Biomedical subjects

M W Robertson

Publications and source records attributed to M W Robertson.

At least 19 recordsLinked to original sources

PubMed2001

Inhibition of antigen-induced mediator release from IgE-sensitized cells by a monoclonal anti-Fc epsilon RI alpha-chain receptor antibody: implications for the involvement of the membrane-proximal alpha-chain region in Fc epsilon RI-mediated cell activation.

The interaction between human IgE and its high affinity receptor, FcepsilonRI, is a critical event in mediating the allergic response. Aggregation of the alpha-chain of FcepsilonRI (FcepsilonRIalpha) occurs via cross-linking of receptor-bound IgE by Ag, resulting in cell activation and the release of mediators of hypersensitivity. Recently, we mapped the epitopes of two anti-FcepsilonRIalpha mAbs, 15/1 and 5H5F8. In contrast to 15/1, mAb 5H5F8 does not inhibit IgE binding to FcepsilonRIalpha. Here we demonstrate both 5H5F8 binding to FcepsilonRI(+) cells as well as a high level of IgE binding to 5H5F8-saturated cells. At the same time 5H5F8 strongly inhibits hexosaminidase release and Ca(2+) flux after Ag triggering from human IgE-sensitized RBL-2H3 cells stably transfected with human FcepsilonRIalpha. Further, 5H5F8 and its Fab inhibit sulfidoleukotriene and histamine release from primary human peripheral blood leukocytes, including cells bearing endogenous IGE: Furthermore, we confirm that 5H5F8 maps to a linear peptide sequence in close proximity to the cell membrane. Two chemically synthesized peptides containing the 5H5F8 epitope sequence PREKY were selected for detailed analysis of 5H5F8 and 5H5F8 Fab binding and were found to produce K(d) values of similar magnitude to that observed for binding to recombinant FcepsilonRIalpha. These peptides may prove useful as targets for the identification of antagonists of FcepsilonRIalpha-mediated biological activity. Moreover, our data indicate that FcepsilonRIalpha-mediated activation may involve a novel alpha-chain epitope in an early step of the cell-triggering pathway leading to cellular activation.

Amino Acid Sequence↗

PubMed2000

Export of the high affinity IgE receptor from the endoplasmic reticulum depends on a glycosylation-mediated quality control mechanism.

The high affinity IgE receptor (FcepsilonRI) is a multisubunit complex comprised of either alphagamma(2) or alphabetagamma(2) chains. The cotranslational assembly of the IgE-binding alpha-chain with a dimer of gamma-chains occurs in a highly controlled manner and is proposed to involve masking of a dilysine motif present at the cytoplasmic C terminus of the FcepsilonRI alpha-chain that targets localization of this subunit to the endoplasmic reticulum (ER). Here, we show that ER quality control modulates export from the ER of newly synthesized alphagamma(2) and alphabetagamma(2) receptors. We demonstrate that the presence of untrimmed N-linked core glycans (Glc(3)Man(9)GlcNAc(2)) on the FcepsilonRI alpha-chain activates the ER quality control mechanism to retain this subunit in the ER, despite the presence of gamma-chains. At the same time, the untrimmed, ER-localized alpha-chain exhibits IgE-binding activity, suggesting that FcepsilonRI alpha-chain folding occurs before constitutive glucose trimming. In additional experiments, we demonstrate that cell surface expression of an alpha-chain C-terminal truncation mutant is also dependent on glucose trimming, but not on gamma-chain coexpression. We suggest that glucosidase trimming of terminal glucose residues is a critical control step in the export of FcepsilonRIalpha from the ER. Finally, we show that the constitutive ER FcepsilonRI alpha-chain, expressed in the absence of the other FcepsilonRI subunits, associates with the ER lectin-like chaperone calnexin, but not the structurally similar ER chaperone calreticulin, presumably through interaction with monoglucosylated alpha-chain ER glycoforms.

Animals↗

PubMed2000

A patient presenting with a pelvic mass, elevated CA-125, and fever.

