Preliminary study of etidronate for prevention of corticosteroid-induced osteoporosis caused by oral glucocorticoid therapy.

Glucocorticoids (GCs) are widely used for the treatment of various diseases, particularly in dermatology. However, there have been few reports about the outcome of treatment for GC-induced osteoporosis in patients with dermatological conditions receiving oral GCs. The present study was undertaken to prospectively evaluate the usefulness of etidronate for preventing steroid-induced osteoporosis in patients on prolonged GC therapy as routine clinical management. In total, 110 patients receiving oral GC therapy were enrolled into the study. Of these, 87 patients were evaluated (44 patients with collagen diseases, 13 patients with autoimmune bullous dermatoses, 19 patients with chronic eczema/dermatitis, 2 patients with toxicoderma/drug eruption and 9 others). Urinary deoxypyridinoline (DPD) was evaluated as a marker of bone resorption, and serum bone-specific alkaline phosphatase (BAP) as a marker of bone formation. Significant increases in urinary DPD were seen in the control group after oral GC therapy had been continued for ≥ 1 year. Treatment with etidronate suppressed this increase. When the patients were stratified according to gender, this improvement was more obvious in women. No significant difference in serum BAP level was found between the two groups. These results suggest that bisphosphonates may be useful for preventing steroid-induced osteoporosis in dermatology patients (particularly women) receiving oral GC therapy.

In vitro metabolism of 2,2',3,4',5,5',6-heptachlorobiphenyl (CB187) by liver microsomes from rats, hamsters and guinea pigs.

The metabolism of 2,2',3,4',5,5',6-heptachlorobiphenyl (heptaCB) (CB187) was studied using liver microsomes of rats, hamsters and guinea pigs, and the effect of cytochrome P450 (CYP) inducers, phenobarbital (PB) and 3-methylcholanthrene (MC), was also investigated. In untreated animals, guinea pig liver microsomes formed three metabolites which were deduced to be 4'-hydroxy-2,2',3,5,5',6-hexachlorobiphenyl (M-1), 4'-hydroxy-2,2',3,3',5,5',6-heptaCB (M-2) and 4-OH-CB187 (M-3) from the comparison of GC/MS data with some synthetic authentic samples. The formation rate of M-1, M-2 and M-3 was 18.1, 36.6, 14.7 pmol h-1 mg protein-1, respectively. Liver microsomes of untreated rats and hamsters did not form CB187 metabolites. In guinea pigs, PB-treatment increased M-1 and M-2 significantly to 1.9- and 3.4-fold of untreated animals but did not affect the formation of M-3. In rats, PB-treatment resulted in the appearance of M-2 and M-3 with formation rates of 87.1 and 13.7 pmol h-1 mg protein-1, respectively, but M-1 was not observed. In hamsters, PB-treatment formed only M-2 at a rate of 29.4 pmol h-1 mg protein-1. On the other hand, MC-treatment of guinea pigs decreased the formation of M-1 and M-2 to less than 50% of untreated animals. MC-microsomes of rats and hamsters produced no metabolites. Preincubation of antiserum (300 microl) against guinea pig CYP2B18 with liver microsomes of PB-treated guinea pigs produced 80% inhibition of M-1 and the complete inhibition of M-2 and M-3. These results suggest that PB-inducible CYP forms, especially guinea pig CYP2B18, rat CYP2B1 and hamster CYP2B, are important in CB187 metabolism and that CB187 metabolism in guinea pigs may proceed via the formation of 3,4- or 3',4'-oxide and subsequent NIH-shift or dechlorination.

Liposteroid (dexamethasone palmitate) therapy for West syndrome: a comparative study with ACTH therapy.

