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Biomedical subjects

C F Lange

Publications and source records attributed to C F Lange.

At least 19 recordsLinked to original sources
PubMed2003

The effect of breathing pattern on nebulizer drug delivery.

The significance of using breathing patterns with simplified functional shapes in vented jet nebulizer research is examined. This study is comprised of three parts: (1) The measurement and analysis of human breathing patterns, (2) the subsequent in vitro testing of the effects of breathing pattern differences using a consistent bench test method, and (3) a computer modeling of these effects on the estimated regional drug deposition in the human lung. Breathing through a Pari LC-Star nebulizer caused statistically significant changes (p < or = 0.05) in measured human breathing patterns when compared to normal breathing. Observed changes included an increase in the tidal volume (34%) and period (39%). Additionally, the average duty cycle shifted 12% towards a more symmetrical breath due to the unequal increase in the inhalation and exhalation times (55% and 28%, respectively). The position of the point of maximum flow in each breath phase shifted towards the beginning and end of the breath for the inhale and exhale by 28% and 48%, respectively. The bench testing revealed that breathing pattern shape variation caused statistically significant differences in nebulizer output only in two cases. Decreasing duty cycles and shifting the point of maximum flow towards the beginning of the breath both result in a decrease in output efficiency. Square flow patterns produced slight but consistently higher output efficiencies (average 2.1% higher) and a constant output particle size over the course of each breath, different from the other non-square patterns. Numerical simulations revealed no significant dosage differences resulting from breathing pattern shape variations. However, square wave patterns consistently produced slight overpredictions in comparison with real nebulizer patterns. In contrast, sine wave patterns were found to produce essentially the same results as nebulizer patterns in both the bench tests and in the deposition simulations. This suggests that sine wave shapes are preferable for simulating breathing when bench testing drug delivery using vented jet nebulizers.

Adult↗
PubMed2001

In vitro aerosol delivery and regional airway surface liquid concentration of a liposomal cationic peptide.

A liposome encapsulation was optimized for the entrapment and aerosol delivery of an alpha-helical cationic peptide, CM3, which had shown good antimicrobial and antiendotoxin activity in vitro. The encapsulation procedure and the phospholipids used were selected to maximize both the encapsulation and nebulization efficiencies, without compromising liposomal integrity during nebulization. The best compromise was found with dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol (3:1 molar ratio), which allowed for peptide encapsulation levels of 730 microg/mL using 30 mM lipid concentration. The aerosol produced with the selected liposomal formulation was subsequently analyzed for determination of size distribution and nebulizer efficiencies. These quantities were used as input for a mathematical lung deposition model, which predicted local lung depositions of the liposomal peptides for three models of lung geometry and breathing patterns: an adult, an 8-year-old child, and a 4-year-old child. The deposition results were then applied to a novel model of airway surface liquid in the lung to assess the concentration of the deposited peptide. The resulting concentration estimates indicate that the minimum inhibitory levels of CM3 can be reached over most part of the tracheobronchial region in the adult model, and can be exceeded throughout the same region in both pediatric model subjects, using a valved jet nebulizer with a 2.5mL volume fill.

Adult↗
PubMed2000

Validating deposition models in disease: what is needed?

To develop theoretical deposition models, assumptions are introduced to make the models computationally affordable. For this reason, experimental (in vivo) validation of such models is needed to give confidence to the assumptions being made. However, for an in vivo deposition experiment to be considered useful for validation of a model, a number of parameters must be measured in the experiment for input to the model. Ideally, these parameters would include time-dependent breathing flow rates during aerosol exposure, properties of the inhaled aerosol as a function of time during the breath (including particle size distribution, aerosol mass fraction, as well as hygroscopic properties, inhaled temperature and humidity if hygroscopicity is important), in addition to anatomical regional deposition data and detailed lung geometry measurements. Furthermore, because of the dependence of extrathoracic filtering on the inlet conditions at the mouth and the complexity of modeling deposition in this region, experimental data on the filtering properties of the mouth-throat are needed. Although some of the above parameters are impractical to measure with current experimental techniques, it would greatly aid the development of deposition models if as many of these parameters as possible were measured in future in vivo deposition experiments. Data exemplifying the importance of measuring the above parameters is discussed.

Aerosols↗
PubMed2000

Lung delivery of aerosolized dextran.

