The gene or not the gene--that is the question: understanding the genetically engineered mouse phenotype.

Embryonic stem cells have had a significant impact on understanding gene function and gene interactions through the use of genetically engineered mice. However, the genetic context (ie, mouse strain) in which these modifications in alleles are made may have a considerable effect on the phenotypic changes identified in these mice. In addition, tissue- and time-specific gene expression systems may generate unanticipated outcomes. This article discusses the history of embryonic stem cells, reviews how mouse strain can affect phenotype (using specific examples), and examines some of the caveats of conditional gene expression systems.

Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice.

Inbred laboratory mouse strains are highly divergent in their immune response patterns as a result of genetic mutations and polymorphisms. The generation of genetically engineered mice (GEM) has, in the past, used embryonic stem (ES) cells for gene targeting from various 129 substrains followed by backcrossing into more fecund mouse strains. Although common inbred mice are considered "immune competent," many have variations in their immune system-some of which have been described-that may affect the phenotype. Recognition of these immune variations among commonly used inbred mouse strains is essential for the accurate interpretation of expected phenotypes or those that may arise unexpectedly. In GEM developed to study specific components of the immune system, accurate evaluation of immune responses must take into consideration not only the gene of interest but also how the background strain and microbial milieu contribute to the manifestation of findings in these mice. This article discusses points to consider regarding immunological differences between the common inbred laboratory mouse strains, particularly in their use as background strains in GEM.

Of mice and microflora: considerations for genetically engineered mice.

The phenotype of genetically engineered mice is a combination of both genetic and environmental factors that include the microflora of the mouse. The impact a particular microbe has on a mouse reflects the host-microbe interaction within the context of the mouse genotype and environment. Although often considered a confounding variable, many host-microbe interactions have resulted in the generation of novel model systems and characterization of new microbial agents. Microbes associated with overt disease in mice have been the historical focus of the laboratory animal medical and pathology community and literature. The advent of genetic engineering and the complex of mouse models have revealed previously unknown or disregarded agents that now oblige the attention of the biomedical research community. The purpose of this article is to describe and illustrate how phenotypes can be affected by microflora by focusing on the infectious diseases present in genetically engineered mouse (GEM) colonies of our collective institutions and by reviewing important agents that are rarely seen in most research facilities today. The goal is to introduce the concept of the role of microflora on phenotypes and in translational research using GEM models.

Pathophysiology of cyclooxygenases in cardiovascular homeostasis.

Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid into prostaglandin H(2) (PGH(2)), which is subsequently converted to the prostanoids PGE(2), PGI(2), PGF(2alpha), and thromboxane A(2). COX has 2 distinct membrane-anchored isoenzymes: COX-1 and COX-2. COX-1 is constitutively expressed in most normal tissues; COX-2 is highly induced by proinflammatory mediators in the setting of inflammation, injury, and pain. Inhibitors of COX activity include conventional nonselective nonsteroidal anti-inflammatory drugs and selective nonsteroidal anti-inflammatory drugs, such as COX-2 inhibitors. The adverse effects of COX inhibitors on the cardiovascular system have been addressed in the last few years. In general, COX inhibitors have many effects, but those most important to the cardiovascular system can be direct (through the effects of prostanoids) and indirect (through alterations in fluid dynamics). Despite reports of detrimental human cardiovascular events associated with COX inhibitors, short, long, and lifetime preclinical toxicology studies in rodents and nonrodents have failed to identify these risks. This article focuses on the expression and function of COX enzymes in normal and pathologic conditions of the cardiovascular system and discusses the cardiovascular pathophysiologic complications associated with COX inhibition.

Alternative splicing of parathyroid hormone-related protein mRNA: expression and stability.

Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that is often dysregulated in cancer. The human PTHrP gene is alternatively spliced into three isoforms, each with a unique 3'-untranslated region (3'-UTR), encoding 139, 173 and 141 amino acid proteins. The regulation of PTHrP mRNA isoform expression has not been completely elucidated, but it may be affected by transforming growth factor-beta1 (TGF-beta1). In this study, we examined differences in the PTHrP mRNA isoform expression in two squamous carcinoma cell lines (SCC2/88 and HARA), an immortalized keratinocyte cell line (HaCaT), and spontaneous human lung cancer with adjacent normal tissue. In addition, the effect of TGF-beta1 on PTHrP mRNA isoform expression and stability was examined. Cell-type specific expression of PTHrP mRNA isoforms occurred between the various cell lines, normal human lung, and immortalized human keratinocytes (HaCaT). PTHrP isoform expression pattern was significantly altered between normal lung tissue and the adjacent lung cancer. In vitro studies revealed that TGF-beta1 differentially altered the mRNA steady-state levels and mRNA stability of the PTHrP isoforms. Protein-RNA binding studies identified different proteins binding to the 3'-UTR of the PTHrP isoforms (139) and (141), which may be important in the differential mRNA stability and response to cytokines between the PTHrP isoforms. The data demonstrate that there is cell-type specific expression of PTHrP mRNA isoforms, and disruption of the normal regulation during cancer progression may in part be associated with TGF-beta1-induced changes in PTHrP mRNA isoform expression and stability.

Messenger RNA stability of parathyroid hormone-related protein regulated by transforming growth factor-beta1.

Humoral hypercalcemia of malignancy (HHM), a paraneoplastic syndrome associated with epithelial cancers, including squamous cell carcinoma (SCC), is due to expression and secretion of parathyroid hormone-related protein (PTHrP). Transforming growth factor-beta1 (TGFbeta1), expressed by many tumors, has been demonstrated in vitro to increase the half-life of PTHrP mRNA. In this study, oral squamous carcinoma cells (SCC2/88) had a two-fold increase in PTHrP mRNA stability (from 45 to 90 min) in response to treatment with TGFbeta1. In order to examine the mechanism of TGFbeta1-mediated PTHrP mRNA stability, a cell-free assay of mRNA degradation was utilized in which the degradation of in vitro-transcribed mRNA incubated with cytoplasmic protein extracts from SCC2/88 treated with vehicle or TGFbeta1 was measured. In this assay, full-length PTHrP mRNA was not significantly stabilized in TGFbeta1-treated samples when compared to vehicle treated samples. However, there was a striking (>5-fold) increase in PTHrP mRNA half-life in TGFbeta1-treated samples when PTHrP mRNA lacked the 3'-untranslated region (3'-UTR). In contrast, the degradation of 3'-UTR-truncated PTHrP mRNA using the cell-free assay was not altered in vehicle-treated samples. UV cross-linking of PTHrP mRNA and cytoplasmic proteins from cells treated with either vehicle or TGFbeta1 revealed numerous mRNA-binding proteins. TGFbeta1 treatment resulting in decreased binding of 33, 31, 27, 20 and 18 kDa binding proteins to the terminal coding region. These studies revealed that TGFbeta1-induced PTHrP mRNA stability might be, in part, the result of cis-acting sequences within the coding region of the PTHrP mRNA.

Expression of cyclooxygenase-1 and 2 in naturally-occurring canine cancer.

The purpose of this work was to determine cox-1 and cox-2 expression by immunohistochemistry in forms of naturally occurring canine cancer in order to identify animal systems for pre-clinical evaluation of cox inhibitors and cox-2 inhibitors in cancer. Canine lymphoma (LSA), prostatic carcinoma (PCA), osteosarcoma (OSA), oral melanoma (MEL), oral squamous cell carcinoma (SCC), oral fibrosarcoma (FSA), mammary carcinoma (MCA), and normal tissues were included. Cox-2 was expressed in epithelial tumors (17 of 26 SCC, 8 of 13 MCA, 5 of 9 PCA cases) and MEL (9 of 15 cases), but was generally absent in normal tissues. Cox-2 expression was minimal or absent in mesenchymal tumors and LSA. Cox-1 was expressed in normal epithelial tissues and in some osteoclast and osteoblast in bone, but was absent in normal lymph node. In conclusion, forms of canine cancer were identified for in vivo studies of the effects of cox inhibitors and selective cox-2 inhibitors on cancer.

The effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage.

Articular cartilage has a limited capacity for repair. We investigated the effect of rhBMP-2 (recombinant human bone morphogenetic protein-2) on the healing of full-thickness osteochondral defects in adult New Zealand White rabbits. A single defect, three millimeters wide by three millimeters deep, was created in the trochlear groove of the right femur in eighty-nine rabbits. The defect was either left empty, filled with a plain collagen sponge, or filled with a collagen sponge impregnated with five micrograms of rhBMP-2. The animals were killed at four, eight, or twenty-four weeks, and the repair tissue was examined histologically and evaluated with use of a grading scale. The defects also were examined immunohistochemically for the presence of type-II collagen at four and eight weeks. The rate of bone repair was evaluated with fluorescent labeling of bone at two and four weeks and with use of fluorescence microscopy at eight weeks. Treatment with rhBMP-2 greatly accelerated the formation of new subchondral bone and improved the histological appearance of the overlying articular surface. At twenty-four weeks, the thickness of the repair cartilage was 70 per cent that of the normal adjacent cartilage and a new tidemark usually had formed between the repair cartilage and the underlying subchondral bone. The average total scores on the histological grading scale were significantly better (p < 0.01) for the defects treated with rhBMP-2 than for the untreated defects (those left empty or filled with a plain collagen sponge) at all time-points. Immunostaining with an antibody against type-II collagen showed the diffuse presence of this cartilage-specific collagen throughout the repair cartilage in the treated defects. The untreated defects demonstrated minimum staining with this antibody.

Repair of articular cartilage defects one year after treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2).

BACKGROUND: Damaged articular cartilage has a limited ability to repair. Operative removal of damaged cartilage and penetration into the subchondral bone to allow population of the defect with progenitor cells can result in filling of the defect with repair tissue. However, this repair tissue often degenerates over time because of its inability to withstand the mechanical forces to which it is subjected. We previously reported that recombinant human bone morphogenetic protein-2 (rhBMP-2) improves the repair of full-thickness defects of cartilage as long as six months postoperatively. We have now extended that study to examine the quality of the repair tissue at one year. METHODS: Full-thickness defects of cartilage were created in the trochlear groove of twenty-five adult New Zealand White rabbits. Eight defects were left empty, eight were filled with a collagen sponge, and nine were filled with a collagen sponge impregnated with five micrograms of rhBMP-2. The animals were killed at fifty-two weeks postoperatively, and the gross appearance of the healed defect was assessed. The repair tissue was examined histologically and was evaluated, according to a grading scale, by four individuals who were blinded with respect to the treatment. The tissue sections were immunostained with antibodies against type-I collagen, type-II collagen, aggrecan, and link protein. The residence time of the rhBMP-2 in the cartilage defect was evaluated in vivo with use of scintigraphic imaging of radiolabeled protein. RESULTS: One year after a single implantation of a collagen sponge containing five micrograms of rhBMP-2, the defects had a significantly better histological appearance than the untreated defects (those left empty or filled with a collagen sponge). The histological features that showed improvement were integration at the margin, cellular morphology, architecture within the defect, and reformation of the tidemark. The total scores were also better for the defects treated with rhBMP-2 than for the untreated defects, but in no instance was the repair tissue identical to normal articular cartilage. The thickness of the cartilage in the defects treated with rhBMP-2 was 70 percent that of the normal cartilage, an observation that was identical to that at twenty-four weeks postoperatively. Immunostaining demonstrated significantly less type-I collagen in the defects treated with rhBMP-2 than in the untreated defects. Immunostaining for other matrix components showed no difference among the treatment groups. The mean residence time of rhBMP-2 in the cartilage defects was eight days with an elimination half-life of 5.6 days. Detectable amounts of rhBMP-2 were present as long as fourteen days after implantation. CONCLUSIONS: The problems associated with operative repair of cartilage include the formation of fibrocartilage rather than normal articular cartilage and the degeneration of that repair tissue over time. Our results demonstrate that the addition of rhBMP-2 to the operative site after creation of a full-thickness defect results in an improvement in the histological appearance and composition of the extracellular matrix at one year postoperatively. If these experimental results translate directly to the clinical situation, it is possible that the addition of rhBMP-2 to existing operative treatments for the repair of cartilage may improve the repair process and may help to maintain the integrity of the repair tissue.

