Control trial and fecal egg count reduction test determinations of nematocidal efficacies of moxidectin and generic ivermectin in recently weaned, naturally infected calves.

An anthelmintic efficacy study was performed with young, naturally infected beef-type calves obtained at local farms. Presumably, the study calves had been recently weaned and had not been treated with a parasiticide prior to study acquisition. After blocking the 24 study calves in accordance with calculated Cooperia spp EPG counts, the calves were randomly allocated within each block to one of four treatment groups, resulting in 6 animals per treatment group (untreated controls, topical ivermectin at the rate of 500 mcg/kg BW [Noromectin Pour-On(®) Norbrook], topical moxidectin at the rate of 500 mcg/kg BW [Cydectin Pour-On(®) Boehringer Ingelheim Vetmedica (BIVM)] and injectable moxidectin at the rate of 200 mcg/kg BW [Cydectin(®) BIVM]. After treatment, the animals were penned by treatment group until necropsy. Fecal, strongyle egg count reduction percentages at 2 days post-treatment were 13, 52, 81 and 93 for control, topical ivermectin, topical moxidectin and injectable moxidectin treatment groups, respectively. In the same order as above, egg count reduction percentages at necropsy (15-18 days post-treatment) were -14, 91, 94 and 97. Based on geometric means for worm burdens quantified at necropsy, anthelmintic efficacies ranged from 96 to 100% for adult Oesophagostomum radiatum, Ostertagia ostertagi, Haemonchus placei and Trichostrongylus axei. Against adult Nematodirus helvetianus, percent efficacies based on geometric means were 56.7, 98.3 and 82.2 for topical ivermectin, topical moxidectin and injectable moxidectin, respectively; an observation that is guarded, as only 5 control animals were infected with adult N. helvetianus. Respective anthelmintic efficacies (%'s) against adult Cooperia oncophora and C. punctata were 93.0 and 73.4 (topical ivermectin), 99.3 and 99.9 (topical moxidectin) and 46.1 and 93.6 (injectable moxidectin). Judging from these data, it appears that treatment of calves soon after weaning with topical moxidectin is effective (>90% efficacy) for all common nematodes in cattle, but injectable moxidectin and topical ivermectin have limited effectiveness against Cooperia spp. With Cooperia spp and H. placei infections, the fecal egg count reduction test and the control trial determinations of anthelmintic effectiveness were in disagreement regarding injectable moxidectin and topical ivermectin.

Preparation and evaluation of sustained-release matrix tablets based on metoprolol and an acrylic carrier using injection moulding.

Sustained-release matrix tablets based on Eudragit RL and RS were manufactured by injection moulding. The influence of process temperature; matrix composition; drug load, plasticizer level; and salt form of metoprolol: tartrate (MPT), fumarate (MPF) and succinate (MPS) on ease of processing and drug release were evaluated. Formulations composed of 70/30% Eudragit RL/MPT showed the fastest drug release, substituting part of Eudragit RL by RS resulted in slower drug release, all following first-order release kinetics. Drug load only affected drug release of matrices composed of Eudragit RS: a higher MPT concentration yielded faster release rates. Adding triethyl citrate enhanced the processability, but was detrimental to long-term stability. The process temperature and plasticizer level had no effect on drug release, whereas metoprolol salt form significantly influenced release properties. The moulded tablets had a low porosity and a smooth surface morphology. A plasticizing effect of MPT, MPS and MPF on Eudragit RS and Eudragit RL was observed via DSC and DMA. Solubility parameter assessment, thermal analysis and X-ray diffraction demonstrated the formation of a solid solution immediately after production, in which H-bonds were formed between metoprolol and Eudragit as evidenced by near-infrared spectroscopy. However, high drug loadings of MPS and MPF showed a tendency to recrystallise during storage. The in vivo performance of injection-moulded tablets was strongly dependent upon drug loading.

