Comparison of global and mode of action-based models for aquatic toxicity.

The ability to estimate aquatic toxicity is a critical need for ecological risk assessment and chemical regulation. The consensus in the literature is that mode of action (MOA) based toxicity models yield the most toxicologically meaningful and, theoretically, the most accurate results. In this study, a two-step prediction methodology was developed to estimate acute aquatic toxicity from molecular structure. In the first step, one-against-the-rest linear discriminant analysis (LDA) models were used to predict the MOA. The LDA models were able to predict the MOA with 85.8-88.8% accuracy for broad and specific MOAs, respectively. In the second step, a multiple linear regression (MLR) model corresponding to the predicted MOA was used to predict the acute aquatic toxicity value. The MOA-based approach was found to yield similar external prediction accuracy (r(2) = 0.529-0.632) to a single global MLR model (r(2) = 0.551-0.562) fit to the entire training set. Overall, the global hierarchical clustering approach yielded a higher combination of accuracy and prediction coverage (r(2) = 0.572, coverage = 99.3%) than the other approaches. Utilizing multiple two-dimensional chemical descriptors in MLR models yielded comparable results to using only the octanol-water partition coefficient (log K(ow)).

Functions of the mismatch repair gene mutS from Acinetobacter sp. strain ADP1.

The genus Acinetobacter encompasses a heterogeneous group of bacteria that are ubiquitous in the natural environment due in part to their ability to adapt genetically to novel challenges. Acinetobacter sp. strain ADP1 (also known as strain BD413) is naturally transformable and takes up DNA from any source. Donor DNA can be integrated into the chromosome by recombination provided it possesses sufficient levels of nucleotide sequence identity to the recipient's DNA. In other bacteria, the requirement for sequence identity during recombination is partly due to the actions of the mismatch repair system, a key component of which, MutS, recognizes mismatched bases in heteroduplex DNA and, along with MutL, blocks strand exchange. We have cloned mutS from strain ADP1 and examined its roles in preventing recombination between divergent DNA and in the repair of spontaneous replication errors. Inactivation of mutS resulted in 3- to 17-fold increases in transformation efficiencies with donor sequences that were 8 to 20% divergent relative to the strain ADP1. Strains lacking MutS exhibited increased spontaneous mutation frequencies, and reversion assays demonstrated that MutS preferentially recognized transition mismatches while having little effect on the repair of transversion mismatches. Inactivation of mutS also abolished the marker-specific variations in transforming efficiency seen in mutS(+) recipients where transition and frameshift alleles transformed at eightfold lower frequencies than transversions or large deletions. Comparison of the MutS homologs from five individual Acinetobacter strains with those of other gram-negative bacteria revealed that a number of unique indels are conserved among the Acinetobacter amino acid sequences.

Prediction of the acute toxicity (96-h LC50) of organic compounds to the fathead minnow (Pimephales promelas) using a group contribution method.

A group contribution method has been developed to correlate the acute toxicity (96-h LC50) to the fathead minnow (Pimephales promelas) for 397 organic chemicals. Multilinear regression and computational neural networks (CNNs) were used for model building. The models were able to achieve a fairly good correlation of the data (r2 > 0.9). The linear model, which included four specific interaction terms, provided a rapid means of predicting the toxicity of a compound. The CNN model was able to yield virtually the same predictions with or without the four interaction terms that were included in the multilinear model.

West Nile virus infection in birds and mosquitoes, New York State, 2000.

West Nile (WN) virus was found throughout New York State in 2000, with the epicenter in New York City and surrounding counties. We tested 3,403 dead birds and 9,954 mosquito pools for WN virus during the transmission season. Sixty-three avian species, representing 30 families and 14 orders, tested positive for WN virus. The highest proportion of dead birds that tested positive for WN virus was in American Crows in the epicenter (67% positive, n=907). Eight mosquito species, representing four genera, were positive for WN virus. The minimum infection rate per 1,000 mosquitoes (MIR) was highest for Culex pipiens in the epicenter: 3.53 for the entire season and 7.49 for the peak week of August 13. Staten Island had the highest MIR (11.42 for Cx. pipiens), which was associated with the highest proportion of dead American Crows that tested positive for WN virus (92%, n=48) and the highest number of human cases (n=10).

