Immunogenicity and protective efficacy of a gE, gG and US2 gene-deleted bovine herpesvirus-1 (BHV-1) vaccine.

The efficacy and safety of a gene-deleted bovine herpesvirus-1 (BHV-1) vaccine was determined in a bovine herpesvirus challenge trial in calves. Three different doses of the vaccine were administered intramuscularly at 10(5), 10(6) and 10(7) PFU/ml and compared to a commercial vaccine and non vaccinated control calves. Challenge was performed by intranasal aerosolization with the Cooper strain of BHV-1 (3 x 10(4) PFU/ml). The non-vaccinated calves shed significantly (P < 0.05) more virus than all other groups on days 4, 8 and 10 post challenge. By day 14 post challenge, antibody titers for BHV-1 of calves vaccinated with 10(7) PFU/ml were significantly (P < 0.05) higher than the commercial or non-vaccinated calves. Clinical scores of non-vaccinated calves were significantly (P < 0.05) higher than all other groups on days 4-14 post challenge. With both radioimmunoprecipitation and competitive enzyme-linked immunosorbent assays (C-ELISA), calves in the gene-deleted vaccine groups mounted comparable specific responses against gB, gC and gD post vaccination as calves in the commercial vaccine group, but in a dose dependent manner. These data suggest that the gene-deleted BHV-1 vaccine tested may be used as an effective vaccine in controlling BHV-1 infections.

Resting energy expenditure in dogs with nonhematopoietic malignancies before and after excision of tumors.

OBJECTIVE: To determine whether apparently resting dogs with nonhematopoietic malignancies have increased resting energy expenditure (REE), compared with clinically normal dogs. ANIMALS: 46 client-owned dogs with nonhematopoietic malignancies and 30 client-owned dogs that were clinically normal. PROCEDURE: Apparently resting, client-owned dogs with nonhematopoietic malignancies before (n = 46) and 4 to 6 weeks after (n = 30) surgical removal of tumors were compared with apparently resting, clinically normal, client owned dogs (n = 30). An open flow indirect calorimetry system was used to determine the following: rate of oxygen consumption (ml/min/kg of body weight); rate of carbon dioxide production (mls/min/kg), REE (kcal/kg/d) and respiratory quotient. Because of the wide range of body weight, REE and oxygen consumption were also expressed per kg of body weight 0.75. RESULTS: Surgical removal of the tumor did not significantly alter any of the variables measured when all dogs with tumors were assessed as a single group, or when the dogs were divided on the basis of having the following types of tumors: carcinomas and sarcomas, osteosarcomas, and mammary adenocarcinomas. None of the data obtained prior to surgical treatment from any of the dogs grouped by tumor type were significantly different from clinically normal dogs. CONCLUSIONS: REE (54.4 +/- 16 kcal/kg/d or 125 +/- 19 kcal/kg0.75/d) and, presumably, caloric requirements of dogs with nonhematopoietic malignancies are not significantly different from those obtained from clinically normal dogs (53.9 +/- 16 kcal/kg/d or 116 +/- 32 kcal/kg0.75/d). Furthermore, these variables do not change significantly when the tumor is excised and the dog is reassessed after 4 to 6 weeks. CLINICAL RELEVANCE: Knowledge that REE in dogs with solid tumors is not significantly different from REE of clinically normal dogs may be of value when planning nutritional treatment for dogs with nonhematopoietic malignancies.

Toxicoses associated with the administration of mitoxantrone to dogs with malignant tumors: a dose escalation study.

Forty-four dogs with histologically confirmed malignant tumors were used in a prospective study to determine the toxicity of the chemotherapeutic agent mitoxantrone, when administered at dosages higher than what has been previously reported for use in dogs. After each dose was administered, dogs were evaluated for signs of toxicosis for 3 weeks or until the dog developed progressive disease, died, or was euthanatized. Forty dogs had been refractory to 1 or more treatment modalities (surgery, n = 26; chemotherapy other than mitoxantrone, n = 17; radiation, n = 2) prior to entering this study. Ten dogs were given mitoxantrone at a dosage of 5.5 mg/m2 of body surface, IV, every 3 weeks (39 total doses); 11 were given mitoxantrone at a dosage of 6.0 mg/m2, IV, every 3 weeks (26 total doses); and 23 were given mitoxantrone at a dosage of 6.5 mg/m2, IV, every 3 weeks (70 total doses). The most common signs of toxicosis were vomiting, anorexia, diarrhea, lethargy, and sepsis secondary to myelosuppression. Two dogs, both of which received the highest dosage, died of complications attributable to mitoxantrone administration. The prevalence of toxicoses was not associated with age, breed, sex, tumor type, number of doses, or dosage. Dogs did develop myelosuppression 7 days after they were given mitoxantrone.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in lipoprotein profiles in dogs with lymphoma.

