Evidence for Clostridium perfringens enterotoxin (CPE) inducing a mitogenic and cytokine response in vitro and a cytokine response in vivo

We investigated some immunogenic properties of Clostridium perfringens enterotoxin (CPE) in vitro using murine J774A macrophages (MPhi) and in vivo using Swiss Webster (SW) mice. CPE was a potent mitogen in vitro, where cell proliferation increased with CPE concentration. CPE was nonmitogenic when MPhi were concurrently incubated with CPE and interferon gamma (IFN-gamma). MPhi incubated in the presence of CPE induced the synthesis of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), but not interleukin-2 (IL-2). In vivo, CPE induced a pro-inflammatory cytokine response with striking production of IFN-gamma, IL-1, and IL-6. Regardless of route of CPE entry, serum cytokine levels generally peaked within 1 h of administration and were maintained for 4-8 h. Although CPE engenders an intense immune response during toxicosis, the toxin does not appear to be a superantigen. Death from CPE-induced shock appears to result from various interrelating immunological mechanisms.     

The effect of Escherichia coli heat-stable (STA) enterotoxin on in vitro uptake of amino acids by small intestine of sucking rats during fluid accumulation

In vitro uptake of 14C-labelled amino acids by segments of small intestine was determined in sucking (2-4-d-old) Wistar rats. Intragastric injections of heat-stable (ST) toxin of enterotoxigenic Escherichia coli (ETEC) were given to produce fluid accumulation, defined as a gut weight: carcass weight value of > 0.085. Continued active uptake of the prototypic amino acids, leucine (by active transport system 1 for monoamino monocarboxylic (neutral) amino acids), lysine (by active transport system 2 for dibasic amino acids), and proline (by active transport system 3 for N-substituted amino acids), persisted during the active fluid accumulation response to ETEC ST toxin. The mean Kt and mean Vmax of the amino acid transport systems were similar in control (non-injected) and ST toxin-injected rats. The present study provides a scientific basis for the use of amino acids in oral rehydration solutions utilizing amino acid transport systems which are linked to absorption of Na (and water) so that reduction in diarrhoeal stools can be achieved, and emphasizes the importance of maintaining feeding during acute diarrhoea to prevent development of malnutrition.     

Effect of adrenaline, noradrenaline and acetylcholine on rabbit and human uterine activity in vivo (author's transl)

Effects of estrogen (abbr. as E), Estrogen + Progesterone (E + P) and autonomic nerve trophic drugs on uterus were investigated in rabbit and human in vivo and the following results were obtained. 1) Spontaneous contraction in cornu and cervix of E treated rabbit were stronger than in those during pregnancy or treated with E + P. 2) 10(-5) g/kg Adrenaline caused a uniform response pattern in cornu and cervix of E treated rabbit, and the pattern could be divided into three phases. Phase I is supposed to be the contraction response through the alpha receptor, and phase II is presumed as the response that comprises a part of element identical with alpha adrenergic response, while phase III is the relaxation response through the beta 2 receptor. 3) Acetylcholine (ACh) 10(-3) g/kg and Neostigmine 10(-5) g/kg caused augmentation of contraction both in cornu and cervix of rabbit, but this effect was blocked by Atropine, suggesting the presence of parasympathetic control in cornu and cervix. 4) In human corpus and cervix, Phenylephrine and ACh augmented contraction but Terbutaline had reducing contraction, showing the similar phenomenon found in rabbit. From this, it was proven that the similar autonomic nerve controls could be present both in rabbit and human.     

