In vitro and in vivo antibiotic susceptibility of Lyme disease Borrelia isolated from the ixodid tick in Japan

Antimicrobial susceptibility of the Lyme disease Borrelia, strain HP1 vi, isolated from the tick ixodes persulcatus in Hokkaido, Japan, was determined in vitro and in vivo. A broth dilution technique was used to determine the minimum inhibitory concentrations (MICs) and minimum borreliacidal concentrations (MBCs) of five antimicrobial agents. Strain HP1 vi was most susceptible to minocycline (MBC, 0.2 micrograms/ml). The other antimicrobial agents tested, aspoxicillin, cefmetazole sodium, imipenem cilastatin sodium, and panipenem betamipron, had higher MBCs of 12.5 micrograms/ml, 25 micrograms/ml, > 25 micrograms/ml, and > 25 micrograms/ml, respectively. In vivo antibiotic susceptibility study using a ddY mouse model demonstrated that minocycline and amoxicillin were effective; minocycline had a lower 50% effective dose (ED50) value (6.25 mg/kg) than amoxicillin (30 mg/kg).     

Fetal alcohol exposure augments the blunting of tumor necrosis factor production in vitro resulting from in vivo priming with lipopolysaccharide in young adult male but not female rats

We previously reported altered responses of thymocytes and splenocytes to mitogen stimulation in fetal alcohol-exposed (FAE) male Sprague-Dawley rats. We also reported enhanced neuroendocrine responses to stressful stimuli in these animals. The experiments we describe herein aimed at testing whether young adult FAE rats manifest a notable dysregulation in the neuroendocrine-immune response to pathogen administration. We tested the effect of in vivo priming of the animal with a low dose of endotoxin [lipopolysaccharide (LPS), 5 micrograms/kg], considered to be suboptimal from the perspective of mounting detectable levels of circulating monokines several hours after administration, upon the production of immunoreactive tumor necrosis factor (TNF-alpha) in response to a further in vitro challenge of peripheral blood mononuclear cells with 2.5 micrograms/ml of LPS 90 min after priming. We show that the response to the LPS pathogen in vitro after priming is significantly blunted (p < 0.01) in male rats exposed prenatally to alcohol, compared with control male animals. FAE female rats and FAE ovariectomized female rats do not show significant differences in the priming response, compared with control animals. We also show that there is no correspondence between plasma corticosterone levels and TNF-alpha production after priming in any of the groups tested.     

First-trimester diagnosis of conjoined twins

The binding of levamisole to total plasma proteins of 6 animal species was determined in vitro by equilibrium dialysis. The percentage of bound drug protein was independent of levamisole concentration within the range studied, 5-50 micrograms/ml (ANOVA). Levamisole was bound to a low extent to plasma proteins of each animal species (19.40-25.91%). There were significant differences in the extent of levamisole binding among species (ANOVA). Owing to the low degree of protein binding and the high volume of distribution of levamisole, the variations in protein binding due to different factors would not be of major clinical importance in its therapeutic application.     

Polyanion induced preferential multinucleation in macrophages at a low level of TNF-alpha secretion

When rat bone marrow macrophages were incubated with acetyl lignin (EP3) in the presence of a 10% solution of fetal bovine serum, the macrophages secreted tumor necrosis factor (TNF-alpha) in a dose-dependent manner. This was followed by macrophage multinucleation. EP3 was found to have a significant effect on TNF-alpha secretion at a minimum dose of 5 micrograms/ml and produced no significant further increase at levels above 50 micrograms/ml, while multinucleation was most active at 10 micrograms/ml. However, multinucleation did not occur at higher concentrations of EP3 (50 micrograms/ml and 100 micrograms/ml). Secretion of TNF-alpha was significantly reduced in the absence of fetal bovine serum, whereas multinucleation was very active, starting after 6 h of incubation. At concentrations of 100 micrograms/ml, sulfonyl lignin (LS) and dextran sulfate (DS) only induced low levels of TNF-alpha secretion from macrophages, but induced active multinucleation. The multinucleation induced by addition of LS or DS was inhibited by further addition of EP3. Thus, macrophage multinucleation was most active when a low level of TNF-alpha was secreted from the macrophages.     