After a 12-hour fast, blood samples were obtained from 31 dogs with previously untreated lymphoma. Blood samples were also collected from 16 of these dogs after up to 5 treatments with doxorubicin (30 mg/m2 intravenously every 3 weeks). All 16 dogs underwent complete remission. Five dogs were re-evaluated after relapse and after overt signs of cancer cachexia had become clinically apparent. Samples were assayed for 8 quantitative parameters: total cholesterol (T-CH) and total triglyceride (T-TG) concentrations, and the concentration of cholesterol and triglyceride in each of the three major lipoprotein fractions, very-low-density lipoprotein (LDL-CH and LDL-TG), and high-density lipoprotein (HDL-CH and HDL-TG). The results were compared with those from 20 healthy control dogs of similar weight and age before and 3 weeks after being given one dose of doxorubicin (30 mg/m2 intravenously). The administration of doxorubicin to control dogs resulted in a significant (P < .05) decrease in T-CH, LDL-CH, and HDL-CH, as well as a significant increase in VLDL-TG and HDL-TG. When compared with untreated controls, untreated dogs with lymphoma had significantly higher concentrations of VLDL-CH, T-TG, VLDL-TG, LDL-TG, and HDL-TG, and significantly lower concentrations of HDL-CH. HDL-TG and VLDL-TG concentrations from dogs with lymphoma were significantly increased above pretreatment values after relapse and development of overt signs of cancer cachexia.(ABSTRACT TRUNCATED AT 250 WORDS)

Repeatability of energy expenditure measurements in clinically normal dogs by use of indirect calorimetry.

Energy expenditure (EE) was determined, using an open-flow indirect calorimetry system in a group of 20 clinically normal, apparently resting, client-owned dogs. Five evaluations were performed over an 8-hour period to determine reliability of the method. The intraclass correlation coefficient was calculated as the ratio of within- and between-subject variances, using repeated-measures ANOVA. When only the middle 3 evaluations were included, the intraclass correlation coefficient was 0.87, indicating good reliability. The first evaluation was higher than the subsequent 4 evaluations for rate of O2 consumption (Vo2/kg and Vo2/kg0.75; (P < or = 0.01), and EE/kg and EE/kg0.75 (P < or = 0.005). The respiratory quotients at the first (P = 0.004) and second (P = 0.013) evaluations were different from the respiratory quotient at the fourth evaluation. Therefore, the first evaluation may not be representative of the actual EE. The mean value of at least 3 subsequent evaluations after an adequate adaptation period (5 to 10 minutes) to the equipment will be useful for predicting energy requirements of apparently resting, clinically normal dogs.

Purified methoxychlor stimulates the reproductive tract in immature female mice.

Methoxychlor is an estrogenic pesticide currently used as a substitute for DDT. The estrogenic effects of 98% pure methoxychlor or base-washed pure methoxychlor were examined on reproductive organs of immature mice and compared to control and estradiol-treated mice. Within 24 hours of birth, neonatal mice received 14 daily intraperitoneal injections of one of the following chemicals: sesame oil only, 10.0 micrograms of 17-beta estradiol, or 0.05 mg, 0.5 mg, or 1.0 mg of 98% pure methoxychlor, or 1.0 mg base-washed 98% pure methoxychlor. Equal doses (1.0 mg) of 98% pure or base-washed methoxychlor stimulated the reproductive organs of immature females such that the effects closely resembled those seen after estradiol treatments. In comparison to control mice, these pesticide treatments also induced precocious vaginal opening, persistent vaginal cornification, increased reproductive tract weights, and epithelial hypertrophy in both the vagina and uterus. However, only estradiol treatments significantly elevated albumin levels in the uterine fluid of the immature mice while the highest methoxychlor doses significantly increased the uterine cell heights when compared to all other groups.

