Recombinant human basic fibroblast growth factor accelerates fracture healing by enhancing callus remodeling in experimental dog tibial fracture.

Effect of recombinant human basic fibroblast growth factor (bFGF) on fracture healing was investigated using a tibial fracture in beagle dogs. Transverse fractures in the middle of the diaphyses were created in the right tibiae and bFGF was injected into the fracture sites at a single dose of 200 micrograms. The time course of changes in callus volume and morphology of the fracture sites were evaluated at weeks 2, 4, 8, 16, and 32 after treatment, and the fracture strength was analyzed at weeks 16 and 32. At week 2, a radiogram of the fracture site showed obvious membranous ossification in the group injected with bFGF. Basic FGF extended the callus area at week 4 and increased the bone mineral content (BMC) in the callus at week 8. bFGF also increased the osteoclast number in the periosteal callus at weeks 2 and 4. In the bFGF group, a maximal increase in the osteoclast index was found at week 4, and an identical increase was recognized in the control group at weeks 8 and 16. These findings strongly suggested that bFGF stimulated not only callus formation but osteoclastic callus resorption. BMC in the bFGF group was followed by a rapid decrease from week 8, while that in the control group was identical from week 4. Fracture strength of the bFGF group showed significant recovery by week 16, and recovery was still evident by week 32. We conclude that bFGF promotes the fracture healing in dogs by the stimulation of bone remodeling.

Stimulation of endosteal bone formation by systemic injections of recombinant basic fibroblast growth factor in rats.

In vivo effects of basic fibroblast growth factor (bFGF) on bone formation was examined in rats. Daily systemic injections of 100 micrograms/kg bFGF for 7 days caused a marked stimulation of endosteal bone formation in both cortical and secondary cancellous bone areas. Histological examinations revealed that the sequence of responses to the injections of bFGF consisted of three phases: an early increase in the number of preosteoblastic cells over the osteoblastic cell layer (days 1-3), recruitment of osteoblasts from preosteoblastic cells (days 3-5), and an increase in new bone formation (days 5-7). These histological changes in the endosteum correlated closely with histomorphometrical parameters of bone formation, and the endosteal mineral apposition rate was almost unaffected during the initial 4 days but was markedly enhanced after this period. Immunohistochemical examinations using antitransforming growth factor (TGF)-beta 1 antibody demonstrated that immunostaining of preosteoblastic cells for TGF-beta already increased 1 day after bFGF treatment. Distribution of TGF-beta in osteoblasts and bone matrices began to increase on day 3, and all the osteoblasts and new bone matrices were intensively immuno-stained on day 7. These results demonstrate that systemic injections of bFGF in rats stimulate endosteal bone formation, and that the stimulation of bone formation is preceded by an initial increase in preosteoblastic cells with later recruitment of osteoblasts from these cells. Because the distribution of TGF-beta in the endosteal cells is increased by bFGF, the effect of bFGF may at least in part be mediated by TGF-beta. However, the precise mechanism of action of bFGF on bone formation remains to be clarified.

Changes of sinusoidal basement membrane collagens in early hepatic fibrosis induced with CCl4 in cynomolgus monkeys.

CCl4 was chronically administrated for 25 months to induce hepatic fibrosis in three cynomolgus monkeys so as to examine the alteration of basement membrane-related collagens during the liver injury. Although type IV collagen was immunohistochemically present along sinusoidal walls before and during the CCl4 administration, basement membrane-associated collagen (BAC), which was recognized with JK-132 monoclonal antibody, appeared around sinusoids at five to ten months of CCl4 administration. We previously developed a sandwich enzyme linked-immunosorbent assay, utilizing two monoclonal antibodies, anti-BAC antibody (JK-132) and anti-type IV collagen antibody (JK-199) [Int Hepatology Commun 1995; 4: 1-8]. The serum level of the collagen complex, which is disulfide-bridged with BAC and type IV collagen, was simultaneously monitored. The serum level of the complex at the initial stage of the examination was 19-34 ng/ml and gradually increased in relation to the intensity of immunofluorescence of BAC and type IV collagen in sinusoids and connective tissues, up to 51-57 ng/ml. The serum collagen complex levels showed a weak correlation with serum hyaluronic acid, a serum marker of hepatic fibrosis. The serum GOT, GPT, ALP and CHE levels did not reflect the alteration of sinusoids or relate to the serum collagen complex level. The increase in BAC around sinusoids and the increase of collagen complex with BAC and type IV collagen in the sera, correlate with early lesional events in hepatic fibrosis.

Parasite infection and Japanese cedar pollinosis in monkeys.