Inhibition of human breast cancer cell proliferation and delay of mammary tumorigenesis by flavonoids and citrus juices

Two citrus flavonoids, hesperetin and naringenin, found in oranges and grapefruit, respectively, and four noncitrus flavonoids, baicalein, galangin, genistein, and quercetin, were tested singly and in one-to-one combinations for their effects on proliferation and growth of a human breast carcinoma cell line, MDA-MB-435. The concentration at which cell proliferation was inhibited by 50% (IC50), based on incorporation of [3H]thymidine, varied from 5.9 to 140 micrograms/ml for the single flavonoids, with the most potent being baicalein. IC50 values for the one-to-one combinations ranged from 4.7 micrograms/ml (quercetin + hesperetin, quercetin + naringenin) to 22.5 micrograms/ml (naringenin + hesperetin). All the flavonoids showed low cytotoxicity (> 500 micrograms/ml for 50% cell death). Naringenin is present in grapefruit mainly as its glycosylated form, naringin. These compounds, as well as grapefruit and orange juice concentrates, were tested for their ability to inhibit development of mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female Sprague-Dawley rats. Two experiments were conducted in which groups of 21 rats were fed a semipurified diet containing 5% corn oil and were given a 5-mg dose of DMBA intragastrically at approximately 50 days of age while in diestrus. One week later, individual groups were given double-strength grapefruit juice or orange juice or fed naringin or naringenin at levels comparable to that provided by the grapefruit juice; in the second experiment, the rats were fed a semipurified diet containing 20% corn oil at that time. As expected, rats fed the high-fat diet developed more tumors than rats fed the low-fat diet, but in both experiments tumor development was delayed in the groups given orange juice or fed the naringin-supplemented diet compared with the other three groups. Although tumor incidence and tumor burden (grams of tumor/rat) were somewhat variable in the different groups, rats given orange juice had a smaller tumor burden than controls, although they grew better than any of the other groups. These experiments provide evidence of anticancer properties of orange juice and indicate that citrus flavonoids are effective inhibitors of human breast cancer cell proliferation in vitro, especially when paired with quercetin, which is widely distributed in other foods.     

Correlation between in vitro and in vivo activity of amoxicillin against Streptococcus pneumoniae in a murine pneumonia model

We studied the relationship between in vitro bacteriological parameters [minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and killing rate, defined as the reduction in the inoculum within 1, 3 or 6 hr] and in vivo activity of amoxicillin against 12 strains of Streptococcus pneumoniae, with penicillin MICs of < 0.01 to 16 micrograms/ml, in a cyclophosphamide-induced neutropenic murine pneumonia model. Dose-response curves were determined for amoxicillin against each strain, and three quantitative parameters of in vivo amoxicillin activity were defined, i.e., maximal attainable antimicrobial effect attributable to the drug [i.e., reduction in log colony-forming units (CFU) per lung, compared with untreated controls], dose required to reach 50% of maximal effect and dose required to achieve a reduction of 1 log CFU/lung. We demonstrated a highly significant correlation between the dose required to reach 50% of maximal effect and MIC (Spearman r = 0.98, P < .0001) or MBC (Spearman r = 0.95, P < .0001) for amoxicillin against strains of S. pneumoniae with a wide range of amoxicillin MICs (0.01-8 micrograms/ml). Significant correlations between the dose required to achieve a reduction of 1 log CFU/lung and MIC (Spearman r = 0.98, P < .0001) or MBC (Spearman r = 0.95, P < .0001) were also observed. In contrast, there were no significant correlations between the maximal attainable antimicrobial effect attributable to the drug and MIC, MBC or killing rate or between killing rate and the dose required to reach 50% of maximal effect or the dose required to achieve a reduction of 1 log CFU/lung. We conclude that in vitro susceptibility test results (MICs and MBCs) correlated well with in vivo amoxicillin activity against pneumococcal strains, including highly penicillin-resistant strains, in this animal model. Furthermore, these data suggest that the estimated MIC breakpoints for amoxicillin against S. pneumoniae would be 2 micrograms/ml for intermediate-resistant and 4 micrograms/ml for resistant, although this remains to be confirmed in clinical studies.     

