Biomedical subjects

E J Miller

Publications and source records attributed to E J Miller.

At least 19 recordsLinked to original sources

PubMed1992

Loco-regional nodal relapse in melanoma.

Two-hundred and seven patients without evidence of disease following lymph node dissection (LND) were stratified into three groups: Group A, lymph node relapse within the site of prior LND; Group B, lymph node relapse in a different, but regional, lymph node group; Group C, no lymph node relapse. Decreased survival was noted in both Groups A and B versus Group C. Prognostic factors were identified as: (i) axial or subungal/volar (subvolar) location and the number of positive lymph nodes at initial LND for nodal relapse within the same lymph node group; (ii) male gender, axial/subvolar location, and the number of histologically positive lymph nodes at initial LND for nodal relapse in a different, but regional lymph node group; (iii) relapse within the initial LND site for a decreased survival. Six of 10 patients with both axial/subvolar primaries and four or more positive lymph nodes developed a relapse within the dissection site post-LND. These prognostic factors describe a subset of patients who would be candidates for postoperative adjuvant local/regional and systemic therapy trials.

Adolescent↗

PubMed1992

Isolation and serine protease inhibitory activity of the 44-residue, C-terminal fragment of alpha 1-antitrypsin from human placenta.

alpha 1-Antitrypsin (AAT) is a potent fluid-phase inhibitor of serine proteases. It forms a tightly bound, stoichiometric complex with these enzymes and is inactivated by cleavage within its reactive center. Evidence is here presented, that the 44-residue C-terminal fragment of AAT, termed SPAAT (short peptide from AAT), is found in human tissue, where it is apparently bound to the extracellular matrix (ECM). The identity of SPAAT was established by amino acid sequence analysis through its 40 N-terminal residues. Placental SPAAT inhibits chymotrypsin, human neutrophil elastase (HNE) and pancreatic elastase, but has no effect on trypsin. Unlike AAT, both placental and chemically-synthesized SPAAT are reversible, competitive inhibitors of chymotrypsin with Kl's of 0.92 and 7.5 microM, respectively. Both AAT and placental SPAAT also bind to diisopropyl fluorophosphate (DFP)-treated HNE as well as cathepsin G. SPAAT may therefore play an important role in the protection of ECM proteins, such as elastin, proteoglycans (PG) and/or collagen, from inappropriate attack by serine proteases.

Amino Acid Sequence↗

PubMed1992

Infection-induced airway fibrosis in two rat strains with differential susceptibility.

Chronic infections play a significant role in the morbidity and mortality of patients with chronic airflow limitation. By stimulating airway inflammation, persistent infection has the potential to cause airway fibrosis. However, in patient this condition is most typically found in lungs damaged by other factors, such as smoking, abnormal secretions, or barotrauma. We report the characterization of Mycoplasma pulmonis infection-induced lung fibrosis in two immunocompetent rat strains with no preexisting lung disease. The fibrosis was predominantly in the airways, as demonstrated by the findings for infected animals of increased airway inflammation, airway fibrosis, and airway wall thickness, which correlated with the collagen content of the lungs. Also, the physiological alterations were the opposite of those found in interstitial fibrosis, with a positive correlation between lung compliance and collagen content. The airway fibrosis was noted earlier and to a greater extent in Lewis rats than in Fisher rats, and this result apparently was related to regulation of the inflammatory response. Airway wall thickness, airway inflammation, and airway fibrosis are commonly reported in tissue specimens from patients with chronic airway diseases and have been shown to correlate with airflow limitation in patients with chronic obstructive pulmonary disease. Thus, this model may be useful in furthering our understanding of the role of chronic infection and airway inflammation in airflow obstruction.

Analysis of Variance↗

PubMed1992

Generation of the neutrophil-activating peptide-2 by cathepsin G and cathepsin G-treated human platelets.

