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Biomedical subjects

C Cullander

Publications and source records attributed to C Cullander.

At least 19 recordsLinked to original sources
PubMed2001

Development of a low-metal adhesive tape to detect and localize metals in or on the stratum corneum at parts per million levels.

Tape-stripping is a well-established method for sampling the stratum corneum (SC). We have developed a tape with low-metal content suitable for use with particle-induced X-ray emission (PIXE), an analytical technique based on X-ray spectrometry. PIXE analysis of tape-stripped samples of SC is a reliable and minimally invasive means of identifying and quantifying metals present at parts per million levels. Assay feasibility and reproducibility was demonstrated using human volunteers. This new tape-strip technique has potential applications in exposure and decontamination assessment, diagnosis of metal dermatitis, forensics, and in environmental research.

Adhesives↗
PubMed2000

A quantitative minimally invasive assay for the detection of metals in the stratum corneum.

A quantitative, minimally invasive tape-stripping assay for the detection of metals on and in skin that also has application to the detection of metallic elements on dry surfaces (where human contact could occur) has been developed. This development included construction, using commercial products, of an approximately 25 microm thick, low-metal content tape suitable both for tape-stripping and elemental analysis. Individual tapes were sequentially applied to the skin surface and then removed, taking with them a sample of the dead outer layer of the skin (stratum corneum). Analysis of such tape strip samples by particle induced X-ray emission (PIXE)--a well-characterized, sensitive, analytical technique based on X-ray spectrometry--identified and accurately quantified the metals in the sample. The assay had elemental sensitivities of approximately 1 ng/cm2 for many metals and analysis of elemental contents could be performed in as little as 5 min. The feasibility of the assay for measuring metals in the stratum corneum was demonstrated on the forearms of healthy human volunteers. Samples from approximately half the subjects were found to contain zirconium, possibly arising from the use of roll-on antiperspirants. The assay has potential as a tool: (1) for risk assessment, (2) to identify exposure levels following possible contact with a hazardous metal, and (3) to determine the effectiveness of cleanup or removal measures.

Humans↗
PubMed1999

Buccal mucosa in vitro experiments. I. Confocal imaging of vital staining and MTT assays for the determination of tissue viability.

Delivery of drugs through the skin and the buccal mucosa has been considered as an alternative to per oral dosing for those substances that are degraded in the gastro-intestinal tract, or are subject to first-pass metabolism in the liver. In the buccal mucosa, contrary to skin, the diffusion barriers are located within living cell layers, hence the physiological state of the tissue is likely to significantly affect in vitro diffusion profiles. In this study, we were interested in assessing the viability of excised buccal mucosa and determining the limits of tissue usage under common in vitro experimental conditions. Using confocal laser scanning microscopy (CLSM), we have shown that optical sectioning of samples exposed to calcein AM and ethidium homodimer-1 (used as 'live' and 'dead' cell probes respectively) can be employed to accurately and reliably determine the viability of buccal mucosa biopsies. The results of the CLSM assay were remarkably consistent with that of an MTT assay. In both studies, viability in PBS at 34 degrees C was lost after about 8 h post-mortem, whereas it could be sustained for up to 24 h in KBR.

Animals↗
PubMed1999

Blockage of skin invasion by schistosome cercariae by serine protease inhibitors.

Invasion of skin by schistosome cercariae is facilitated by a serine protease secreted from the acetabular cells of cercariae in response to skin lipid. Specific inhibitors of the protease, when applied to human skin in formulations designed to retain the inhibitor on and in the upper stratum corneum layers, block cercarial invasion of human skin. Both peptide-based, irreversible inhibitors and non-peptide, reversible inhibitors block cercarial invasion when applied in a propylene glycol:isopropyl alcohol (3:1) formulation in vitro. Arrest of cercarial invasion could be achieved even after immersion of treated skin in water for 2 hr. Peptide-based irreversible inhibitors in the presence of three different Topicare Delivery Compounds optimized arrest of cercarial invasion. The three Topicare Delivery Compounds applied alone prevented 80-100% of cercarial invasion. With inclusion of the inhibitor, there was 97-100% inhibition in vitro. The optimal formulation with inhibitor was then applied to the tails of BALB/c mice, and the mice were exposed to 120 cercariae by tail immersion. With the carrier lotion alone, there was a 50% reduction in worm burden and a 70% reduction in egg burden. When inhibitor was included, an 80% reduction in worm burden and a 92% reduction in egg burden was observed.