Schistosoma mansoni oviposition in vitro reflects worm fecundity in vivo: individual-, parasite age- and host-dependent variations

In an attempt to determine whether in vitro oviposition of adult S. mansoni reflects the fecundity status of worms in vivo, Mongolian gerbils and ICR, BALB/c and SCID mice were infected with about 100 cercariae and examined on an individual basis, 5-12 weeks later, for worm burden, counts of eggs in liver and small intestine, and for the rate of egg deposition of ex-vivo female worms cultured in vitro, singly or in pairs, over a 3-5 day incubation period. The percentage of egg-laying female worms and the number of eggs laid/female after 3 days in culture showed, like worm fecundity in vivo, wide inter-worm variability, especially in 5-, 6- and 12-week-old worms; varied significantly with the age of the parasite with a maximum level attained by worms of approximately 8 weeks of age; and differed in worms recovered from different host species and strains. The data taken together indicate that measuring the egg-producing ability of S. mansoni in vitro reflects the fecundity status of worms in vivo and additionally provides likely explanations for hitherto poorly understood findings on schistosome fecundity.     

Effect of testosterone propionate treatment on thyrotropin secretion of young and old rats in vitro

The aim of this study was to evaluate the influence of androgens on TSH secretion during aging in Dutch rats. Male young (2 months) and old (16-21 months) rats were castrated (Cx) or sham-operated (C) and received testosterone propionate (TP--4 mg/Kg B.W., i.m., 7 days) or vehicle. Female adult (3 months) and old (12 and 17 months) intact rats received TP or corn oil in the same dose. The rats were decapitated, trunk blood was collected and anterior pituitaries were dissected out for in vitro incubation. In Cx young male rats, only TSH pituitary content showed lower levels than in their controls. Cx TP-treated rats showed higher serum TSH and in vitro basal and TRH-induced TSH secretion, but TP only partially reversed the decrease in pituitary TSH promoted by castration. The old male rats showed lower basal in vitro TSH secretion and pituitary TSH content. In Cx old male rats, serum and basal in vitro TSH concentrations were higher than those of old controls and TP treatment further increased basal in vitro TSH secretion, as well as, stimulated TRH-induced TSH secretion. Interestingly, TP had no effect on intact young or old male rats. However, in intact old female rats, TP stimulated in vitro TSH secretion but, as observed in the intact male, TP had no effect on adult female rats. These results suggest a stimulatory role of testosterone on TSH secretion of young and old male rats. Thereafter, it seems that the testes of old rats secrete some testicular factor that inhibits TSH secretion. However, in male rats with normal testosterone levels TP treatment did not increase further TSH secretion, but in old female rats it had a stimulatory effect.     

The mite allergen and allergoid stimulation of histamine secretion by mast cells

Rat peritoneal mast cells were incubated with serum from highly mite-sensitive patients. It was demonstrated that exposure of passive sensitized mast cells to allergen from mites Dermatophagoides farinae induced the release of histamine. Exposure of mast cells to 10 micrograms/ml and 50 micrograms/ml mite allergen resulted in an increase of histamine secretion to 48% of the basal level. The allergoid (formaldehyde-modified mite allergen) had poor histamine-releasing activity compared to allergen. The allergoid (50 micrograms/ml) induced a 2.5-fold decrease in histamine release. The allergen at the same concentrations and the same release as allergen in dose 0.1 microgram/ml.     