To evaluate the relationship between helminthic parasite infection and sensitization to Japanese cedar (Cryptomeria japonica; CJ) pollen allergens in Japanese monkeys (Macaca fuscata), we examined the parasite infection, presence of anti-pollen allergen IgE and development of pollinosis. Serum samples and fecal specimens were taken from 169 monkeys in five troops, and the presence of IgE antibody for CJ pollen allergen and of helminthic parasite eggs in their feces was examined. Of the 169 monkeys, helminthic parasite eggs from 138 monkeys were detected. The frequency of anti-CJ IgE (19%) and the level of total IgE did not differ significantly between the monkeys with and without parasite eggs. We examined the presence of anti-CJ IgE and pollinosis symptoms in 31 monkeys of a troop; six of the monkeys demonstrated anti-CJ IgE and pollinosis symptoms. Five of these six monkeys had parasite eggs. We found that the monkeys that demonstrated anti-CJ IgE and pollinosis symptoms had helminthic parasite infections. These data suggest that helminthic parasite infection does not reduce the development of clinical signs of CJ pollinosis in Japanese monkeys.

D-glucose transport and glycolytic enzyme activities in erythrocytes of dogs, pigs, cats, horses, cattle and sheep.

The activities of D-glucose transport (D-GT) and the glycolytic enzymes hexokinase (HK) and pyruvate kinase (PK), were measured in the erythrocytes of dogs, pigs, cats, horses, cattle and sheep. The erythrocytes of dogs had the highest activities of D-GT, HK and PK, significantly higher than the activities in the erythrocytes of the herbivores. The activities of D-GT and HK in cat erythrocytes were significantly lower than in those of dogs. The differences between the activities of D-GT in the erythrocytes of the different species followed the differences in activities of HK but not those in the activities of PK or in the blood glucose concentrations. It is considered that the activity of HK provides a convenient measurement of the relative rates of glucose oxidation in erythrocytes.

Annual changes of testis size, seminiferous tubules and plasma testosterone concentration of wild Sika deer (Cervus nippon yesoensis Heude, 1884) in Hokkaido.

Testis size, seminiferous tubules and plasma testosterone concentrations showed conspicuous annual changes in Sika deer of Hokkaido, Japan. The onset of the spermatogenic process occurred in July or August. Spermatogenic activity had already reached its height in late October, at the beginning of the rutting season, and had begun to decline in late December. Spermatogenesis had stopped in February or March. Plasma testosterone concentrations showed very high levels in late October and early November, but was almost at the basal level in February, March, June and December. The wide individual variation of the plasma levels in October suggest pulsatile secretions of testosterone.

Removal of cat major allergen (Fel d I) from futon (Japanese bedding) with a home washing machine.

We evaluated the removal of a cat major allergen (Fel d I) from futons (Japanese bedding) with the use of a large-sized home washing machine. Before and after washing a futon that had been used in a home with a cat, a small amount of cotton was collected from the futon and Fel d I was extracted from the cotton. The levels of Fel d I were assayed by a sandwich enzyme-linked immunosorbent assay (ELISA). We found that washing reduced the Fel d I level in futons by more than 95%. In conclusion, washing of futons is an effective method for elimination of their cat allergens.

[The artificial fecundation in Japanese monkeys (Macaca fuscata) by induced ovulation and artificial insemination in nonmating season].

Induced ovulation trials by PMSG-hCG administration were conducted in eleven female Japanese monkeys showing neither bleeding nor ovulation in nonmating season. The ovulation was confirmed by the laparoscopic observation in nine of the 11 females. Artificial inseminations were performed in these 9 females by the injection of semen collected by the penile electrode approach. The semen was injected into the uterine cervix in 6 females or the uterine cavity in 3 females. A gestational sac was confirmed on the ultrasonic diagnostic apparatus 19 days after ovulation in one of the 3 females inseminated into the uterine cavity. The pregnancy, however, could not be maintained.

[Periovulatory time courses of serum LH in the Japanese monkey (Macaca fuscata)].

Serum LH, E2-17 beta and progesterone concentration were measured in 16 cycles of 15 female Japanese monkeys. Three of the 16 cycles were ascertained to be anovulatory. Ten of the 13 ovulatory cycles showed LH peaks varying from 25 to 280 ng/ml. However, in remaining 3 cycles, LH peak could not be determined, probably because of a lag of blood-sampling schedule. E2-17 beta peaks were detected 0-30 hrs before LH peak in 8 cycles, but 13 or 20 hrs after LH peak in 2 cycles. Time-intervals from LH peak to ovulation ranged 0-47 hrs 30 min. No correlation was detected between concentrations of LH and progesterone in the luteal phase.