2-Propanol↗
PubMed1998

The secretory granular cell: the outermost granular cell as a specialized secretory cell.

The contents of epidermal lamellar bodies (LB) are delivered selectively to the intercellular spaces at the stratum granulosum (SG)-stratum corneum (SC) interface. We assessed the subcellular basis for LB secretion first by confocal microscopy, following labeling with Nile red or NBD-ceramide, which reveals a tubulo-reticular membrane system within the apical cytosol of the outermost SG cell layer under basal conditions, changing to a more peripheral staining pattern when secretion is stimulated. Ultrastructural study demonstrates that this network is composed of a widely disbursed trans-Golgi-like network (TGN), associated with arrays of contiguous LB, and deep invaginations of the SG-SC interface. Under basal conditions, limited fusion of apically directed LB leads to deep, interconnected invaginations of the apical plasma membrane, resulting in the formation of an extensive, honeycomb extension of the SG-SC interface. Still deeper invaginations and more extensive organelle fusion develop after the epidermis is acutely permeabilized by either acetone treatment, sonophoresis, or iontophoresis. Finally, nascent LB appear to bud off cisternae of the TGN, a process that appears to accelerate after barrier disruption. The deep invaginations of the SG-SC interface; the wide distribution of the TGN within the apical cytosol; the association of nascent LB with the TGN; and the rapid fusion of LB with these invaginations, deep within the cytosol, account for (i) the polarized secretion of LB from the apex of the outermost SG cell, and (ii) the rapid LB-secretory response to barrier perturbations. Finally, our results point to the outermost SG cell as a uniquely specialized secretory cell. We propose the term "secretory granulocyte" to encompass the specialized features of these cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed1998

Determination of the pH gradient across the stratum corneum.

The objective of this work was to determine the pH gradient profile across hairless mouse stratum corneum (SC) using ratiometric laser scanning confocal microscopy imaging and direct pH measurement with a flat surface electrode. Dual-emission ratiometric imaging used the fluorophore, carboxy seminaphthorhodafluor-1, which displays a pH-dependent shift in its emission spectrum. The assay developed was unsuccessful, however, because (i) the pKa of seminaphthorhodafluor (approximately 7.5) makes it insufficiently sensitive to the normal pH gradient (4-7.4) across mammalian SC, and (ii) the unexpectedly high buffering capacity of the skin precluded a meaningful calibration of the system. In the second method, pH measurements with a flat surface electrode were recorded as the hairless mouse SC was progressively tape stripped. In the superficial SC, the pH remained relatively constant (approximately 5.9); further removal of the barrier resulted in a steady increase in pH to approximately 7. In conclusion, improved methodology is clearly required to characterize precisely the pH profile across the SC.

Animals↗
PubMed1998

Light microscopy of living tissue: the state and future of the art.

Since its introduction over a decade ago, confocal microscopy has found wide applicability in the microscopy of thick specimens and living tissue because of its ability to obtain images from deep inside the sample without interference from scattered or out-of-focus light. Three new instruments that are capable of imaging optically dense specimens such as the skin are considered here: a modified tandem-scanning confocal, the video-rate laser-scanning confocal microscope (both of which were developed specifically to examine skin in vivo), and the two-photon laser-scanning microscope, a design which is "inherently confocal." The tandem-scanning and video-rate confocals use visible and infra-red light, respectively, to acquire reflection images, whereas the two-photon scanner is a fluorescence microscope. The advantages and drawbacks of each of these instruments is considered.