BACKGROUND: Tuberculous peritonitis is a rare event which can mimic advanced stage ovarian cancer. A pelvic mass and an elevated CA-125 is suggestive of an ovarian malignancy; however, benign conditions may be discovered, especially in the premenopausal patient. CASE: A patient with a pelvic mass, ascites, and an elevated CA-125 underwent an exploratory laparotomy for presumed ovarian cancer. Final pathology revealed pelvic tuberculosis without any pulmonary involvement. Acid-fast bacilli were confirmed with polymerase chain reaction in the surgical specimen. DISCUSSION: Pelvic tuberculosis is an uncommon gynecologic condition that presents with ascites, a pelvic mass, and fever. An elevated CA-125 is not specific for ovarian malignancy.

Adult↗

PubMed1999

Surgical management of endometrial adenocarcinoma using laparoscopically assisted staging and treatment.

BACKGROUND: Because of inaccuracies in clinical staging, endometrial adenocarcinoma is now a surgically staged disease. This study was done to determine the safety and efficacy of a laparoscopically assisted approach in the treatment and staging of this disease. METHODS: Using a retrospective chart review, we identified demographic characteristics, mean blood loss, operative findings, and complications of patients who had laparoscopically assisted staging and treatment for endometrial carcinoma from 1992 to 1997. RESULTS: Of 34 patients, 28 had laparoscopic surgical staging that included pelvic and para-aortic lymph node assessment, peritoneal washings, bilateral salpingo-oophorectomy, and total vaginal hysterectomy; 23 patients (82%) had stage I disease, 2 (7%) had stage II disease, and 3(11%) had stage III disease. Complications included herniation through a 5 mm port site, necessitating small bowel resection, and a fatal myocardial infarction 10 days postoperatively. CONCLUSION: Laparoscopic staging and treatment of endometrial carcinoma is appropriate in a select group of patients.

Adenocarcinoma↗

PubMed1998

Response to salvage treatment in recurrent ovarian cancer treated initially with paclitaxel and platinum-based combination regimens.

OBJECTIVE: The aim of this study was to evaluate the response to salvage treatment in recurrent ovarian cancer treated initially with paclitaxel-based chemotherapy. METHODS: A retrospective review of patients with recurrent ovarian cancer treated with surgical debulking and paclitaxel-based chemotherapy was performed. All cases received second-line treatment with a response evaluated by clinical or surgical means. Data analysis was conducted using the SAS statistical package. RESULTS: Fifty cases of advanced stage disease were available for review. Patients received paclitaxel and cisplatin or carboplatin with a 72.0% response rate. The median time to recurrence after primary treatment was 6 months. Second-line treatment included cisplatin or carboplatin (50%), Taxol (10%), or lutetium (22%), an intraperitoneal radiolabeled monoclonal antibody targeted to TAG-72. A 52.0% clinical response to salvage treatment was detected. With a median follow-up of 7 months, 68.0% of patients had experienced recurrence or progression of their disease. The median time to second recurrence was 5 months. Cases sensitive to initial paclitaxel-containing chemotherapy responded to any of the salvage treatments more frequently than chemotherapy-resistant tumors (88.5% versus 11.5%, P < 0.05). CONCLUSIONS: Recurrent ovarian cancer patients initially treated with paclitaxel-based chemotherapy frequently responded to salvage treatment. However, the duration of response was brief, and hospitalization for treatment-related side-effects was common. Tumor response to initial paclitaxel/platinum treatment was predictive of future response to second-line agents. Current salvage therapies appear to provide little benefit in cases of tumors resistant to primary chemotherapy.

Adult↗

PubMed1998

Endothelial cell vehicles for delivery of cytotoxic genes as a gene therapy approach for carcinoma of the ovary.

Human umbilical vein endothelial cells (HUVECs) were evaluated for utility as a vector to achieve a bystander effect and killing of ovarian carcinoma cell lines. After demonstrating that HUVECs could be transduced with the reporter gene LacZ encoded by an adenoviral vector, appropriate cell killing of the AdCMVHSV-TK-transduced HUVECs was exhibited after treatment with 20 microM ganciclovir. Mixing experiments were then performed to determine whether the transduced HUVECs would demonstrate a bystander effect with the ovarian cancer cell lines. When 50% AdCMVHSV-TK-transduced HUVECs were mixed with untransduced ovarian cancer cells, > 70% of all cells were killed. Finally, s.c. and i.p. injections of herpes simplex-thymidine kinase-expressing HUVECs and SKOV3ip1 tumor cells were performed to evaluate the effects of HUVECs in in vivo models. These studies showed a decrease in tumor growth s.c. as well as a statistically significant survival prolongation (P < 0.05) in the i.p. model. These findings suggest that endothelial cells may be used as a vehicle for the delivery of cytotoxicity (bystander effect) in molecular chemotherapy.