[Hypothalamo-pituitary-ovarian function in female Japanese monkeys (Macaca fuscata) in the non-mating season].

We investigated the mechanism of reduction of hypothalamo-pituitary-ovarian function in female Japanese monkeys in the non-mating season. We administered PMSG and LH-RH to 19 females in the non-mating and in the mating season. When PMSG was administered every day for 12 to 14 days to seven monkeys in the non-mating season, follicle development was observed together with an increase in serum estradiol-17 beta (E2), but there was no rise in serum LH in two animals and little increase in five others. Follicle involution began about ten days after PMSG administration, and no ovulation occurred. These findings show that secretion of LH by the pituitary in response to positive feedback by the E2 secreted by the follicles which had developed was in adequate to induce ovulation. Serum LH levels increased markedly in five females after a single iv injection of LH-RH in the mating season, but not at all in the non-mating season, even when five-times the dose was administered. These data show that the LH-secreting function of the pituitary is definitely reduced in the non-mating season. When LH-RH was administered to seven monkeys following PMSG administration, an LH surge was observed in all animals, and ovulation occurred in four animals. These findings suggest that one reason for the reduction in pituitary-ovarian function in the non-mating season was a decline in LH-RH secretion by the hypothalamus.

[Hypothalamo-pituitary-testicular function in male Japanese monkeys (Macaca fuscata) in non-mating season].

We examined seasonal changes in serum testosterone and LH, and investigated mechanisms of reduction of testicular function in non-mating season in male Japanese monkeys. Blood samples were collected monthly throughout one year in 5 males, and hCG and LH-RH were administered to 5 and 3 animals, respectively, in non-mating and mating season. Serum testosterone and LH were measured by RIA. Serum testosterone showed the obvious seasonal changes, being low in March to August and high in October to February. Also we found the seasonal changes in LH being low in May to August and high in September to February. When hCG was administered to 5 monkeys in mating season (January), serum testosterone levels increased markedly 30 to 180 min after single administration and showed further increases after continuous administrations for four days. However, in non-mating season (August), serum testosterone showed a little increase for the same treatment as in mating season. These data showed that the reserve ability of testosterone secretion in testis in non-mating season decreased obviously in contrast to mating season. The increase of serum LH and testosterone levels in 3 males in non-mating season (July) after LH-RH single administration were less than those in mating season (December). In intermediate period (September) between non-mating season and mating season, LH showed higher level than that in mating season but the testosterone level was the same extent as in non-mating season.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effects of repeated restraint stress at 30-minute intervals during 24-hour on serum testosterone, LH and glucocorticoids levels in male Japanese monkeys (Macaca fuscata)].

We investigated effects of frequent blood collections under the compulsory restraints on serum testosterone, LH and glucocorticoids in adult male Japanese monkeys. Blood samples were withdrawn from three animals at 30-min intervals and two animals at 4-hr intervals during 24 hr. Serum testosterone and LH were measured by a radioimmunoassay, and glucocorticoids was determined by a competitive protein binding assay. Sampling at 4-hr intervals during 24 hr revealed diurnal changes in serum testosterone and glucocorticoids. Levels of testosterone were high throughout the night and low at the day time, conversely serum glucocorticoids were high levels in the morning and low in the evening. On the other hand, 30-min intervals sampling, serum testosterone levels decreased and glucocorticoids levels increased, respectively, immediately after the start of blood sampling. And then, low in testosterone and high in glucocorticoids levels were continually maintained. But serum LH levels had the pulsatile pattern and did not change markedly during 24 hr. After ACTH administration in five animals, serum glucocorticoids levels increased markedly and also testosterone levels increased slightly, but LH levels did not change. These data indicated that the every 30-min restraint stress caused the increment of glucocorticoids levels and the suppression of testosterone levels, but did not affect the serum LH levels. The increased glucocorticoids might inhibit the testicular steroidogenesis, without suppressing the LH secretion from the pituitary.

Birth rate and mortality rate of infants with congenital malformations of the limbs in the Awajishima free-ranging group of Japanese monkeys (Macaca fuscata).

The birth rate and mortality rate of infants with congenital malformations of the limbs were examined in the Awajishima free-ranging group of Japanese macaques (Macaca fuscata). Of the 606 infants born between 1978 and 1995, 86 (14.2%) were malformed. The male-female ratio did not differ between malformed and normal infants. Most kin-groups included females who gave birth to malformed infants at least once. The mortality rate within the first year after birth for malformed infants (28.2%) was significantly higher than that for normal infants (10.0%). However, this indicates that more than 70% of malformed infants were able to survive for the first year of life, even though they were unable to cling to their mother's ventrum due to their limb deformities. This finding indicates that maternal care-taking is sufficient to enable malformed infants to survive during the early stages of development and that clinging by the infant is not necessary for the display of maternal care.

