolate stimulation, there is an increase in the number of N-acetyllactosamine residues and terminal alpha-D-galactosyl end groups. The blood group B, and H type 1 determinants--DGa1 alpha 1,3[LFuc alpha 1,2]DGa1 beta 1,3(or 4)DGlcNAc and LFuc alpha 1,2DGa1 beta 1,3DG1cNAc, respectively, as well as DGa1 alpha 1,3DGa1 beta 1,3(or 4)DGlcNAc--may be considered to be possible receptors for the E. europaea lectin. These glycoconjugates, present on the surface of peritoneal exudate cells, provide new chemical markers for studying the differentiation of resident peritoneal cells.

Agglutination Tests↗
PubMed1986

Macrophages express a plasma membrane receptor for basement membrane laminin.

Laminin, a noncollagenous extracellular matrix glycoprotein, can serve as an attachment factor for epithelial and endothelial cells to basement membrane collagen. The authors have observed that inflammatory macrophages have the capacity to bind exogenous laminin in a time-, temperature- and dose-dependent manner. The laminin binds to these cells via a specific plasma membrane receptor which is detectable on several macrophage subpopulations as well as isolated macrophage membranes. The isolated receptor is a trypsin-sensitive protein with an apparent molecular weight of 70,000 which binds laminin with relatively high affinity, Kd approximately equal to 4 X 10(-9) M. The inflammatory macrophages attach preferentially to a substrate of laminin compared to Type I or Type IV collagen. Thus, the authors postulate that during tissue inflammation macrophages utilize the laminin receptor to bind to vascular basement membranes.

Animals↗
PubMed1985

A potential role for the extracellular matrix glycoprotein laminin in macrophage-tumor-cell interactions.

Although cell surface molecules are thought to be involved in macrophage (MO)-tumor-cell recognition, the nature of these molecules remains unknown. In this study we have shown that the glycoprotein laminin may facilitate macrophage-tumor-cell binding. Macrophage binding to tumor cells was assessed by measuring the adherence of radiolabelled 3-MCA2 induced malignant fibrosarcoma cells to syngeneic peritoneal MOs. Addition of exogenous laminin promoted the binding of a weakly metastatic subline of these tumor cells by 31-68%. These weakly metastatic tumor cells express negligible endogenous cell-surface laminin but display specific cell-surface receptors for binding soluble laminin. Exogenous laminin promoted MO binding of these tumor cells whether it was present during the assay or whether the tumor cells were pretreated with the laminin. This increase in binding was blocked by anti-laminin antibody. In contrast, MO binding of a strongly metastatic variant of the same tumor was not enhanced by the addition of exogenous laminin. This highly malignant fibrosarcoma line already expressed endogenous cell-surface laminin. Since the MOs were found to specifically bind 125I-laminin, the interaction between laminin-bearing tumor cells and MOs may be mediated via a specific MO plasma membrane receptor. Thus, the expression of cell-surface laminin and its receptors on both tumor cells and MOs may provide a mechanism for promoting MO-tumor-cell binding.

Animals↗
PubMed1983

Macrophages express cell surface laminin.

Laminin, a non-collagenous extracellular connective tissue glycoprotein, was detected on the surface of mouse peritoneal macrophages. As determined by indirect immunofluorescence, as many as 60% of peritoneal macrophages elicited with thioglycollate expressed cell surface laminin. Only 14% of resident cells displayed detectable laminin. The expression of laminin increased with time post-injection. Concomitant with laminin expression, macrophages also displayed a receptor for the IB4 isolectin from Griffonia simplicifolia. This lectin, which binds methyl-alpha-D-galactopyranoside, may also react with the carbohydrate moeity of laminin. A small population of macrophages displayed both laminin and surface fibronectin. Unlike the difference in laminin expression between resident and thioglycollate-stimulated cells, there was no difference in cell surface fibronectin between these cell populations. Since laminin has been found to mediate cell attachment in other systems, expression of this molecule on the surface of stimulated macrophages may be important in cell-cell and cell-matrix adhesive properties of these cells.

Animals↗
PubMed1983

Effects of serum from patients with system lupus erythematosus on leukocyte cytotoxicity: modulation of NK activity and ADCC.

The ability of sera from patients with systemic lupus erythematosus (SLE) to alter the natural killer (NK) cell activity and antibody dependent cell mediated cytotoxicity (ADCC) of peripheral blood mononuclear cell populations from normal donors was determined employing 4-h 51Cr release assays. SLE sera significantly lowered the NK activity of both whole mononuclear cell populations (MN) (26 +/- 3% decrease) (mean +/- SEM) and lymphocyte enriched populations (24 +/- 5%) toward K562 targets when compared to results with sera from normal donors. The continuous presence of serum was not required for this effect since MN effector cells preincubated with sera and then washed also exhibited diminished NK activity. SLE sera decreased only slightly lymphocyte-mediated ADCC to either erythrocyte (human 0 + RBC) or tumor cell (human CEM T lymphoblast) targets. ADCC by isolated monocytes, however, was significantly diminished by SLE sera (54 +/- 5%). These data suggest that in patients with SLE, serum factors may be responsible for alteration of both NK activity and ADCC.

Antibody-Dependent Cell Cytotoxicity↗