Animals↗

PubMed1998

Gene therapy for ovarian carcinoma.

Originally conceived and applied for the treatment of inherited monogenetic defects such as adenosine deaminase deficiency and cystic fibrosis, gene therapy was later applied to the treatment of cancer. Such a genetic strategy seemed rational given the recognition that cancer typically develops in a multistep process involving alterations of a number of different genes as demonstrated in familial polyposis and colorectal cancer through the work of Vogelstein et al. Because of the numerous alterations that may result in the eventual development of cancer, there is no obvious single choice for a therapeutic gene. Although one may view this as an obstacle, it also allows for a variety of possible therapeutic interventions. This review focuses on the known genetic defects that occur in ovarian cancer, the gene therapy strategies suggested by such defects, and the approaches under current development for the treatment of this disease. As such, this work also describes some of the approved human gene therapy protocols. Finally, an overview of the problems and directions for future growth and research is presented.

Drug Resistance, Multiple↗

PubMed1997

Affinity of the interaction between Fc gamma receptor type III (Fc gammaRIII) and monomeric human IgG subclasses. Role of Fc gammaRIII glycosylation.

Binding of the Fc region of IgG antibodies to low affinity Fc gamma receptors (Fc gammaR) triggers important effector functions in the immune system. The type IIIb Fc gammaR (Fc gammaRIIIb or CD16) is a heavily glycosylated protein anchored to the membrane of neutrophils by a glycosylphosphatidylinositol link. This receptor contributes to cell activation by IgG immune complexes. To better understand the nature of the ligand-receptor association, we have studied the affinity and kinetics of the interaction between human IgG subclasses and two soluble forms of Fc gammaRIIIb (sFc gammaRIIIb or sCD16) corresponding to the 188 N-terminal amino acids of the extracellular region of the receptor, a glycosylated one made in eucaryotic cells (euc.sCD16) and a non-glycosylated one (proc.sCD16) made in Escherichia coli. Experiments using a BIAcore instrument, to measure protein binding in real time, showed that monomeric human IgG1 and IgG3, but not IgG2, IgG4, IgA and divalent antigen-binding fragments (F(ab')2) of IgG1, bound to immobilized euc.sCD16 with an affinity constant (K(A)) of 1.3 +/- 0.6 x 10(6) M(-1) and 2.6 +/- 0.4 x 10(5) M(-1), respectively. The affinity constant of proc.sCD16 for human IgG1 was in the same range (1.1 +/- 0.2 x 10(6) M(-1)), whereas that for human IgG3 was twofold higher (4.2 +/- 0.4 x 10(5) M(-1)). The specificity of the non-glycosylated receptor for human IgG subclasses bound to Sepharose was IgG1 > IgG3 >> IgG4 >>> IgG2. Thus, the extracellular polypeptide of Fc gammaRIIIb dictates the interaction of the receptor with IgG subclasses although glycosylation plays an inhibitory role in the interaction with human IgG3.

Animals↗

PubMed1996

Peptides derived from IgE heavy chain constant region induce profound IgE isotype-specific tolerance.

(BALB/c x SJL)F1 mice, perinatally injected with peptide-N-glyconase F-treated, deglycosylated IgE heavy chain or recombinant IgE heavy chain (CH epsilon 2-CH epsilon 4), were profoundly inhibited in antigen-specific IgE production. There exist minimally two tolerogenic IgE peptides, residing in the CH epsilon 2 and CH epsilon 4 domains. Peptide I, generated by V8 protease, comprises 39 amino acids within CH epsilon 2, beginning at amino acid 103. Peptide E begins at amino acid 312 of the CH epsilon 4 domain and extends through the CH epsilon 4 domain. The total lack of antigen-specific IgE responses in IgE peptide-treated mice was not due to overproduction of interferon-gamma, nor lack of interleukin (IL)-4, as predicted by the Th2/IL-4 paradigm for IgE production. IgE-tolerant mice exhibited comparable levels of circulating anti-IgE antibodies to those of PBS-treated control mice. IgG obtained from sera of both sources failed to inhibit IgE responses in vitro. Moreover, IgE responses of spleen cells from IgE peptides-treated mice were restored by CD4+ T cells from PBS-treated control mice. We hypothesize that regulation of antigen-specific IgE responses is mediated by CD4+ T cells which normally recognize IgE peptides on IgE precursor B cells, and can be rendered tolerant by perinatal IgE peptide treatment.

