In situ zymography: a molecular pathology technique to localize endogenous protease activity in tissue sections.

Proteases play important roles in modulating a wide range of cellular functions, in the regulation of biologic processes, and in the pathogenesis of various diseases. Several molecular techniques are available to identify and characterize proteases in cells and tissues. Most of these techniques do not provide information on the activity of proteases in tissues. In situ zymography (ISZ) is a relatively low-cost technique that uses specific protease substrates to detect and localize specific protease activities in tissue sections. Used in combination with other techniques, ISZ provides data that further our understanding of the role of specific proteases in various pathologic and physiologic conditions. This review describes the general principle of ISZ and highlights the past and future applications of this technique in molecular pathology.

Selective cyclooxygenase-2 inhibition does not affect the healing of cutaneous full-thickness incisional wounds in SKH-1 mice.

BACKGROUND: The inducible cyclooxygenase-2 (COX-2) enzyme is upregulated in inflammatory diseases, as well as in epithelial cancers, and has an established role in angiogenesis and tissue repair. OBJECTIVE: Because of these physiological effects and the widespread use of the selective COX-2 inhibitor, celecoxib, we wanted to determine if inhibition of COX-2 would affect incisional skin wound healing. METHODS: Using a cutaneous full-thickness, sutured, incisional wound model in hairless SKH-1 mice, we evaluated the role of COX-2 in the wound healing process by comparing the effects of a nonselective COX inhibitor, diclofenac, with a selective COX-2 inhibitor, SC-791. Healing was monitored for up to 28 days postincision histologically and for recovery of wound strength. RESULTS: COX-2 expression was observed over the first week of healing, peaking at day 3 and was not affected by treatment with the selective COX-2 or nonselective COX inhibitors. Infiltrating macrophages, as well as keratinocytes and dermal fibroblasts at the wound site, expressed COX-2. Neither selective COX-2, nor nonselective COX inhibition had a significant effect on the macroscopic or microscopic morphology of the wounds, whereas dexamethasone treatment resulted in epidermal and granulation tissue atrophy. In addition, neither selective COX-2, nor nonselective COX inhibition altered keratinocyte proliferation and differentiation, dermal angiogenesis or the recovery of wound tensile strength, whereas dexamethasone reduced the tensile strength of the wounds by 30-38% throughout the healing period. CONCLUSIONS: These data indicate that selective COX-2 inhibition does not affect the healing of surgical skin wounds.

Gastrointestinal stromal tumors in equids.

Eleven gastrointestinal neoplasms from 10 aged horses and 1 pony were examined grossly, his tologically, immunohistochemically, and (in two cases) ultrastructurally. Clinical signs were associated with two neoplasms, and the other nine tumors were incidental findings at laparotomy or necropsy. The neoplasms were solitary (9/11) or multifocal (2/11), well demarcated, serosal or mural masses of stomach (1), jejunum (1), ileum (3), cecum (5), and/or colon (2). Microscopic examination revealed discrete spindle cells arranged in compact patterns with fascicles and whorls or cribriform pattern with fascicles and rare palisades, often with a myxoid interstitial matrix. Three tumors infiltrated between the muscularis interna and the muscularis externa at the myenteric plexi. All neoplasms were vimentin positive, 3/11 were S-100 positive, 2/11 were muscle actin positive, and no neoplasm was positive for glial fibrillary acid protein, desmin, factor VIII, chromogranin, or neuron-specific enolase. Of the two tumors studied ultrastructurally, one contained an admixture of smooth muscle cells and cells resembling Schwann cells, and the second was populated by homogeneous fusiform mesenchymal cells separated by homogeneous matrix. Gastrointestinal stromal tumors (GIST) have been recognized in humans, more recently in dogs and nonhuman primates, and now in equids. Most of these tumors are comprised of a loosely arranged network of spindled cells separated by myxoid matrix. GIST may be composed of myogenic, neurogenic, combined myogenic and neurogenic, and undifferentiated mesenchymal cells.

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.

Effect of the storage period of paraffin sections on the detection of mRNAs by in situ hybridization.