IgE reactivity and cross-reactivity of Japanese monkeys (Macaca fuscata) to Japanese cedar (Cryptomeria japonica) and cypress (Chamaecyparis obtusa) pollen allergens.

BACKGROUND: The natural occurrence of Japanese cedar (Cryptomeria japonica, CJ) pollinosis has been reported in Japanese monkeys (Macaca fuscata). However, the reactivity to Japanese cypress (Chamaecyparis obtusa, CO) pollen allergens in these monkeys has not yet been reported. OBJECTIVES: The present study was designed to investigate the reactivity to CO pollen allergens in monkeys sensitized to CJ pollen allergens. METHODS: Serum samples from 40 monkeys naturally sensitized to CJ pollen allergens were collected from four troops. We measured the specific IgE to CO pollen allergens and examined the reactivity to the allergens by intradermal test. Cross-reactivity between CJ and CO pollen allergens was examined by ELISA inhibition method. Furthermore, we examined the sensitivity to the allergens by histamine release assay from leucocytes. RESULTS: All 40 monkeys had specific IgE to crude and purified major allergens (Cha o 1) of CO pollen. The monkeys showed a positive reaction to CO pollen allergens in the intradermal test. Allergenic cross-reactivity between Cha o 1 and Cry j 1 (a major allergen in CJ pollen) was also observed. Specific histamine release to both the major allergens was noted in two monkeys with CJ pollinosis. CONCLUSION: Japanese monkeys sensitized to Japanese cedar pollen allergens also demonstrate reactivity to Japanese cypress pollen allergens.

Sensitization to grass, ragweed, mugwort pollen allergens in Japanese monkeys (Macaca fuscata): preliminary report.

BACKGROUND: The natural occurrence of Japanese cedar (CJ, Cryptomeria japonica) pollinosis has been reported in Japanese monkeys (Macaca fuscata). Furthermore, most of these monkeys with CJ pollinosis have immunoglobulin (Ig) E sensitization to Japanese cypress (Chamaecyparis obtusa) pollen. However, specific IgE to other pollens has not yet been reported. OBJECTIVES: The present study was designed to investigate IgE sensitization of Japanese monkeys to grass, ragweed, and mugwort pollen. METHODS: Serum samples from 47 monkeys as a general population in one troop were collected at random. We measured specific IgE to grass, ragweed and mugwort pollen. Next, 10 monkeys with CJ pollinosis from the same troop were also examined for their IgE sensitization to grass, ragweed, and mugwort pollen. RESULTS: Of 47 monkeys, 13 (28%) had specific IgE to CJ pollen, 15 (32%) to grass pollen, five (11%) to ragweed pollen, and three (6%) to mugwort pollen. Furthermore, CJ pollinosis monkeys seemed to be sensitized to these pollen allergens with higher frequency; of 10 monkeys, 10 (100%) had specific IgE to CJ pollen, six (60%) to grass pollen, four (40%) to ragweed pollen, and two (20%) to mugwort pollen. CONCLUSION: Japanese monkeys had specific IgE to grass, ragweed, and mugwort pollen in addition to CJ pollen.

The incidence of japanese cedar pollinosis and sensitization to the pollen allergens among Japanese monkeys in a troop.

The natural occurrence of Japanese cedar (Cryptomeria japonica; CJ) pollinosis has been reported in Japanese monkeys (Macaca fuscata), an appropriate animal model for developing antipollinosis therapies. However, there has been no study on the incidence of Japanese cedar pollinosis in monkeys. To evaluate the incidence of CJ pollinosis in Japanese monkeys, we investigated the presence of pollinosis symptoms among monkeys in a troop, and the response to CJ allergens in pollinosis monkeys. We examined the presence of pollinosis symptoms in 272 monkeys in a troop throughout the CJ pollination season (February to April). Of the 272 monkeys, 21 (7.7%) showed pollinosis symptoms during the CJ pollen season. Blood samples were taken from the 21 monkeys that showed pollinosis symptoms and were tested for the presence of immunoglobulin E (IgE) antibody for CJ allergens. All 21 monkeys with CJ pollinosis had anti-CJ IgE. Of the 21 monkeys, peripheral blood mononuclear cells (PBMC) could be taken from 12, all of which showed CJ allergen-specific PBMC proliferation. The incidence of CJ pollinosis in a troop was 7.7%. The monkeys with CJ pollinosis demonstrated specific IgE and PBMC proliferation for CJ allergens.