Animals↗

PubMed1995

Glycosylation of human truncated Fc epsilon RI alpha chain is necessary for efficient folding in the endoplasmic reticulum.

The high affinity immunoglobulin E (IgE) receptor is an alpha beta gamma 2 tetrameric complex. The truncated extracellular segment (alpha t) of the heavily glycosylated alpha chain is sufficient for high affinity binding of IgE. Here we have expressed various alpha t mutants in eukaryotic and prokaryotic cells to analyze the role of glycosylation in the folding, stability, and secretion of alpha t. All seven N-linked glycosylation sites in alpha t are glycosylated and their mutations have an additive effect on the folding and secretion of alpha t. Mutation of the seven N-glycosylation sites (delta 1-7 alpha t) induces misfolding and retention of alpha t in the endoplasmic reticulum. Similarly, tunicamycin treatment reduces substantially the folding efficiency of wild-type alpha t. In contrast, no difference in folding efficiency is detected between wild-type alpha t and delta 1-7 alpha t expressed in Escherichia coli. In addition, maturation of N-linked oligosaccharides and addition of O-linked carbohydrates are not required for either the transport or the IgE-binding function of alpha t. Furthermore, complete enzymatic deglycosylation does not affect the stability and the IgE-binding capacity of alpha t. Therefore, glycosylation is not intrinsically necessary for proper folding of alpha t but is required for folding in the endoplasmic reticulum. Our data are compatible with the concept that specific interactions between N-linked oligosaccharides and the folding machinery of the endoplasmic reticulum are necessary for efficient folding of alpha t in eukaryotic cells.

Amino Acid Sequence↗

PubMed1994

Effects of prenatal cocaine exposure upon postnatal development of neostriatal dopaminergic function.

Pregnant rats were injected twice daily with 20 mg/kg cocaine (or saline) from gestational day 10 to parturition. Brains from offspring were examined with quantitative receptor autoradiography [D1 receptor (D1R), D2 receptor (D2R) and dopamine transporter (DAT)] and quantitative in situ hybridization [D1R mRNA, D2R mRNA, preproenkephalin (PPE) mRNA] for markers of neostriatal dopaminergic function. Prenatal cocaine exposure did not alter postnatal development of striatal D1R sites, but D1R mRNA levels were reduced by a third at days 14 and 35. D2R sites were increased over control in lateral striatum by day 6, and remained elevated through postnatal day 35. Total D2R mRNA was increased over control in both medial and lateral striatum at 7 and 14 days but was equal to control at 35 days. Prenatal cocaine exposure increased DAT density at postnatal days 1 through 5, but reduced it at days 14 and 35; PPE mRNA expression was reduced at days 7, 14 and 35. Many of these results are similar to those found in experimental animals and humans following cocaine withdrawal.

Animals↗

PubMed1993

Phage and Escherichia coli expression of the human high affinity immunoglobulin E receptor alpha-subunit ectodomain. Domain localization of the IgE-binding site.

The high affinity IgE receptor is a multisubunit complex that participates in IgE-dependent activation of mast cells and basophils. The IgE-binding portion of the receptor resides exclusively in the alpha-subunit and specifically within the 180 residues of the mature extracytoplasmic portion. In this study the contiguous two-domain human alpha-subunit has been displayed on the surface of a filamentous bacteriophage in a form that specifically binds both human and murine IgE but not other isotypes. Phage display of the individual immunoglobulin-like domains reveals that the IgE-binding portion resides exclusively in the COOH-terminal domain and that this domain appears to bind IgE with lower affinity than the comparably displayed two-domain fragment. Phage display results using a chimeric rat-human alpha-subunit fragment suggest that structural elements within the NH2-terminal domain are necessary to impart high affinity IgE binding activity to the alpha-chain ectodomain. The two-domain human receptor fragment was also expressed in a soluble form from Escherichia coli and purified in one step by affinity chromatography. The solution binding of the purified receptor fragment to IgE was measured by fluorescence quench which afforded an approximate equilibrium affinity constant of 2 x 10(9) M-1 together with a stoichiometry of 1 receptor molecule/molecule of IgE. The complementary approach of phage display and E. coli expression used in this study represents a useful strategy for further analysis of discrete receptor epitope(s) that contribute to IgE binding activity.