Dexamethasone palmitate (liposteroid) was used for the treatment of West syndrome and compared with adrenocorticotropic hormone (ACTH) therapy. A single intravenous injection of liposteroid (0.25 mg/kg) was administered seven times in 3 months (total dosage = 1.75 mg/kg) to five symptomatic patients with West syndrome, aged 4-11 months. ACTH (0.025 mg/kg/day) was administered intramuscularly for 6 weeks according to the conventional therapy in Japan (total dosage = 0.625 mg/kg) to five symptomatic patients with West syndrome, aged 6-10 months. Nodding spasm and hypsarrhythmia on EEG disappeared in all patients in the liposteroid therapy group within four doses; however, partial seizures and focal spikes on EEG reappeared in three patients 2 months after the end of liposteroid therapy. In the ACTH therapy group, nodding spasm and hypsarrhythmia on EEG similarly disappeared during treatment in all patients, but nodding spasm reappeared 2 months after therapy in two patients and partial seizures reappeared in one patient 3 months after therapy. No notable adverse reactions occurred in the liposteroid group, but transient dysfunction of the thyroid and anterior pituitary gland and increased levels of serum cortisol were experienced in the ACTH group. These results suggest that glucocorticoid incorporated in a lipid emulsion is useful for the treatment of West syndrome.

Capsaicin does not inhibit the intracellular calcium handling process in rat ventricular papillary muscle.

1. We studied the effects of capsaicin, a pungent agent extracted from red pepper, on rested-state contraction (RSC) of isolated rat ventricular papillary muscles. 2. The RSC was induced by stimulation, after a rest interval of 5 sec to 10 min, after the twitch tension of the muscle preparation stimulated at the regular stimulus frequency of cycle lengths of 5, 1 or 0.2 sec attained the steady state. 3. Drug effects were evaluated on the RSC in the presence of capsaicin 10(-5) M, caffeine 10(-2) M or ryanodine 10(-7) M, respectively. 4. All drugs inhibited the RSC but to different degrees. Ryanodine was the most effective in inhibiting RSC, followed by caffeine and capsaicin, in that order. However, the inhibitory mode varied, depending on the drugs. 5. These findings suggest that capsaicin may not inhibit the function of intracellular Ca2+ store in rat cardiac muscle.

Inhibition of contractile tension by capsaicin in isolated rat papillary muscle.

1. We examined effects of capsaicin (10(-9) - 10(-5) M), a pungent agent extracted from red pepper, on the contractile tension of rat ventricular papillary muscles stimulated at various cycle lengths (0.2, 0.5, 1, 2 and 5 sec). 2. Capsaicin produced a marked concentration-dependent decrease in the amplitude, the rate of rise (dp/dt) and the rate of relaxation (dr/dt) of the tension. 3. However, the half relaxation time and the time to peak tension of the tension were slightly affected by the agent. 4. The negative inotropic effect of capsaicin was stimulus cycle length (CL) dependent. In particular, IC50 (50% inhibitive concentration) of the agent in the amplitude of the tension was stimulus CL dependent. That is, the values of IC50 were around 10(-7) M at longer CLs (1, 2 and 5 sec), and the value of IC50 at CL 0.2 sec was 4 x 10(-6) M. 5. These capsaicin-induced negative inotropic effects were reversible. Other studies from our laboratory show that the negative inotropic effects may be largely due to a decrease in Ca2+ current.

Statistical analysis of functional region(s) of the TSH receptor.

In this study, we apply recently developed methods to evaluate the thyrotropin receptor (TSH-R). These methods are called deviation (DEV) model, deviation decrease (DD) and DEV/DD analyses, and are based on deviation of amino acid sequences. A 3-dimensional structure model of TSH-R was graphically constructed, and found to possess a large central cavity (donut-like structure). The N-terminus was found to be in the center of the whole extracellular structure and to form a part of the bottom of the cavity. High DEV values indicate deviated amino acid compositions in the protein and were seen in 7 regions, 6 of which were found to be in regions with hydrophilic and acrophilic character. On the basis of the analysis of intra-molecular cis-acting relationships, 7 pairs of regions were presumed to be closely related. Further, when 3 exoplasmic loop lesions were analyzed similarly, 3 other regions were shown to have a close relationship with the cell surface. DEV/DD values were applied to predict the interface of TSH-R with trans-acting molecules such as TSH-R antibody or TSH. The regions in association with trans-acting molecules were seen in 14 regions, 11 of which included the high DEV regions. Both of the TSH-R specific regions in the N- and C-terminal side, especially the latter, were found to be the major components.

