Effect of administering activated lymphocytes originated from the dam on the immune cell reaction in Holstein calves.

Injection of lymphokine activated killer (LAK) cells is known as useful for activation of cellular immune system. Although the effect of LAK cells has been clarified in human or mice, this effect on function of immune cells has not been examined in calves. Healthy ten Holstein calves were injected with the LAK cells 2 days after birth (LAK Group), and another eight calves were observed as controls (Control Group). All calves received the colostrum formulation on the day of birth, and then, were inoculated with a live attenuated vaccine of bovine herpesvirus (BHV)-1 at 2 (the first vaccination) and 6 (the second vaccination) weeks after birth. Peripheral blood of their dam obtained 3 weeks before calving was used for preparation of LAK cells. Blood samples were taken prior to vaccine inoculation and 3 days after the first inoculation, as well as 3 and 6 days after the second vaccination from all calves. Numbers of CD8+ and CD21+ cells increased significantly after the second vaccination in the LAK Group compared with Control Group. The present study suggested the improved effect of injecting LAK cells originated from dams on immune cells function of young calves after BHV-1 live vaccine.

Improvement of androgenetic alopecia with topical Sophora flavescens Aiton extract, and identification of the two active compounds in the extract that stimulate proliferation of human hair keratinocytes.

BACKGROUND: Androgenetic alopecia (AGA) is a hair loss disorder that commonly affects middle-aged men. To date, the properties of a number of natural or synthetic substances have been investigated for their ability to improve the condition. AIM: To evaluate the hair growth-promoting activities of an extract from the root of Sophora flavescens Aiton. METHODS: We used a human hair keratinocyte proliferation assay and ex vivo organ cultures of human hair follicle to examine the potential of the extract to stimulate hair growth via anagen elongation. We isolated the compounds promoting the growth of epithelial cells, and determined their chemical structures. A randomized, double-blinded, placebo-controlled clinical study for S. flavescens extract was carried out for 6 months with patients with AGA. RESULTS: The extract stimulated the proliferation of hair keratinocytes at a concentration of 0.1 ng/mL, while 100 ng/mL of the extract had a marked effect on hair shaft elongation in an organ culture of human hair follicle. Cell proliferation assay-directed fractionation led to the identification of two pterocarpan derivatives, L-maackiain and medicarpin, as active compounds that promote the proliferation of human hair keratinocytes. Studies in human subjects showed that improvement in the inspected alopecia scores in the lotion plus extract group were significant over a period of 6 months (P < 0.01). CONCLUSIONS: S. flavescens root extract is effective for the treatment of AGA. The isolated two pterocarpans might have important role in this effect.

Hematologic changes associated with the appearance of eccentrocytes after intragastric administration of garlic extract to dogs.

OBJECTIVE: To determine whether dogs given garlic extract developed hemolytic anemia and to establish the hematologic characteristics induced experimentally by intragastric administration of garlic extract. ANIMALS: 8 healthy adult mixed-breed dogs. PROCEDURE: 4 dogs were given 1.25 ml of garlic extract/kg of body weight (5 g of whole garlic/kg) intragastrically once a day for 7 days. The remaining 4 control dogs received water instead of garlic extract. Complete blood counts were performed, and methemoglobin and erythrocyte-reduced glutathione concentrations, percentage of erythrocytes with Heinz bodies, and percentage of eccentrocytes were determined before and for 30 days after administration of the first dose of garlic extract. Ultrastructural analysis of eccentrocytes was performed. RESULTS: Compared with initial values, erythrocyte count, Hct, and hemoglobin concentration decreased to a minimum value on days 9 to 11 in dogs given garlic extract. Heinz body formation, an increase in erythrocyte-reduced glutathione concentration, and eccentrocytes were also detected in these dogs. However, no dog developed hemolytic anemia. CONCLUSIONS AND CLINICAL RELEVANCE: The constituents of garlic have the potential to oxidize erythrocyte membranes and hemoglobin, inducing hemolysis associated with the appearance of eccentrocytes in dogs. Thus, foods containing garlic should not be fed to dogs. Eccentrocytosis appears to be a major diagnostic feature of garlic-induced hemolysis in dogs.

The cause of the predilection of Babesia gibsoni for reticulocytes.