Hox D3 expression in normal and impaired wound healing.

BACKGROUND: We have previously shown that Hox D3 and Hox B3 can promote angiogenesis. As angiogenesis is essential for wound healing, we examined expression of these genes in the vasculature following wounding in normal and genetically diabetic adult mice with impaired healing. METHODS: In situ hybridization was performed on tissues taken 0, 1, 4, 7, and 14 days following administration of linear wounds in wild-type and genetically diabetic mice. Expression of Hox D3 and Hox B3, angiogenesis, and synthesis of type I collagen were assessed in the wound. RESULTS: Hox B3 was expressed in endothelial cells (ECs) of both medium and small vessels in unwounded tissue, whereas little Hox D3 was detected in resting ECs. Hox D3 expression was significantly upregulated by 1 day after wounding in ECs of vessels immediately adjacent to the wound site, and expression was maintained for at least 7 days. In the diabetic mice, expression of Hox B3 was similar to that of wild-type mice. In contrast, expression of Hox D3 in ECs was significantly lower and delayed during wound repair in diabetic mice. In cultured microvascular ECs, Hox D3 selectively induced high levels of collagen I mRNA expression. Hox D3-deficient wounds of diabetic animals also displayed a reduction in expression and deposition of type I collagen. CONCLUSIONS: These results suggest that reduced angiogenesis and type I collagen in diabetic mice with impaired wound healing may be related to deficient Hox D3 expression, and restoring Hox D3 expression may enhance angiogenesis and wound repair.

Mercury-reactive lymphocytes in peripheral blood are not a marker for dental amalgam associated disease.

OBJECTIVES: The popular press and publications associated with alternative medicine increasingly report that chronic ill health, particularly myalgic encephalitis like conditions, are associated with mercury amalgam fillings. There are no scientifically proven definitive tests to support these claims. One of the more scientific tests in vogue is to assess the level of blood-borne mercury-reactive lymphocytes and to conclude that patients with high levels have developed a hypersensitivity reaction to mercury. The objective of this study was to determine the diagnostic value of this test. METHODS: This study represents an open comparison of mercury-reactive lymphocyte levels in healthy control individuals with those in patients complaining of symptoms associated with adverse effects of dental metal amalgam fillings. The healthy control group consisted of 51 male and female individuals, aged between 12 and 82 years, with and without dental amalgam fillings. The patient group consisted of 70 male and female individuals, aged between 12 and 87 years, and with the exception of one patient, with three or more mercury amalgam fillings of more than 1 year's duration. In vitro lymphocyte responses to mercury, and to nickel, as an example of a metal commonly associated with hypersensitivity reactions, and to more conventional protein antigens were determined. RESULTS: In the blood of patients and controls, there were similar levels of specifically reactive lymphocytes to all of the in vitro stimulating agents, but there were significantly higher numbers of sub-normal and non-responders within the patient group. CONCLUSIONS: The incidence and quantity of mercury-reactive lymphocytes in the blood are not pathogenic markers of illness associated with dental metal amalgams, but may rather reflect exposure to mercury. The clinical relevance of the decreased in vitro lymphocyte responses in the patient group needs further investigation.

Expression of heat shock proteins in a linear rodent wound.

Heat shock proteins (hsps) are ubiquitous and known to be expressed in all organisms. These stress proteins are likely to be induced in the wound environment and may play a critical role in the overall process of wound repair. Linear incisions were made in Sprague-Dawley rats. Serial skin biopsies were taken, the dermis and epidermis were separated and a protein lysate made. The expression of hsp 72, 47, and 32 were analyzed by Western blotting and immunohistochemistry. There were distinct patterns of expression of hsp 72, 47, and 32 in the wound. In unwounded dermis, there was no constitutive expression of any of the heat shock proteins studied. In the epidermis, there was constitutive expression of hsp 32 and 72, but not hsp 47. With wounding, all hsps exhibited increased expression both in the dermis and epidermis. These patterns of protein expression are suggestive of the individual heat shock proteins' molecular function, such as hsp 72's role as an indicator of cellular stress and injury, hsp 47's role in collagen synthesis, and hsp 32's role as an antioxidant.

The initial coverage and impact of the pneumococcal and influenza vaccination program for at-risk indigenous adults in Far North Queensland.

