Adhesion and invasion of Streptococcus pneumoniae to primary and secondary respiratory epithelial cells.

The interaction between Streptococcus pneumoniae (S. pneumoniae) and the mucosal epithelial cells of its host is a prerequisite for pneumococcal disease development, yet the specificity of this interaction between different respiratory cells is not fully understood. In the present study, three areas were examined: i) The capability of the encapsulated S. pneumoniae serotype 3 strain (WU2) to adhere to and invade primary nasal­derived epithelial cells in comparison to primary oral­derived epithelial cells, A549 adenocarcinoma cells and BEAS­2B viral transformed bronchial cells; ii) the capability of the unencapsulated 3.8DW strain (a WU2 derivative) to adhere to and invade the same cells over time; and iii) the ability of various genetically­unrelated encapsulated and unencapsulated S. pneumoniae strains to adhere to and invade A549 lung epithelial cells. The results of the present study demonstrated that the encapsulated WU2 strain adhesion to and invasion of primary nasal epithelial cells was greatest, followed by BEAS­2B, A549 and primary oral epithelial cells. By contrast, the unencapsulated 3.8­DW strain invaded oral epithelial cells significantly more efficiently when compared to the nasal epithelial cells. In addition, unencapsulated S. pneumoniae strains adhered to and invaded the A459 cells significantly more efficiently than the encapsulated strains; this is consistent with previously published data. In conclusion, the findings presented in the current study indicated that the adhesion and invasion of the WU2 strain to primary nasal epithelial cells was more efficient compared with the other cultured respiratory epithelial cells tested, which corresponds to the natural course of S. pneumoniae infection and disease development. The target cell preference of unencapsulated strains was different from that of the encapsulated strains, which may be due to the exposure of cell wall proteins.

Fluorescence spectroscopy of H-ras transfected murine fibroblasts: a comparison with Monte Carlo simulations.

Autofluorescence properties of tissues have been widely used to diagnose various types of malignancies. In this study, we measured the autofluorescence properties of H-ras transfected murine fibroblasts and the counterpart control cells. The pair of cells is genetically identical except for the transfected H-ras gene. We applied Monte Carlo simulations to evaluate the relative contributions of Rayleigh and Mie scattering effects towards fluorescence in an in vitro model system of normal and H-ras transfected fibroblasts. The experimental results showed that fluorescence emission intensity was higher for normal cells than the malignant counterpart cells by about 30%. In normal cells, linearity in emission intensity was observed for cell densities of up to 1.0 x 10(6) cells/ml whereas for transformed cells it was up to 1.4 x 10(6) cells/ml. Nuclear volume changes give good account for the differences in the intrinsic fluorescence between normal and malignant cells. The Monte Carlo (MC) code, newly developed for this study, explains both predominant experimental features: the large fluorescence intensity differences between the transfected and the corresponding control cells as well as the phenomena of the red shift in the excitation spectra as a function of cell density. The contribution of Rayleigh scattering was found to be predominant compared to Mie scattering.

Combined ultrasonic and enzymatic debridement of necrotic eschars in an animal model.

Several methods are used to debride burn eschars, however, most are ineffective for ischemic eschars. We investigated a novel combination of enzymatic and ultrasonic debridement for ischemic eschars. A previously described ischemic flap model in rats was used to compare the time to flap debridement or perforation of enzymatic (Debrase, a derivative of bromelain), ultrasonic, or combined debridement (Hybrid Debridement Technology). We also evaluated the effects of ultrasonic intensity, probe size, probe housing, and operation mode (pulsatile vs. continuous) on the time to full eschar perforation. Ultrasonic and enzymatic debridement alone did not result in flap perforation even after 15 minutes. Combined ultrasonic and enzymatic debridement resulted in flap perforation within 2 to 5 minutes in the four flap zones (P < 0.001 for all four flap zones compared with ultrasound alone). The most rapid debridement was observed with an ultrasonic intensity of 3.2 W/cm, applied using a 4.9 cm probe. The temperature elevation associated with ultrasonication was controlled by perfusion of fresh Debrase solution and alternating the ultrasound energy. Combination of ultrasonic and enzymatic debridement of ischemic flap eschars in rats with Debrase is more rapid and effective than either method alone.

