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Biomedical subjects

L Hazlett

Publications and source records attributed to L Hazlett.

12 recordsLinked to original sources
PubMed1999

Expression of CD134 (0X-40) on T cells during the first 100 days following allogeneic bone marrow transplantation as a marker for lymphocyte activation and therapy-resistant graft-versus-host disease.

CD134 (OX-40) is an activation-associated antigen which functions as a costimulatory receptor for CD4+ T cells. In order to determine the expression of CD134 during immune recovery following allogeneic bone marrow transplantation (BMT), we measured its expression on T cells and T cell subsets during the first 100 days following BMT in 26 patients. CD4+CD134+ T could be seen approximately 14 days following BMT cells in patients who did not develop GvHD which required therapy (n = 20). The percentage of CD4+CD134+ cells continued to increase up to the fourth week following BMT to a maximum of 40-50% of CD4+ T cells (normal = 1-8%). Two patients who developed Grade I-II GvHD and who responded to treatment with pulsed high-dose methylprednisolone (MPD) showed a decline of approximately 40% in CD4+CD134+ T cells was seen within 48 hours of treatment. Four patients who developed GvHD that was not responsive to MPD and who later developed high IV GvHD showed increasing CD4+CD134+ T cells up to 85% of the CD4+ T cells. Additionally, rapid increases in CD134+ T cells following antibody-based T cell therapy were associated with GvHD recurrence. In no cases was the percentage of CD134+ CD4+ T cells predictive of clinical GvHD. In this exploratory study, we have shown that CD134, although not predictive of the initial onset of GvHD, may be a useful tool for monitoring the response to early GvHD therapy and identification of patients at risk for reemergence of GvHD who may benefit from anti-T cell therapy. Cytometry (Comm. Clin. Cytometry) 38: 238-243, 1999.

Biomarkers↗
PubMed1998

Phenotypic and functional reconstitution of peripheral blood lymphocytes following T cell-depleted bone marrow transplantation from partially mismatched related donors.

Myeloablative chemotherapy followed by transplantation of a T cell-depleted bone marrow graft from a partially mismatched related donor provides a potentially curative option for patients with leukemia and other disorders of hematopoiesis, although the patient is faced with a period of sustained immunodeficiency as well as pharmacologic immunosuppression as a result of prophylaxis against graft-versus-host disease. Thirty patients who received one to three antigen T cell-depleted mismatched grafts were evaluated for immune reconstitution. The percentage and numbers of cells expressing lymphocyte subset antigens were determined by flow cytometry at 14, 28, 60, 100, 180, 270 and 365 days post-BMT and at 6 month intervals thereafter. Lymphocyte reconstitution was characterized by the early appearance of natural killer cells and a low percentage of both T and B cells. During the first year after BMT, the number of NK cells remained constant while T and B cells gradually returned to normal numbers and proportions. Response to the lymphocyte mitogen phytohemagglutinin returned to normal over the course of 2 years, while the response to concanavalin A was slightly depressed and the response to pokeweed mitogen became supranormal at about 1.5 years and continued to increase. These data suggest the need for long-term immunophenotypic monitoring as well as prolonged infection surveillance and prophylaxis.

B-Lymphocytes↗
PubMed1996

Ultrastructural, immunohistological and biochemical characterization of cultured mouse corneal epithelial cells.

We developed a method to culture mouse corneal epithelium. Cultured cells tested by 1-D-SDS-PAGE exhibited protein mobility patterns similar to freshly isolated epithelia. Western blots with antibodies broadly recognizing cytokeratins showed a similar pattern for both fresh and cultured cells, but only the fresh sample stained with J7, specific for a 55-kD 'corneal' cytokeratin. Cultured cells examined at confluency by transmission electron microscopy exhibited desmosomal contacts typical of epithelia. The ability to culture mouse corneal epithelial tissue will be useful for studies requiring large amounts of material by reducing animal numbers.

Animals↗
PubMed1995

In the immature mouse, Pseudomonas aeruginosa pili bind a 57-kd (alpha 2-6) sialylated corneal epithelial cell surface protein: a first step in infection.