The neutrophil-activating peptide-2 (NAP-2) is a cytokine that is generated by the proteolytic cleavage of a precursor protein and that causes neutrophil degranulation and chemotaxis. NAP-2 precursors are produced in platelets and are normally found in the circulation. We showed that NAP-2 is generated by the action of neutrophil cathepsin G on two of the precursors, the connective tissue-activating peptide-III (CTAP-III) and beta-thromboglobulin (beta-TG). However, neutrophil elastase degraded the precursors to inactive peptides. The specific binding of cathepsin G to platelets caused the platelets to secrete NAP-2, and cathepsin G bound to the platelets could still generate NAP-2 from its precursor proteins. In addition, activated neutrophils in the presence of platelets generated NAP-2 from its precursors and caused platelets to secrete NAP-2. These studies demonstrate a unique mechanism for the activation of neutrophils through the interaction of neutrophils, platelets, and NAP-2 precursors that are released either by activated platelets or are present in circulation. It is therefore possible that NAP-2 may be generated at sites where aggregations of neutrophils and platelets occur in vessels such as pulmonary capillaries in patients with the adult respiratory distress syndrome and coronary arteries in patients with evolving myocardial infarctions.

Blood Platelets↗

PubMed1992

Elevated levels of NAP-1/interleukin-8 are present in the airspaces of patients with the adult respiratory distress syndrome and are associated with increased mortality.

The adult respiratory distress syndrome (ARDS) is characterized by increased neutrophils within the airspaces of the lungs. In order to determine if neutrophil activating protein (NAP)-1/interleukin-8 (NAP-1/IL-8) could be an important cause of neutrophil influx and activation in ARDS, we examined fluid, which was either directly aspirated or lavaged with saline from the lungs of patients with ARDS. NAP-1/IL-8 was present in significantly higher concentrations in the fluids of patients with ARDS compared with control subjects. There was a significant correlation between the percentage of neutrophils in the lavage fluids and the NAP-1/IL-8 concentration (r2 = 0.74). Furthermore, the NAP-1/IL-8 concentration of the pulmonary edema fluid was equivalent to the optimal concentration required to induce neutrophil chemotaxis in vitro. Although not all of the chemotactic activity of the edema fluid was removed by an anti-NAP-1/IL-8 affinity column, the data established that NAP-1/IL-8 is an important neutrophil chemotaxin in the airspaces of patients with ARDS. In addition, those patients with very high concentrations of NAP-1/IL-8 in their bronchoalveolar lavage fluids had a higher mortality rate than those patients with lower concentrations of NAP-1/IL-8. The correlation between NAP-1/IL-8 concentration and mortality is not paralleled by total protein concentration and mortality.

Adolescent↗

PubMed1991

Treatment of squamous cell carcinoma of the anal canal.

Progress in the prognosis of patients with squamous cell carcinoma of the anal canal has followed the use of multimodality therapy. From 1977 to 1985, 42 patients with squamous cell carcinoma of the anal canal were treated with mitomycin C (15 mg/m2) and 5-fluorouracil (750 mg/m2) on day 1, 5-FU (750 mg/m2/d) alone on days 2 to 5, and radiation therapy (3000 cGy) on days 7 to 28. They were evaluated 4 to 6 weeks after completion of the chemotherapy/radiation therapy protocol and received local excision, abdominoperineal resection, or both. Patient follow-up times ranged from 7 to 161 months, with a mean follow-up time of 71 months. Pathologic examination showed no residual carcinoma in 19 (45%) patients. The authors could not predict, based on clinical evaluation, which patients would have a complete response. Of the patients with a complete clinical response, 44% had tumor in the pathology specimen. Wide local excision was the most common initial operation (23 of 42 patients; 55%), with five of these patients subsequently requiring abdominoperineal resection. Anal continence was retained in 18 of 42 (43%) patients. Eleven patients experienced recurrent disease: six local recurrences, one distant, and four both local and distant. The 5-year overall survival rate was 82% and the 5-year disease-specific survival rate was 87%. There were no treatment related deaths. Preoperative tumor size was the only factor significantly related to survival. Contrary to other reports, tumor in the pathology specimen did not adversely affect long-term survival. Hence, patients should be treated after chemotherapy/radiation therapy with surgical therapy sufficient to control local disease.

Adult↗

PubMed1991

The principal site of glycation of human complement factor B.