Multiple drug allergy in patients with cutaneous adverse drug reactions diagnosed by in vitro drug-induced interferon-gamma release.

BACKGROUND: Multiple drug allergy syndrome is a rarely reported clinical condition characterized by an adverse reaction to more than one different class of pharmacologically and structurally unrelated drugs. The pathogenesis may involve immediate-type or delayed-type hypersensitivity. OBJECTIVES: To further characterize patients with MDA in terms of the type of CADR, drug intake and clinical drug suspicion. METHODS: The study group comprised 12 patients (6 males, 6 females) with CADRs showing in vitro drug-induced IFNgamma release for multiple drugs, suggesting the presence of MDA. The diagnostic role of in vitro IFNgamma release in identifying the culprit drugs was evaluated in terms of clinical data and the results of in vivo tests (withdrawal and/or challenge tests) with the offending drugs. RESULTS: Clinical relevance was attributed to in vitro drug-induced IFNgamma release towards multiple drugs in this series of 12 patients with a variety of CADRs, implying MDA. The results of in vivo tests for the offending drugs confirmed the diagnosis. The main causative agents responsible were antibiotics and non-steroidal anti-inflammatory drugs. CONCLUSIONS: The study further supports the role of a T cell-mediated mechanism in the pathogenesis of MDA. The in vitro drug-induced IFNgamma release test may serve as a laboratory tool to identify the culprit drugs associated with this allergy.

Bubble growth within the skin by rectified diffusion might play a significant role in sonophoresis.

Low frequency ultrasound has successfully been used for enhancing transdermal transport of a variety of different molecules. This phenomenon is referred to as sonophoresis. Several attempts have been made to investigate the enhancing mechanism in order to modulate the overall process. In this study we assess whether rectified diffusion is a process that occurs within the skin, which could eventually lead to channeling and thereby to transdermal sonophoresis. The model presented in this paper is based on the following postulate: gas bubbles are randomly distributed within the lipid bilayers of the stratum corneum (SC). As the skin is subjected to ultrasound, gas bubbles grow by rectified diffusion. During this period, bubbles may merge with the outer or inner boundaries of the SC, or merge with neighboring bubbles. Eventually, channels are created, allowing drugs to easily penetrate through the most significant barrier to transdermal delivery, the SC. As a result, transdermal transport rate is enhanced. In this work, a mathematical model has been formulated, in which permeability enhancement of the SC is linked to channels, possibly created by means of rectified diffusion. Sonophoresis may result from various mechanisms that act in synergy. The present model predicts that rectified diffusion might be one of the factors that lead to sonophoresis during ultrasound treatment.

The nature of ultrasound-SLS synergism during enhanced transdermal transport.

Ultrasound and sodium lauryl sulfate (SLS) exhibit a synergistic effect on transdermal transport, when applied simultaneously on the skin. The synergistic mechanism is not fully understood. Previous studies have shown that application of ultrasound simultaneously with SLS, results in enhanced mass transfer and improved penetration and dispersion of the surfactant. In this study we demonstrate that simultaneous application of ultrasound and SLS leads to modification of the pH profile of the stratum corneum. This pH modification within the stratum corneum's microenvironment, can affect both the structure of the lipid layers and the activity of SLS as a chemical enhancer due to its improved lipophilic solubility. The altered pH profile that results in improved SLS lipophilic solubility, together with improved SLS penetration and dispersion, can explain the synergistic enhancing effect of ultrasound and SLS on transdermal transport.

Clinical implications of in vitro drug-induced interferon gamma release from peripheral blood lymphocytes in cutaneous adverse drug reactions.