PURPOSE: To test the hypothesis that in the unscarified immature eye, Pseudomonas aeruginosa pili bind glycoprotein receptors, one or more of which are surface associated. METHODS: Several methods--including radioiodination of bacterial pili and surface-associated corneal epithelial proteins (CEPs), solid-phase binding assays, carbohydrate detection, and immunoblotting techniques in which periodate oxidation and preincubation of blots with purified pili, neuraminidase, sialic acid, other sugars, and SNA and MAA lectins--were used to identify and characterize host proteins. Some of these proteins in the immature mouse corneal epithelium interacted with bacterial pili. RESULTS: Seven proteins, with molecular weights from 14 to 66 kd were identified that strongly bound PAK/PR1 pili. To determine if any protein(s) was cell surface localized, corneal epithelial surface membrane proteins were radioiodinated and examined using a pilus overlay assay and lectin analysis. Only one protein of 57 kd was cell surface labeled and bound pili in an overlay assay. This protein was alpha (2-6) sialylated, as shown by SNA binding. Furthermore, SNA lectin was able to block pilus binding to CEPs. 125I labeling of pili and a solid-phase binding assay confirmed that pili bind to CEPs and, further, that binding could be competitively inhibited by excess unlabeled pili and that the receptors appeared saturable. GlycoTrack reagents were used to show that the epithelial proteins of the postnatal day 5 (P5) mouse cornea were glycosylated. Removal of carbohydrates by preincubation of blots with periodate, or combining pili with sialic acid, eliminated pili binding. Pretreatment of blots with either neuraminidase (N'ase) to decrease and/or remove sialic acid residues, or pretreatment with SNA lectin with specificity for alpha (2-6) linked sialic acid to galactose, also diminished pili binding to CEPs. Other sugars or MAA lectin, specific for sialic acid alpha (2-3) linked to galactose, had no inhibitory effect. CONCLUSIONS: These data show that a 57-kd surface membrane protein bound pili in the immature cornea and that for both this protein and the other nonsurface proteins, sialic acid alpha (2-6) linked to galactose was important in receptor recognition by the pilus adhesion. The 57-kd protein is putatively important in the initial interaction of pili with the unwounded ocular epithelium and may be the initial pathogenic event in this model.

Animals↗
PubMed1992

Proteoglycans from osteoarthritic human articular cartilage influence type II collagen in vitro fibrillogenesis.

Collagen fibrils were formed in the presence of dermatan sulfate (DSPG) and high density (HDPG) proteoglycans isolated from human adult knee femoral articular cartilage. Eroded cartilage had a higher percentage of DSPGs in the extracted proteoglycans than normal cartilage (p = .018). The dermatan sulfate proteoglycans (DS-PGI and DS-PGII) were detected in normal and osteoarthritic cartilage. DSPGs compared to HDPG inhibited in vitro collagen fibrillogenesis producing a longer lag phase (p less than .05) and a slower rate of fibril formation (p less than .05). DSPGs from eroded osteoarthritic cartilage alone or in combination with HDPG produced a longer lag phase than DSPGs from normal cartilage alone or in combination with HDPG (p less than .05). The inhibition of fibrillogenesis by DSPGs suggests that collagen fibril formation in vivo may be abnormal due to the influence of molecular changes in proteoglycan as well as an increased proportion of DSPGs occurring in osteoarthritic cartilage. Abnormal fibril formation may produce a weakened cartilage matrix, thus contributing to an accelerated process of cartilage degeneration in osteoarthritis.

Cartilage, Articular↗
PubMed1991

Pseudomonas aeruginosa: a probe to detail change with age in corneal receptor(s).

This in vitro study used Pseudomonas aeruginosa as a probe to examine the change with age of the host receptor(s) facilitating bacterial adherence to cornea in the immature eye and to pinpoint in time, when the host receptor(s) was no longer able to be recognized by the bacterium. Scanning EM was used to quantitate adherent organisms at the corneal surface of mice of select ages (5-37 postnatal days, P, and 28 months) at 15, 30 and 60 min following bacterial application. Organisms were most numerous at the surface of the 5-P eye at all time periods. In 9-21-P mice, no bacterial binding was observed until 30 min, and bacterial adherence modestly increased at 60 min after bacterial application. In the 37-P and in aged 28-month-old mice, no binding to cornea was observed until 60 min after bacterial application. Statistical analysis of these data revealed significant differences in the mean number of bacteria binding to cornea between age groups.

Aging↗
PubMed1991

Characterization of pseudomonal adherence to unwounded cornea.