Fecal egg count reduction and control trial determinations of anthelmintic efficacies for several parasiticides utilizing a single set of naturally infected calves.

In this study, a single set of naturally infected calves was used for the conduct of a fecal egg count reduction test (FECRT) immediately followed by a control trial; all, to evaluate the efficacies of several commonly used, non-generic anthelmintics. Ten animals were allocated into each of the five treatment groups; untreated control (UTC), injectable 1% moxidectin given at 0.2 mg kg(-1)BW (MXD), injectable 1% ivermectin given at 0.2 mg kg(-1)BW (IVM), 9.06% oxfendazole given orally at 4.5 mg kg(-1)BW (OXF), and 10.0% fenbendazole given orally at 5.0 mg kg(-1)BW (FBZ). Confinement of animals to clean, concrete-floored pens was initiated on day -7 and continued until animal necropsy (2 animals were necropsied per treatment group per day on days 35-39 for nematode collections). All treatments were given on day 0, and the FECRT was conducted on all animals until necropsy. From days 2 to 14, FECR %'s for the combined strongyle egg counts were >or=90% for all anthelmintic groups. At the time of necropsy, FECRT %'s for the combined strongyle egg counts continued to be >or=90% for all treatments with the exception of IVM (84%). After adjustment of the strongyle egg counts in accordance with coproculture larvae percentages, FECRT %'s at the time of necropsy for Haemonchus, Ostertagia and Cooperia were found to be >or=94% for MXD and OXF, but <90% for FBZ (Ostertagia) and IVM (Haemonchus and Cooperia). At necropsy, more than six of the ten untreated animals were infected with Ostertagia ostertagi (adults, EL(4) and LL(4)), and adult Haemonchus placei, Trichostrongylus axei, Cooperia oncophora, C. surnabada and C. punctata. Based on geometric means: all of the above populations were removed by >or=96% by MXD; were removed by >or=90% by IVM except for O. ostertagi LL(4) (81.9%), C. oncophora and C. surnabada adults (77.4%) and C. punctata adults (84.8%); were removed by >or=90% by OXF except for O. ostertagi adults, EL(4) and LL(4) (89.9, 70.2 and 48.1%, respectively); and were removed by >or=90% by FBZ except for O. ostertagi adults, EL(4) and LL(4) (72.5, 0.0 and 21.9%, respectively). Judging from the above data, FECR and control trial results can be extremely similar given the proper experimentation and, despite varied degrees of nematode resistance, targeted nematode burdens commonly carried by Midwestern beef cattle are effectively removed by the parasiticides that are available today.

Interleukin 6 secreted from adipose stromal cells promotes migration and invasion of breast cancer cells.

Excessive adiposity has long been associated with increased incidence of breast cancer in post-menopausal women, and with increased mortality from breast cancer, regardless of the menopausal status. Although adipose tissue-derived estrogen contributes to obesity-associated risk for estrogen receptor (ER)-positive breast cancer, the estrogen-independent impact of adipose tissue on tumor invasion and progression needs to be elucidated. Here, we show that adipose stromal cells (ASCs) significantly stimulate migration and invasion of ER-negative breast cancer cells in vitro and tumor invasion in a co-transplant xenograft mouse model. Our study also identifies cofilin-1, a known regulator of actin dynamics, as a determinant of the tumor-promoting activity of ASCs. The cofilin-1-dependent pathway affects the production of interleukin 6 (IL-6) in ASCs. Depletion of IL-6 from the ASC-conditioned medium abrogated the stimulatory effect of ASCs on the migration and invasion of breast tumor cells. Thus, our study uncovers a link between a cytoskeleton-based pathway in ASCs and the stromal impact on breast cancer cells.

Adenovirus-delivered DKK3/WNT4 and steroidogenesis in primary cultures of adrenocortical cells.