BACKGROUND: Drug-specific T cells are involved in the pathogenesis of cutaneous adverse drug reactions (CADRs). OBJECTIVE: We sought to evaluate the diagnostic role of in vitro drug-induced release of interferon gamma in CADRs. METHODS: The study group consisted of 36 patients with CADRs after intake of 106 drugs that were classified into 3 categories of drug suspicion: high, possible, and low. The control group consisted of 22 individuals taking a similar profile of 54 drugs, without CADRs. In vitro drug-induced interferon gamma release was determined by enzyme-linked immunosorbent assay in culture supernatants after incubation of peripheral blood lymphocytes with parent drug compounds. The in vitro tests were conducted after the acute phase of the CADRs. In vitro tests were compared with in vivo withdrawal and/or challenge drug tests. RESULTS: Positive interferon gamma tests were recorded in 77.8% of the patients for 49.0% of the drugs. The proportion of positive interferon gamma tests was directly associated with the degree of drug suspicion (64.4%, 36.4%, and 27.8% for high, possible, and low, respectively). There were significantly more positive tests for high- compared with low-suspicion drugs (P=.001). The degree of agreement between the results of the in vitro interferon gamma release tests and the in vivo tests was intermediate to good (kappa=0.47). CONCLUSION: In vitro drug-induced interferon gamma release may help to identify the responsible drug in CADRs.

Effect of rat salivary glands extracts on the proliferation of cultured skin cells--a wound healing model.

OBJECTIVE: Salivary gland secretions play an important role in promotion of wound healing. The healing of intra- or extra-oral wounds is delayed in desalivated rats. However, the specific role of each salivary gland in promoting wound healing is unknown. This study was aimed to investigate the effect of crude extracts of rat salivary glands on a simplified in vitro wound healing model. DESIGN/METHODS: Cultured human keratinocytes (HaCat) and murine fibroblasts (3T3) were subjected to 48 h serum starvation, and were later activated by extracts of rat salivary glands, 1-10 mug protein/ml of each gland. The resultant cellular metabolic activity of the activated cells was determined 24 h later, measuring reduction of XTT by mitochondrial enzymes, and calculated relatively to positive controls [optimal supplementation of 10% fetal calf serum (FCS)], and negative controls (starved non-supplemented cells). RESULTS: The relative stimulatory effect of parotid (P) extract on the cells was significantly lower than either submandibular (SM) or sublingual (SL) extracts. Under the assumption that physiologically, the cells are exposed to the combined effect of saliva secreted from all the glands, different combinations of the extracts were presented to the cells. The relative stimulation was maximal following treatment with the three glands extracts (P + SM + SL) and exceeded the effect of 10% FCS. CONCLUSION: The results suggest that each salivary gland has a specific effect on wound healing and the combination of the three extracts has an additive effect but no the sum of all individual glands. This model might be useful to study the wound healing effect of salivary glands.

Multiple effects of arginine vasopressin on prostaglandin E2 synthesis in fibroblasts.

Recent evidence supports the viewpoint that vasopressin, a neurohypophyseal peptide, should be also considered as a neuroendocrine modulator of immune and inflammatory responses. In this work we investigated the role of vasopressin in the regulation of prostaglandin E(2) synthesis by human dermal fibroblasts. Recombinant human interleukin-1 beta increased prostaglandin E(2) synthesis in fibroblasts about sixfold. The prostaglandin E(2) response to interleukin-1 beta was attenuated by lower concentrations of vasopressin (10(-10)-10(-9) M). By contrast, higher concentrations (10(-8)-10(-7) M) of vasopressin effected significant enhancement of the interleukin-1 beta-induced prostaglandin E(2) synthesis. In a similar way, vasopressin (10(-8)-10(-7) M), in the absence of interleukin-1, significantly increased prostaglandin E(2) production. An inhibitory effect of lower concentrations of vasopressin was also observed on basal production of prostaglandin E(2). The effects of vasopressin on basal and interleukin-1 beta-induced prostaglandin E(2) synthesis were antagonized by selective vasopressin receptor antagonists. The findings presented here disclose a novel modulatory role of vasopressin on prostaglandin E(2) synthesis in human dermal fibroblasts and suggest a possible role of vasopressin in the regulation of inflammation.

Effect of donor age on cultivation of human oral mucosal keratinocytes.