In this study we evaluated whether storing non-deparaffinized sections can affect the detection of specific mRNAs by radioactive in situ hybridization (ISH). Using a standard ISH protocol, we hybridized serial sections of paraffin blocks stored for different periods of time with (33)P-labeled riboprobes specific for rat Type III collagen and matrix metalloproteinase-2 (MMP-2). Signal intensities were evaluated using a phosphorimager and by blinded microscopic examination. For slides hybridized with the Type III collagen riboprobe, signal intensities measured with the phosphorimager or evaluated by microscopic examination were negatively correlated with the storage period of the sections. For slides hybridized with the MMP-2 riboprobe, differences in signal intensity could be detected, albeit inconsistently, with the phosphorimager, although microscopic examination consistently indicated stronger signals in freshly sectioned slides compared to slides stored for 2 weeks or more. We concluded that it was preferable to use recently prepared sections for trying to locate mRNAs in paraffin-embedded tissues by ISH. In addition, our results suggest that quantifying signal intensity using a phosphorimager is feasible for abundant mRNAs or when large differences in expression are anticipated.(J Histochem Cytochem 49:927-928, 2001)

The use of computer-assisted image analysis in the evaluation of preclinical tumor efficacy models.

Quantitation in histopathology generates numerical or rank-order data amenable to statistical analysis, but can be very labor-intensive. The advent and coupling of computerized image analysis systems and optimized immunostaining procedures has not only decreased the laborious aspect of counting, but has also improved reproducibility and reliability by overcoming inter- and intra-observer variation. Still, a certain degree of manual intervention is required and a good understanding of the underlying biology is mandatory for proper design of the sampling system. Here, we focus on subcutaneous tumor models to illustrate the use and benefits of computer-assisted image analysis in quantitative histopathology. In particular, we discuss the quantitation of biological parameters frequently assessed in biology and toxicology: cell proliferation, apoptotic and oncotic necrosis, and angiogenesis. For each of these parameters, specific sampling procedures and labeling techniques are discussed based on current molecular and cellular concepts.

Parathyroid hormone-related protein as a growth regulator of prostate carcinoma.

Parathyroid hormone-related protein (PTHrP) is produced by prostate carcinoma cells and tumors, but little is known of its role in prostate carcinogenesis. The goal of this study was to evaluate PTHrP expression in the regulation of prostate carcinoma growth using human and animal models. PTHrP expression was assessed in prostate cancer cell lines in vitro. Seven of nine cell lines produced PTHrP, and increased expression was seen during cell proliferation. The MatLyLu rat prostate carcinoma model was used to determine the effects of PTHrP overexpression on prostate tumor growth. PTHrP overexpression did not alter proliferation of the cells in vitro. However, when PTHrP-overexpressing cells were injected into rat hind limbs, primary tumor growth and tumor size were significantly enhanced as compared with control cells. To evaluate PTHrP in human prostate carcinoma patients, immunohistochemistry was performed on metastatic bone lesions. Immunolocalization of PTHrP protein was found in the cytoplasm and nucleus of cancer cells in the bone microenvironment. Because nuclear localization of PTHrP has been associated with an inhibition of apoptosis, the ability of full-length PTHrP to protect prostate cancer cells from apoptotic stimuli was examined. Cells transfected with full-length PTHrP showed significantly increased cell survival after exposure to apoptotic agents as compared with cells producing no PTHrP (plasmid control) or cells transfected with PTHrP lacking its nuclear localization signal. To determine the mechanism of action of PTHrP in prostate cancer cells, the parathyroid hormone/PTHrP receptor status of the cells was determined. These cell lines did not demonstrate parathyroid hormone/PTHrP receptor-mediated binding of iodinated PTHrP or steady-state receptor message by Northern blot analysis, but they did have a detectable receptor message by reverse transcription-PCR analysis. In summary, PTHrP is expressed in many prostate cancer cell lines in vitro and in metastatic bone lesions in vivo. PTHrP expression positively influences primary tumor size in vivo and protects cells from apoptotic stimuli. These data suggest that PTHrP plays an important role in the promotion of prostate tumor establishment and/or progression.

Dalmeny disease in an alpaca (Lama pacos): sarcocystosis, eosinophilic myositis and abortion.

Disseminated eosinophilic myositis was diagnosed in an alpaca that had been imported to the USA from Peru 5 years earlier. The myositis was associated with macroscopically visible large sarcocysts that were characterized histologically by septate compartments containing bradyzoites, and ultrastructurally by cyst walls composed of anastomosing villous protrusions. Two hours before death, the alpaca aborted an 8-month-gestation fetus, but no lesions were found in the uterus, placenta or fetus. Additional macroscopical findings included haemoabdomen and myofibre haemorrhage, degeneration and necrosis. It is believed that this is the first described case of clinical disease associated with a Sarcocystis sp. (probably S. aucheniae) in camelids.