Animals↗

PubMed1991

Heterogeneous IgE glycoforms characterized by differential recognition of an endogenous lectin (IgE-binding protein).

IgE is highly glycosylated, but the function of the oligosaccharide side chains is largely unknown. The previous discovery of an animal lectin, IgE-binding protein (epsilon BP), affords an opportunity to study potential carbohydrate-dependent effector functions of IgE. epsilon BP is a beta-galactoside-specific lectin with binding affinity for IgE and is now known to be equivalent to carbohydrate-binding protein 35 and the Mac-2 Ag; thus, it may have multiple functions in addition to IgE binding. We have previously shown that rat r epsilon BP recognizes sialidase-treated human myeloma IgE to a much greater extent than the untreated IgE. In contrast, human epsilon BP binds essentially equivalently to a monoclonal murine IgE with or without sialidase pretreatment. To validate a possible role for epsilon BP in the IgE system, we investigated the pattern of recognition of epsilon BP for various polyclonal human IgE samples. We show that polyclonal IgE derived from four individuals with hyper-IgE syndrome or atopic dermatitis recognizes epsilon BP and that there is individual variation in the proportion of IgE recognized by epsilon BP, ranging from greater than 60% for one sample to almost undetectable levels in another. We conclude that epsilon BP does indeed recognize polyclonal IgE and that this recognition is modulated by sialylation of IgE oligosaccharides. Furthermore, there exist different IgE glycoforms, varying in the degree of sialylation, and these are distributed in a distinct manner in different individuals.

Antigens, Differentiation↗

PubMed1991

Postnatal development of D1 dopamine receptors in the medial prefrontal cortex, striatum and nucleus accumbens of normal and neonatal 6-hydroxydopamine treated rats: a quantitative autoradiographic analysis.

The postnatal development of dopamine (DA) D1 receptors in the medial prefrontal cortex (mPFC), striatum (STR) and nucleus accumbens (NAC) of control and perinatally 6-hydroxydopamine (6-OHDA) lesioned rats was examined using quantitative autoradiography of 3H-SCH 23390 binding. D1 receptors are present at one week and increase only slightly to a stable level by 2 weeks in the STR and NAC. Their ontogeny is not altered by intracisternal injection of 6-OHDA 5 days after birth. A biphasic pattern of appearance of D1 receptors was found in the mPFC. D1 receptors are present in the mPFC at 1 week, increase 3-fold by 2-3 weeks, and then decline at 4 and 6 weeks. 6-OHDA lesions do not significantly alter this pattern. At all postnatal ages. D1 receptor binding in the mPFC exhibits a laminar distribution with increased receptor density in deep cortical layers (V, VI) compared to more superficial cortical layers (I, II). Both superficial and deep layers of D1 receptors in the mPFC show similar postnatal developmental patterns. DA turnover rates are consistently about 10-fold higher in frontal pole compared to remainder of forebrain at all postnatal ages. Early 6-OHDA lesions increase DA turnover in forebrain, but lead to a persistent reduction in DA turnover in frontal pole by 2 weeks of age.

Animals↗

PubMed1991

Apparent synaptic dopamine deficiency induced by withdrawal from chronic cocaine treatment.

The effects on motor behavior and forebrain dopamine (DA) synaptic function of withdrawal from chronic cocaine treatment were examined with simultaneous activity monitoring and microdialysis in nucleus accumbens. Rats exhibited behavioral sensitization to daily 30 mg/kg i.p. cocaine. After 18 days of daily cocaine and 7 days of withdrawal, dialysate DA and homovanillic acid (HVA) levels were reduced 36-38%, consistent with a synaptic DA deficiency.

Animals↗

PubMed1991

Development of D1 and D2 dopamine receptors and associated second messenger systems in fetal striatal transplants.