Dermatology↗
PubMed1998

Formation of the epidermal calcium gradient coincides with key milestones of barrier ontogenesis in the rodent.

The epidermal permeability barrier forms late in gestation, coincident with decreased lipid synthesis, increased lipid processing, and development of a mature, multi-layered stratum corneum. Prior studies have shown that changes in the epidermal Ca++ gradient in vivo regulate lamellar body secretion and lipid synthesis, and modulations in extracellular Ca++ in vitro also regulate keratinocyte differentiation. We asked here whether a Ca++ gradient forms in fetal epidermis in utero, and whether its emergence correlates with key developmental milestones of barrier formation and stratum corneum development. Using either ion precipitation or proton induced X-ray emission analysis of fetal mouse and rat skin, we showed that a Ca++ gradient is not present at gestational days 16-18, prior to barrier formation, and that a gradient forms coincident with the emergence of barrier competence (day 19, mouse; day 20, rat) prior to birth. These results are consistent with a role for Ca++ in the regulation of key metabolic events leading to barrier formation. Whether the calcium gradient is formed actively or passively remains to be determined.

Animals↗
PubMed1998

Acute barrier perturbation abolishes the Ca2+ and K+ gradients in murine epidermis: quantitative measurement using PIXE.

Epidermal permeability barrier homeostasis requires the delivery of lipids and hydrolytic enzymes by lamellar body exocytosis from the uppermost granular cells, a process that is upregulated following barrier disruption. As lamellar body secretion is controlled by ionic concentrations, especially Ca2+ and K+, we used a quantitative technique, microbeam proton-induced X-ray emission, to measure Ca2+, K+, Cl-, and P concentrations before and after acute barrier perturbation by acetone applications. We found a steep gradient of Ca2+ in normal tissue, peaking in the outer stratum granulosum, which disappeared after barrier disruption, and partially reformed as the barrier recovered. A similar gradient, peaking somewhat lower in the epidermis (i.e., at the stratum granulosum-stratum corneum interface), was found for K+. Epidermal concentrations of K+ also decreased after barrier abrogation, although to a lesser extent than Ca2+. In contrast, P and Cl- demonstrated distribution gradients at baseline, which remained unchanged after barrier disruption. These studies quantitate the levels of Ca2+, K+, Cl-, and P within specific epidermal cell layers at baseline, and in relation to changes in permeability barrier integrity. Ca2+ and K+, but not Cl- or P, decrease after barrier disruption, consistent with these two ion's role in barrier repair.

Animals↗
PubMed1997

Disruption of barrier function in neonatal skin associated with adhesive removal.

OBJECTIVE: Patients in the neonatal intensive care unit require life support and monitoring equipment that must be securely attached to the skin; removal or replacement often causes skin trauma. In this study, we compared the effects of application and removal of three different adhesives on the skin barrier function of premature neonates. The effects were measured by transepidermal water loss (TEWL), colorimetric measurements, and visual inspection. DESIGN: Thirty neonates, between 26 and 40 weeks of gestational age and with birth weights ranging from 690 to 3000 gm, were enrolled in the study during the first week of life. Pieces of plastic tape (1 cm2), pectin barrier, and hydrophilic gel were applied to previously undisturbed sites on the back. A fourth site was used as a control. We measured TEWL, colorimetric readings, and visual inspection scores of skin irritation and stripping at each of the four sites serially: before adhesive application, 30 minutes after adhesive removal, and 24 hours later. RESULTS: Thirty minutes after adhesive removal, TEWL, colorimetric measurements, and visual inspection scores were all significantly higher at the sites of plastic tape and pectin barrier removal than at the control and gel adhesive sites (p < 0.01), demonstrating greater disruption of skin barrier function with removal of the plastic tape and pectin barrier. When the neonates were divided into three groups on the basis of birth weight (< 1000 gm [n = 10], 1000 to 1500 gm [n = 11], and > 1500 gm [n = 9], the same pattern of greater disruption in skin barrier function, as measured by TEWL, was observed in each birth weight group. Twenty-four hours after adhesive removal, TEWL of the plastic tape and pectin barrier sites were not significantly different from the control site, indicating recovery of skin barrier function. CONCLUSIONS: This study demonstrates that a single application and removal of two commonly used adhesives, plastic tape and pectin barrier, disrupts skin barrier function in neonates of varying gestational ages.