Head and neck squamous cell carcinoma: measurement of plasma parathyroid hormone-related protein and serum and urine calcium concentrations.

OBJECTIVES: Parathyroid hormone-related protein (PTHrP) is expressed by squamous cell carcinomas. Our first objective was to examine the stability of PTHrP in normal human plasma. Our second objective was to determine whether plasma PTHrP could be used in patients with head and neck squamous cell carcinoma (HNSCC) as an indicator of tumor burden or relapse. STUDY DESIGN AND SETTING: Blood and urine samples from 55 HNSCC patients undergoing tumor resection at The Ohio State University were measured for plasma PTHrP (1-86) concentration, serum ionized calcium concentration, and urine calcium/creatinine ratio. RESULTS: Two of 55 HNSCC patients had detectable levels of plasma PTHrP. Serum ionized calcium concentrations and urinary calcium/creatinine ratios were within normal limits in all patients. CONCLUSIONS: Plasma PTHrP was not a valuable indicator of tumor presence or recurrence in our patient population. SIGNIFICANCE: Plasma PTHrP is not a useful marker of tumor presence or recurrence in patients with stage II to IV or recurrent HNSCC.

Idiopathic systemic granulomatous disease and macrophage expression of PTHrP in a miniature pony.

Idiopathic systemic granulomatous disease, which has been reported in horses, cattle and human beings, is characterized by perivascular granulomatous and lymphoplasmacytic inflammation in many organ systems. Diagnosis is based on the exclusion of possible viral, fungal or bacterial causes. The disease was identified in a miniature pony with widespread lymphoplasmacytic and granulomatous inflammation, special staining techniques having revealed no evidence of any aetiological agent. Skin lesions, which were severe, consisted of hyperkeratosis and serocellular crust formation, with inflammatory infiltrates in a perivascular to diffuse pattern in both the superficial and deep dermis. Inflammatory infiltrates were also present in lymph nodes and around the blood vessels in most organs. Immunohistochemically, both CD3-positive T lymphocytes and BLA36-positive B lymphocytes were identified in the inflammatory infiltrates, and macrophages were immunolabelled for parathyroid hormone-related protein, a factor associated with hypercalcaemia in human beings with granulomatous diseases.

Congenital defects in newborn foals of mares treated for equine protozoal myeloencephalitis during pregnancy.

Three weak, recumbent neonatal foals with skin lesions, including a thin wooly coat, were born to mares being treated for equine protozoal myeloencephalitis. Mares received sulfadiazine or sulfamethoxazole-trimethoprim, pyrimethamine, folic acid, and vitamin E orally. Foals were anemic, leukopenic, azotemic, hyponatremic, and hyperkalemic. Serum folate concentrations in the 3 foals and 2 mares were lower than those reported in the literature for clinically normal brood mares. Treatment was unsuccessful. For each foal, necropsy revealed lobulated kidneys with thin cortices and a pale medulla, and the spleen and thymus were small. Histologic examination revealed marked epidermal necrosis without inflammatory cells, thin renal cortices, renal tubular nephrosis, lymphoid aplasia, and bone marrow aplasia and hypoplasia. These observations indicate that oral administration of sulfonamides, 2,4-diaminopyrimidines (pyrimethamine with or without trimethoprim), and folic acid to mares during pregnancy is related to congenital defects in newborn foals.

The influence of daylength, body weight, and age on the reproductive ability of broiler breeder cockerels.