This study was conducted to determine why Babesia gibsoni replicates well in reticulocytes. First, B. gibsoni was cultivated in resealed erythrocyte ghosts loaded with either erythrocyte or reticulocyte lysate, and in reticulocyte ghosts loaded with either erythrocyte or reticulocyte lysate. The parasites multiplied well in the erythrocyte or reticulocyte ghosts loaded with reticulocyte lysate compared to the other resealed cells loaded with erythrocyte lysate. Second, the parasites were cultivated in erythrocytes in culture medium supplemented with either erythrocyte or reticulocyte lysate. The parasites multiplied better in reticulocyte lysate-containing cultures than in erythrocyte lysate-containing cultures. Finally, the parasites were cultivated in erythrocytes in culture medium supplemented with glutamate, aspartate, asparagine, glycine, isoleucine, proline, taurine or GSH, which were present in higher concentrations in reticulocytes than in erythrocytes. Supplementation of the culture medium with glutamate and GSH resulted in enhancement of the multiplication of the parasites, while the other amino acids did not enhance the multiplication. These results indicated that the high levels of the multiplication of B. gibsoni in reticulocytes are partly due to the high concentrations of glutamate and GSH in reticulocytes.

Identification of a receptor for reg (regenerating gene) protein, a pancreatic beta-cell regeneration factor.

Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114). Rat and human Reg gene products, Reg/REG proteins, have been demonstrated to stimulate islet beta-cell growth in vitro and in vivo and to ameliorate experimental diabetes. In the present study, we isolated a cDNA for the Reg protein receptor from a rat islet cDNA library. The cDNA encoded a cell surface 919-amino acid protein, and the cells into which the cDNA had been introduced bound Reg protein with high affinity. When the cDNA was introduced into RINm5F cells, a pancreatic beta-cell line that shows Reg-dependent growth, the transformants exhibited significant increases in the incorporation of 5'-bromo-2'-deoxyuridine as well as in the cell numbers in response to Reg protein. A homology search revealed that the cDNA is a homologue to a human multiple exostoses-like gene, the function of which has hitherto been unknown. These results strongly suggest that the receptor is encoded by the exostoses-like gene and mediates a growth signal of Reg protein for beta-cell regeneration.

Rumen bacteria are involved in the onset of onion-induced hemolytic anemia in sheep.

The mechanism of onion-induced hemolytic anemia in ruminants was investigated. The ether-extract obtained from the mixture of rumen fluid and onion juice incubated at 38.5 degrees C for 9 hr induced oxidative damage in sheep erythrocytes in vitro, indicating the production of certain oxidants in the mixture. The increase of the oxidative effect in the mixture was inhibited completely by the removal of rumen microorganisms and partly by treatment with antibiotics and by oxygen gas. The sheep fed onions (50 g/kg body weight/day) for 15 days developed more severe Heinz body hemolytic anemia than did the sheep fed the equivalent amount of onions with 5 g/day ampicillin sodium salt. The results indicated that certain rumen bacteria appear to be involved in the onset of onion-induced hemolytic anemia in sheep.

Reduced glutathione accelerates the oxidative damage produced by sodium n-propylthiosulfate, one of the causative agents of onion-induced hemolytic anemia in dogs.

The oxidative effects of sodium n-propylthiosulfate, one of the causative agents of onion-induced hemolytic anemia in dogs, were investigated in vitro using three types of canine erythrocytes, which are differentiated by the concentration of reduced glutathione and the composition of intracellular cations. After incubation with sodium n-propylthiosulfate, the methemoglobin concentration and Heinz body count in all three types of erythrocytes increased and a decrease in the erythrocyte reduced glutathione concentration was then observed. The erythrocytes containing high concentrations of potassium and reduced glutathione (approximately five times the normal values) were more susceptible to oxidative damage by sodium n-propylthiosulfate than were the normal canine erythrocytes. The susceptibility of the erythrocytes containing high potassium and normal reduced glutathione concentrations was intermediate between those of erythrocytes containing high concentrations of potassium and reduced glutathione and normal canine erythrocytes. In addition, the depletion of erythrocyte reduced glutathione by 1-chloro-2, 4-dinitrobenzene resulted in a marked decrease in the oxidative injury induced by sodium n-propylthiosulfate in erythrocytes containing high concentrations of potassium and reduced glutathione. The generation of superoxide in erythrocytes containing high concentrations of potassium and reduced glutathione was 4.1 times higher than that in normal canine erythrocytes when the cells were incubated with sodium n-propylthiosulfate. These observations indicate that erythrocyte reduced glutathione, which is known as an antioxidant, accelerates the oxidative damage produced by sodium n-propylthiosulfate.