Magnitudes and mutual orientations of dipolar and shielding interaction tensors determined from the orientation dependence of spinning sidebands of slowly rotating powder samples

Rifampicin (RMP), antituberculous drug, has been controversially described for many years as an immunosuppressant. The goal of this work was to determine the influence of RMP on selected parameters of the immune response in vivo and in vitro. In vivo (in B6AF1 mice) the influence of long-term treatment on primary humoral response and cellular response was evaluated. Drug dose was 50 mg/kg. In vitro (using peripheral blood of volunteers) the influence of RMP on mitogen induced proliferation, metabolic activity of granulocytes and leukocyte induced angiogenesis by diverse subpopulation of mononuclear cells was examined. The concentrations tested were 7 and 70 micrograms/ml. RMP slightly stimulated production of anti-SRBC antibodies and suppressed cellular response in mice, decreased PHA and ConA induced proliferation in higher concentration and strongly inhibited chemiluminescence at concentration used. RMP inhibited also leukocytes induced angiogenesis.     

Sub-additive effect of the combination of radiation and cisplatin in cultured murine and human cell lines

The use of cisplatin (CDDP) as a potential radiosensitizer in tumors is controversial. Reports about CDDP interaction with radiation range from high radiosensitization to a clear sub-additive effect. We examined the effect of the combination of different concentrations of CDDP with radiation in murine mammary adenocarcinoma (EMT-6) and human ovarian carcinoma (OV-1063) cell lines. CDDP was given in the dose range of 0.01-3.0 micrograms/ml and radiation in the dose range of 1-6 Gy. A methylene blue assay of cell density was used for the evaluation of cell survival and rate of proliferation in 96-microwell plates. The validity of this assay for evaluation of cell survival was verified by colony-forming assay and radiolabeled thymidine uptake. The dose response to CDDP for both OV-1063 and EMT-6 cells lines was examined; the ID50 was 0.06 and 0.9 micrograms/ml respectively. A sub-additive effect of the combination of radiation with CDDP was clearly observed in the two cell lines tested; the increase in dose of each modality resulted in a decrease of the relative contribution on the effect of the other. These findings question the rationale of combining CDDP with radiation for the enhancement of tumor response, since with the increase in the dose of either modality the additional effect of the other decreases.     

Isolation of a cytotoxin from L-form Salmonella typhimurium

A cytotoxic protein was isolated from the sodium dodecyl sulphate (SDS)-solubilized extract of the stable L forms of Salmonella typhimurium by ion-retardation chromatography, ion-exchange chromatography, isoelectric focusing and gel filtration. The purified toxin, with a molecular mass of 32 kDa and with isoelectric point of 6.4, was thermolabile and trypsin-sensitive. Against mouse macrophages, its cytolytic effect was detectable in vitro at concentrations higher than 0.7 micrograms/ml, with a complete lysis obtained at 5 micrograms/ml. In contrast, it stimulated C3H/HeJ macrophages in the dose range of 0.1-0.5 micrograms/ml to allow the cell to respond to endotoxin, resulting in the significant production of tumor necrosis factor alpha. By Northern blot analysis, this effect was detectable at a dose as low as 0.01 micrograms/ml. These findings suggest that the transformation of bacillary S. typhimurium into L forms in vivo may induce alterations in host resistance against murine typhoid.     