The ability of nebulizers to deliver dextran (nominal molecular mass, 4,000 g/mol) to the lung as an inhaled aerosol is evaluated by in vitro experimental methods and mathematical models. Dextran in isotonic saline was aerosolized by four nebulizer types (Pari LC STAR, Hudson T-Updraft II, Acorn II, and Sonix 2000) at dextran concentrations </= 400 mg/ml and with 2.5- and 4-ml volume fills. Aerosols inhaled during breath simulation were characterized by in-line phase Doppler anemometry, filter collection, osmometry, and gravimetry. Mathematical models were used to estimate amounts of the characterized aerosols depositing in the different regions of lung models, and mathematical models of mucous thickness were then developed to estimate initial concentrations of the depositing dextran in the mucus of each conducting airway generation. Models of three subjects (4 yr old, 8 yr old, and adult) were used. The high viscosity of the dextran solutions tested (up to seven times that of water) negatively impacts nebulization, and results in poor performance with most delivery systems tested. Our results suggest that airway mucosal dextran concentrations associated with efficacy in previous animal and in vitro models are achievable with reasonable delivery times (</= 12 min) with only one of the delivery systems/formulations tested: the Pari LC STAR nebulizer, using a 2. 5-ml volume fill and a dextran concentration of 200 mg/ml.

Administration, Inhalation↗
PubMed2000

Overcoming the adverse effect of humidity in aerosol delivery via pressurized metered-dose inhalers during mechanical ventilation.

The well-known problem of reduced drug delivery that occurs when heated, humid air is used with pressurized metered-dose inhalers (pMDIs) and spacers in intubated settings is carefully studied with Airomir using an in vitro model under controlled conditions of temperature and humidity. A better understanding of the physical processes leading to the aforementioned drop in performance is obtained, and a method is devised to circumvent the problem without having to reduce the temperature or humidity of the ventilator circuit. The present study shows that the mole fraction of water vapor in the ventilation air (and not the temperature) is the major factor behind the sharp drop in the amount of drug delivered to the lung. However, the presence of water vapor does not affect performance because of hygroscopic growth. Instead, it influences the initial atomization process and the early stages of aerosol generation. Removal of these negative effects can be achieved by using a larger spacer that allows longer times for the aerosol to evaporate, as is demonstrated in the present study.

Aerosols↗
PubMed1996

Epitope mapping of homologous and cross-reactive antigens by monoclonal antibodies to streptococcal cell membrane (mAb to SCM).

An approach to epitope mapping of a series of anti-streptococcal cell membrane (SCM) mAbs is described. Evaluations by enzyme-linked immunosorbent assay (ELISA) of one control mAb HB-35 and 13 different anti-SCM mAbs were made on homologous SCM antigen and human basement membrane antigens isolated from glomeruli (GBM) and lung (LBM). These anti-SCM mAbs were previously shown to be cross-reactive in a variety of systems with both GBM and LBM. The binding capacities were measured for all 14 mAbs on ELISA plates sensitized with SCM antigen or the cross-reactive GBM or LBM antigens, at 5 micrograms/ml or approximately 20 pM/well. From the 50% binding capacity dilution the pM of mAb bound/pM antigen-well was calculated which translated into an estimate of the ratio-number of epitopes bound. Observations with the homologous and cross-reactive antigens showed multiple reactive epitope ratios to eight mAbs whereas the other five yielded a ratio value of one or two on the tested antigens. Plates blocked with a specific dilution of one mAb evaluated the binding by a second mAb providing both binding and specificity data. One mAb (I-F-3) blocked all the other anti-SCM mAbs on all three antigen plates. An additive effect was noted by three mAbs, I-G-8, II-C-4 and II-D-8 with most of the other mAbs. The order of placement, however, made distinctive differences; II-F-4 showed an additive or enhancement effect on I-B-5 but no reciprocal effect was seen. A similar effect was made with I-G-8 and II-C-4 or I-F-7. Possible interpretations are that each mAb is binding different epitopes each fully exposed, and the order of placement of the mAbs makes no difference. Where an enhanced effect was observed it is suggested that the binding of the first mAb changed the conformation of the antigen, thereby opening and exposing additional epitope(s) to the second antibody. Or, in contrast, where the second mAb was blocked by the first, a fixing of the protein conformation is suggested thereby occluding the other epitope, as seen with I-F-3 and II-C-4. These epitope mapping procedures confirmed that all 13 anti-SCM mAbs were binding at different epitopes. The nature of basement membrane collagens and how this relates to post-streptococcal sequelae will be discussed.

Antibodies, Bacterial↗
PubMed1995

Immunochemical studies of streptococcal cell membrane antigens immunologically related to glomerular basement membrane.