The Wnt family molecules Dickkopf-3 (DKK3) and WNT4 are present at higher concentrations in the zona glomerulosa than in the rest of the adrenal cortex. In order to study direct effects of these proteins on adrenocortical cell function, we created adenoviruses encoding human DKK3 and WNT4. When added to cultured human adrenocortical cells, DKK3 inhibited aldosterone and cortisol biosynthesis, either alone or together with cyclic AMP. WNT4 increased steroidogenesis when added alone but decreased it in the presence of cyclic AMP. A control adenovirus encoding GFP had no effect. RNA was prepared from cultured cells and was assayed by real-time PCR. CYP11A1 (cholesterol side-chain cleavage enzyme), HSD3B2 (3beta-hydroxysteroid dehydrogenase type II), CYP17 (17 alpha-hydroxylase), CYP21 (21-hydroxylase) and CYP11B1 (11 beta-hydroxylase) mRNAs were all increased by cyclic AMP, whereas CYP11B2 (aldosterone synthase) was unaffected. DKK3 decreased cyclic AMP-stimulated CYP17. WNT4 increased both CYP17 and CYP21 in the absence of cyclic AMP. Both DKK3 and WNT4 increased the level of CYP11B2. These data show that these Wnt signaling molecules have multiple actions on steroidogenesis in adrenocortical cells, including effects on overall steroidogenesis (aldosterone and cortisol biosynthesis) and distinct effects on steroidogenic enzyme mRNA levels. The co-localization of DKK3 and WNT4 in the glomerulosa and their stimulation of CYP11B2 imply an action on glomerulosa-specific function.

Zonal expression of dickkopf-3 and components of the Wnt signalling pathways in the human adrenal cortex.

The mechanisms underlying the differentiation of the adrenal cortex into zones are unclear. Microarray studies on RNA from microdissected zona reticularis (ZR) and zona fasciculata/zona glomerulosa (ZF/ZG) derived from adult human adrenal glands showed that a gene of the dickkopf family (DKK), DKK3, is differentially expressed in the zones. The Dickkopf proteins are morphogens involved in Wnt signalling. Northern blotting showed higher DKK3 transcript levels in ZF/ZG than ZR samples. In situ hybridization on adult human adrenal gland sections showed that DKK3 expression was much higher in the ZG than in the ZF or ZR. DKK3 expression was also higher in the medulla. We screened for expression of other members of the DKK family and the related Wingless-type mouse mammary tumor virus integration site gene family (WNT), frizzled (FZD), and dishevelled (DVL) gene families. Among dickkopf family members, only DKK3 was expressed at a detectable level in both human and mouse adrenocortical RNA samples. Consistent with previously published data on the effects of Wnt4 gene disruption in the mouse, we found only WNT4 expression within the WNT family in both human and mouse RNA. Northern blotting showed that WNT4 was expressed at a higher level in ZF/ZG cells than in ZR. The higher level of DKK3 and WNT4 expression in ZF/ZG cells was confirmed by real-time PCR. In the frizzled and dishevelled families we found FZD1, FZD2 and DVL3 transcripts in human adrenocortical RNA, and FZD2 and DVL3 in mouse adrenocortical RNA. These data show that a variety of genes of the Wnt signalling pathways are expressed in the adrenal cortex. The zonal distribution of DKK3 expression suggests that it could be involved in zonal differentiation or growth.

The human melanocyte: a model system to study the complexity of cellular aging and transformation in non-fibroblastic cells.