Concentration of alpha 1-acid glycoprotein in dogs with malignant neoplasia.

Serum alpha 1-acid glycoprotein (alpha 1AG) concentrations were determined in 55 dogs with previously untreated, histologically confirmed, high-grade lymphoblastic lymphoma, and in 34 dogs with histologically confirmed nonhematopoietic malignancies (13 dogs with carcinomas and 21 dogs with sarcomas). Serum concentrations were again determined in 32 dogs with lymphoma that were in complete remission 3 weeks after 1 dose of doxorubicin (30 mg/m2 of body surface, i.v.) and in 22 dogs that were still in complete remission 3 weeks after a fourth dose of doxorubicin. For comparison, serum alpha 1AG concentrations were measured in 19 clinically normal (control) dogs of similar weight and age. Eight of the control dogs were given 1 dose of doxorubicin (30 mg/m2, i.v.), and serum alpha 1AG concentrations were measured 3 weeks later. In control dogs, mean serum alpha 1AG concentration after treatment with doxorubicin was not significantly different from mean concentration before treatment. Mean alpha 1AG concentrations in untreated dogs with lymphoma, in dogs with sarcomas, and in dogs with carcinomas were all significantly higher than mean concentration for untreated control dogs. In addition, the mean concentration for dogs with osteosarcomas was significantly higher than mean concentration for untreated control dogs. There were no significant differences in mean serum alpha 1AG concentrations among dogs with different clinical stages of lymphoma (stage IIIa, stage IVa, stage Va). However, mean serum alpha 1AG concentrations were significantly increased for dogs with stages IIIa, IVa, and Va lymphoma, compared with mean concentration for untreated control dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevalence of nephrotoxicosis associated with a four-hour saline solution diuresis protocol for the administration of cisplatin to dogs with naturally developing neoplasms.

We evaluated the development of nephrotoxicosis in 64 dogs with malignant neoplasia given cisplatin during 4-hour saline solution diuresis. Cisplatin (70 mg/m2 of body surface area, IV, q 21 d) was given to 8 dogs once, 22 dogs twice, 9 dogs 3 times, and 25 dogs 4 times. For each treatment, cisplatin was given over a 20-minute period after saline (0.9% NaCl) solution was administered IV for 3 hours at a rate of 25 ml/kg/h. After cisplatin infusion, saline solution diuresis was continued at the same rate for 1 hour. Before each treatment with cisplatin, the dogs were evaluated by conducting a physical examination, CBC; and analysis of serum urea nitrogen and creatinine concentrations, and in most cases, serum phosphorus concentration and urine specific gravity were determined. Exogenous creatinine clearance also was evaluated in 8 dogs prior to 1 (n = 8), 2 (n = 8), 3 (n = 6), and 4 (n = 4) treatments. Five (7.8%) of 64 dogs developed clinically evident renal disease after two (n = 3) and three (n = 2) doses of cisplatin. Two of the 5 dogs had preexisting diseases of the urinary tract prior to the start of treatment. Survival time in dogs that developed renal disease (median, 114 days; range, 26 to 273 days) was similar to that of all dogs in this study (median, 145 days; range, 5 to 586 days), with 30 dogs still alive at the conclusion of the study. Three of the 5 dogs that developed renal disease were alive at the conclusion of the study, 1 died of tumor-related causes, and another died as a direct result of nephrotoxicosis. There was a significant (P < 0.05) decrease in median neutrophil counts and a significant (P < 0.05) increase in median creatinine concentrations prior to the third and fourth treatments, compared with pretreatment values.(ABSTRACT TRUNCATED AT 250 WORDS)

Xylosylation is an endoplasmic reticulum to Golgi event.