Big trees from little genomes: mitochondrial gene order as a phylogenetic tool

Mixed acetylboswellic acids, pentacyclic triterpenes extracted from the gum resin of Boswellia serrata Roxb., significantly inhibited the ionophore-stimulated release of the leukotrienes (LT) B4 and C4 from intact human polymorphonuclear neutrophil leukocytes (PMNLs), with IC50 values of 8.48 micrograms/ml and 8.43 micrograms/ml, respectively. Purified acetyl-11-keto-beta-boswellic acid was about three times more potent as inhibitor of the formation of both LTB4 (IC50 = 2.53 micrograms/ml) and LTC4 (IC50 = 2.26 micrograms/ml) from human PMNLs in the same assay. The comparative agent MK 886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]- 2,2-dimethylpropanoic acid, L-663,536, CAS 118, 414-82-7) was about 10 to 100-fold more active than the boswellic acids in inhibiting the formation of 5-lipoxygenase products in human PMNLs, with IC50 values of 0.0068 microgram/ml (LTB4) and 0.49 microgram/ml (LTC4). After daily intraperitoneal dosage the extract of mixed acetylboswellic acids (20 mg/kg) significantly reduced the clinical symptoms in guinea pigs with experimental autoimmune encephalomyelitis (EAE) between days 11 and 21. However, the inflammatory infiltrates in the brain and the spinal cord were not significantly less extensive in the treated animals than in the respective control group. The multiple intraperitoneal application of boswellic acids did not inhibit the ionophore-challenged ex vivo release of leukotrienes B4 and C4 from PMNLs separated from the blood of guinea pigs with EAE. The boswellic acids have thus been characterized as selective, non-redox and potent inhibitors of the biosynthesis of leukotrienes in vitro.     

In vitro cytotoxicity test for estimating non-ocular irritation dose of ophthalmic solutions

The in vitro cytotoxicity test for estimating the non-ocular irritation dose of ophthalmic solutions was investigated. In the in vitro test, normal human epidermal keratinocytes (NHEK) in a confluent monolayer were incubated for 48 hr in a medium with test compounds. The concentration of a test compound which causes a 50% reduction in NHEK viability was determined as IC50 by MTT colorimetric assay. For comparison, the in vivo rabbit ocular irritation tests were carried out by the standard Draize method. The maximum concentration, which did not show any ocular irritation, was determined as DS0. The results showed the correlation coefficient between the IC50 values and the DS0 values for 19 test compounds to be 0.82. However, the correlation coefficients for 10 compounds, which have IC50 values of less than 300 micrograms/ml, and for 7 alcohols were 0.99. The IC50-DS0 correlation curves obtained could be utilized as the critical concentrations for ocular irritation. These results suggest that our in vitro/in vivo test can estimate non-ocular irritation dose of the ophthalmic preparations in advance of the in vivo tests.     

Inhibitory effects of Na2SeO3 on Tca8113 cells in vitro and in vivo

In the study, Na2SeO3 effectively limited the growth and proliferation of Tca8113 cells both in vitro and in vivo. The response was dependent on the dose, the starting administered time, exposed length of selenium and density of inoculated Tca8113 cells. At 1 microgram/ml dose of selenium, there were remarkable inhibitory effects while no detectable inhibitory effect to L929 cells. 1 microgram/ml dose for less than 24 hours, the growth and proliferative ability of Tca8113 cells was reversal whereas more than that period, was irreversal. In vivo experiment, the morbidity of transplanted tumors was remarkably depressed with Se-intraperitoneal injection. At 60 micrograms/ip dose, the weight of nude mice were not reduced and the pathological changes in liver and kidney had not found.     

A new diabetes model induced by neonatal alloxan treatment in rats

Anti-leishmanial activity of chloroform and methanol extracts of Vernonia amygdalina, a plant widely used in Ethiopia for the treatment of parasitic infections, has been assessed in vitro on Leishmania aethiopica. Amastigotes were more sensitive to V. amygdalina than promastigotes. The chloroform extract had a stronger parasiticidal activity, with median effective doses (ED50) of 18.5 micrograms/ml and 13.3 micrograms/ml for promastigotes and amastigotes, than the methanol extract with ED50 of 74.4 micrograms/ml and 45.8 micrograms/ml respectively. Cytotoxicity caused by V. amygdalina to host cells, the human leukaemia monocyte THP-1 cell line, as determined by the methyl tetrazolium assay, resulted in a median lethal dose (LD50) of 19.6 micrograms/ml for the chloroform extract and 243.4 micrograms/ml for the methanol extract. In comparison, the ED50 and LD50 of pentamidine, a standard anti-leishmanial drug, were 0.5 micrograms/ml and 1.4 micrograms/ml respectively. These results indicate that V. amygdalina displays potent anti-leishmanial activities and warrants further investigation.     