Synergistic anti-influenza virus A (H1N1) activities of PM-523 (polyoxometalate) and ribavirin in vitro and in vivo

A Kegin-type polyoxometalate, PM-523, in combination with ribavirin, was tested for its therapeutic effectiveness against influenza virus (FluV) A (H1N1) infection in tissue culture and in mice. PM-523 [(PriNH3)6H [PTi2W10O38(O2)2] x H2O, where Pri is isopropanol] and ribavirin individually inhibited FluV A-induced cytopathic effects in Madin-Darby canine kidney (MDCK) cells at median effective concentrations (EC50s) of 30 and 34 microM, respectively, and at 70% effective concentrations (EC70s) of 48 and 72 microM, respectively. On the other hand, a combination of PM-523 and ribavirin at a ratio of 1:16 exhibited lower EC50s and EC70s than each compound used singly, and combination indices were less than 1. A wide range of combinations of PM-523 and ribavirin at ratios of from 1:128 to 1:1 exhibited additive or synergistic anti-FluV effects in MDCK cells. When these compounds were tested for their anti-FluV A activities in vivo by aerosol exposure of mice which had been infected with a lethal dose of FluV A by an intranasal route, a 1:16 combination of PM-523 and ribavirin was found to have a significantly better therapeutic effect than a single dose of either compound used singly with respect to both the survival rate of the mice and the virus titer in the lungs of the infected mice. PM-523 was effective for the treatment of experimental FluV infection, and in combination with ribavirin, PM-523 exhibited enhanced anti-FluV effects in vitro and in vivo compared with the effect of PM-523 alone.     

Effect of serotonin on unidirectional ion fluxes in rat intestine in vivo

AIMS: A stimulating intestinal secretory effect is described in vitro and an inhibition with selective inhibitors of the different receptors of serotonin (5-HT), in vivo. But a direct effect, in vivo, in fully vascularized and innervated intestine has not yet been clearly evidenced. We studied the effect of 5-HT in anesthetized rats with ligated loops. This work, performed at 4 intestinal levels, allowed a comparison with the effects of a known stimulant of intestinal secretion, VIP, and a specific inhibitor of Na/H exchange, dimethylamiloride (DMA). RESULTS: 5-HT induced an inhibition of epithelial Na influx in agreement with the inhibition of Na/H exchanger, an inhibition of the influx of Cl, partially passive absorption following Na by paracellular route. A decrease of Na and Cl efflux was induced by 5-HT in duodenum, jejunum and ileum while in colon, a stimulation was obtained by intraluminal but not intravenous route. CONCLUSION: Even though 5-HT induced a liquid accumulation in all intestinal segments, the effect differed according to the intestinal level, either inhibition of absorption in the small intestine, or stimulation of secretion in the colon. The comparison of the effect of 5-HT with that of DMA shows that the inhibition of absorption is not only due to Na/H exchanger inhibition.     

The molecular chaperone calnexin interacts with the NSP4 enterotoxin of rotavirus in vivo and in vitro

Calnexin is an endoplasmic reticulum (ER)-associated molecular chaperone proposed to promote folding and assembly of glycoproteins that traverse the secretory pathway in eukaryotic cells. In this study we examined if calnexin interacts with the ER-associated luminal (VP7) and transmembrane (NSP4) proteins of rotavirus. Only glycosylated NSP4 interacted with calnexin and did so in a time-dependent manner (half-life, 20 min). In vitro translation experiments programmed with gene 10 of rhesus rotavirus confirmed that calnexin recognizes only glycosylated NSP4. Castanospermine (a glucosidase I and II inhibitor) experiments established that calnexin associates only with partly deglucosylated (di- or monoglucosylated) NSP4. Furthermore, enzymatic removal of the remaining glucose residues on the N-linked glycan units was essential to disengage the NSP4-calnexin complex. Novel experiments with castanospermine revealed that glucose trimming and the calnexin-NSP4 interaction were not critical for the assembly of infectious virus.     

Ubiquitin-mediated degradation of tyrosine aminotransferase (TAT) in vitro and in vivo

Cells coexpressing lymphoid and myeloid cell surface markers have been described for various leukemias and non-Hodgkin's lymphomas. It is unclear whether these mixed lineage characteristics are due to malignancies of early progenitor cells or alternatively to malignant cells with lineage infidelity. Recently, it has been shown that cells coexpressing lymphoid and myeloid markers can be generated from peripheral blood lymphocytes from normal individuals as well. In this review, consequences of this surprising fact are discussed.     