OBJECTIVES: To describe the initial coverage and impact of a pneumococcal and influenza vaccination program for at-risk Indigenous adults in Far North Queensland that formally commenced in 1996. DESIGN: Ascertainment of vaccine coverages, and prospective laboratory surveillance of invasive pneumococcal disease occurring in Indigenous adults in the region. MAIN OUTCOME MEASURES: Coverages of the first doses of both vaccines administered since 1995, and the incidence of invasive pneumococcal disease in Indigenous adults in the region between 1993-2000. RESULTS: Most (96% and 73%) of the Indigenous adults > or = 50 years of age received influenza and pneumococcal vaccines, respectively, for the first time between 1995-2000. Assuming that either 33% or 50% of Indigenous adults 15-49 years of age in Far North Queensland were eligible for vaccination, then either 109% or 72% of this population received influenza vaccine, and either 75% or 50% received pneumococcal vaccine, respectively, for the first time between 1995-2000. The incidence of vaccine-preventable invasive pneumococcal disease fell from 111 (95% confidence interval [CI] 77-154) cases per 100,000 per year in 1993/94 to 28 (95% CI 13-53) cases per 100,000 per year in 1999-2000 (p<0.05). CONCLUSION: Although there was a significant decline in the incidence of invasive pneumococcal disease, the vaccine coverages after five years of the program were suboptimal. Because of the difficulties in targeting the 15-49 years age group and because of unrecognised risk factors, we suggest that a universal Indigenous adult pneumococcal and influenza vaccination program should be considered.

Neutralizing vascular endothelial growth factor activity inhibits thyroid cancer growth in vivo.

BACKGROUND: Without angiogenesis, tumor growth is limited to a few millimeters, the limit of diffusion. Vascular endothelial growth factor (VEGF) is an endothelial-specific mitogen and a major regulator of angiogenesis. METHODS: To investigate the relationship between VEGF and thyroid tumor angiogenesis, we xenografted human dermal matrix inoculated with FTC-133 cells into nude mice or directly injected FTC-133 cells subcutaneously. To block the function of VEGF, the neutralizing anti-VEGF monoclonal antibody A.4.6.1 (mAb A.4.6.1) was injected intraperitoneally twice weekly. As control, an antibody of the same isotype (Ab 5B6) or phosphate buffer saline solution (PBS) was used. To evaluate the dermal matrix as a model for angiogenesis studies, recombinant human VEGF was inoculated into the dermal matrix pocket and xenografted into mice. RESULTS: In the dermal matrix angiogenesis model, the number of blood vessels paralleled the concentration of recombinant human VEGF and was highest at 100 ng/mL. Mice that were treated with the mAb A4.6.1 developed fewer blood vessels (mean, 6.6 per HPF) than control mice (18 per HPF in Ab 5B6 and 22 per HPF in PBS; P <.01). Tumors from mice that were treated with mAb A.4.6.1 were much smaller (mean +/- SD, 0.09 +/- 0.02 gm) at 5 weeks, compared with the tumors treated with Ab 5B6 (5.38 +/- 1.15 gm) or PBS (4.0 +/- 0.72 gm; P <.001). CONCLUSIONS: VEGF is produced by the follicular thyroid cancer cell line and stimulates angiogenesis and growth of thyroid cancer. This stimulation can be blocked by mAb A.4.6.1.

Cranial nerve VII region of the traumatized facial skeleton: optimizing fracture repair with the endoscope.