Presence of heterogeneous thyroid-stimulating antibodies in sera from individual Graves' patients as shown by synthesized thyrotropin receptor peptide application: evidence showing two independent epitopes and a possible recognition of two epitopic regions by one antibody molecule.

To define the epitope(s) of stimulating thyrotropin receptor antibody (TSH-R-Sab), we synthesized 19 oligopeptides covering almost all amino acids of the extracellular domain of the human TSH-R and studied these effects on the inhibition of one TSH-R-Sab activity. Four of the 19 peptides encompassing residues 31-50 (P31-20), 91-119 (P91-29), 287-304 (P287-18) and 354-367 (P354-14) were found to show significant TSH-R-Sab inhibition and to have similar effects on the other three Graves' immunoglobulins. When these peptides were applied in combination with P354-14 only P287-18 revealed additional effects but the other two combinations did not. Furthermore, sequential addition of these peptide pairs confirmed the additional effects of P287-18 and P354-14. Sequential peptide-affinity gel studies were then performed. Most of the TSH-R-Sab activity in the unabsorbed fraction from P287-18 gel was absorbed to a subsequent P354-14 gel and the eluted fraction from P287-18 mostly remained unabsorbed by the P354-14 gel. On the other hand, most of the unabsorbed fraction from P91-29 gel remained unabsorbed even by the subsequent P354-14 gel. When a P354-14 affinity gel-purified TSH-R-Sab immunoglobulin was labeled and evaluated for its binding to FRTL-5 cells, additions of original immunoglobulin, P354-14 and P91-29 resulted in significant inhibition of the binding but P287-18 did not affect either. From these results, it was concluded that most of the individual Graves' immunoglobulins contain at least two heterogeneous moieties with TSH-R-Sab activity, one of which binds P354-14 and the other binds P287-18. Further, P354-14 and P91-29 were indicated to bind the same molecule of TSH-R-Sab immunoglobulin.

Persistence of a single electropherotype and serotype (G6P5) of bovine rotavirus in calves on a closed dairy farm from 1990 to 1993.

A virologic survey was conducted on calves with diarrhea associated with bovine rotavirus (BRV) on a closed dairy farm. The BRV was detected from 32 of 219 (14.6%) fecal specimens repeatedly collected from 56 calves born during the years 1992-1993, regardless of whether they had diarrhea. Most of the 32 strains were isolated from fecal specimens obtained from 2- to 6-week-old calves. After electrophoresis of double-stranded viral RNA from the 32 strains, genomic RNA migration patterns were similar to those of the predominant BRV strains isolated at the same farm during the years 1990-1991. All representative strains were identified as G serotype 6 (G6) and P type 5 (P5) by results of the virus-neutralization test and polymerase chain reaction procedure. Thus, BRV had no change in genomic RNA electropherotypes and serologic antigenicities in a closed dairy herd over a period of several years.

Thyroid-stimulating antibodies in sera from patients with Graves' disease are heterogeneous in epitope recognition.

Two synthetic peptides, P354-14 (amino acid nos. 354 to 367) and P338-16 (nos. 338 to 353), corresponding to the partial amino acid sequences of the hTSH receptor structure were studied for their ability to bind specifically serum IgGs from patients with Graves' disease and to inhibit thyroid stimulating TSH receptor antibody (TSH-R SAb) activity. IgG binding was measured by an ELISA using sera from 102 Graves', 20 Hashimoto patients, and 9 normal subjects. Both peptides showed significantly increased IgG binding of Graves' sera compared with those of Hashimoto and normal sera. There was a significant correlation (r = 0.529) between the amount of IgG bound by the two peptides, but neither of these values correlated well with their TSH-R SAb activity nor thyrotropin-binding inhibitor TSH receptor antibody (TSH-R IAb) activity. TSH-R SAb inhibiting effects of these peptides were then analysed by measuring TSH-R SAb activity after incubation with the peptides. Among eight Graves' IgGs tested the TSH-R SAb activity of three was inhibited by both peptides, two were inhibited only by P354-14 and three were not affected by either. These TSH-R SAb inhibiting effects were dose-dependent and reproducible. To confirm these findings, a peptide-sepharose gel affinity absorption study was performed. Eleven Graves' IgGs were applied to both peptide gels and the TSH-R SAb activity of the unabsorbed fraction was measured. The TSH-R SAb activity of five IgGs was strongly absorbed only by P354-14 and five others were absorbed by both peptides to an almost similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)