Sequence diversity of SIV(Mne) Nef in vivo and in vitro

The immune system and nervous system are intimately related. In addition to neuroendocrine mechanisms, neuropeptides have a variety of effects on immune cells and are responsible at least in part for neurogenic inflammation. The presence of neuropeptides in the skin has been well documented. The influence of neuropeptides on Langerhans cells is the focus of this paper. The physical presence and effects of calcitonin gene-related peptide on Langerhans cells is emphasized. Discussion also includes the putative inflammatory and immunologic roles of vasoactive intestinal peptide, substance P, neurotensin, neuropeptide Y, and somatostatin in the skin.     

Effect of D-glucose on intestinal permeability and its passive absorption in human small intestine in vivo

BACKGROUND: Based on studies in animals, it has been proposed that carrier-mediated D-glucose absorption markedly enhances passive permeability of the jejunal mucosa, allowing the majority of D-glucose absorption to proceed passively. In this study, we evaluated this hypothesis in the human jejunum in vivo. METHODS: Using the constant perfusion, nonabsorbable marker technique, permeability of jejunal mucosa was assessed by measuring the ratio of diffusion rates of urea/L-xylose and mannitol/L-xylose. Passive D-glucose absorption was quantitated using L-glucose and mannitol as probes for D-glucose. RESULTS: Addition of D-glucose to perfused solutions did not change the diffusion ratios, indicating that D-glucose has no effect on the size of channels for passive diffusion across the jejunal mucosa. The fraction of total D-glucose absorption that could be attributed to a passive mechanism averaged 5%. In the human ileum in vivo, we detected no evidence of passive D-glucose absorption. CONCLUSIONS: Carrier-mediated D-glucose absorption does not increase passive permeability of human jejunal mucosa to solutes with molecular radii between 2.6 and 4.0 A. The amount of D-glucose absorbed passively from the human jejunum is trivial compared with the overwhelmingly dominant mechanism, carrier-mediated transport. Our results do not support the concept that sodium-dependent nutrient transport increases tight junction permeability.     

Acidity near eroding polylactide-polyglycolide in vitro and in vivo in rabbit tibial bone chambers

Eroding poly(DL-lactide-co-glycolide)(PDLLG) washers were observed chronically in vitro and in vivo following loading in a bone chamber tibial implant (BCI). Images were recorded using intravital microscopy of the implanted rabbit and direct pH measurements were obtained of the tissue in the bone chamber using a combination probe-reference microelectrode. While statistically significant pH differences were found between the control (unloaded) and experimental (PDLLG-bearing) BCIs, they were only of the order of 0.2 pH units. This value proved to be physiologically insignificant as no statistically significant difference in bone defect healing, as indicated by angiogenesis, was detected. It was shown that the measured small pH changes during PDLLG washers erosion would result whether the buffer was phosphate-buffered saline replaced weekly or interstitial fluid subject to vascular exchanges.     

Electrophysiologic effects of civamide (zucapsaicin) on canine cardiac tissue in vivo and in vitro

Mucins are highly expressed in many different human cancers and numerous murine monoclonal antibodies (MAbs) to human mucins, particularly Mucin 1 (MUC1), have been produced. However, no such antibodies to murine mucin 1 (muc1) have been described and we now describe 6 different antibodies produced to murine muc1 and to human MUC1 cytoplasmic tail, either by immunising rats, or muc1 o/o mice with synthetic peptides or a fusion protein composed of glutathione-s-transferase (GST) linked to the tandem repeat region of muc1. The antibodies to both the extracellular tandem repeat region and to the cytoplasmic tail were found to react with mucin-containing murine tissues such as breast, stomach, colon, ovary, kidney and pancreas, and the staining patterns were similar to those found in humans. The reagents reacted specifically with muc1 peptides and tissues; however, some cross reactivity with other mucin-derived peptides was noted, particularly those containing the amino acid sequence TSS. Three different epitopes (TSS, TAVLSGTS and LSGTSSP) of the M30, M70 and MFP25 MAbs were detected. Of interest was the finding that some of the antibodies reacted with murine lymphocytes; it was not clear whether these reactions were due to mucin 1 on mouse lymphocytes (MUC1 was considered to be absent from human lymphocyte), or due to cross reaction with a sialic adhesion molecule on lymphocytes. The antibodies should prove valuable reagents when studying differentiation and expression in murine glandular tissues and the ontogeny of mucin-secreting tumours.     