Angiotensin II type 2 receptor blockade amplifies the early signals of cardiac growth response to angiotensin II in hypertrophied hearts.

BACKGROUND: We have previously shown that the acute molecular growth response of new protein synthesis and protein kinase C activation in response to angiotensin II (Ang II) is altered in left ventricular (LV) hypertrophy compared with normal hearts. We have also shown an upregulation of Ang II type 2 (AT2) receptors in hypertrophied hearts relative to controls. Activation of AT2 receptors is proposed to counteract growth effects of AT1 receptor in response to Ang II. Thus, we tested the hypothesis that in hypertrophied hearts, the AT2 receptor mediates inhibitory effects on the new cardiac protein synthesis in response to acute Ang II stimulation. METHODS AND RESULTS: Flaccid buffer-perfused adult normal and hypertrophied rat hearts were perfused with Ang II 10(-8) mol/L plus prazosin 10(-7) mol/L or Ang II plus the AT2 blocker PD 123319 5x10(-7) mol/L. New protein synthesis was measured by the rate of [3H]phenylalanine incorporation into the LV proteins. In normal hearts, Ang II (n=8) increased the rate of [3H]phenylalanine incorporation by 74+/-27% (P<0.05 versus no drug). Treatment with PD123319 (n=8) did not increase protein synthesis compared with Ang II alone (32+/-11% versus Ang II alone, P=NS). In hypertrophied hearts, Ang II alone (n=6) increased the rate of [3H]phenylalanine incorporation only by 23+/-13% (P=NS versus no drug). In contrast, treatment with PD123319 (n=7) induced a 76+/-21% increase in new LV protein synthesis compared with Ang II alone (P<0.05). AT2 receptor blockade in Ang II-stimulated hypertrophied hearts was associated with enhanced membrane protein kinase C translocation and reduced LV cGMP content. CONCLUSIONS: These data support the hypothesis that in adult hypertrophied rat hearts, inhibition of cardiac AT2 receptors, which are upregulated in chronic LV hypertrophy, amplifies the immediate LV growth response to Ang II. This appears to be related to augmented Ang II-stimulated PKC activation and suppression of cGMP signaling.

Re-evaluation of the culture condition of polymorphonuclear cells for the study of apoptosis induction.

The culture conditions of human peripheral blood polymorphonuclear leukocytes (PMN) in the study of apoptosis induction were re-evaluated. The changes in the relative viable cell number of PMNs after tumor necrosis factor (TNF) treatment were colorimetrically investigated using a cell counting kit. The relative potency of PMNs to produce the superoxide anion (O2-) was measured as the reduction of color intensity by addition of superoxide dismutase (SOD). When the PMNs were cultured in conventional RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), the stimulation effect of TNF on O2- generation by PMNs was observed only for the first 6 hours. When FBS was replaced with human serum, the effect of TNF was maintained for longer incubation periods. Prolonged incubation of PMNs spontaneously produced large DNA fragments, and the extent of DNA fragmentation was relatively smaller in human serum-containing medium. TNF, LPS, hyperthermia or potassium thiocyanate slightly accelerated the production of large DNA fragments, as well as the induction of trace amounts of internucleosomal DNA cleavage in PMNs, which became detectable only after concentration by fractional isopropanol precipitation. The present study suggests the importance of the use of human serum rather than conventional FBS for the study of apoptosis induction in PMNs.

Effects of Chinese herbal medicines on DNA-synthesizing enzyme activities in mammary tumors of mice.

Sho-saiko-to (SST) and Juzen-taiho-to (JTT), Japanese modified Chinese herbal prescriptions, suppressed the activities of thymidylate synthetase and thymidine kinase involved in de novo and salvage pathways for pyrimidine nucleotide synthesis, respectively, in mammary tumors of SHN mice with the reduction of serum prolactin level. These results indicate that SST and JTT may have the antitumor effects on mammary tumors.