Parathyroid hormone-related protein is a positive regulator of keratinocyte growth factor expression by normal dermal fibroblasts.

Parathyroid hormone-related protein (PTHrP), an important factor in the pathogenesis of humoral hypercalcemia of malignancy, is produced by many normal tissues, including the skin, where it regulates keratinocyte growth and differentiation and dermal fibroblast function. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, is a secretory product of stromal cells and functions as a mediator of epithelial cell growth and differentiation. Phenotypes of the skin in several transgenic mouse models, in which the KGF and PTHrP genes have been overexpressed or disrupted, suggest that these two factors interact in vivo to regulate homeostasis of the skin. In this study, we investigated the effects of KGF on PTHrP secretion and expression by normal human foreskin keratinocytes (NHFK) and the effects of PTHrP on KGF secretion and expression by normal human dermal fibroblasts (NHDF) in vitro. N-terminal PTHrP(1-36) increased KGF secretion, protein expression and mRNA expression by NHDF in a dose-dependent manner, however, KGF did not regulate PTHrP expression and secretion by NHFK. By flow cytometry, PTHrP also increased the percentage of NHDF producing KGF. Our results indicate that PTHrP produced by keratinocytes is a potential paracrine regulator of KGF expression by dermal fibroblasts in vivo. This paracrine regulation may explain, in part, the epidermal atrophy seen in the PTHrP null mice and epidermal hyperplasia seen in transgenic mice overexpressing PTHrP in their basal keratinocytes. Our results also suggest that PTHrP is an important mediator for the healing of skin wounds and growth of neoplasms of squamous origin.

Skeletal metastasis of prostate adenocarcinoma in rats: morphometric analysis and role of parathyroid hormone-related protein.

BACKGROUND: Prostate cancer frequently metastasizes to bone, where it induces osteoblastic lesions. Parathyroid hormone-related protein (PTHrP), a product of normal and neoplastic prostate cells, may promote growth and bone metastasis of certain types of cancer. In this study, we investigated the: 1) pathogenesis and morphology of bone metastases in the MATLyLu rat prostate adenocarcinoma model, and 2) effect of PTHrP overexpression on tumor growth and incidence of bone metastasis. METHODS: MATLyLu cells were stably transfected with a PTHrP expression vector or control plasmid. PTHrP expression was determined in vitro by immunoradiometric assay and Northern blot analysis. MATLyLu cells were injected into the left ventricle of Copenhagen rats to induce bone metastases. Histology and radiography were used to quantify the size and number of bone metastases. Serum alkaline phosphatase isoenzyme concentrations and histomorphometric analysis were used to evaluate bone formation and resorption. RESULTS: All rats developed osteolytic metastases in long bones and vertebrae. There was no evidence of increased intramedullary bone formation. PTHrP overexpression by MATLyLu cells was not associated with any difference in the incidence of bone metastasis, size of metastatic foci or tumor-cell proliferation. CONCLUSIONS: The MATLyLu intracardiac injection model of prostate carcinoma is an aggressive tumor model with a high incidence of osteolytic skeletal metastases, and is not altered by increased PTHrP production by neoplastic prostate epithelial cells.

Spatial and temporal expression of parathyroid hormone-related protein during wound healing.