Effect of chronic glucagon-administration on the digestive and absorptive function of rat small intestine in vivo

Mitomycin C-induced sister chromatic exchange and quadriradial formation were studied in chromosomes from Muntiacus muntjac fibroblasts and A9, a transformed mouse cell line. We present the first direct cytological evidence for the induction of true somatic recombination involving homologous chromosomes. Analysis of the quadriradials from Muntjac cells indicates a non-random distribution with respect to the chromosomes involved and with respect to the points of conjunction. Sister chromatid exchange and quadriradial formation may reflect the outcome of repair processes involving a high frequency of homologous exchanges in the interphase nucleus.     

Effect of dantrolene sodium on the contractility of rabbit jejunum in vitro

The effect of dantrolene sodium on the spontaneous contractions of rabbit jejunum was studied in vitro. Dantrolene sodium (4.5 x 10(6) to 4.5 10(4) M) reversibly decreased the amplitude of contractions in a dose-dependent manner. ED50 was found to be about 7.9 x 10(-5) M. Its effect was biphasic in that a period of potentiation preceded that of suppression of contractions. Lowering or increasing (2.5 fold in each direction) the calciumm concentration of bathing media did not affect the suppression of contraction caused by dantrolene sodium to any significant degree. Caffeine but not quinine was found to be able to restore the activity of the intestine to normal after a 50% inhibition caused by dantrolene sodium. Dantrolene sodium, verapamil and nifedipine were able to shift the dose-response curves of calcium in potassium-polarized rabbit jejunum to the right and pA2 values were found to be 4.18, 7.76 and 8.47 respectively. These data indicate that the effect of dantrolene on smooth muscle is mediated via inhibition of calciu movement across the membrane.     

Effect of fatty acids on thyroid function tests in vitro and in vivo

Addition of long-chain fatty acids to serum increased thyroxine (T4), measured by a competitive protein binding assay, and triiodothyronine (T3) uptake by Sephadex or resin (T3U tests). This is compatible with the assumption that fatty acids compete with thyroxine for binding sites on T4-binding proteins. When equimolar concentrations of various saturated and unsaturated fatty acids were added to serum it was observed that the effectiveness in raising tests based on protein binding of thyroid hormones incrreased serum T3 determined by radioimmunoassay (RIA). T4(RIA) was not significantly influenced by either saturated or unsaturated fatty acids. Serum T4(CPB) rose during storage at 22degreesC and 37degreesC but was stable at 4degreesC and --20degreesC for periods up to two weeks. The proportional increase in T4(CPB) and free fatty acids (FFA) indicated that this phenomenon was due, at least partly, to the interference from FFA formed during storage of the serum. There was also a small, significant increase in T3U, T3(RIA) and CT4I (a free thyroxine estimate) after storage of serum at room temperature or higher for one to two weeks. Serum T4(RIA) did not alter during two weeks of storage. In five subjects with raised serum FFA after eating a fat meal followed by a heparin injection an increase in T4(CPB), T3U, T3(RIA) and CT4I that was proportional to the increase in FFA was observed. This effect on the thyroid tests was small until the increase in FFA concentration exceeded 2 mmol/l. T4(RIA) did not respond to the increase in FFA. In ten patients with raised levels of FFA due to uncontrolled diabetes T4(CPB), T4(RIA) and T3(RIA) decreased while T3U increased. These unexpected alterations were probably related to the severe, chronic illness in these patients. Increased FFA in vivo seem to be of little importance for the interpretation of thyroid tests in clinical practice.     