Accumulating amino acid sequence data have made it increasingly evident that many essential complement proteins have potentially modifiable lysine residues in putative critical functional regions. Evidence is now presented that glucose is covalently attached to lysine-266 of purified human complement Factor B as a result of glycation. Purified B was treated with NaB3H4, which reduces such bound glucose to a mixture of radiolabelled hexitols. Amino acid analysis revealed the expected radiolabelled hexitol-lysine epimers. In addition, fluorography of dried gels resolving the major high-molecular-mass h.p.l.c.-fractionated CNBr-cleavage peptides of NaB3H4-reduced B indicated that this radioactivity was specifically associated with the 15 kDa fragment derived from the N-terminal region of fragment Bb. Amino acid sequence analysis suggested that the C-terminal lysine (residue 266 of B) of the N-terminal Lys-Lys doublet of this peptide is preferentially modified. If such glycation can subsequently be shown to occur in vivo, then perhaps this modification might also be found to affect the functional activity of B and offer a potential explanation for some of the immunopathological complications of diseases exposing key plasma proteins, such as this active-site-containing proteinase of the multimeric alternative-complement-pathway C3/C5 convertases, to long-term high concentrations of glucose, such as the decreased resistance to infection and impaired chemotaxis and phagocytosis characteristic of diabetes.

Amino Acid Sequence↗

PubMed1991

Quantitation of type I, III, and V collagens in human tissue samples by high-performance liquid chromatography of selected cyanogen bromide peptides.

A method to determine the proportions of the major fiber-forming collagens (types I, III, and V) in noncartilaginous human tissues is presented. The procedure relies on direct solubilization of tissue collagen as cyanogen bromide peptides. The peptides are subjected to cation exchange chromatography followed by gel permeation chromatography in a manner consistent with the rapid resolution and quantitation of relatively low-molecular-weight marker peptides for each collagen. The marker peptides utilized for type I, III, and V collagens are alpha 1 (I)-CB2, alpha 1 (III)-CB2, and alpha 1 (V)-CB1, respectively. Quantitation of the peptides is attained as a function of ultraviolet absorbance during gel permeation chromatography. The nature of the marker peptides, the use of high-performance liquid chromatography techniques, and quantitation of the peptides by ultraviolet absorbance renders the method suitably rapid, sensitive, and accurate for routine evaluations of collagen composition. The utility of the method is illustrated in the presentation of analyses on specimens of placental membranes and blood vessel walls.

Chromatography, Gel↗

PubMed1991

Immunolocalization of types V and XI collagen in cartilage using monoclonal antibodies.

Monoclonal antibodies produced against pepsin-solubilized newborn rat skin type V collagen [alpha 1(V)]2 alpha 2(V), and chondrosarcoma type XI collagen [alpha 1(XI) alpha 2(XI) alpha 3(XI)] are used to localize the collagens in sections of the chondrosarcoma as well as the normal rat knee joint by indirect immunofluorescence. Immunostaining for type V collagen shows strong cellular staining of chondrocytes; while the interstitial matrix as well as the lacunae are not stained. In contrast, antitype XI stains not only chondrocytes, but the extracellular compartments as well. In ELISA, rat anti-type XI collagen reacts with its native antigen, but does not cross-react with native types I, II, III, or V collagen from rat. The distinct locations of type V and XI collagens in cartilaginous tissue suggest varied functional roles for these constituents in the tissue.

Animals↗

PubMed1991

Use of antibodies in the study of protein structure and function in lung diseases.

Proteins normally fold in a variety of three-dimensional structures. This variety of forms accounts for a multiplicity of functions. One of the major undertakings of modern biochemistry is to determine the structure of a given protein and in doing so learn about its function. Much of the effort in the field of protein chemistry is currently focused on defining areas or domains within the molecule that are responsible for its function. Antibodies specific for functional domains on a protein have been powerful tools in these studies and have provided a great deal of the current knowledge related to the location and function of well-defined structural areas within the protein molecules. The use of antibodies in the study of proteins has considerably advanced our knowledge of both the structure of proteins and their interaction with other molecules. In reviewing the use of antibodies for structure and/or function analysis, we have tried not only to review the techniques and applications involving antibodies but also to show how useful antibody reagents may be designed and prepared. In this review, some of the newer uses of antibodies in modern biology will be described, and a few illustrations of each will be provided.

Amino Acid Sequence↗

PubMed1990

A second fibronectin-binding region is present in collagen alpha chains.

