Biomedical subjects

Mitchell Klausner

Publications and source records attributed to Mitchell Klausner.

5 recordsLinked to original sources

PubMed2006

Antimicrobial barrier of an in vitro oral epithelial model.

OBJECTIVE: Oral epithelia function as a microbial barrier and are actively involved in recognizing and responding to bacteria. Our goal was to examine a tissue engineered model of buccal epithelium for its response to oral bacteria and proinflammatory cytokines and compare the tissue responses with those of a submerged monolayer cell culture. DESIGN: The tissue model was characterized for keratin and beta-defensin expression. Altered expression of beta-defensins was evaluated by RT-PCR after exposure of the apical surface to oral bacteria and after exposure to TNF-alpha in the medium. These were compared to the response in traditional submerged oral epithelial cell culture. RESULTS: The buccal model showed expression of differentiation specific keratin 13, hBD1 and hBD3 in the upper half of the tissue; hBD2 was not detected. hBD1 mRNA was constitutively expressed, while hBD2 mRNA increased 2-fold after exposure of the apical surface to three oral bacteria tested and hBD3 mRNA increased in response to the non-pathogenic bacteria tested. In contrast, hBD2 mRNA increased 3-600-fold in response to bacteria in submerged cell culture. HBD2 mRNA increased over 100-fold in response to TNF-alpha in the tissue model and 50-fold in submerged cell culture. Thus, the tissue model is capable of upregulating hBD2, however, the minimal response to bacteria suggests that the tissue has an effective antimicrobial barrier due to its morphology, differentiation, and defensin expression. CONCLUSIONS: The oral mucosal model is differentiated, expresses hBD1 and hBD3, and has an intact surface with a functional antimicrobial barrier.

Cells, Cultured↗

PubMed2006

Ensuring quality of in vitro alternative test methods: Current practice.

In Vitro toxicology methods are being validated and adopted by regulatory agencies for use as alternatives to animal testing. Such methods may use ex vivo tissues or bioconstructs, some of which may be proprietary. Users of the data from these methods need to be reassured that the assays or assay components used in their studies provide consistent, good quality data over time, matching the standards achieved during the validation process. This paper presents an overview of approaches currently used by representatives of a manufacturer and a contract testing laboratory to ensure that the results from in vitro alternative methods are reproducible and of high quality over time. These approaches include full characterization of cells or tissues, sampling of each lot of manufactured bioconstructs for performance, and regular use of controls and benchmark chemicals to provide assurance of consistency of assay performance.

Animal Testing Alternatives↗

PubMed2005

Organotypic human vaginal-ectocervical tissue model for irritation studies of spermicides, microbicides, and feminine-care products.

A three-dimensional organotypic vaginal-ectocervical (VEC) tissue model has been developed to test the irritation of topically applied spermicides, microbicides, and vaginal-care products. The in vitro tissue model was reconstructed using normal VEC epithelial cells and is well stratified, containing differentiated basal, suprabasal, intermediate, and superficial cell layers similar to in vivo tissue. The intermediate and superficial cell layers contain glycogen, and the expression of cytokeratins 13 and 14 in the tissue also parallels that of native tissue. The MTT viability assay and histological assessment were used to test inter-lot and intra-lot reproducibility. The MTT average intra-lot coefficient of variation (CV) was less than 10% and the time required to reduce tissue viability by 50% (ET-50) following application of 1% Triton X-100 averaged 1.25+/-0.24h (n=23) upon completion of the 11-day culture period and 1.30 h+/- 0.19 for the same tissues stored overnight at 4 degrees C on agarose gels. The utility of the VEC model for irritation studies was examined by testing commercially available products using the MTT assay and histological assessment. The average ET-50 values ranged between 1.8 and 2.7h for feminine washes, 3.9-6.7 h for spermicides, 6.8-18 h for anti-itch creams, and >18 h for douches, lubricants, and anti-fungal creams. Studies of cytokines released from VEC cultures following product application showed that elevated concentrations of IL-1alpha and IL-1beta were associated with toxicity of test materials. In conclusion, the VEC tissue model is a highly reproducible, non-animal means to assess the irritation of contraceptives, microbicides, and vaginal-care products.

Adult↗

PubMed2004

Intranasal delivery of recombinant human parathyroid hormone [hPTH (1-34)], teriparatide in rats.

The aim of this study was to explore the nasal route as an alternative to daily subcutaneous injections of hPTH (1-34). Anesthetized rats were surgically prepared and nasally dosed with aqueous solutions of hPTH (1-34). Plasma samples were assayed by radioimmunoassay and data generated fit to two-(intravenous) and one-(intranasal) compartment pharmacokinetic models using WinNonlin. The toxicity of hPTH (1-34) solution administered to the rats was assessed by screening its effect on transepithelial electrical resistance, potential difference, paracellular marker permeation, tissue viability, and protein leakage using the EpiAirway tissue model. The intranasal absorption of hPTH (1-34) was rapid; the absorption rate constants (alpha) were 33.2+/-24 h(-1) [without bovine serum albumin (BSA)] and 9.8+/-5.1 h(-1) (with 1% BSA). The maximum plasma concentrations (Cmax): 151+/-24 pg/mL (without BSA) and 176+/-37 (with 1% BSA) were attained within approximately 15 min. The intranasal bioavailabilities (Fabs) were 12.1+/-3.4% (without BSA) and 17.6+/-1.5% (with 1% BSA). The hPTH (1-34) formulation administered to the rats had no detrimental effect on the EpiAirway tissue epithelial electrical parameters and functional integrity. Based on the results of this study, the nasal route appears to be a prospective alternative to subcutaneous injections of hPTH (1-34).

Administration, Intranasal↗

PubMed2003

Gene expression profile of tissue engineered skin subjected to acute barrier disruption.

The main function of the skin is to protect the body from infection, dehydration, and other environmental insults by creating an impermeable barrier of cornified cell layers, the stratum corneum. In contrast to cells in culture, tissue-engineered skin equivalents contain well-developed basal, spinous, granular, and cornified cell layers providing an excellent model to study the tissue response to barrier disruption. After 7 d of culture at the air-liquid interface the barrier of the tissues was disrupted by short exposure to acetone and the global gene expression profile of the tissues was evaluated using DNA microarrays. We found that tissue-engineered skin responds to barrier disruption by a two-wave dynamic response. Early on, the cells upregulate signal transducing, stress, proliferation, and inflammation genes to protect the tissue and possibly to communicate the damage to the immune system and neighboring tissues. At later times, pro-inflammatory cytokines and some growth-related genes are significantly reduced but enzymes that participate in lipid synthesis increase, suggesting that the epidermal cells attempt to restore the lost barrier. Quantitative immunostaining for the proliferation antigen Ki67 revealed that barrier disruption by acetone increased proliferation by 4-fold in agreement with the microarray data and previous in vivo studies. Our work suggests that functional genomics may be used in tissue engineering to understand tissue development, wound regeneration, and response to environmental stimuli. A better understanding of engineered tissues at the molecular level may facilitate their application in the clinic and as biosensors for toxicologic testing.

Acetone↗