The melanocyte is a neural crest-derived cell that localizes in humans to several organs including the epidermis, eye, inner ear and leptomeninges. In the skin, melanocytes synthesize and transfer melanin pigments to surrounding keratinocytes, leading to skin pigmentation and protection against solar exposure. We have investigated the process of replicative senescence and accompanying irreversible cell cycle arrest, in melanocytes in culture. As was found in other cell types, progressive telomere shortening appears to trigger replicative senescence in normal melanocytes. In addition, senescence is associated with increased binding of the cyclin-dependent kinase inhibitor (CDK-I) p16(INK4a) to CDK4, down-regulation of cyclin E protein levels (and consequent loss of cyclin E/CDK2 activity), underphosphorylation of the retinoblastoma protein RB and subsequent increased levels of E2F4-RB repressive complexes. In contrast to fibroblasts, however, the CDK-Is p21(Waf-1) and p27(Kip-1) are also down-regulated. These changes appear to be important for replicative senescence because they do not occur in melanocytes that overexpress the catalytic subunit of the enzyme telomerase (hTERT), or in melanomas, which are tumors that originate from melanocytes or melanoblasts. In contrast to unmodified melanocytes, hTERT overexpressing (telomerized) melanocytes displayed telomerase activity, stable telomere lengths and an extended replicative life span. However, telomerized melanocytes show changes in cell cycle regulatory proteins, including increased levels of cyclin E, p21(Waf-1) and p27(Kip-1). Cyclin E, p21(Waf-1) and p27(Kip-1) are also elevated in many primary melanomas, whereas p16(INK4a) is mutated or deleted in many invasive and metastatic melanomas. Thus, the molecular mechanisms leading to melanocyte senescence and transformation differ significantly from fibroblasts. This suggests that different cell types may use different strategies to halt the cell cycle in response to telomere attrition and thus prevent replicative immortality.

Telomere shortening and decline in replicative potential as a function of donor age in human adrenocortical cells.

Telomere shortening is the cause of replicative senescence of mammalian cells in culture and may be a cause of cellular aging in vivo. Some tissues clearly show telomere shortening during aging in humans, but the relationship between replication history and telomere length is obscured by complex relationships between stem cells and more differentiated cell types. Previous experiments on the adrenal cortex and human adrenocortical cells in culture indicate that the proliferative biology of this tissue is relatively simple; cell division occurs continuously throughout life, without evidence for a distinct stem cell compartment. In this tissue we investigated the relationship between telomere biology and replicative senescence by measuring replicative capacity and telomere length as a function of donor age. Cells cultured from adrenal tissue from donors of different ages showed a strong age-related decline in total replicative capacity, falling from about 50 population doublings for fetal cells to an almost total lack of division in culture for cells from older donors. Telomere restriction fragment (TRF) length was analyzed in the same sets of cells and decreased from a value of about 12 kb in fetal cells to approximately 7 kb in cells from older donors. The latter value is consistent with that in fibroblasts which have reached replicative senescence. Furthermore, there was a good correlation in individual donor samples between TRF length and replicative capacity in culture. To confirm the relationship between telomere length, telomerase, and replicative capacity, we measured telomere length in cells before and after infection with a retrovirus encoding hTERT, the catalytic component of human telomerase. The adult adrenal cortex does not have telomerase activity; cells after transduction with the hTERT retrovirus had high telomerase activity. Whereas control cells underwent a replication-dependent shortening in telomeres during long-term growth in culture, hTERT-modified cells maintained telomere length and are probably immortalized. Symmetric cell division in human adrenocortical cells, occurring slowly over the life span, is associated with progressive telomere shortening and may result in proliferative defects in vivo in old age, which could partly account for the age-related changes in the structure and function of the human adrenal cortex.

Telomerase activity in primary cultures of normal adrenocortical cells.

Telomerase activity was measured in isolated cells from bovine and human adrenal cortex, in cells in primary culture, in cells in later passages in culture, and in cells genetically modified by expression of hTERT (human telomerase reverse transcriptase). Telomerase activity in freshly isolated bovine adrenocortical cells and in human adrenal cells from donors of various ages (6-79 years) was very low or undetected. However, primary bovine adrenocortical cell cultures were strongly positive for telomerase activity, and primary human adrenocortical cell cultures were weakly positive. Both cell types proliferate in primary culture but proliferation of bovine cells is much more vigorous. When primary bovine cells were subcultured to make successively secondary and tertiary cultures, telomerase activity declined strongly, and was undetected by the third passage. There was only a slight decrease in growth rate over this period. Levels of the telomerase RNA component did not change with passage number when assessed by semi-quantitative competitive RT-PCR. When both bovine and human cells were infected with a retrovirus encoding hTERT, telomerase activity in the cells was very high. We conclude that in the adrenal cortex, as in some other tissues, TERT expression is regulated and upregulation of telomerase activity is associated with rapid proliferation in primary culture. Telomerase activity is not maintained, and introduction of TERT is required for stable telomerase activity and for immortalization.