CD8+ T lymphocytes are the major cell population involved in the early gamma interferon response and resistance to acute primary Toxoplasma gondii infection in mice.

Gamma interferon (IFN-gamma) is known to be a major mediator influencing host defense against Toxoplasma (T.) gondii. To evaluate lymphocyte populations involved in this cytokine-mediated early resistance to T. gondii, the effects of in vivo administration of monoclonal antibodies (MAbs) against T-cell subsets and anti-asialo GM1 antibody on the course of infection and IFN-gamma response were investigated in mice infected acutely with this parasitic protozoan. A single injection of anti-CD8 MAb on day -1 or day 4 severely exacerbated the infection, in accordance with a marked suppression of endogenous IFN-gamma production. Moreover, the administration of anti-IFN-gamma MAb on day 0 but not later than day 4 resulted in a total abrogation of resistance to T. gondii, suggesting that endogenous IFN-gamma produced during the first several days of infection is critical for the generation of antitoxoplasmal resistance in mice. In contrast, no significant increase in mortality was observed when injected with either anti-CD4 MAb or anti-asialo GM1 antibody on day -1, while these antibodies reduced significantly the ability of mice to produce IFN-gamma. Indeed, simultaneous depletion of CD4+ and CD8+ cells had no greater suppressive effect on host defense and endogenous IFN-gamma production than depletion of CD8+ cells alone. Together, these results suggest that CD8+ T cells play a central role for resolution of acute toxoplasmosis by participating in endogenous IFN-gamma production. The possible role of early produced IFN-gamma in the development of protective immune response to T. gondii is also discussed.

A case of myelodysplastic syndrome progressing to acute myelocytic leukemia in which adult-onset Still's disease had occurred 6 years before.

A patient with adult-onset Still's disease who presented with myelodysplastic syndrome (MDS) after a course of 6 years is reported. To our knowledge, this is the first such reported case. The patient died of acute myelocytic leukemia. The possibility that cyclosporine contributed to the onset of MDS is discussed.

Preventive administration of bovine colostral immunoglobulins for foal diarrhea with rotavirus.

Foal diarrhea due to serotype 3 rotavirus broke out on a foal-raising farm in the years 1987 and 1989. In 1989, all of the foals, regardless of whether or not they suffered from diarrhea, received bovine colostral immunoglobulin (Ig) powder orally for 3 to 5 days during the epidemic. The morbidity of the diarrhea was lower than that observed in 1987, when the Ig powder was not administered to foals. These data suggested that the administration of Ig powder might partially prevent foal diarrhea with rotavirus infection.

A minor prevalent strain in a severe outbreak of foal diarrhea associated with serotype 3 rotavirus.

An epizootic of foal diarrhea due to serotype 3 rotavirus (RV) was observed in 89 of 168 cases (53%) during the period from March to July in 1987. A total of 51 strains of RV were isolated from the 62 diarrheal feces examined, and one isolate (CH-3) showed a unique electropherotype of viral RNA which differed from the others that widely prevailed on this farm. No positive reaction was observed between strain CH-3 and each of the antisera against serotypes 1 to 12 of human and animal RV in neutralization tests. However, dsRNAs of the CH-3 virus were hybridized with a probe prepared from a strain of equine serotype 3 RV.

Clinical importance of CD7 expression in acute myelocytic leukemia. The Japan Cooperative Group of Leukemia/Lymphoma.