Parathyroid hormone-related protein is produced by many normal tissues including the skin, where it regulates growth and differentiation of keratinocytes. To define better the role of parathyroid hormone-related protein in the skin, we investigated the spatial and temporal expression of parathyroid hormone-related protein and mRNA by immunohistochemistry and in situ hybridization during the healing of skin wounds, and the effects of topical administration of a parathyroid hormone-related protein agonist [parathyroid hormone-related protein (1-36)] and a parathyroid hormone-related protein antagonist [parathyroid hormone (7-34)] on the healing rate and morphology of the wounds. Wounds were produced on the back of guinea pigs with a 4 mm punch, and wound sites were collected at different time points during the healing process. Parathyroid hormone-related protein was expressed in normal skin by all viable keratinocyte layers, hair follicles, and adnexae. Following injury, migratory keratinocytes at wound margins and the newly restored epidermis expressed increased levels of parathyroid hormone-related protein. The remodeling phase was associated with progressive restoration of the pattern of parathyroid hormone-related protein expression in normal epidermis. Granulation tissue myofibroblasts and infiltrating macrophages also expressed parathyroid hormone-related protein. In vitro studies using THP-1 cells (a promonocytic cell line) confirmed that macrophages expressed parathyroid hormone-related protein, especially after activation. Topical application of parathyroid hormone related protein (1-36) or parathyroid hormone (7-34) did not result in significant changes in the healing rate and morphology of the wounds. These findings demonstrated that, in addition to keratinocytes, myofibroblasts and macrophages also represent sources of parathyroid hormone-related protein during the healing of skin wounds. Although the data suggest a role for parathyroid hormone-related protein in the healing of skin and in the restoration of epidermal homeostasis, parathyroid hormone-related protein does not appear to be required for proper re-epithelialization in response to injury, potentially because of redundancy in epidermal growth and wound healing, as has been shown for other paracrine and autocrine growth factors of the epidermis.

Hypereosinophilic syndrome with Hodgkin's-like lymphoma in a ferret.

Hodgkin's-like lymphoma involving the lung, mediastinum, liver, kidneys and mesenteric lymph nodes was diagnosed in a ferret. The diagnosis was based on the presence of an admixture of CD3+ small lymphocytes with smaller numbers of macrophages, eosinophils, and large, pleomorphic, frequently multinucleated, Reed-Sternberg-like cells which were immunoreactive to BLA.36 monoclonal antibody. In addition, the liver, pancreas, small intestine and lungs were infiltrated with moderate to large numbers of eosinophils, forming eosinophilic granulomas with occasional deposition of Splendore-Hoeppli material, supporting a diagnosis of hypereosinophilic syndrome. The concurrent diagnosis of hypereosinophilic syndrome and Hodgkin's-like lymphoma in this ferret provides further support to the concept that, in animals, multisystemic eosinophilic infiltrates may be caused by the abnormal proliferation of T lymphocytes, as has been demonstrated in man.

Dependence of humoral hypercalcemia of malignancy on parathyroid hormone-related protein expression in the canine anal sac apocrine gland adenocarcinoma (CAC-8) nude mouse model.

Circulating parathyroid hormone-related protein (PTHrP) is the primary humoral factor in dogs with spontaneous humoral hypercalcemia of malignancy (HHM) and adenocarcinomas derived from apocrine glands of the anal sac. A canine apocrine adenocarcinoma model of HHM in nude mice (CAC-8) was developed and characterized. After 32 passages in vivo, a spontaneous variant of the tumor (CAC-8 Lo Ca) that has altered cellular morphology and that fails to induce HHM in tumor-bearing nude mice has been discovered. The hypercalcemic and nonhypercalcemic tumor lines were compared by tumor weight, effect on body weight, serum calcium concentration, plasma PTHrP concentration, histopathology, expression of PTHrP protein by radioimmunoassay and immunohistochemistry, and expression of PTHrP mRNA by in situ hybridization and northern blot analysis. Messenger RNA expression for other factors and cytokines known to alter PTHrP secretion or bone resorption in vivo, including tumor necrosis factor alpha (TNF alpha), interleukin (IL)-1, IL-6, and transforming growth factor beta (TGF beta), were also measured in the adenocarcinomas. There was no significant difference in weight of individual tumors. Nude mice bearing the CAC-8 (Lo Ca) tumor maintained normal body weight as compared with non-tumor-bearing control mice. In contrast, mice with the CAC-8 (Hi Ca) tumor had markedly decreased body weights. The CAC-8 (Hi Ca) tumor-bearing mice had severe hypercalcemia (mean = 13.4 mg/dl) and increased plasma concentrations of PTHrP (30.4 pM), whereas the CAC-8 (Lo Ca) tumor-bearing mice had a mean serum calcium concentration of 10.1 mg/dl and mildly increased PTHrP concentrations (5.7 pM) as compared with control mice (9.0 mg/dl and 1.0 pM, respectively). The original tumor (CAC-8 [Hi Ca]) is a well-differentiated adenocarcinoma, whereas the variant tumor (CAC-8 [Lo Ca]) is a solid carcinoma with both polygonal and spindle-shaped cells. The CAC-8 (Lo Ca) tumor had decreased PTHrP mRNA expression and protein synthesis. Messenger RNA expression of TGF beta, TNF alpha, IL-1, and IL-6 was similar in both tumors and was consistent with the central role of PTHrP in the induction of hypercalcemia in this animal model.