The subcellular site of xylosylation, the first carbohydrate modification of the core protein that initiates glycosaminoglycan chain synthesis, was characterized in situ. Methods were developed to combine electron microscopic (EM) autoradiography and the radiolabeling of semi-intact chondrocytes. In the accompanying paper, Kearns et al. (Kearns, A. E., Vertel, B. M., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11097-11104) presented biochemical and subcellular fractionation studies that utilized semi-intact chondrocytes and radiolabeled UDP sugars to overcome obstacles to the direct analysis of xylosylation. The results suggested that xylosylation begins in the endoplasmic reticulum (ER) and continues in the Golgi. The site of xylosylation was not specified further due to the limitations of subcellular fractionation techniques. The studies described in this report were undertaken to localize these modifications directly in situ. Semi-intact cell preparations were optimized for ultrastructural preservation by modifications of permeabilization methods utilizing nitrocellulose filter overlays. Biochemical analysis demonstrated the exclusive incorporation of UDP-xylose into the cartilage chondroitin sulfate proteoglycan (aggrecan) core protein and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) into the highly modified proteoglycan monomer. Immunolocalization studies showed the equivalence of cytoplasmic subcompartments in normal and semi-intact chondrocytes at the levels of light and electron microscopy. Once the biochemical and morphological equivalence of intact and semi-intact cells was established, EM autoradiographic studies were pursued using UDP-[3H]xylose and [35S]PAPS. Based on both qualitative and quantitative data, silver grains resulting from incorporated sulfate were concentrated in the perinuclear Golgi, while those resulting from incorporated xylose were found at the cis or forming face of the Golgi and in vesicular regions of the peripheral cytoplasm associated with the late ER. These data support the view that xylose addition begins in a late ER compartment and continues in intermediate compartments, perhaps including the cis-Golgi.

Energy expenditure in dogs with lymphoma fed two specialized diets.

BACKGROUND: Cancer-caused cachexia has been reported to be caused in part by an increase in energy expenditure, and diets with nutrient profiles containing 30-50% nonprotein calories as fat instead of carbohydrate may exacerbate this state of inefficient energy utilization. METHODS: Indirect calorimetry was performed on 22 dogs with high-grade lymphoblastic lymphoma that were randomized into a blind study and fed isocaloric amounts of a high-fat diet (Diet A) or a high-carbohydrate diet (Diet B) before and after remission was attained with up to five doses of doxorubicin chemotherapy (30 mg/m2 intravenously). Indirect calorimetry was also performed on 30 normal dogs for comparison. RESULTS: During the initial evaluation period, the resting energy expenditure (REE/kg0.75, P < 0.05) and respiratory quotient (RQ, P < 0.05) were significantly lower than in the controls. Six weeks after the start of the study, the REE/kg0.75 and oxygen consumption (VO2/kg0.75) were significantly (P < 0.05) lower in both groups of dogs with lymphoma compared with the controls. The RQ determined 6 weeks after the start of the study for the dogs fed Diet A was significantly (P < 0.05) lower compared with that in the controls evaluated at the same time. When the two groups of dogs with lymphoma were compared with each other, there was no significant difference in any of the outcomes. The REE/kg0.75 and VO2/kg0.75 values were significantly lower (P < 0.05) in the group fed Diet A after the third evaluation period compared with the second evaluation. The REE/kg0.75 and VO2/kg0.75 values were significantly lower (P < 0.05) in the group given Diet B at the fourth evaluation period compared with the fifth. CONCLUSIONS: These data suggest that energy expenditure of dogs with lymphoma decreases transiently in response to chemotherapy and remission, but these values are less than those determined in normal dogs and not altered significantly by diet.

Nanomelic chondrocytes synthesize, but fail to translocate, a truncated aggrecan precursor.

Cartilage extracellular matrix (ECM) is composed primarily of type II collagen and large, link stabilized aggregates of hyaluronic acid and chondroitin sulfate proteoglycan (aggrecan). Maturation and function of these complex macromolecules are dependent upon sequential processing events which occur during their movements through specific subcellular compartments in the constitutive secretory pathway. Failure to complete these events successfully results in assembly of a defective ECM and may produce skeletal abnormalities. Nanomelia is a lethal genetic mutation of chickens characterized by shortened and malformed limbs. Previous biochemical studies have shown that cultured nanomelic chondrocytes synthesize a truncated aggrecan core protein precursor that disappears with time; however, the protein does not appear to be processed by the Golgi or secreted. The present study investigates the intracellular trafficking of the defective aggrecan precursor using immunofluorescence, immunoelectron microscopy and several inhibitors. Results indicate that nanomelic chondrocytes assemble an ECM that contains type II collagen, but lacks aggrecan. Instead, aggrecan precursor was localized intracellularly, within small cytoplasmic structures corresponding to extensions of the endoplasmic reticulum (ER). At no time were precursor molecules observed in the Golgi. In contrast, normal and nanomelic chondrocytes exhibited no difference in the intracellular or extracellular distribution of type II procollagen. Therefore, retention of the aggrecan precursor appears to be selective. Incubation of chondrocytes at 15 degrees C resulted in the retention and accumulation of product in the ER. After a return to 37 degrees C, translocation of the product to the Golgi was observed for normal, but not for nanomelic, chondrocytes, although the precursors disappeared with time. Ammonium chloride, an inhibitor of lysosomal function, had no effect on protein loss, suggesting that the precursor was removed by a non-lysosomal mechanism, possibly by ER-associated degradation. Based on these studies, we suggest that nanomelic chondrocytes are a useful model for examining cellular trafficking and sorting events and the processes by which abnormal products are targeted for retention or degradation. Further investigations should provide insight into the mechanisms underlying chondrodystrophies and other related diseases.