The interactions of plasma fibronectin with alpha chains or cyanogen bromide fragments of collagen types I and II have been studied using a variety of techniques. Affinity chromatography of cyanogen bromide-cleaved type II collagen on immobilized fibronectin revealed the binding of cyanogen bromide fragment CB12 in addition to the previously characterized CB10. Using fluorescence polarization, we analyzed the interaction between the collagen peptides and fluorescein isothiocyanate-labeled 42-kDa gelatin-binding fragment of fibronectin in solution. Dissociation constants for the binding of CB10 and CB12 to the fibronectin fragment were calculated as 0.38 and 0.94 microM, respectively, indicating a lower affinity for the uncharacterized site. However, as with CB10, CB12 was able to compete effectively with the intact alpha chain for bindinng to fibronectin. Additionally, both CB10 and CB12 absorbed to tissue culture surfaces were each able to support fibronectin-dependent cell adhesion. Finally, the regions of alpha 2(I) homologous to CB12 and CB10 were found to be active in fibronectin binding, demonstrating the presence of two fibronectin-binding regions in this collagen chain.

Amino Acid Sequence↗

PubMed1990

Amino acid analysis of collagen hydrolysates by reverse-phase high-performance liquid chromatography of 9-fluorenylmethyl chloroformate derivatives.

A recently described procedure for amino acid analyses has been modified and adapted for use in quantitating the unique mixture of products commonly found in hydrolysates of the collagens. The method involves precolumn derivatization of hydrolysates with 9-fluorenylmethyl chloroformate (FMOC-CL), chromatographic separation of the derivatives and excess reagent on a reverse-phase column, and quantitation based on the fluorescent properties of the derivatives. The method takes advantage of the ease with which stable derivatives are formed with the FMOC reagent. Using a ternary gradient system, a complete amino acid analysis with good resolution of all components can be performed within 35 min. The sensitivity of the method is comparable to levels attained by other derivatives and the fluorescence response of each derivative is linear over the total range of 1-800 pmol. Given these parameters, the method allows complete amino acid analyses to be performed on 100 ng of collagen corresponding to a single picomole of a collagen chain (Mr 100,000).

Amino Acids↗

PubMed1990

Craniofacial osseous restoration with osteoinductive proteins in a collagenous delivery system.

Implants fabricated from mixtures of extracted bovine bone proteins and highly purified human type I collagen have been tested for efficacy in the restoration of osseous defects. Experimental implants composed of osteoinductive proteins and collagen, and control implants composed of collagen alone were placed in 15 x 15 mm cranial defects in New Zealand white rabbits. Implants harvested at 10, 12 and 14 week intervals were subjected to histological evaluation as well as quantitative analysis utilizing a computer morphometric planimeter. Defects filled with control implants displayed highly variable repair with a mean of 31% bony healing. Experimental implants were consistently effective in inducing significant bony regeneration with a mean of 91% repair within the time range of the study. In light of these results, it is apparent that an osteoinductive-collagenous implant system represents an efficacious treatment modality in osseous reconstruction and augmentation.

Animals↗

PubMed1990

Liberation of a neutrophil enzyme-releasing peptide from the surface of human alveolar macrophages.

The human alveolar macrophage product, enzyme-releasing peptide (ERP), has a molecular mass of 8,000 Da, and releases azurophilic and specific granule constituents from neutrophils. A murine monoclonal anti-ERP antibody (12E10H), previously used to show a lack of antigenic identity between ERP and C5a, interleukin 1, tumor necrosis factor, and gamma-interferon, showed no cross-reactivity with interleukin 8. 12E10H and a fluorescein-labeled second antibody were used to visualize ERP on the macrophage surface. ERP was removed from alveolar macrophages by a 3-min incubation with 5 X 10(-7) M bovine pancreatic trypsin at 37 degrees C. The washed trypsinized cells could readhere to plastic and exclude trypan blue. Dilution of the trypsin-derived ERP released myeloperoxidase from cytochalasin-B-treated neutrophils dose dependently. The enzyme-releasing ability of the trypsin-derived material was removed by immunoprecipitation using antibody 12E10H bound to Staphylococcal protein A Sepharose 4B. The estimated molecular mass of the trypsin-derived ERP (by molecular sieve chromatography on HPLC) was approximately 8,500 Da. Other proteases (plasmin, thrombin, and cathepsin G) also released ERP from the cell surface, but the ERP was not an active secretagogue for neutrophils. However, macrophages cultured with protease inhibitors did not show decreased ERP accumulation in the medium. Our data indicate that ERP exists on the surface of human alveolar macrophages and can be released by proteases found within the lung environment in some disease states.