Effect of transforming growth factor-beta1 on parathyroid hormone-related protein secretion and mRNA expression by normal human keratinocytes in vitro.

Parathyroid hormone-related protein (PTHrP) is produced by a wide range of neoplastic and normal cells, including keratinocytes where it may regulate growth and differentiation. Transforming growth factor-beta (TGF-beta) is a growth factor produced by many cells, including keratinocytes where it regulates epidermal homeostasis. TGF-beta has been reported to be cosecreted with PTHrP in some neoplasms and to stimulate PTHrP production by neoplastic keratinocytes. However, the effects of TGF-beta on PTHrP production by normal keratinocytes are not well characterized. In this study, we investigated the effects of endogenous and exogenous TGF-beta on PTHrP production by normal human foreskin keratinocytes. PTHrP secretion, mRNA expression, and mRNA transcription in vitro were determined by N-terminal radioimmunoassay, ribonuclease protection assay, and transient transfections. PTHrP production and secretion of latent TGF-beta activity were greatest in proliferating keratinocytes prior to and at confluence of monolayer cultures. TGF-beta1 increased PTHrP mRNA expression by normal keratinocytes in a dose-dependent manner with maximal stimulation at 6-1 2 h after treatment. In addition, keratinocytes treated with a monoclonal anti-TGF-beta antibody expressed decreased levels of PTHrP mRNA. The increased levels of PTHrP mRNA following TGF-beta1 treatment were owing, at least partly, to an increase in PTHrP mRNA stability. TGF-beta1 failed to activate transcription of the luciferase reporter gene driven by either the human or mouse PTHrP promoters. In conclusion, TGF-beta1 functions as a paracrine or autocrine regulator of PTHrP production in normal human keratinocytes, and this may play a role in the regulation of keratinocyte proliferation or differentiation.

Parathyroid hormone-related protein expression and secretion in a skin organotypic culture system.

Parathyroid hormone-related protein (PTHrP), an important factor in the pathogenesis of humoral hypercalcemia of malignancy, is produced by many normal tissues, including the epidermis, where it is thought to play a role in the regulation of keratinocyte growth and differentiation. Most in vitro studies of normal keratinocytes use monolayer cell cultures, which have limitations, including the inability to reproduce the stratified structure of the epidermis. The objective of this study was to investigate PTHrP production and secretion, and mRNA expression in skin organotypic cultures. The cultures consisted of an artificial dermis with differentiating keratinocytes grown at the air-liquid interface. Immunohistochemical assessment of cytokeratins 14 and 10/13, involucrin, and proliferative cell nuclear antigen (PCNA) demonstrated that keratinocytes differentiated in a manner similar to keratinocytes in normal epidermis. PTHrP expression was demonstrated in all viable layers of the epidermis, as well as in some fibroblasts of the collagen lattice by immunohistochemistry and in situ hybridization. Since most fibroblasts expressed alpha-smooth muscle actin, these cells were interpreted to be consistent with myofibroblasts. PTHrP expression by myofibroblasts suggests a possible role for PTHrP in the regulation of contractibility of these cells. PTHrP was also detected in conditioned media for 50 days. In conclusion, because of its superior tissue morphology and ability to induce organized keratinocyte differentiation, this culture system will be an excellent model to study the role of PTHrP in pathologic and physiologic processes involving the epidermis in vitro.

Stromal and epithelial cells of the canine prostate express parathyroid hormone-related protein, but not the PTH/PTHrP receptor.