Evaluation of single-agent chemotherapy for treatment of clinically evident osteosarcoma metastases in dogs: 45 cases (1987-1991).

A study was undertaken to determine the effect chemotherapy had when used to treat 45 dogs with measurable metastatic osteosarcoma. The primary tumor was histologically confirmed as an osteosarcoma in each case. Thirty-nine dogs had the primary tumor surgically removed. Twenty-four of these dogs were treated adjunctively with cisplatin (70 mg/m2 of body surface, IV, q 3 weeks; median 2 doses, range 1 to 6 doses) prior to the onset of metastasis. The remaining 6 dogs from which the primary tumor was not surgically removed were diagnosed as having metastatic osteosarcoma in addition to the primary tumor on initial examination. The median time from initial examination until the development of metastatic disease was 115 days (range, 27 to 1,199 days). The location of the metastatic disease was lungs (31 dogs), bone (3 dogs), soft tissue (1 dog), and multiple sites including lungs, bone, and soft tissue sites (10 dogs). The metastatic lesions were confirmed by pretreatment biopsy (n = 8) or cytologic evaluation (n = 2) in 10 cases and at necropsy in 27 cases. The remaining 8 cases were diagnosed radiographically as multiple metastatic lesions in the lungs consistent with metastatic osteosarcoma. The metastatic disease was treated with cisplatin in 31 dogs (70 mg/m2, IV, q 3 weeks; median 2 doses, range 1 to 4 doses), doxorubicin in 11 dogs (30 mg/m2, IV, q 3 weeks; median 2 doses, range 1 to 3 doses), and mitoxantrone in 3 dogs (5 mg/m2, IV, q 3 weeks; median 2 doses, range 1 to 3 doses).(ABSTRACT TRUNCATED AT 250 WORDS)

Subcompartments of the endoplasmic reticulum.

The endoplasmic reticulum (ER) is the largest continuous endomembrane structure in the cytoplasm. It may be viewed as a series of unique subcompartments. In this review, we examine the rough ER, nuclear envelope and several smooth ER subcompartments. Consideration is given to the characteristic properties and functions of the ER and its domains, and to the formation and maintenance of subcompartments. Associations within the ER membrane bilayer, and with constituents of the cytoplasm and the ER lumen, contribute to the formation of domains and lead to the establishment of subcompartments that reflect specialized functions and vary according to the physiologic state and phenotype of the individual cell. Although the structural complexity of some ER subcompartments (such as the sarcoplasmic reticulum) is highly elaborate, the ER remains a dynamic organelle, subject to assembly and disassembly, capable of extensive remodelling and active in exchange with other organelles through mechanisms of membrane transport.

Use of a biological extract of Serratia marcescens to decrease doxorubicin-induced myelosuppression in dogs.