N,N-dimethylglycyl-amido derivative of minocycline and 6-demethyl-6-desoxytetracycline, two new glycylcyclines highly effective against tetracycline-resistant gram-positive cocci

The in vitro activities of the N,N-dimethylglycyl-amino derivative of minocycline (DMG-MINO) and 6-dimethyl-6-dexoxytetracycline (DMG-DMDOT), members of a new generation of tetracyclines, were evaluated by an agar dilution method and were compared with those of tetracycline and minocycline against 224 tetracycline-resistant and 73 tetracycline-susceptible recent clinical isolates of gram-positive cocci, including multiple-antibiotic-resistant methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae. The MICs of DMG-MINO and DMG-DMDOT were up to 500- to 2,000-fold lower than those of tetracycline against methicillin-resistant S. aureus and Streptococcus pneumoniae (MIC for 50% of strains tested [MIC50], < 0.06 microgram/ml). Against Streptococcus groups A, B, C, and G and Enterococcus faecalis, the MIC50 was 0.5 microgram/ml. MIC50s were greater only for coagulase-negative staphylococci (2 micrograms/ml). These data indicate that DMG-MINO and DMG-DMDOT are very potent drugs, and further in vitro and in vivo studies are warranted.     

Selection for high levamisole resistance in Haemonchus contortus monitored with an egg-hatch assay

To investigate the characteristics of selection for levamisole resistance in Haemonchus contortus, the consecutive nematode generations of an in vivo selection were monitored with a newly developed egg-hatch assay. The in vivo selection was started with a population not previously exposed to any anthelmintics (SHS). At first, the levamisole resistance progressed gradually in successive nematode generations by treating sheep with increasing doses of levamisole, the initial dose being 1 mg kg-1. Treatment with 5 mg kg-1 levamisole resulted, however, in a steep increase of resistance. The selection was ended after six generations, since a level of 30 mg kg-1 levamisole, which is not far from the toxic level for sheep, was reached. The final population, RHS6, was studied in a controlled test. Treatment of RHS6-infected sheep with 30 mg kg-1 levamisole caused an 80% decrease of faecal egg output, and a reduction of 34% in worm numbers. It was remarkable that only the number of female adults was reduced. RHS6 showed a reduced viability, but a fertility similar to the starting population SHS.     

Oxyntomodulin reduces hydromineral transport through rat small intestine

Glicentin (GLIC) and oxyntomodulin (OXM) are released from the ileum and colon during digestion. Both hormones reduce fluid and proton secretion in the stomach. The luminal concentration of sodium and chloride underlying the nutrient absorption, the effect of OXM on electrolyte transport through the small intestine, was assessed in vivo using ligated loops and in vitro using Ussing chambers. In vivo, a zero transport state, estimated by the net water, chloride, and sodium fluxes, was observed when an 80 mM NaCl normoosmolar solution (274 mosm) was administered intraluminally. Active secretion was observed with hyperosmotic challenge (474 mosm). The amplitude of this active secretion increased 2.5- to 3-fold when an electrogenic challenge (NaCl 40 mM) was substituted to the hyperosmotic one. OXM (800 fmol/ml plasma) did not modify the basal transport in the duodenum or in the jejunum (t = 45 min). When active secretion was induced by the hyperosmotic challenge, OXM (200 fmol/ml plasma) had no effect on duodenal or jejunal transport (t = 50 min). When active secretion was induced by an electrogenic challenge, OXM (300 fmol/ml plasma) preferentially reduced the hydromineral transport in jejunum. In vitro, OXM also induced a reduction in the ion transport towards the jejunal lumen (EC50 = 20 pM), the amplitude of which depended upon the integrity of the tetrodotoxin-sensitive neurons. In conclusion, OXM was able to reduce the large secretion induced in rat jejunum in vivo by an electrogenic gradient. In vitro, the antisecretory effect of OXM was partly mediated by the neurons present in the intrajejunal wall.