Stereotactic implantation of fetal brain regional anlage into adult host brain ("brain transplantation") appears to be an increasingly viable strategy for therapy of neurodegenerative diseases. We have studied implantation of fetal striatum into adult striatum, previously lesioned by neurotoxic amino acid injection, as a model for transplantation therapy of Huntington's disease. The beginning of behavioral recovery to apomorphine is not apparent until 6.5 months after implantation. By 4 months after implantation cerebral blood flow through the implants appears equal to that in the intact contralateral striatum. At this time, cerebral glucose utilization is reduced in the implants but increases following apomorphine treatment. The development of D1 and D2 dopamine (DA) receptors is markedly deficient in the striatal grafts at both 4 and 6.5 months after implantation. Very little D2 radioligand binding was observed in the grafts at either time point; D1 receptors appeared in a patchy fashion by 6.5 months at densities approaching normal striatum. In situ hybridization of D2 dopamine receptor mRNA demonstrated robust hybridization signal in normal striatum and accumbens but no signal in 6.5-month-old striatal grafts. Adenylate cyclase (AC) activity, examined with high-affinity [3H]forskolin binding, also appeared in patches similar to D1 receptors at 6.5 months. In contrast, protein kinase C activity, labeled with [3H]phorbol ester, was very apparent in the grafts at both time points. Higher and generally homogenous densities of muscarinic cholinergic receptors, assessed with [3H]QNB binding, develop in the grafts, but there appear to be few functioning cholinergic terminals, as measured by [3H]hemicholinium binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1991

mRNA variants encoding multiple forms of the high-affinity IgE receptor alpha subunit in transformed and nontransformed mast cells.

Multiple mRNA species encoding several predicted forms of the high-affinity IgE receptor alpha subunit (Fc epsilon RI-alpha) have been previously characterized from rat basophilic leukemia cells. Using the polymerase chain reaction procedure, we have extended these findings to show that one Fc epsilon RI-alpha mRNA variant, characterized by a 163-bp deletion within the coding sequence, exists in normal rat connective tissue mast cells as well as in both transformed and non transformed murine mast cell lines. In addition, a partial murine Fc epsilon RI-alpha genomic clone, spanning the internal-deletion sequence, has been identified, and from analysis of this sequence a mechanism of alternative pre-mRNA splicing is proposed. Finally, mRNA variants have been translated in a cell-free system and the protein products partially characterized.

Amino Acid Sequence↗

PubMed1990

Expression of biologically active recombinant rat IgE-binding protein in Escherichia coli.

IgE-binding protein (epsilon BP) is a protein which has affinity for IgE and was originally identified in rat basophilic leukemia (RBL) cells. Subsequently, it was found to be the rat homolog of CBP35, a murine beta-galactoside-specific lectin. This protein is also designated as L-34 and RL-29 and studied independently by several laboratories. More recently, CBP35 (epsilon BP) was found to be equivalent to Mac-2, a surface marker on activated macrophages. Using rat epsilon BP cDNA, we have succeeded in expressing recombinant epsilon BP in Escherichia coli. Milligram quantities of homogeneous epsilon BP could be obtained from bacterial lysate in a one-step affinity purification procedure utilizing lactosyl-Sepharose 4B and elution with a lactose gradient. The recombinant epsilon BP (r epsilon BP) binds mouse IgE and retains reactivity to anti-peptide antibodies specific for a sequence within rat epsilon BP. The purified r epsilon BP exhibits binding activity to various saccharides, with affinity for N-acetyllactosamine greater than thiodigalactoside greater than lactose much greater than D-galactose greater than L-arabinose, an order identical to that exhibited by native epsilon BP isolated from RBL cells. The recombinant lectin displayed hemagglutination activity when tested with rabbit erythrocytes. Although epsilon BP shares sequence homology to other lectins containing S-type (thiol-dependent) carbohydrate-recognition domains, r epsilon BP is resistant to air oxidation and does not require reducing agents for maintaining its activity. Furthermore, the single cysteine residue appears to be unexposed and can be alkylated only when the protein is denatured in 5.6 M guanidinium hydrochloride. The availability of a source for a large quantity of epsilon BP should facilitate the analysis of biological function(s) and structure-activity relationships of this lectin.

Animals↗