This study examined binding of Pseudomonas aeruginosa to unwounded postnatal day (P) 5 immature mouse cornea. To determine whether the receptor molecule was protein or lipid in nature, the eyes were incubated in vitro with trypsin or lipase before bacteria were applied. Trypsin significantly increased bacterial binding, whereas lipase had no effect. To determine if enhanced binding after trypsin indicated exposure of a lipid or a protein receptor, the eyes were treated sequentially with trypsin, then lipase. Subsequent binding did not differ significantly from trypsin alone, indicating that the exposed receptor was not a lipid. Incubation of the P 5 eye with neuraminidase to remove sialylated residues also significantly enhanced binding at all times, and premixing of the inoculum with the enzyme before adherence testing increased binding at 15 and 30 min. The effects of a monosialoganglioside (GM1) and gangliotetraosylceramide (asialo GM1) were also examined. Incubation of eyes with GM1 or asialo GM1 produced no significant inhibition of bacterial binding, but premixing of the bacterial inoculum with GM1 or asialo GM1 before corneal application transiently decreased adherence. Fibronectin (FN) treatment of the P 5 eye, premixing FN with the bacterial inoculum before its ocular application, or similar treatment with N-acetylneuraminic acid (NANA) transiently inhibited binding. These data demonstrate that pseudomonal binding to the unwounded eye is not lipase sensitive and is enhanced by trypsin treatment which exposes a lipase insensitive receptor and by neuraminidase which removes sialylated residues. It is not inhibited by pretreatment of the eye with either GM1 or asialo GM1 and is transiently inhibited by pretreatment of the eye or the inoculum with NANA or FN.

Animals↗
PubMed1988

Study of experimental cataract produced by sugar alcohols in the organ-cultured mammalian ocular lens.

Freshly isolated rabbit lenses were cultured in isosmolar TC-199 medium or hyperosmolar medium containing 180 mM sorbitol or mannitol. These experiments were performed to investigate the probable effects of hyperosmolar media on lens clarity and the ability of lens fiber cells to synthesize membrane intrinsic protein, MP-26. The data from these experiments show that incubation in hyperosmolar medium causes depressed MP-26 synthesis, whereas the presence of sugar alcohols in the culture medium induced anterior and posterior subcapsular opacities. The cation levels of lenses incubated in iso- and hyperosmolar medium were also measured. Data from these experiments revealed that although the experimental lenses display prominent opacities, their cation levels are generally similar to those of control lenses. It is proposed that the observed lens opacities are due to the presence of sugar alcohols in the culture medium and not to hyperosmolar shock.

Animals↗
PubMed1987

Examination of mouse exorbital lacrimal gland after exposure to 2-chloroacetophenone vapor.

The compound 2-chloroacetophenone has been used to stimulate tear secretion from human subjects, yet the morphological response of the lacrimal gland to this agent has not been experimentally determined. This study used light and electron microscopic techniques to examine the effect of this agent with time on the morphology of the mouse exorbital lacrimal gland. In brief, exposure to 2-chloroacetophenone vapor causes rapid exocytosis of acinar cell granules as well as vacuolation of the secretory and ductal epithelial cells. Concomitantly, intralobular ductal epithelial cells which are normally cuboidal in shape, enlarge in size and release electron-dense granules into the duct lumen. Within 15 min after exposure, acinar cells show a preponderance of cells containing only pale granules and the ductal epithelium returns to a more cuboidal shape. By 60 min after exposure, the gland is not readily distinguishable from the unexposed, control gland.

Animals↗
PubMed1986

Development of ocular mucin: scanning EM analysis.

Ocular mucin (mucus) development in the mouse was examined using several different fixatives and scanning electron microscopy. One of the fixatives, a picric acid--paraformaldehyde-glutaraldehyde (PA-P-G) preparation which fixes mucin at the adult mouse corneal surface, first demonstrated mucin at the surface of the pup cornea and conjunctiva at postnatal day 10. The mucus was scant, strand-like as well as granular in morphology and did not form smooth patches as in the adult. Thus, mucin is present at the ocular surface well before the mouse eye opens (day 14-15). Its presence is essential to ensure tear film stability, corneal wettability and possibly to protect the eye from bacterial and viral insult.

Animals↗
PubMed1985

Actinlike material in Pseudomonas aeruginosa.

Actinlike material was obtained from disrupted Pseudomonas aeruginosa cells by a modification of the method of Hancock and Nikaido (J. Bacteriol. 136:381-390) for isolating outer membrane vesicles. Pelleted membranes were dissolved in Laemmli sample buffer and electrophoresed in parallel lanes with purified rabbit skeletal muscle actin. The bacterial preparation migrated similarly to rabbit skeletal muscle actin. A doublet band was detectable by affinity-purified antiactin antibody in a passive transfer immunoblot. Molecular weight of the actinlike protein doublet was 60,000 to 63,000 as determined by linear regression analysis of Bio-Rad low-molecular-weight standards run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transmission electron microscopy of the actinlike material in high salt concentrations revealed 10- to 14-nm filaments of various lengths. Despite its ability to form filaments and to react with a polyclonal rabbit antiactin antibody, the bacterial filaments did not bind the S-1 fragment of heavy meromyosin.

Actins↗