Stages of change and weight loss among rural African American women.

OBJECTIVE: Obesity is a prevalent public health problem in the United States, especially for rural African American women, and causes increased morbidity and mortality. The purpose of this analysis was to determine whether the transtheoretical stages of change model was generalizable to weight loss intention among overweight and obese rural African American women and to identify important predictors of the stages of change. RESEARCH METHODS AND PROCEDURES: The study was conducted in two rural counties in central Virginia. A population-based sample of 200 women under the age of 40 completed questionnaires concerning weight loss behavior and beliefs about weight. Ordinal logistic regression was used to predict stage of change. RESULTS: A total of 142 of the 200 women (71%) were overweight or obese (body mass index of > or =25) and were classified into a stage of change. Overall, 30% of respondents were in the precontemplation stage, 15% in the contemplation stage, 48% in the preparation stage, 4% in the action stage, and 3% in the maintenance stage. Education, what friends think about weight, body mass index, and a scale of the positive aspects of weight loss were significant predictors of the stage of change (p < 0.05). CONCLUSIONS: Several predictors of stage were the same as those found in studies of other health behaviors, and this research provides support for applying a stages of change model for weight loss intention among rural African American women. Two predictors in particular, significance of what friends think about weight and a scale of the positive aspects of weight loss, have implications for health education initiatives and social support in weight loss interventions.

Contrasting roles of p57(KIP2) and p21(WAF1/CIP1/SDI1) in transplanted human and bovine adrenocortical cells.

Cell transplantation provides a way to compare the regulation of cell proliferation in the same cell type in cell culture and in a vascularized tissue structure in a host animal. The cyclin-dependent kinase inhibitors p57(KIP2), p21(WAF1/CIP1/SDI1) and p27(KIP1) have been extensively studied in cell culture but their role in growth control in tissues is less well understood. In the present experiments we compared the behavior of cell cycle inhibitors in human and bovine adrenocortical cells in culture and following cell transplantation in scid mice. p57 was expressed in the majority of cells in the intact human adrenal cortex. However, double immunofluorescence showed that cells that are in the cell cycle are p57(-) adrenocortical cells, p57 and p27 levels were not affected by inhibition of growth at high cell density, whereas p21 was higher in dividing than growth-inhibited cells. However, p21 was also high in senescent adrenocortical cells. After transplantation of human adrenocortical cells in scid mice, p57 and p27 were observed in most cells in the transplant tissue. Over time the number of p21(+) cells decreased greatly in human adrenocortical cells, but not in bovine adrenocortical cells. This difference correlated with lower levels of cell division (assessed by Ki-67 or incorporation of bromodeoxyuridine) in the human cells in transplant tissues in comparison to bovine cells. The differences between human and bovine cells were observed both when cells were transplanted beneath the kidney capsule and when cells were injected subcutaneously in collagen gel. We conclude that the behavior of p57, but not p21, is consistent with a role as a physiological mediator of proliferative quiescence in the adrenal cortex. The high level of p21 in dividing adrenocortical cells in culture, and in bovine adrenocortical cells in transplant tissues, may be a response to conflicting positive and negative growth influences. Cells may enter the cell cycle under the influence of a strong positive mitogenic signal, but coexisting negative growth stimuli trigger a p21-dependent block to further progression through the cell cycle. This model suggests that bovine adrenocortical cells respond to positive growth stimuli in transplant tissues but human cells lack this response.