Suppression by kampo medicines in preneoplastic mammary hyperplastic alveolar nodules of SHN virgin mice.

Sho-saiko-to (SST), Keishi-bukuryo-gan (KBG), and Shakuyaku-kanzo-to (SKT) are Japanese modified traditional Chinese herbal medicines (Kampo medicines) consisting of 7, 5, and 2 medical plants, respectively. It is known that the hyperplastic alveolar nodule (HAN) is a representative preneoplastic state in the mammary glands of mice. We examined the effects of SST, KBG, and SKT on the formation and growth of HAN in a high-mammary-tumor strain of SHN virgin mice. Oral administration of SST for 60 days beginning at 90 days of age reduced the number and area of HAN and mammary thymidylate synthetase activity with a reduction of serum prolactin level. There was little difference between the other experimental groups and the control in the formation and growth of HAN and the enzyme activities. These results indicate that SST may have a preventive effect on malignant mammary transformations.

The Bo-RBC-SCID mouse model for evaluating the efficacy of anti-theilerial drugs.

We have previously developed a mouse model which allowed the proliferation of Theileria sergenti in severe combined immunodeficiency (SCID) mice with circulating bovine erythrocytes (Bo-RBC). In the present study, this model was utilized to test the efficacy of anti-theilerial drugs. Bo-RBC-SCID mice were created by giving periodic transfusions of T. sergenti-free Bo-RBC, and subsequently infecting with T. sergenti. Three anti-protozoal compounds, Pamaquine (Yamanouchi Pharmaceutical Co. Ltd), Ganaseg (Japan CIBA-GEIGY Ltd) and Buparvaquone (Coopers Animal Health Ltd), were subcutaneously administered into the mice at doses recommended for cattle therapy. Blood examinations demonstrated that all three drugs significantly reduced the level of parasitemia although Ganaseg was effective only at a dose five times higher than that recommended for cattle therapy. Administration of the drugs neither caused any sign of acute toxicity nor changed the rate of Bo-RBC in the SCID mice's circulating blood cells. The results indicate that the Bo-RBC-SCID mouse model may offer a useful in vivo system for evaluating the efficacy of anti-protozoal drugs against T. sergenti.

Seroepidemiology of mycoplasmal pneumonia of swine in Japan as surveyed by an enzyme-linked immunosorbent assay.

Antibodies to Mycoplasma hyopneumoniae were surveyed by enzyme-linked immunosorbent assay (ELISA) on serum, colostrum and milk samples collected from sows and on sera of growing/finishing pigs in Japan. Only one of 196 specific-pathogen-free sows induced a low ELISA value, while 72% of 411 sows from conventional herds were seropositive. A seropositive rate in the conventional sows gradually decreased with an increase in farrowing frequency or with age. In 3267 growing/finishing pigs ranging in age from one to six months, a seropositive rate increased remarkably from the age of 4 months onwards, reaching the maximum at the age of 6 months. A survey conducted on 42 conventional farms revealed that the highest seroconversion occurred when pigs were 4 months of age. The level of maternal antibodies was proportional to that of the dam's colostral antibodies. After maternal antibodies waned, active immunity in newborn piglets from dams with high colostral antibodies appeared earlier and higher than that in piglets from dams with low colostral antibodies. In 950 slaughter pigs, there was a correlation between seropositiveness and the presence of pneumonic lesions, but the ELISA value did not correlate with the degree of the lesions. Pigs that were raised under unfavorable environmental conditions developed pneumonic lesions more frequently than pigs rearing under better conditions, regardless of their immune status. These results suggested that M. hyopneumoniae and some secondary respiratory pathogens may have been involved in the development of these pneumonias.

Release of lipoteichoic acid from Staphylococcus aureus by treatment with cefmetazole and other beta-lactam antibiotics.

The effect of cefmetazole on the growth together with the release of cellular lipoteichoic acid from cefazolin-resistant strains of Staphylococcus aureus was compared with that of cefazolin, cefotiam, cefoxitin and cefuroxime. Bacteriolytic actions were measured by turbidity and bactericidal actions were followed by viable cell count. Release of cellular lipoteichoic acid was measured by the radioactivity in the supernatant of the cultures. Cefmetazole exerted more potent effects on the bacterial growth and induced more marked release of cellular lipoteichoic acid from resistant strains as compared with other beta-lactams.