BACKGROUND: Parathyroid hormone-related protein (PTHrP), a principal factor in the pathogenesis of humoral hypercalcemia of malignancy, is also widely expressed in many normal tissues, including human prostatic epithelial cells. The role of PTHrP in the prostate is not known, but may include regulation of cell growth and differentiation or calcium secretion into prostatic fluid. The dog is a valuable animal model for human prostatic diseases. The objective was to investigate the expression of PTHrP and the PTH/PTHrP (type 1) receptor in primary cultures of canine stromal and epithelial prostatic cells. METHODS: Expression and secretion of PTHrP and the PTH/PTHrP receptor was measured in homogeneous primary cultures of canine prostatic stromal and epithelial cells using immunohistochemistry, Northern blots, radioimmunoassay, RT-PCR, and receptor stimulation assays. RESULTS: Epithelial and stromal cells expressed and secreted abundant PTHrP, but PTH/PTHrP receptor expression was not detected in either cell type. CONCLUSIONS: PTHrP expression by stromal and epithelial prostatic cells and the absence of the PTH/PTHrP (type I) receptor suggest that some functions previously proposed for PTHrP in the prostate are unlikely. The separation procedure presented is a valuable tool for studying the role and regulation of PTHrP in the prostate.

Multisystemic, eosinophilic, epitheliotropic disease with intestinal lymphosarcoma in a horse.

Multisystemic, eosinophilic, epitheliotropic disease and intestinal lymphosarcoma were diagnosed in a Paso Fino mare that presented with anorexia and weight loss. The stomach, ileum, cecum, colon, pancreas, and lungs were infiltrated by large numbers of eosinophils forming prominent eosinophilic granulomas, as well as lymphocytes and plasma cells. Two jejunal masses composed of solid sheets of neoplastic lymphocytes were present. In contrast to the regions of inflammation, the infiltrates in these masses did not contain plasma cells, eosinophils, and eosinophilic granulomas. Immunohistochemically, the neoplastic lymphocytes expressed CD3 but not CD20 or kappa and lambda light chains, supporting a diagnosis of T-cell lymphosarcoma. Concurrent diagnoses of hypereosinophilic syndrome and lymphosarcoma in this horse and several humans suggest that the multisystemic eosinophilic and lymphoplasmacytic infiltrates were caused by the clonal proliferation of T-lymphocytes that secreted interleukin-5 triggering differentiation and activation of eosinophils.

Influence of extracellular matrix macromolecules on normal human keratinocyte phenotype and parathyroid hormone-related protein secretion and expression in vitro.

Parathyroid hormone-related protein (PTHrP) is produced by a wide range of neoplastic and normal cells, including keratinocytes where it may be involved in the regulation of cellular growth and differentiation. There is evidence that the nature of the extracellular matrix (ECM) influences gene expression and cell phenotype. The objective of this study was to investigate the phenotype of normal human keratinocytes grown on different types of ECM (basement membrane components or collagen type I), as well as the expression and secretion of PTHrP. Normal keratinocytes grown on basement membrane extract (Matrigel) actively reorganized the matrix and formed networks of cells with traction of the matrix. This was associated with linear arrays of intracellular microtubules and formation of prominent actin stress fibers and was suppressed by treatment with colchicine or cytochalasin B, confirming the role of the cytoskeleton in this process. In addition, growth on Matrigel was associated with increased PTHrP nuclear translocation, secretion, and mRNA expression compared to growth on collagen where keratinocytes exhibited decreased proliferation and increased differentiation. PTHrP, as a paracrine keratinocyte factor, did not appear to mediate the morphologic changes, since they were not altered by treatment with neutralizing anti-PTHrP antibodies. It was concluded that different types of ECM influenced the morphologic, functional, and proliferative characteristics of keratinocytes, as well as the level of PTHrP expression and secretion in vitro.

Systemic candidiasis in a cheetah (Acinonyx jubatus).

Systemic candidiasis, with involvement of the spleen, liver, kidneys, and lymph nodes, was diagnosed in a geriatric captive cheetah (Acinonyx jubatus). The animal had a long clinical history of intermittent chronic gastritis associated with Helicobacter acinonyx and chronic renal failure, both of which were repeatedly treated with broad-spectrum antimicrobial therapy. Following euthanasia, a postmortem examination showed numerous microabscesses and granulomas composed of degenerate eosinophils and containing asteroids or Splendore-Hoeppli material throughout the body. Yeast, pseudohyphae, and infrequently branching septate hyphae, demonstrated with special stains, were identified as a Candida sp. by fluorescent antibody testing. Low genetic variation in cheetahs may increase their susceptibility to infectious agents. Additional factors contributing to the overgrowth and dissemination of Candida sp. in this case may have included changes in the bacterial flora of the alimentary tract as a result of repeated antimicrobial therapy and alterations in the topography of the alimentary mucosa caused by chronic gastritis.