Antibodies, Monoclonal↗

PubMed1990

Retroperitoneal accessory spleen.

We report a lower abdominal mass found to be a retroperitoneal accessory spleen. The vascular supply originated from retroperitoneal vessels independent of the splenic, testicular, and spermatic vessels.

Adult↗

PubMed1990

Pseudohypertestosteronemia during danazol therapy. A case report.

Several steroid radioimmunoassay kits, particularly testosterone, cross-react with danazol, producing false elevations of serum steroid levels. A patient taking danazol simultaneously developed signs of virilization and an apparent 15-fold elevation in her serum testosterone. Discontinuation of danazol resulted in a prompt return to physical and biochemical normality.

Adult↗

PubMed1989

Immune response of patients with congenital coagulation disorders to hepatitis B vaccine: suboptimal response and human immunodeficiency virus infection.

Seventy-eight patients with congenital coagulation disorders were treated with hepatitis B vaccine either subcutaneously or intradermally. All the children (eight vaccinated subcutaneously and eight vaccinated intradermally) responded. Seventeen of 19 (90%) anti-HIV-negative adults vaccinated subcutaneously and 14/25 (56%) anti-HIV-negative adults vaccinated intradermally showed an immune response. At 24 months, the anti-HBs level was greater than 10 IU/l in all children vaccinated subcutaneously, 83% of children vaccinated intradermally, 77% of adults vaccinated subcutaneously, and 55% of adults vaccinated intradermally. Eight of 15 (53%) adult patients who were anti-HIV positive were also anti-HBc positive before vaccination and 6/8 (75%) failed to produce an amnestic response to vaccine. Subcutaneous vaccination with regular monitoring of anti-HBs levels and appropriate boosting is recommended.

Adolescent↗

PubMed1989

Characterization of mammalian type IX collagen fragments from limited pepsin digests of a transplantable swarm rat chondrosarcoma.

Collagenous fragments from type IX molecules have been solubilized by limited pepsin proteolysis of a transplantable rat chondrosarcoma and isolated by selective salt precipitation. Chromatography of the solubilized precipitate on CM-cellulose under nondenaturing conditions yielded three fractions. When examined by polarimetry, the material in all three fractions revealed native collagen helical structure with melting points which ranged from 31-37 degrees C. When the fractions were denatured and rechromatographed on a column of agarose beads, the most acidic fraction eluted as 13-kDa polypeptides with and without prior reduction and alkylation. In contrast, the second and third fractions eluted as 100-kDa and 30-kDa polypeptides prior to reduction, but on reduction and alkylation produced reducible products of 34 kDa and 10 kDa, respectively. The general compositional features of the three fractions closely resemble comparable collagenous fragments of type IX collagen from other species. The denaturation products of the 13-kDa nonreducible, the 30-kDa reducible, and the 100-kDa reducible fractions were sequentially purified by CM-cellulose and reversed-phase chromatography to resolve the chain constituents. The isolated 10-kDa, 13-kDa, and 34-kDa chains were cleaved with CNBr, and the cleavage products identified by gel-permeation chromatography. Two 13-kDa polypeptides, 13K2 and 13K3, which did not contain any methionyl residues and were not cleaved with CNBr, were digested with trypsin, and the peptide digests were resolved by reversed-phase chromatography. Comparisons of the CNBr and tryptic cleavage products demonstrate that the three major collagenous fragments are composed of three unique polypeptides. A partial amino acid sequence of an 8-kDa CNBr peptide derived from a purified 10-kDa peptide (10K1) matches identically the amino acid sequence derived from a cDNA sequence in the rat alpha 1(IX) chain (Kimura et al., 1989). These studies, then, present convenient procedures useful in the isolation of mammalian type IX collagen fragments and describe features of the rat molecule, indicating that it is similar to the avian counterpart with respect to chain composition and general molecular structure.

Amino Acid Sequence↗