Pursuing an autoimmune model for the etiology of poststreptococcal glomerulonephritis, protein antigens isolated from the cytoplasmic membrane of nephritogenic group A Type 12 Streptococcus pyogenes were immunochemically characterized using antistreptococcal cell membrane (SCM) monoclonal antibody (MAb) cross-reactive with glomerular basement membrane (GBM). Low molecular weight (9.2, 7.0, 4.7, 2.3 kDa) HPLC-purified SCM polypeptide antigens were characterized by competitive inhibition and equilibrium dialysis. Competitive inhibition of the MAb, by different sized SCM polypeptide antigens showed an inverse relationship between the size of these antigens and the molar amount required to obtain 50% inhibition of the MAb, confirming previous observations that suggested that these SCM antigens exhibit increasing epitope concentration with increasing size, that is constant epitope density. The observed changes in epitope concentration correlated with differences in the valence and affinity of the MAb as determined by equilibrium dialysis. The Kds of the MAb for 9.2-, 7.0-, 4.7-, and 2.3-kDa SCM antigens ranged from 7.42 x 10(-7) to 1.15 x 10(-5). The experimentally determined MAb valence for these antigens was 2 for the 9.2-kDa antigen and approached 10 for the smaller antigens. Finally, the similarity of these SCM antigens was reflected in similar amino acid compositions; of note, these data agreed with the compositions previously reported for sized GBM antigens. Concentrations of Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, and Leu paralleled increasing epitope concentration. Apparent N-terminal blocking prevented sequencing of these peptides, but these immunochemical data suggest that intact SCM antigen recognized by the anti-SCM MAb consists of repeating epitopes, an observation consistent with the cytoplasmic membrane source of the antigen.

Animals↗
PubMed1995

Tracking the in vivo localization of streptococcal cell membrane (SCM) monoclonal antibodies: potential model for post-streptococcal sequelae.

Placement of ten different anti-streptococcal monoclonal antibody (mAb) secreting hybridoma cells, as well as positive and negative controls into animals and sacrificing on a daily basis showed a difference in the tissue sites of accumulating mAb as noted by fluorescent antibody testing. Initial binding of the mAb was noted by day two on the kidney GBM in all the animals. Striated muscle tissues tested positive starting at day nine with only four of the mAbs, by which time the GBM was strongly positive. Fluorescent antibody testing of heart and skeletal muscle from animals in which one of the polyreactive IgM mAb secreting hybridoma cell lines was placed showed a distinctive staining of the Z-lines. Indirect fluorescent and immunoperoxidase testing as well as competitive blocking experiments confirmed the reactivity of this mAb for the Z-line of heart and skeletal muscle. Immunodots and Western blots along with direct and blocking assays confirmed that the critical cross-reactive Z-line antigen was alpha-actinin, supporting the concept that this anti-SCM mAb was reactive both in vivo and in vitro. These results confirm the cross-reactive nature of anti-SCM antibodies for mammalian tissue and bear important implications on the etiology of post-streptococcal glomerulonephritis and rheumatic heart disease.

Animals↗
PubMed1994

Localization of [14C] labeled anti-streptococcal cell membrane monoclonal antibodies (anti-SCM mAb) in mice.

Six different hybridomas secreting anti-SCM mAb and one control mAb were placed into adult mice along with [14C] amino acids for biosynthetically labeling. After sacrifice, the 14C mAb ascites along with serum, heart, kidney, lung and skeletal muscle were recovered. Tissue associated specific radio-activity (SpAc) and microscopic structural analyses were performed. Confirmation of mAb specificity was by both immunodot blots as well as Western blot analysis. Peritoneal injection of measured doses of anti-SCM mAb yielded tissue SpAc confirming their in vitro specificities. Two of the mAb showed strong reactivity to both renal (GBM) and lung basement membrane (LBM), two were mainly GBM reactive and two showed polyreactivity with a marked reactivity to a Z-line antigen. Autoradiographic light microscopy confirmed that the anti-SCM mAbs bound to both GBM and LBM and to Z-line antigen. Titrated doses of the mAb yielded autoradiographic confirmation in which the grain number on the GBM and LBM increased with increasing dose of mAb for all mAb except the control. This effect was not seen in the muscle tissues but anatomical localization at the Z-line was consistent. The major significance of these studies is the demonstration that circulating antibodies to SCM can react in vivo with normal mammalian antigens adding confirmation to the in vitro specificity of these cross-reactive anti-SCM mAbs.

Animals↗
PubMed1993

Experimental autoimmune pinealitis in the rat: ultrastructure and quantitative immunocytochemical characterization of mononuclear infiltrate and MHC class II expression.