Fifteen dogs were given doxorubicin, IV, at a dosage of 30 mg/m2 of body surface. A commercially available biological extract of Serratia marcescens (BESM) was administered SC to 9 of these dogs (0.04 mg/kg of body weight every third day, n = 2; 0.08 mg/kg every other day, n = 2; and 0.08 mg/kg daily, n = 5), beginning the day after administration of doxorubicin, in an attempt to find an optimal dosage and schedule of administration of BESM to reduce the duration and severity of chemotherapy-induced myelosuppression. Nine additional dogs were randomized into 3 groups of 3 dogs to receive 1 of the following dosages of BESM SC: 0.08, 0.16, and 0.32 mg/kg. Serum was harvested immediately prior to treatment and at 2, 4, 6, 8, 12, 24, 48, and 72 hours from this latter group of dogs for subsequent analysis of canine granulocyte colony-stimulating factor (G-CSF) by enzyme immunoassay. Increasing the dosage and schedule of administration of BESM reduced the duration and severity of doxorubicin-induced myelosuppression. Neutrophil counts of the group of dogs given BESM daily at a dosage of 0.08 mg/kg and the controls were evaluated statistically. The neutrophil count increased significantly (P < 0.05) above pretreatment values in BESM-treated dogs after day 7. Median neutrophil counts of the BESM-treated dogs were never significantly lower than pretreatment values, whereas the median counts of the dogs treated with doxorubicin alone were significantly below normal for 6 days (days 7-12).(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a one-hour saline diuresis protocol for administration of cisplatin to dogs.

A study was undertaken to determine the toxic effects of cisplatin, an antineoplastic agent, when administered immediately after a 1-hour saline diuresis. Four treatments with cisplatin (70 mg/m2 of body surface, q 3 wk) were administered IV to 6 healthy dogs over a 20-minute period after 0.9% NaCl (saline) solution was administered IV for 1 hour at a volume of 132 ml (kg)0.75. Each dog vomited at least once within 8 hours after each treatment was administered. Clinical status, body weight, and food consumption were normal throughout the 12-week study for 5 of the 6 dogs. The sixth dog developed acute renal failure and became acutely blind and deaf within 3 days after the fourth treatment with cisplatin. Serum electrolyte, creatinine, and urea nitrogen values remained within established normal limits in all dogs immediately prior to each treatment, and in 5 of 6 dogs evaluated 3 weeks after the final treatment. The serum creatinine value (3.3 mg/dl) obtained from the Beagle euthanatized 2 weeks after the fourth treatment was above established normal values. Despite normalcy for all but 1 of the creatinine values, serum creatinine concentration obtained 3 weeks after the final treatment with cisplatin was significantly (P = 0.0001) higher than pretreatment values. When compared with data from all other evaluation periods, significant decreases in glomerular filtration rate, as determined by exogenous (P less than or equal to 0.0001) and endogenous (P less than or equal to 0.0001) creatinine clearance testing, were identified 3 weeks after the fourth treatment with cisplatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of recombinant canine granulocyte colony-stimulating factor to decrease myelosuppression associated with the administration of mitoxantrone in the dog.

Ten dogs were given mitoxantrone at a dose of 5 mg/m2 body surface area intravenously. Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered subcutaneously daily for 20 days after an infusion of mitoxantrone in five of these dogs to determine the effect of the hematopoietic growth factor on the duration and severity of myelosuppression. The median neutrophil counts dropped below normal (less than 3,000/uL) for 2 days in the dogs that received rcG-CSF, and for 5 days in the dogs that received only mitoxantrone. Four of five dogs not treated with rcG-CSF and none of those receiving rcG-CSF developed serious neutropenia (less than 1,500/uL). The neutrophil counts were significantly (P less than 0.05) higher in the rcG-CSF treated dogs at all time points except before the administration of the colony-stimulating factor, and the sixth day after the mitoxantrone was administered. These findings demonstrate that rcG-CSF is capable of reducing the duration and severity of mitoxantrone-induced myelosuppression.

Basal lamina components are concentrated in premuscle masses and at early acetylcholine receptor clusters in chick embryo hindlimb muscles.