June-hatched broiler breeder cockerels of two strains were evaluated for reproductive performance in mating pens from 22 to 48 weeks of age. Two light programs with maximum daily daylengths of 15.5 or 16.5 hr were studied. An increase in the light day to 16.5 hr did not significantly affect the male reproductive traits except in number of completed matings and percent packed sperm volume (PSV). The males on 16.5 hr of light had significantly fewer completed matings than did the males on 15.5 hr. Strain 1 males on 16.5 hr produced significantly more spermatozoa at 35 and 40 weeks of age than males on 15.5 hr of light. Male body weights of both strains were significantly lower at 30 weeks of age on the longer daylength. Body weights of cockerels in all treatment groups increased with aging. The range in male body weight markedly influenced the percentage of producing cockerels with aging. During the late egg cycle, the number of producing males increased as body weight increased. The overall mean age of peak in the percentage of males in semen production was 44 weeks of age, which was 7 weeks later than peak egg production. Hatchability was significantly lower for male Strain 2, which had a higher percent PSV and a higher number of attempted matings than did Strain 1. Body weight was significantly correlated with male vent feather-wear at all ages and with the percentage of producing males at 48 weeks of age. The percentage of producing males was correlated with vent feather-wear, evert score, semen volume, and PSV. These results emphasize the importance of body weight selection for optimum breeding flock performance.

Pre-clinical evaluation and efficacy studies of a melanin-binding IgM antibody labeled with 188Re against experimental human metastatic melanoma in nude mice.

PURPOSE: Currently there is no satisfactory treatment for metastatic melanoma. Radioimmunotherapy (RIT) uses the antigen-antibody interaction to deliver lethal radiation to target cells. Recently we established the feasibility of targeting melanin in tumors with 188-Rhenium ((188)Re)-labeled 6D2 mAb to melanin. Here we carried out pre-clinical development of (188)Re-6D2 to accrue information necessary for a Phase I trial in patients with metastatic melanoma. RESULTS: TCEP proved to be effective in generating a sufficient number of -SH groups on 6D2 to ensure high radiolabeling yields with (188)Re and preserved its structural integrity. (188)Re-6D2 was quickly cleared from the blood with the half-life of approximately 5 hrs and from the body--with the half-life of 10 hr. The doses of 0.5, 1.0 and 1.5 mCi significantly (p < 0.05) slowed down A2058 tumor growth in nude mice, also causing release of melanin into the extracellular space which could provide additional target for repeated treatments. Transient effects of RIT on WBC and platelet counts resolved by Day 14 post-treatment. EXPERIMENTAL DESIGN: Tris(2-Carboxyethyl) Phosphine Hydrochloride (TCEP) was evaluated as potential agent for generation of -SH groups on 6D2 mAb. TCEP-treated 6D2 mAb was radiolabeled with (188)Re and its radiochemical purity and stability was measured by ITLC and HPLC and its immunoreactivity--by melanin-binding ELISA. The pharmacokinetics, therapeutic efficacy and acute hematologic toxicity studies were performed in nude mice bearing lightly pigmented A2058 human metastatic melanoma tumors. CONCLUSIONS: We have developed radiolabeling and quality control procedures for melanin-binding (188)Re-6D2 mAb which made possible currently an on-going Phase I clinical trial in patients with metastatic melanoma.

Cyclooxygenase-2 expression in the cornea of dogs with keratitis.

Cyclooxygenase-2 (COX-2) can be overexpressed at inflammatory sites, leading to the generation of proinflammatory prostanoids. Selective inhibitors of COX-2 have potential use in treating inflammatory conditions including ophthalmic diseases in veterinary medicine. Keratitis is considered the most common inflammatory eye disease in dogs. In this study we evaluated the expression of COX-2 in normal dog eyes and in dog eyes with keratitis by immunohistochemistry using isoform-specific antibodies. In the normal eye (n = 4), no COX-2 immunoreactivity was observed in the cornea. In keratitis, COX-2 (n = 12) expression was observed in all corneal layers (epithelium, stromal cells, and endothelium). COX-2 immunoreactivity was also noted in the stromal and epithelial cells of the iris and the stromal cells of the trabecular meshwork. These data indicate that COX-2 may play a pathophysiologic role in keratitis and suggest potential therapeutic implications of prostaglandin modulation in inflammatory eye diseases.