Lewis rats immunized with Peptide M (an oligopeptide epitope of the S-antigen protein) developed experimental autoimmune uveoretinitis (EAU) and experimental autoimmune pinealitis (EAP). Temporal changes in mononuclear infiltrate to the pineal gland were quantitated by computer image analysis of sections immunostained with monoclonal antibodies to specific mononuclear populations. T helper/inducer cells (W3/25+) and monocyte/macrophages (OX-42+) were elevated during the early phases of inflammation (day 15) while cytotoxic/suppressor T cells (OX-8+) were elevated at days 15 and 21. Expression of MHC class II (OX-6) was markedly enhanced on pineal glia, but was not present on vascular endothelia during EAP. Ultrastructurally, many capillaries exhibited thickenings of the endothelia and basal lamina. EAP had little effect on the fine structure of pinealocytes and glia and there was little evidence of cellular destruction by day 21, in contrast to the extensive retinal destruction resulting from EAU. These findings suggest fundamental differences between EAU and EAP related to mechanisms of antigen processing/recognition in autoimmune diseases. Our study further indicates the importance of EAP as a model to investigate neuroendocrine-immune interactions.

Animals↗
PubMed1991

Hybridomas to specific streptococcal antigen induce tissue pathology in vivo; autoimmune mechanisms for post-streptococcal sequelae.

A gross examination of organs from approximately 100 mice which were producing ascites fluids toward a series of streptococcal reactive monoclonal hybridomas showed, in some animals, what appeared to be autoimmune-like findings. A pattern of major lung pathology was associated with specific clones. These specific hybridomas led to the development of an experimental autoimmune animal model mimicking a Goodpasture's syndrome. Tissue injury was induced in mice, on a dose dependent basis, by the injection of monoclonal antibody generated against streptococcal cell membrane (SCM) antigens. A more severe onset of the pathology, also on a dose dependent bases, was induced by placement of the anti-SCM mAb secreting hybridoma cells into the peritoneal cavity of the host. Severity of observed lesions was dependent upon the number of cells injected (10(5), 5 x 10(5), 10(6) or 10(7], as well as the animals' sex. Severe and total hemorrhagic lungs were seen in animals challenged with 1 x 10(6) hybridomas cells when sacrificed on the tenth day. In all cases the lesions were greater in the female litter mate than the male. Gross and histologic observations were confirmed by lung/body weight ratios. Pulmonary hemorrhage ranged from slight, when mAb was injected at a low dose of 24 micrograms/g, to severe when 96 micrograms/g was injected. Reported findings were based on the review of approximately 300 mice. Immunochemical evaluations and ELISAs confirmed the ability of these anti-SCM mAb to react with glomerular basement membrane (GBM) antigens as well as lung basement membrane (LBM). Mitogenic experiments indicated that the parent immunogen (SCM) used to generate immunocytes was non-stimulatory to lymphocytes.

Animals↗
PubMed1990

Use of solid-phase C1Q to remove soluble antigen/antibody complexes in an inhibition ELISA for streptococcal cell membrane antigens.

Solid-phase C1q was used to remove antigen/antibody complexes in an inhibition ELISA for low molecular weight streptococcal cell membrane (SCM) polypeptide antigens. To selectively fix IgM monoclonal antibody bound to antigen, binding was carried out in C1q-coated ELISA plates; transfer of supernatants to SCM-coated plates for ELISA permitted measurement of residual antibody. When inhibition occurred in the presence of C1q, the maximal binding was 72-98%. In the absence of C1q the maximum apparent binding was only 45-50%, which we attribute to displacement of the initially bound SCM antigen by solid phase SCM antigen. Removal of antigen/antibody complexes by solid-phase Clq during inhibition assays may facilitate analysis of low affinity antigen/antibody interactions.

Antibodies, Monoclonal↗
PubMed1989

Isolation and partial characterization of antigens from basement membranes and streptococcal cell membrane (SCM) employing anti-SCM monoclonal antibody.

Monoclonal antibodies (mAb) against streptococcal cell membrane (SCM) antigen were used to identify specific cross-reactive peptides prepared by trypsin digestion of purified glomerular basement membrane (GBM) and lung basement membrane (LBM). Anti-SCM mAb-coupled HPLC columns were used to affinity isolate soluble LBM, GBM, and SCM antigens which then were sized by HPLC. Alternatively, SCM, GBM, and LBM digests were subjected to an initial separation by HPLC into component polypeptides, followed by affinity purification and ELISA of these fractions using anti-SCM mAb. Comparison of the antigenic reactivities by ELISA of the sized polypeptides on a nanomolar basis permitted the estimation of their individual relative epitope densities. The results for SCM antigens showed increasing epitope density with increasing molecular size, which suggests that intact SCM consists of repeating epitopes. Low mol. wt GBM polypeptides in nanogram amounts inhibited mAb binding to SCM, indicating that these small GBM polypeptides may similarly contain more than a single cross-reactive epitope. The identification of these cross-reactive epitopes in LBM and GBM has important implications for the etiology of post-streptococcal sequelae.