As an initial step in characterizing the function of basal lamina components during muscle cell differentiation and innervation in vivo, we have determined immunohistochemically the pattern of expression of three components--laminin, proteins related to agrin (an acetylcholine receptor (AChR)-aggregating protein), and a heparan sulfate proteoglycan--during the development of chick embryo hindlimb muscles. Monoclonal antibodies against agrin were used to purify the protein from the Torpedo ray and to characterize agrin-like proteins from embryonic and adult chicken. In early hindlimb buds (stage 19), antibodies against laminin and agrin stained the ectodermal basement membrane and bound to limb mesenchyme with a generalized, punctate distribution. However, as dorsal and ventral premuscle masses condensed (stage 22-23), mesenchymal immunoreactivity for laminin and agrin-like proteins, but not the proteoglycan, became concentrated in these myogenic regions. Significantly, the preferential accumulation of these molecules in myogenic regions of the limb preceded by 1-2 days the appearance of muscle-specific proteins, myoblast fusion, and muscle innervation. All three basal lamina components were preferentially associated with all AChR clusters from the time we first observed them on newly formed myotubes at stage 26. Localization of these antigens in three-dimensional collagen gel cultures of limb mesenchyme, explanted prior to innervation of the limb, paralleled the staining patterns seen during limb development in the embryo. These results indicate that basal lamina molecules intrinsic to limb mesenchyme are early markers for myogenic and synaptic differentiation, and suggest that these components play important roles during the initial phases of myogenesis and synaptogenesis.

Tartrate-resistant acid phosphatase activity in tibial osteoclasts and cells elicited by ectopic bone and suture implants in normal and osteopetrotic rats.

Bone-induced multinucleated cells have been suggested as surrogates for the study of osteoclastic lineage and function. This study evaluates this proposal by comparing acid phosphatase localization in tibial osteoclasts (in situ) with that of cell populations elicited by subcutaneous implantation of devitalized trabecular bone chips from two week old rats and suture into normal and osteopetrotic (ia) rats, emphasizing tartrate-resistant acid phosphatase, an osteoclastic marker. The ia rat mutation of osteopetrosis is characterized by defective osteoclasts which typically express enhanced TRAP activity when compared to normal; ia macrophage populations do not share the same osteoclastic defect and demonstrate normal amounts of acid phosphatase reactivity. The majority of the acid phosphatase activity expressed by implant-elicited mononuclear cells was tartrate sensitive. An increase in the percentage of tartrate-sensitive, but not TRAP-positive, mononuclear cells was observed during the 14-day implantation period, suggesting the mononuclear cells did not undergo osteoclastic differentiation. Both normal and ia osteoclasts contained high concentrations of TRAP reaction product (++) while bone- and suture-induced multinucleated cells examined at 14 days post-implantation were negative (0) or mildly (+) TRAP reactive. We conclude that devitalized bone matrix implanted at this ectopic site is capable of the formation of TRAP-positive (+) multinucleated cells, but when compared on the basis of strength of TRAP activity, the bone-induced multinucleated cells do not resemble active osteoclasts, but are similar to suture-elicited macrophage polykaryons. Therefore, we suggest caution in the use of bone-induced multinucleated cells as surrogates for the study of osteoclasts and normal bone resorption. Instead, these cells may represent a population of cells involved in pathological bone loss due to inflammation.

Cellular response to ectopically implanted silk sutures and osteopetrotic bone.

Faulty osteoclasts, characteristic of the incisors-absent (ia) rat mutation of osteopetrosis, cause a resorptive defect which results in the persistence of immature, highly mineralized bone matrix. We implanted osteopetrotic bone subcutaneously into normal and ia rats to determine if ia bone could induce functionally active and morphologically identifiable osteoclasts at the implant surface. Assays of 45Ca released from the preparations showed that normal and ia recipients were capable of equivalent cell-mediated release of Ca over a 2-week implant period, indicating that the ia resorptive defect was not reproduced at the subcutaneous site. Freeze-thawed osteopetrotic bone released twice as much 45Ca as normal bone. This difference was eliminated by collagenase treatment. Cellular profiles were similar in both normal and ia animals regardless of the implant preparation. At 3 days after implantation, both bone and suture were surrounded by mononuclear cells. By 14 days, multinucleated cells appeared at the implant surfaces. Morphological comparison of implant-induced multinucleated cells and tibial osteoclasts indicated that bone-elicited multinucleated cells lacked the ruffled borders characteristic of normal osteoclasts or the extensive clear zones typical of ia osteoclasts, but more closely resembled suture-induced macrophage-polykaryons. We conclude that ectopically implanted ia bone as compared to normal bone elicits a different functional response from structurally similar cell populations. Bone-elicited multinucleated cells could not be classified as active osteoclasts despite evidence of release of 45Ca. Release of labeled Ca was probably due to the action of mononuclear phagocytes and macrophage-polykaryons rather than to osteoclastic resorption.