Forty patients (9 females and 31 males; mean age 41.9 years) with CD7+ acute myelocytic leukemia (AML) were investigated; they were classified into the following subgroups according to French-American-British classification: 15 M1, 18 M2, 3 M4, and 4 M5. Leukemic cells from all the patients were negative for T-cell-specific antigens, surface CD3, and T-cell-receptor molecules. The sex and age distributions were different from those of CD7- AML patients (P < .01). Hepatomegaly and central nervous system involvement were also frequent in the CD7+ AML patients. The phenotype of and responsiveness to hematopoietic growth factors by the leukemic cells showed their immaturity, as evidenced by frequent expression of CD34, HLA-DR, and TdT, and the greatest growth response to interleukin-3. No particular karyotypic abnormality was shown. One hundred eighty AML patients were treated with a therapeutic regimen routinely used for AML. The CD7+ AML patients showed a significantly lower response than CD7- AML patients (P < .01), and had a poorer prognosis (P < .01). CD7+ AML patients with M1 or M5b had unfavorable responses to the therapeutic regimen in comparison with patients with M2, M4, or M5a. In addition, 3 of 4 CD7+ CD2+ AML patients, who did not respond to the therapy, were induced into complete remission with an acute lymphoblastic leukemia therapy. The results presented here indicate the diagnostic importance of CD7 positivity in AML, suggesting that the cellular and clinical characteristics of CD7+ AML are sufficient for it to be recognized as a distinct category of AML.

Cytidine deaminase activity in abnormal pregnancy.

OBJECTIVE: To determine the usefulness of cytidine deaminase (CTD) activity in the prediction of abnormal pregnancy and the prognosis of fetal outcome. METHOD: The CTD activities in 367 pregnant women and 17 placentas and their cord sera were determined. The CTD activity was estimated to determine the amount of ammonia liberated during conversion of cytidine into uridine. The Kruskal-Wallis test and paired t-test were employed for statistical comparisons. RESULT: The placentas contained extremely high levels of CTD activity, but the cord serum did not. The mean value of CTD activity in abnormal pregnancy women was significantly higher than in normal pregnancy women. The two cases with a CTD activity of 40 U or more had the worst infant prognosis. CONCLUSION: The CTD activity in abnormal pregnancies was excessively elevated due to a damaged placenta under progressive deterioration. This CTD assay was simple and had predictive value for the prognosis of an infant of an abnormal pregnancy.

Antibody responses of Japanese horses to influenza viruses in the past few years.

A total of 305 horse sera collected in the Hidaka district of Hokkaido in the years 1988-90 were tested for the presence of hemagglutination-inhibition (HI) antibodies to A/equine/Newmarket/1/77 (H7N7), A/equine/Tokyo/2/71 (H3N8) and A/equine/Kentucky/1/81 (H3N8, Kentucky) strains of equine influenza (EI) virus. Antibodies to the 3 strains were detected in hardly of the 45 sera from 2-years-old horses which were collected before vaccination. Many of the 51 horses, after vaccination with inactivated EI virus, had HI antibodies to the 3 strains in 37 to 88 per cent. However, the number of positive reactors among the horses aged 3 to 23 years gradually decreased with the advance in age. In particular, no antibody response was found in the 60 horses over 9 years of age, except for 3 cases with HI antibody of low titer, with the Kentucky strain which has recently been prevalent among the horse populations in many countries. In a complement-fixing test, antibodies to the soluble antigen of type A influenza virus were detected in the sera collected from the horses which were exposed to an outbreak of EI virus infection, but not in the sera from the vaccinated horses examined.

Enhancement by recombinant human interleukin 2 of host resistance to Toxoplasma gondii infection in pregnant mice.