OBJECTIVE: Cranial nerve VII (CN VII) is anatomically positioned adjacent to the condylar neck of the mandible and arch of the midface. Fracture treatment of this region of the facial skeleton remains controversial because of difficult surgical access. Conservative management rarely achieves anatomic fracture repair and can result in irreversible structural deformity and dysfunction. Traditional operative methods require access through very visible facial incisions and risk injury to the facial nerve. We report endoscopic methods of facial fracture repair in the region of CN VII that achieve excellent fracture reduction and stabilization, with minimal risk of facial nerve injury by using hidden incisions. METHODS: A consecutive series of 65 endoscopically assisted facial fracture repairs were analyzed. Endoscopic repairs of the condylar neck (n = 40) of the mandible were performed through an intraoral incision. Endoscopic arch repairs (n = 25) of the midface were performed through a preauricular incision. Outcomes were evaluated by postoperative fracture reduction on radiographs, occlusion, interincisal jaw opening, and facial nerve function. RESULTS: Thirty-seven of 40 condylar neck mandible fractures went on to anatomic bone union, whereas 3 of 40 had either incomplete fracture reduction or re-fracture through the plate. There was one temporary palsy of CN VII that completely resolved spontaneously. Jaw opening exceeded 40 mm by the 8th postoperative week. Computed tomographic images demonstrated anatomic arch repair in all 25 endoscopically repaired cases. Six of seven endoscopically repaired Le Fort III facial fractures went on to restoration of their premorbid occlusion. One of seven had an excellent restoration of the occlusal interface but a cant to the occlusal plane. Two of seven had improved but incomplete restoration of the malar prominence and enopthalmos ipsilateral to the side of endoscopic arch repair. Eight of 25 endoscopic arch repairs developed temporary paralysis of the frontal branch of CN VII that recovered completely by the 10th postoperative week. CONCLUSION: We have developed novel endoscopically assisted techniques to facilitate repair of facial fractures in the region of CN VII. These techniques have been successfully applied to accurately restore the facial skeleton to its preinjury anatomic position in the region of CN VII with minimal risk of facial paralysis by using limited and well-hidden incisions.

Interleukin-8 mediates injury from smoke inhalation to both the lung endothelial and the alveolar epithelial barriers in rabbits.

Although prior studies have shown that smoke inhalation causes lung endothelial injury and formation of pulmonary edema, there is no information about the effect of smoke inhalation on the function of the alveolar epithelial barrier. Therefore, the primary objective of this study was to determine the effect of smoke-induced lung injury on the alveolar epithelial barrier in a rabbit experimental model. The second objective was to investigate whether pretreatment with a monoclonal anti-interleukin (IL)-8 antibody prevented alveolar epithelial barrier injury after smoke inhalation. Anesthetized rabbits were tracheotomized and were insufflated with cooled smoke generated from burning cotton cloth (75 breaths). In some experiments, anti-IL-8 antibody or an irrelevant antibody (2 mg/¿g) was given intravenously 5 min before insufflation of cotton smoke. Smoke inhalation caused a significant increase in the alveolar epithelial permeability to protein and a 40% reduction in the fluid transport capacity of the alveolar epithelium. Pretreatment with anti-IL-8 antibody, but not with an irrelevant-isotype antibody, significantly reduced the smoke-mediated increase in bidirectional transport of protein across the alveolar epithelium, and restored alveolar liquid clearance to a normal level. The results of the study show that smoke inhalation causes injury to both the alveolar epithelial barrier and the lung endothelium, and that IL-8 is an important mediator of this injury.

Phenotypic expression of PCR-generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation.

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37 degrees C. Of recombinant clones that failed to express xylE at 37 degrees C, about 10% expressed the gene when grown at 22 degrees C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.

Diet-induced protection against lipopolysaccharide includes increased hepatic NO production.

The host response to Gram-negative infection includes the elaboration of numerous proinflammatory agents, including tumor necrosis factor alpha (TNFalpha) and nitric oxide (NO). A component of the hepatic response to infection is an elevation in serum lipids, the so-called "lipemia of sepsis," which results from the increased production of triglyceride (TG)-rich lipoproteins by the liver. We have postulated that these lipoproteins are components of a nonadaptive, innate immune response to endotoxin [lipopolysaccharide (LPS)] and have previously demonstrated the capacity of TG-rich lipoproteins to protect against endotoxicity in rodent models of sepsis. Herein we report the capacity of a high-fructose diet to protect against LPS, most likely by inducing high circulating levels of endogenous TG-rich lipoproteins. The protective phenotype included the increased production of NO by hepatic endothelial cells. Rats, made hypertriglyceridemic by fructose feeding, experienced decreased LPS-induced mortality (P < 0.03) and systemic TNFalpha levels (P < 0.05) as compared with normolipidemic (chow-fed) controls. The increased survival was associated with elevated levels of inducible NO synthase (NOS2) mRNA levels and NO production (82 +/- 26 vs 3 +/- 3 nmol nitrite/10(6) cells, P < 0.001) by hepatic endothelial cells. Nonselective NOS inhibitors reversed the protective phenotype in vivo and readily decreased NO production by cultured endothelial cells from hypertriglyceridemic rats in vitro. This study suggests that a high-fructose diet can protect against endotoxicity in part through induction of endogenous TG-rich lipoproteins and hepatic endothelial cell NO production. This is the first report of diet-induced hyperlipoproteinemia and subsequent protection against endotoxemia.