Adhesives↗
PubMed1997

Iontophoresis of poly-L-lysines: the role of molecular weight?

PURPOSE: (1) To determine the extent of iontophoretic transport as a function of molecular weight (MW) of the penetrant; and (2) to visually and quantitatively characterize the iontophoretic transport pathways (follicular (F) versus nonfollicular (NF) of the fluorescently-labeled poly-L-lysines employed. METHODS: A series of fluorescently-labeled poly-L-lysines (FITC-PLLs) [4 KDa, 7 KDa and 26 KDa] were used to study the extent and distribution of iontophoretic skin penetration as a function of MW using laser scanning confocal microscopy (LSCM). RESULTS: It was found that, relative to the passive controls, and under the electrical conditions considered, iontophoresis greatly enhanced the penetration of the 4 KDa analog, slightly elevated the delivery of the 7 KDa FITC-PLL, but had no effect on the transport of the larger 26 KDa FITC-PLL. Quantitative analyses of LSCM images revealed that iontophoresis increased transport via F pathways only slightly more than that through NF pathways for the 4 KDa and 7 KDa FITC-PLL molecules. CONCLUSIONS: It is visually apparent that the iontophoretic transport pathways taken are importantly determined by the physicochemical properties (including size and charge) of the penetrant. The results presented here demonstrate an inverse dependence of iontophoretic delivery upon the MW of the penetrant.

Animals↗
PubMed1997

Confocal laser scanning microscopic visualization of the transport of dextrans after nasal administration to rats: effects of absorption enhancers.

PURPOSE: To visualize the transport pathway(s) of high molecular weight model compounds across rat nasal epithelium in vivo using confocal laser scanning microscopy. Furthermore, the influence of nasal absorption enhancers (randomly methylated beta-cyclodextrin and sodium taurodihydrofusidate) on this transport was studied. METHODS: Fluorescein isothiocyanate (FITC)-labelled dextrans with a molecular weight of 3,000 or 10,000 Da were administered intranasally to rats. Fifteen minutes after administration the tissue was fixed with Bouin. The nasal septum was surgically removed and stained with Evans Blue protein stain or DiIC18(5) lipid stain prior to visualization with the confocal laser scanning microscope. RESULTS: Transport of FITC-dextran 3,000 across nasal epithelium occurred via the paracellular pathway. Endocytosis of FITC-dextran 3,000 was also shown. In the presence of randomly methylated beta-cyclodextrin 2% (w/v) similar transport pathways for FITC-dextran 3,000 were observed. With sodium taurodihydrofusidate 1% (w/v) the transport route was also paracellular with endocytosis, but cells were swollen and mucus was extruded into the nasal cavity. For FITC-dextran 10,000 hardly any transport was observed without enhancer, or after co-administration with randomly methylated beta-cyclodextrin 2% (w/v). Co-administration with sodium taurodihydrofusidate 1% (w/v) resulted in paracellular transport of FITC-dextran 10,000, but morphological changes, i.e. swelling of cells and mucus extrusion, were observed. CONCLUSIONS: Confocal laser scanning microscopy is a suitable approach to visualize the transport pathways of high molecular weight hydrophilic compounds across nasal epithelium, and to study the effects of absorption enhancers on drug transport and cell morphology.

Absorption↗
PubMed1997

Localization of aminopeptidase activity in freshly excised human skin: direct visualization by confocal laser scanning microscopy.