Cultured mucosal grafts (CMG) are among recently developed biological grafting materials to cover large oral mucosal defects following resection of mucosal pathology. This study evaluates the effect of donor's age on the cultivation process of oral mucosal keratinocytes for grafting. Human mucosal epithelial cells were utilized and classified into three donor age groups: 3-30 years (14 patients); 31-60 years (9 patients); and >60 years (6 patients) (11 males and 18 females). Isolation and cultivation of oral mucosal keratinocytes were according to Rheinwald and Green [Cell 6 (1975) 331], originally developed for epidermal keratinocytes. Isolated primary cell lines were seeded and cultivated. Propagation of cell lines ("passages"), time period required to reach confluence, yield of cells and plating efficiency were recorded. All cells propagated well up to the fourth passage. Thereafter, a decline was observed and was more distinct with age. Period to confluence was longer among the old age group. Yield of cells in fourth passage was high among the young age group and decreased with age. Plating efficiency in passages 4-6 decreased with age. These results suggest that age-related changes in cultivation of oral keratinocytes are not general phenomena, but rather limited to the donor age of 60 years and above. In this age group all the parameters studied were adversely affected. Oral mucosal keratinocytes may be a useful model for oral aging.

Autologous cultured mucosal graft to cover large intraoral mucosal defects: a clinical study.

PURPOSE: The study evaluated the performance of cultured mucosal grafts (CMG) for large intraoral mucosal defects caused by surgical excision of mucosal pathology. PATIENTS AND METHODS: Eleven patients (10 men and 1 woman; mean age, 52.4 +/- 14.1 years) were treated using CMG following mucosal defects. A biopsy specimen (0.2 to 0.5 cm(2)) was taken from a clinically healthy oral mucosa a few weeks before the surgery. Mucosal epithelial cells were cultured in vitro over a feeder layer of fibroblasts. Usually, within 3 to 4 weeks, multilayered sheets (50 to 250 cm(2)) were generated. The cultured sheets were placed on the mucosal defects (48.4 +/- 21.7 cm(2); 8 to 70 cm(2)) and anchored to the adjacent tissue with sutures. RESULTS: One week after surgery, the CMG survived and adhered to the wound bed. Three weeks postoperatively, the grafted site was smooth and keratinized, without infection or scar contraction. Three months postoperatively, the grafted area was covered with a healthy mucosa, indistinguishable from the adjacent mucosa. CONCLUSION: CMG is a useful grafting material for large intraoral mucosal defects.

Mutation of TBCE causes hypoparathyroidism-retardation-dysmorphism and autosomal recessive Kenny-Caffey syndrome.

The syndrome of congenital hypoparathyroidism, mental retardation, facial dysmorphism and extreme growth failure (HRD or Sanjad-Sakati syndrome; OMIM 241410) is an autosomal recessive disorder reported almost exclusively in Middle Eastern populations. A similar syndrome with the additional features of osteosclerosis and recurrent bacterial infections has been classified as autosomal recessive Kenny-Caffey syndrome (AR-KCS; OMIM 244460). Both traits have previously been mapped to chromosome 1q43-44 (refs 5,6) and, despite the observed clinical variability, share an ancestral haplotype, suggesting a common founder mutation. We describe refinement of the critical region to an interval of roughly 230 kb and identification of deletion and truncation mutations of TBCE in affected individuals. The gene TBCE encodes one of several chaperone proteins required for the proper folding of alpha-tubulin subunits and the formation of alpha-beta-tubulin heterodimers. Analysis of diseased fibroblasts and lymphoblastoid cells showed lower microtubule density at the microtubule-organizing center (MTOC) and perturbed microtubule polarity in diseased cells. Immunofluorescence and ultrastructural studies showed disturbances in subcellular organelles that require microtubules for membrane trafficking, such as the Golgi and late endosomal compartments. These findings demonstrate that HRD and AR-KCS are chaperone diseases caused by a genetic defect in the tubulin assembly pathway, and establish a potential connection between tubulin physiology and the development of the parathyroid.

Autoimmune progesterone dermatitis manifested as erythema annulare centrifugum: Confirmation of progesterone sensitivity by in vitro interferon-gamma release.

Autoimmune progesterone dermatitis was manifested as deep-type erythema annulare centrifugum. Sensitivity to progesterone was studied in vivo by means of intradermal and patch tests, which revealed immediate-type and delayed-type hypersensitivity, respectively. Sensitivity to progesterone was further confirmed in vitro by results of an interferon-gamma release test, which imply a possible role for T(H)1-type cytokines in autoimmune progesterone dermatitis.

Accreditation of skin from a methanol-poisoned victim for banking and grafting.