Invasion of human follicular thyroid carcinoma cells in an in vivo invasion model.

Models that demonstrate histological invasion of extracellular matrix barriers by tumor cell lines are useful for assessing new methods to treat or prevent tumor metastasis. An in vivo invasion model using acellular human dermal matrix has been described in a murine squamous cell carcinoma line. The present study examined the application of this tumor invasion model to another epithelial cell line derived from a different species. A human follicular thyroid carcinoma cell line, known to be invasive by other assays, was grown on the dermal-epidermal basement membrane surface of human acellular dermal matrix in culture and then grafted in athymic mice. Immunohistochemical staining of type IV collagen was used to identify the basement membrane and invasion was determined as penetration of the basement membrane by tumor cells. Identification of the human tumor cells in the in vivo grafts was done by in situ hybridization with species specific probes. FTC-133 tumor cells did not invade the matrix after 4 weeks of growth in in vitro culture, but there was extensive loss of the basement membrane and infiltration of the tumor cells into the dermis after 2 weeks growth in vivo. This study suggests that the in vivo dermal matrix model of invasion is applicable to a broad range of epithelial carcinoma cell lines to study their capability to penetrate basement membrane. A model such as this may be useful for studying the local effects of genetic manipulations of implanted tumor cell populations, leading to the development of therapeutic agents that block invasion.

Staging of colorectal cancer: biology vs. morphology.

PURPOSE: An accurate determination of the extent or staging of a disease is critical, because it provides the basis for making therapeutic decisions. Staging is a collaborative effort by the surgeon and the pathologist. Radioimmunoguided surgery has been evaluated for its ability to help surgeons determine the extent of disease during surgery, when management decisions have the most impact on patient care. This study was done to compare radioimmunoguided surgery "biostaging" with traditional pathologic staging (TNM) as predictors of survival in patients undergoing curative resections for colorectal cancer. METHODS: Ninety-seven patients with colorectal cancer were prospectively enrolled in radioimmunoguided surgery protocols. Evaluation of follow-up survival data was performed. All patients underwent exploratory laparotomy and radioimmunoguided surgery with resection of their primary colorectal tumor. Survival data were analyzed with the Kaplan-Meier method with log-rank comparisons. RESULTS: Of 97 patients enrolled in the study, 59 were evaluable and completely resectable by radioimmunoguided surgery. Mean follow-up was 62 months, with a range of 34 to 89 months. By traditional staging 13 patients were pStage I, 18 patients were pStage II, and 28 patients were pStage III. By radioimmunoguided surgery biostaging, 24 patients were radioimmunoguided surgery-negative whereas 35 patients were radioimmunoguided surgery-positive. Survival rates by pathologic stage approached a significant difference, but did not, as of the conclusion of the study period, reach it (P = 0.12). Survival rates based on radioimmunoguided surgery status demonstrated a highly significant difference (P = 0.0002). CONCLUSIONS: Radioimmunoguided surgery biostaging provides new information intraoperatively on cancer staging that has not been available before. This may lead to new strategies for therapy that can be individualized and optimized for each patient with cancer.

Intraorbital squamous epithelial cyst: an unusual complication of Silastic implantation.

Thin Silastic sheet alloplasts (Dow Corning, Midland, MI, U.S.A) are commonly used to reconstruct posttraumatic orbital floor defects. Complications associated with orbital Silastic implantation include infection, migration, and extrusion. The authors report an unusual case of an intraorbital, squamous, epithelial-lined cyst appearing as progressive vertical globe dystopia and proptosis occurring after Silastic reconstruction of a traumatic orbital floor defect.

Vascular endothelial growth factor expression is higher in differentiated thyroid cancer than in normal or benign thyroid.