Transfection by polyethyleneimine-coated microspheres.

UNLABELLED: Polyethyleneimine (PEI) can be used as a DNA delivery mechanism in cell culture and in vivo. Cells can be transfected by using surface-bound PEI, as well as by PEI/DNA microparticles. In the present experiments we extended these observations by preparing microspheres with covalently attached PEI. Blends of poly(epsilon-CBZ-L-lysine) mixed with poly(D,L-lactic-co-glycolic acid) were formed into microspheres using a double-emulsification/solvent evaporation procedure. CBZ (carbobenzoxy) groups on the surface of microspheres were removed by Li(0) /liquid ammonia reduction. Surface amino groups were used for covalent attachment of PEI and other molecules. Silica microspheres with bonded-phase PEI were also used. Microspheres were mixed with plasmid DNA encoding green fluorescent protein and added to cultured cells. PEI-coated microspheres transfected cultured Caco cells and MH-S alveolar macrophages. Expression of the transfected DNA increased over several days. MH-S cells phagocytosed PEI-coated silica microspheres, which were shown to reside in an acidic subcellular compartment. This was demonstrated by conjugating a pH-sensitive fluorescent dye (seminaphthofluorescein, SNAFL) to the microsphere surface. Transfection of MH-S cells was increased when plasmid DNA was complexed with histone on the surface of the microspheres. CONCLUSION: PEI-coated microspheres have potential as a DNA delivery device with advantages of the unique properties of PEI and ease of surface chemical modification.

Differentially expressed genes in zona reticularis cells of the human adrenal cortex.

The zona reticularis (ZR) cell in the human adrenal cortex is responsible for the secretion of dehydroepiandrosterone, but its biology, origin, and putative decrease in number during aging are poorly understood. In the present experiments, we investigated to what extent ZR and zona fasciculata (ZF) cells differ in patterns of gene expression. Both cell types were purified by microdissection from adult adrenal cortex specimens. After a brief period in culture, RNA was harvested from the cells and used to prepare radioactively labeled probes following amplification by PCR. Probes were used in hybridizations of arrays of cDNAs on nylon membranes (PCR products or plasmids obtained from an adrenal cDNA library). Analysis of hybridization intensities showed that 17 of the 750 genes studied differed in expression by more than 2-fold. Several genes expressed at higher levels in ZR cells encode components of the major histocompatibility complex or enzymes involved in peroxide metabolism. Members of the tubulin gene family were expressed at higher levels in ZF cells. Differential expression of four of the genes was confirmed by Northern blotting. These differences show that although ZR and ZF cells are similar in gene expression, ZR cells have a gene expression pattern related to the unique biology of this cell type.

Cutaneous window for in vivo observations of organs and angiogenesis.

BACKGROUND AND OBJECTIVE: The continuous observation in experimental animals of internal organs and processes, such as wound healing and angiogenesis, has been achieved using a variety of transparent windows and chambers. Our objective was to develop procedures for these observations using disposable material for the window and simple surgical techniques. METHODS: For observation of wound healing in the mouse kidney, the kidney was externalized and a wedge was excised. An oval window of polyvinyl chloride film was sutured in place in the skin over the wound. The progress of healing of the wound was observed through the window over 10 days. For observation of angiogenesis, adrenocortical cells were transplanted beneath fascia and muscle and a window was sewn into the skin above the site of transplantation. RESULTS: Clear observations could be made using these cutaneous windows over the period of the experiments. Healing of a wound in the kidney was photographed and measured. The growth of new blood vessels over the site of adrenocortical cell transplantation was observed. CONCLUSIONS: Continuous in vivo observations of organs such as the kidney and processes such as angiogenesis can be made in experimental animals using this simple technique.

Clinical implications of body image among rural African-American women.