The incidence of Japanese cedar pollinosis and sensitization to the pollen allergens among Japanese monkeys in a troop.

The natural occurrence of Japanese cedar (Cryptomeria japonica; CJ) pollinosis has been reported in Japanese monkeys (Macaca fuscata), an appropriate animal model for developing antipollinosis therapies. However, there has been no study on the incidence of Japanese cedar pollinosis in monkeys. To evaluate the incidence of CJ pollinosis in Japanese monkeys, we investigated the presence of pollinosis symptoms among monkeys in a troop, and the response to CJ allergens in pollinosis monkeys. We examined the presence of pollinosis symptoms in 272 monkeys in a troop throughout the CJ pollination season (February to April). Of the 272 monkeys, 21 (7.7%) showed pollinosis symptoms during the CJ pollen season. Blood samples were taken from the 21 monkeys that showed pollinosis symptoms and were tested for the presence of immunoglobulin E (IgE) antibody for CJ allergens. All 21 monkeys with CJ pollinosis had anti-CJ IgE. Of the 21 monkeys, peripheral blood mononuclear cells (PBMC) could be taken from 12, all of which showed CJ allergen-specific PBMC proliferation. The incidence of CJ pollinosis in a troop was 7.7%. The monkeys with CJ pollinosis demonstrated specific IgE and PBMC proliferation for CJ allergens.

Seasonal changes of humoral and cellular immune responses to Japanese cedar (Cryptomeria japonica) pollen allergens in Japanese monkeys (Macaca fuscata) with pollinosis.

The natural occurrence of Japanese cedar [Cryptomeria japonica (CJ)] pollinosis has been reported in Japanese monkeys (Macaca fuscata). The present study was designed to investigate seasonal changes in immunological reactions to CJ pollen allergens in monkeys with CJ pollinosis. Blood samples were collected from six monkeys with CJ pollinosis before and after CJ pollen season. Seasonal changes in specific IgE and IgG to major allergens (Cry j 1 and Cry j 2) were observed before and after CJ pollen season. The humoral responses decreased significantly before CJ pollen and increased after CJ pollen season. Similar seasonal changes in peripheral blood mononuclear cells proliferative responses to CJ allergens were observed before and after CJ pollen season. These humoral and cellular immune responses might serve as a biomarker for assessing new immunotherapies for monkeys with pollinosis.

Analysis of sequential immunoglobulin E-binding epitope of Japanese cedar pollen allergen (Cry j 2) in humans, monkeys and mice.

BACKGROUND: Japanese cedar (Cryptomeria japonica; CJ) pollinosis has been reported to occur naturally in Japanese monkeys (Macaca fuscata) as well as in humans. Most human patients and monkeys with pollinosis have specific IgE for Cry j 2, a major allergen of CJ pollen. OBJECTIVE: The main purpose of this study was to identify IgE B cell epitopes of Cry j 2 using a synthetic peptide in humans, monkeys and mice. METHODS: We synthesized 38 overlapping peptides that span the entire length of Cry j 2. We examined the B cell epitopes of Cry j 2 that are recognized by IgE in the sera of human patients and monkeys with pollinosis and immunized mice using synthetic peptides of Cry j 2. We also examined the reaction of Cry j 2-specific mouse monoclonal IgG antibodies to the peptides. Furthermore, we conducted a histamine release assay with leucocytes from a pollinosis patient using human serum albumin (HSA) conjugated with the peptides as a B cell epitope. RESULTS: We found that 16 of the 20 pollinosis patients who had specific IgE to Cry j 2 also exhibited IgE reaction with some Cry j 2 peptides. Of these 16 patients, 10 exhibited IgE reaction with Cry j 2 peptide no. 13 (121GQCKWVNGREICNDRDRPTA140). Five of the seven monkeys with CJ pollinosis exhibited a reaction with peptide no. 13. Furthermore, IgE in mice immunized with Cry j 2 and two mouse monoclonal IgG antibodies reacted with peptide no. 13. Peptide no. 13-conjugated HSA showed the release of histamine from basophils. Furthermore, to determine the minimum epitope in peptide no. 13, we conducted an enzyme-linked immunosorbent assay inhibition test. The core of the epitope in humans, monkeys and mice was 124KWVNGREI131. CONCLUSION: We found that 124KWVNGREI131 is an important B cell epitope recognized by IgE in humans, monkeys and mice.