Vascular endothelial growth factor (VEGF) is an angiogenic factor, and its expression has been rarely demonstrated in thyroid tumors. We, therefore, investigated the expression of VEGF messenger RNA (mRNA) and production of VEGF protein in cell lines from human primary and metastatic follicular (FTC-133, FTC-236, and FTC-238), papillary (TPC-1), Hürthle cell (XTC-1), and medullary thyroid cancers (MTC-1.1 and MTC-2.2), and in human thyroid tissues (papillary, follicular, medullary, and Hürthle cell cancers, follicular adenomas, and Graves' thyroid tissue) by Northern blot, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) studies. All thyroid cell lines expressed a 4.2-kilobase VEGF mRNA. The VEGF mRNA levels were higher in the thyroid cancer cell lines than in primary cultures of normal thyroid cells, and higher in thyroid cancers of follicular than those of parafollicular cell origin. The VEGF mRNA levels were similar in primary and metastatic thyroid tumors. Immunohistochemical staining and Northern blot analysis of the cell lines correlated positively, thus thyroid cancer cell lines stained more intensely than normal thyroid cells and follicular tumor cells more intensely than parafollicular tumor cells. Again, no difference was noted in VEGF staining between primary and metastatic thyroid tumors. Deparafinized sections of papillary, follicular, and Hürthle cell cancers also stained much stronger than those of medullary thyroid cancers, benign, or hyperplastic (Graves' disease) thyroid tissue. Thyroid cancer cell lines (XTC-1 > TPC-1 > FTC-133 > MTC-1.1) also secreted more VEGF protein as measured by ELISA than did normal thyroid cells. VEGF secretion of cell lines derived from primary and metastatic thyroid tumors were similar. VEGF mRNA is therefore expressed, and VEGF protein is secreted by normal, hyperplastic, and neoplastic thyroid tissues. The higher levels of VEGF expression in differentiated thyroid cancers of follicular cell origin suggests a role in oncogenesis.

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a prospective consortium and its most effective isolate Serratia marcescens.

The biotransformation of hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) has been observed in liquid culture by a consortium of bacteria found in horse manure. Five types of bacteria were found to predominate in the consortium and were isolated. The most effective of these isolates at transforming RDX was Serratia marcescens. The biotransformation of RDX by all of these bacteria was found to occur only in the anoxic stationary phase. The process of bacterial growth and RDX biotransformation was quantified for the purpose of developing a predictive type model. Cell growth was assumed to follow Monod kinetics. All of the aerobic and anoxic growth parameters were determined: micro(max), K(s), and Y(x/s). RDX was found to competitively inhibit cell growth in both atmospheres. Degradation of RDX by Serratia marcescens was found to proceed through the stepwise reduction of the three nitro groups to nitroso groups. Each of these reductions was found to be first order in both component and cell concentrations. The degradation rate constant for the first step in this reduction process by the consortium was 0.022 L/g cells . h compared to 0.033 L/g cells . h for the most efficient isolate. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 515-522, 1997.

Human acellular dermal matrix as a novel model of malignant epithelial cell invasion.

Cancer invasion and metastasis are associated with matrix degradation. We describe a novel in vivo model of invasion by squamous epithelial neoplastic cells derived from transgenic mice grown on acellular human dermis. Human dermis was subjected to multiple freeze-thaw cycles to render it acellular, maintaining the basement membrane of the former dermal-epidermal junction. Cells representing discrete stages of a multistep transgenic mouse model of epidermal carcinogenesis (neonatal transgenic keratinocytes, moderately/poorly differentiated squamous cell carcinoma, and lymph node metastasis) were seeded onto the basement membrane surface, grown in culture for 4 days, grafted in a subpannicular pocket of athymic mice, and harvested after 3 weeks. Histological analysis demonstrated that neonatal transgenic keratinocytes did not degrade the basement membrane or invade the underlying dermis. In contrast, malignant cells derived from both a moderately differentiated squamous carcinoma and a lymph node metastasis were highly invasive. Immunohistochemical analysis revealed collagenase only in nests of invading malignant cells in contact with the dermal matrix, but not in the tumor mass remaining above the basement membrane, suggesting that this proteinase may be required for stromal invasion. This novel model recapitulates the events seen in malignant invasion: transgenic keratinocytes are unable to penetrate the dermis while cells from a moderately differentiated carcinoma and from lymph node metastasis consistently invade.