Antibodies, Monoclonal↗
PubMed1987

The isolation of cross-reactive monoclonal antibodies: hybridomas to streptococcal antigens cross-reactive with mammalian basement membrane.

Based upon the assumption that post-streptococcal sequelae are the result of cross-reactive antibodies, hybridomas were prepared from the spleens of mice immunized with Group A type 12 streptococcal cell membranes (SCM) specifically to screen for such cross-reactive antibodies. One fusion produced a cell population displaying antibodies reactive to both SCM and glomerular basement membrane (GBM) antigens as demonstrated by ELISA technique. Ascites produced by this cell population also showed reactivity to lung basement membrane (LBM). Limiting dilution procedures have produced 15 monoclonal hybrids with both anti-SCM and anti-GBM activity. Confirmation of the cross-reactive and monoclonal nature of the antibody was accomplished by both direct and indirect competitive ELISA. These observations have established that unique cross-reactive antibody-secreting hybrid cells with reactivity to both SCM and basement membrane (BM) antigens can be isolated by standard cloning procedures.

Animals↗
PubMed1986

Age effects on the reactivity of antistreptococcal cell membrane antisera to murine glomerular basement membrane. In vitro versus in vivo analysis.

Antisera to the streptococcal cell membrane (SCM) were evaluated for their reactivity to murine glomerular basement membrane (GBM) in four strains of mice. Animals were studied on a daily basis from birth through 3 months and weekly thereafter through 18 months. Paired animals were compared for in vivo binding of antibody versus an indirect fluorescent antibody technique on fresh kidney sections. The findings demonstrated a granular type GBM staining for all anti-SCM which were positive. Nonspecific background staining accompanied most of the indirect fluorescent antibody sections tested while being totally absent for the direct fluorescent test on tissue from in vivo challenge of the primary antibody. The in vitro testing showed tissue from young mice (0-6 days old) to be most reactive, while the strongest reactivity was seen in the age group of 10-20 days for in vivo testing. These cross-reactive antibodies, i.e., GBM-binding anti-SCM, are best evaluated by in vivo methods where tissue is taken 4 days after antiserum injection. Animals of the age range 6-8 weeks were often negative, indicating that this age range selected for many studies may not be the most favorable one via either in vitro or in vivo studies.

Aging↗
PubMed1985

Identification of possible calcium dependent antigens in lithium diiodosalicylate extraction of group A, type 12 streptococcal cell membranes.

Group A, type 12 streptococcal cell membranes were extracted by aqueous lithium diiodosalicylate (LIS) and the extract treated with trifluorotrichloroethane (Genetron). An initial component was isolated (GLCM) which was soluble in aqueous buffer, but was heavily contaminated with LIS. Dialysis of GLCM vs. 0.01 M Tris-EDTA (TE) buffer yielded a weakly antigenic component, TE-GLCM, with negligible contamination by LIS. Subsequent dialysis of TE-GLCM vs. isotonic calcium chloride produced two fractions, a soluble one (CMS) and a precipitate (CMP). It was demonstrated that CMS possessed immunological characteristics distinct from TE-GLCM and CMP. CMS was shown to be a calcium dependent antigen, and immunologically related to human glomerular basement membrane (GBM) antigens.

Amino Acids↗
PubMed1984

Transfer factor and repeated otitis media.

The effect of transfer factor (TF) was investigated in 12 children with repeated otitis media. These patients were immunologically compared to a control group of 23 age-matched healthy children. Levels of immunoglobulins, total and "active" T-cells, and phagocytic activity of granulocytes and monocytes were evaluated in the 12 children prior to, during, and after TF therapy. Percentages of "active" T cells and absolute numbers of "active" T and total T cells, which were initially low in the patient group, increased significantly after TF therapy to statistically match those of the healthy control group. The percentage of phagocytic monocytes in patients after therapy did not differ from healthy children; however, the percentage of phagocytic granulocytes remained depressed significantly. The levels of IgG, IgA, and IgM were unaffected by the therapy although the IgA and IgM were higher in the patient population throughout the study. After therapy, one-half of the patient population remained asymptomatic for a 1-year period and the others had markedly reduced attack rates.

Child↗