The lymphokine production by pregnant mice infected with a lethal dose of Toxoplasma gondii was evaluated in comparison with that by virgin mice infected with a sublethal dose of this protozoan parasite. Splenocytes taken from mice before and on the day after infection produced considerable amounts of IL-2 in response to concanavalin A (Con A) stimulation, but the titers rapidly declined in both pregnant and virgin mice as infection progressed. A trace amount or undetectable level of IL-2 was produced by splenocytes from acutely infected mice when stimulated with Toxoplasma lysate antigen (TLA). In contrast to the kinetics of IL-2 production, the levels of IFN-gamma produced by splenocytes cultured with Con A or TLA increased steadily in the later stage of infection in both pregnant and virgin mice. Thus, the response to Con A or TLA of splenocytes to produce IL-2 and IFN-gamma differed strikingly in acute toxoplasmosis in mice. The administration of rHuIL-2 resulted in a significant decrease in the mortality of pregnant mice infected with a lethal dose of Toxoplasma. The combination of rHuIL-2 and rMuIFN-gamma increased the survival rate slightly but not significantly compared with pregnant mice receiving either rHuIL-2 or rMuIFN-gamma. Moreover, exogenously administered rHuIL-2 enhanced the production of both IFN-alpha and IFN-gamma in the bloodstreams of pregnant mice, in accordance with the decreased mortality. These results indicate that IL-2 may play a significant role in modulating the host defense against Toxoplasma infection in pregnant mice.

Antibody responses of swine to type A influenza viruses in the most recent several years.

A serological survey was conducted on 4,080 swine sera collected for the years 1985-90. The swine sera positive to A/New Jersey/8/76 (swine type H1N1) strain were observed in annual (10-20%) and monthly (20-40%) incidences during the observation period except for occasional months. Antibodies to recent human H1N1 viruses in swine were recognized in relation to the human H1N1 influenza epidemics. Antibody responses of swine to human H3N2 strains appeared irrespective of human epidemics with the virus in the years 1985-87. However, in 1988 almost no antibodies to three human H3N2 isolates of 1983-88 were observed for this year except a few months though the human epidemic occurred in the area. Although in 1989-90 many swine had antibodies to the three strains in the percentage of 3 to 35, no antibody to the latest isolate, A/Hokkaido/20/89 (H3N2), was found for almost all the months of both years. These findings differed markedly from the possible relationship between the prevalence of H3N2 virus-antibodies in swine and the human influenza epidemics, which were described previously in many reports including our studies.

Correlation between increased susceptibility to primary Toxoplasma gondii infection and depressed production of gamma interferon in pregnant mice.

To explore a possible mechanism of pregnancy-associated suppression of T cell-mediated immunity to Toxoplasma gondii, acquired resistance and gamma interferone (IFN-gamma) production in pregnant mice were compared with those in virgin mice after infection with the S-273 strain of this protozoan parasite. The 50% lethal dose of this strain was less than 200 tachyzoites for pregnant mice and 2,800 organisms for virgin controls. Toxoplasma-induced production of both IFN-alpha and IFN-gamma in the bloodstream of pregnant mice was significantly depressed as compared with that in virgin controls. The administration of recombinant murine IFN-gamma (rMuIFN-gamma) resulted in a significant decrease of mortality and parasitic growth in the organs of pregnant mice infected with a lethal dose of S-273 strain tachyzoites. Thus, the impairment of T cell-mediated immune responses was evident in pregnant mice from the impaired IFN-gamma-generating capacity and poor survival rate after primary infection with Toxoplasma. When mice with chronic Toxoplasma infection were injected with specific antigen, the resultant production of IFN-gamma was also significantly suppressed during pregnancy. However, there was no direct correlation between the serum levels of IFN-gamma and susceptibility to reinfection, since the mortality rate of chronically infected pregnant mice after the challenge with the high virulent RH strain was not significantly higher than that of virgin controls.

Efficacy of colostral immunoglobulins for therapeutic and preventive treatments of calf diarrhea.

Pooled colostral milk samples were collected from apparently healthy Holstein cows. Immunoglobulin (Ig) was partially purified from the colostral milk and concentrated by freeze drying. The Ig powders had neutralizing antibody titers of 1:6,250 to 1:31,250 against bovine rotavirus (RV), and reacted with four RV proteins of VP2, VP4, VP6, and VP7. No therapeutic effect of Ig powder showed on clinically serious diarrhea with RV in dairy and beef calves. However, the Ig powder had a preventive effect on the diarrhea in beef calves when it was given soon after calving.