The aim of this study was to localize and visualize aminopeptidase activity within freshly excised, dermatomed human skin without perturbation of its histologic integrity. The use of confocal laser scanning microscopy (CLSM) is introduced as a novel approach by which to monitor the degradation of suitable substrates in the skin. The fluorescence of the metabolites originating from the cleavage of the aminopeptidase probe bis-Leu-rhodamine 110 (Leu2-R11O) was interpreted to reflect the local aminopeptidase activity in the tissue. To separate the kinetics of diffusion and degradation of Leu2-R110, a lateral application mode was introduced: the probe was applied at the cutting plane of a mechanical cross-section of the sample, and optical cross-sections were made parallel to the cutting plane of the mechanical section. By this means, simultaneous and equal access of the substrate to the various strata and domains of the skin was achieved. The observations revealed that the fluorescence, i.e., aminopeptidase activity, was evenly distributed throughout the viable part of the epidermis, with enhanced fluorescence ("hot spots") in the upper layers of the stratum granulosum, while dermis and stratum corneum showed considerably less aminopeptidase activity. Independent studies with hair follicles (obtained from trypsin-separated stratum corneum) also showed aminopeptidase activity, mostly at the root sheath. Because of the advantage of direct visualization and localization of enzymatic activity in intact tissue, the lateral application mode of substrate administration in combination with CLSM may be beneficial to further elucidate the location and intensity of metabolic activity in other living tissues as well.

Aminopeptidases↗
PubMed1997

Assessment of cornea viability by confocal laser scanning microscopy and MTT assay.

PURPOSE: Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study design and ocular-toxicity assessment. By using simultaneous vital staining by calcein AM (CAM) and ethidium homodimer-1 (EH-1), as "live" and "dead" probes, respectively, we developed a confocal laser scanning microscopy (CLSM) assay to determine epithelial and endothelial viability and estimate cornea thickness. METHODS: New Zealand White rabbit corneas were stored in phosphate-buffered saline (PBS) or Optisol at 4 degrees C or at room temperature. At various times, corneas were stained with an EH-1/CAM solution and observed, without further treatment, by CLSM. Storage effects on the cornea were also assessed by using an MTT assay. RESULTS: Stromal swelling, shedding of the upper epithelial layers, and severe endothelial damage were observed after 4 h in PBS at room temperature. After 8 h, lower epithelial cell death was observed, along with loss of endothelial structure. Corneas stored in similar conditions in Optisol were indistinguishable from controls. Storage in Optisol at 4 degrees C affected the superficial layers of the corneal epithelium similarly at both 7 and 14 days. Extensive epithelial shedding and wing-cell death were observed at 25 days, but the basal layer remained approximately 50% healthy. Significant endothelial cell loss was observed at 25 days. MTT results were consistent with CLSM data in the medium-term storage study only. CONCLUSIONS: This CAM/EH-1 CLSM fluorescence assay is a sensitive index of viability in cornea, and thus may prove useful in investigations in which maintenance of vital functions in different cell layers is critical.

Animals↗
PubMed1996

Imaging regions of transport across human stratum corneum during high-voltage and low-voltage exposures.

Scanning confocal fluorescence microscopy was used to image localized regions of calcein transport across human stratum corneum during constant low-voltage (iontophoresis) and pulsed high-voltage exposures. Following an electrical protocol, imaging revealed regions of fluorescence which were interpreted as sites where transport of a fluorescent probe (calcein) into the stratum corneum had taken place. Electrically-assisted transport of calcein, whether enhanced by iontophoresis or high-voltage pulsing, appears to occur through intercellular and, to some extent, transcellular pathways into localized regions of stratum corneum that are not associated with appendages. Uniquely associated with the highest voltage pulses used (300 V across the skin) was the appearance of small, brightly fluorescent areas containing nonfluorescent interiors, i.e., fluorescent "rings". We present evidence which suggests that the dark interiors represent sites through which transport occurred during pulsing, but where calcein was no longer present at the time of imaging. Transport of charged microspheres into the stratum corneum was also observed.

Biological Transport↗