OBJECTIVE: To increase understanding of body image among rural, African-American women through open-ended interviews. DESIGN: Individuals' perceptions of body image were investigated using open-ended, in-depth interviews that were tape-recorded, transcribed, and analyzed to identify common themes and to compare thematic data across three body mass index categories (obese, overweight, and normal). SETTING: University-affiliated rural community health center. PARTICIPANTS: Twenty-four African-American women, aged 21 to 47 years. MAIN RESULTS: Respondents reported the following common themes: dissatisfaction with current weight; fluctuating levels of dissatisfaction (including periods of satisfaction); family and social pressure to be self-accepting; and social and physical barriers to weight loss. The interviews revealed ambivalence and conflicts with regard to body image and weight. Among these women, there was strong cultural pressure to be self-accepting of their physical shape, to "be happy with what God gave you," and to make the most of their appearance. CONCLUSIONS: The pressure to be self-accepting often conflicted with these obese women's dissatisfaction with their own appearance and weight. Although the respondents believed they could lose weight "if [they] put [their] mind to it," those women wanting to lose weight found that they lacked the necessary social support and resources to do so. The conflicts stemming from social pressures and their own ambivalence may result in additional barriers to the prevention of obesity, and an understanding of these issues can help health care providers better address the needs of their patients.

Transfection of cells mediated by biodegradable polymer materials with surface-bound polyethyleneimine.

Poly(epsilon-CBZ-L-lysine) can be mixed with biodegradable polymers such as poly(D,L-lactic-co-glycolic acid) or poly(L-lactic acid) and formed into films, foams, or microspheres. Surface amino groups may then be deprotected with acid or lithium/liquid ammonia. The amino groups serve as a method to modify the surface by attachment of other molecules. In the present experiments, we show that these polymer materials, as films or foams, may be surface modified by the attachment of polyethyleneimine (PEI). Plasmid DNA attached to the PEI can transfect cells plated on the surface over several days. Covalent atachment of PEI was required for transfection to be efficient. PEI was also attached to surface-bound collagen on cell culture plates and was shown to mediate transfection.

Formation of functional tissue from transplanted adrenocortical cells expressing telomerase reverse transcriptase.

We report the first use of human telomerase reverse transcriptase (hTERT) expression in experimental xenotransplantation. Previously, we showed that bovine adrenocortical cells can be transplanted into severe combined immunodeficient (SCID) mice, and that these cells form functional tissue that replaces the animals' own adrenal glands. We cotransfected primary bovine adrenocortical cells with plasmids encoding hTERT, SV40 T antigen, neo, and green fluorescent protein. These clones do not undergo loss of telomeric DNA and appear to be immortalized. Two clones were transplanted beneath the kidney capsule of SCID mice. Animals that received cell transplants survived indefinitely despite adrenalectomy. The mouse glucocorticoid, corticosterone, was replaced by the bovine glucocorticoid, cortisol, in the plasma of these animals. The tissue formed from the transplanted cells resembled that formed by transplantation of cells that were not genetically modified and was similar to normal bovine adrenal cortex. The proliferation rate in tissues formed from these clones was low and there were no indications of malignant transformation.

Transplantation of normal and genetically modified adrenocortical cells.

Cell transplantation techniques have been applied to the study of the biology of the adrenal cortex and to adrenocortical cell proliferation, differentiation, and senescence. Primary bovine adrenocortical cells, primary human adrenocortical cells and genetically modified bovine adrenocortical cells have been transplanted. Successful methods include transplantation of cells beneath the kidney capsule and several subcutaneous cell transplantation procedures. In successful transplants the cells form a functional vascularized tissue structure that allows the host animals to survive adrenalectomy. We show here that subcutaneous cell transplantation does not depend on embedding cells in collagen gel before introduction into the host animal. Subcutaneous transplants secrete both cortisol and aldosterone. However, the variability of plasma aldosterone levels indicates that the factors that determine glomerulosa-type and fasciculata-type cell function in transplant tissues are not well understood.