BACKGROUND: Acute poisoning is a contraindication for organ and tissue donation. In this study the suitability of skin from a methanol-poisoned (MP) donor for future grafting and keratinocytes culturing was investigated. METHODS: A patient was admitted with a methanol blood level of 2.7 mg/mL, which became undetectable after 4 days of treatment with 4-methylpyrazole (fomepizole). Upon declared brain death and family consent, organs and skin were harvested. For approving MP skin for grafting, the following parameters were studied: viability and plating efficiency of MP keratinocytes, integrity of MP skin after cryopreservation, and its performance as xenografts on wounds in a pig model. Nonpoisoned (NP) controls included skin of matching age, cryopreservation period, and NP keratinocytes. RESULTS: No significant differences were observed for any parameter between NP and MP samples. Furthermore, in vitro exposure of NP keratinocytes and fibroblasts to <10 mg/mL methanol inhibited their growth by <20%, with an extrapolated LD50 of 100 mg/mL. A parallel exposure to formaldehyde, a spontaneous metabolite of methanol, yielded LD50 of 20 microg/mL and eradication of viability at 300 microg/mL. CONCLUSIONS: These results indicate that skin from a carefully monitored MP donor is suitable for banking toward massive burns and skin losses. This methodology may be applied to approve skin harvested from other types of poisoned donors for banking and future grafting.

Skin Donors Positive for HBV Core Antibodies - to Bank or Not to Bank? An Overview from a Regional Tissue Bank.

A prompt transplantation of skin allografts on patients with severe, large body area burns is a preferred treatment, but depends on a suitable supply of tissue donors. Limiting factors include donors' identification, families consent, and following the standards - exclusion due to assessed transmissible diseases. To increase the current rate of skin donations to our regional skin bank, we reviewed the data of all potential organ donors, identified at Soroka University Medical Center from October 1997 to December 2000 and evaluated the causes for exclusion, especially due to HBV serological profile. 114/168 (67.9%) patients did not meet the indicated standards for organ donation, among which 20/114 patients (17.5%) positive for anti-HBc (anti-HBc(+)). 54/168 persons were declared brain dead, with consents obtained from 21 families. To discuss the intriguing approval of skin from potential donors with anti-HBc(+) serology, the literature was reviewed, specifically - the reported outcomes of organ transplants from anti-HBc(+) donors, updates of HBV and skin, available tests, and finally a look for a safe commendable algorithm. The results suggested that HBV might be replicating in the skin, but proven communication of HBV has not been reported following grafting skin from anti-HBc(+) donors. Unlike other procured organs and tissues, grafted banked skin is a temporary cover, storable up to six years, under appropriate conditions. Hence, banking of skin from anti-HBc(+) donors might be considered for future grafting of patients with identical serological profiles, presumably immune to a subsequent HBV infection, until a further re-evaluation of the standards. This procedure is anticipated to increase the potential of organ and tissue donations, specifically skin.

Application of Banked Autologous Mucosal Cells to Graft Large Intraoral Mucosal Defects.

Purpose: This study was aimed to investigate the conditions that affect the generation of autologous cultured mucosal grafts (CMG) to promote their clinical application for large intraoral defects, following surgical excision of mucosal pathology. Specific parameters studied were the effect of patient's age and cell banking on the in vitro development of CMG, and on their clinical performance in vivo.Patients and methods: Twelve patients (10 M, 2F; mean age of 50.7+/-14.3 years) with intraoral mucosal pathologies were included in this study. Autologous mucosal cells derived from 0.2-0.5 cm(2) biopsies, taken from clinically normal oral mucosa, were cultured in vitro and banked, as cell suspensions, in liquid nitrogen. Two to three weeks before the scheduled surgery, multilayered CMG were developed according to the anticipated area of the excised tissue (8-100 cm(2)), from banked or proliferating autologous cells (for 7/12 and 5/12 patients, respectively). Following excision, CMG sheets were placed on the mucosal defects and anchored to the adjacent tissue with sutures, which were removed a week later.Results: Both the production of CMG, and their clinical performance were unaffected significantly by patient's age. Three weeks postoperatively, the grafted sites were smooth and keratinized, without infection or scar contraction, with complete and comparable healing among the two groups, treated with banked and non-banked cells.Conclusion: This study indicates that CMG is a proper dressing for large intraoral mucosal defects of various etiologies, for a large range of patients' age, using either banked or non-banked cells.