In vivo and in vitro action of endothelin-1 on goat cerebrovascular bed

This study concerned the effects and mechanisms of action of endothelin-1 on the cerebral circulation. Cerebral blood flow was electromagnetically measured in awake goats. Endothelin-1 (0.01-0.3 nmol) produced dose-dependent decreases in this flow (maximal reduction = 34%) and increases in cerebrovascular resistance (maximal increase = 74%) (P < 0.01). IRL 1620 (Suc-[Glu9, Ala11,15]endothelin-1-(8-21), agonist for endothelin ET(B) receptors, 0.01-0.3 nmol) slightly decreased cerebral blood flow. The effects of endothelin-1, but not those of IRL 1620, on cerebral blood flow were diminished by 50% during infusion of the antagonist for endothelin ET(A) receptors, BQ-123 (cyclo-(D-Asp-Pro-D-Val-Leu-Trp), 2 nmol min(-1)), but not affected during infusion of the antagonist for endothelin ET(B) receptors, BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-Leucyl-1-(met hoxycarbonyl)-D-tryptophyl]-Dnorleucine monosodium), 2 nmol min(-1)). Intravenous administration of NW-nitro-L-arginine methyl ester (L-NAME, 47 mg kg(-1)) or NW-nitro-L-arginine (L-NNA, 47 mg kg(-1)) reduced basal cerebral blood flow by 39 and 33%, increased cerebrovascular resistance by 108 and 98% and mean arterial pressure by 23 and 17%, and decreased heart rate by 27 and 25%, respectively (all at least P < 0.05). The increases in cerebrovascular resistance (as absolute values) induced by endothelin-1 were not affected during either L-NAME or L-NNA (as absolute values and percentages). Intravenous administration of meclofenamate (5 mg kg(-1)) did not change the cerebrovascular effects of endothelin-1 and IRL 1620. In isolated goat cerebral arteries under control, resting conditions, endothelin-1 (10(-11)-10(-7) M) induced concentration-dependent contractions (EC50 = 4.78 X 10(-9) M; maximal contraction = 3177+/-129 mg), whereas IRL 1620 (10(-11)-10(-7) M) produced no effect. This contraction produced by endothelin-1 was competitively blocked by BQ-123 (10(-7)-3 X 10(-6) M), and was not affected by BQ-788 (10(-6) and 10(-5) M). L-NAME (10(-4) M), meclofenamate (10(-5) M), indomethacin (10(-5) M), L-NAME (10(-4) M) plus meclofenamate (10(-5) M) and phosphoramidon (10(-4) M) did not affect the contraction in response to endothelin-1. Endothelium removal increased the response to endothelin-1, as well as the BQ-123 antagonism against endothelin-1 (pA2 values, 7.62 vs. 6.88; P < 0.01). In both intact and de-endothelized arteries precontracted with prostaglandin F2alpha endothelin-1 induced a further contraction, and IRL 1620 caused no effect. These results suggest that: (1) endothelin-1 produces cerebral vasoconstriction by activating endothelin ET(A) receptors probably located in smooth muscle; (2) endothelin ET(B) receptors, nitric oxide and prostanoids might be not involved in the cerebrovascular action of endothelin-1, and (3) endothelium removal may increase cerebrovascular reactivity by increasing sensitivity of endothelin ET(A) receptors to endothelin-1.     

cis-diamminedichloroplatinum(ii) resistance in vitro and in vivo in human embryonal carcinoma cells

In the embryonal carcinoma cell line Tera and its 3.7-fold cis-diamminedichloroplatinum(II) (CDDP)-resistant subline, Tera-CP, parameters were studied that might have changed in relation to induction of CDDP resistance. Phenotypes of both lines were embryonal carcinoma. Karyotypes were related with a decreased mean number of chromosomes and fewer copies of the short arm of chromosome 12 in Tera-CP. Tera-CP showed cross-resistance for melphalan and 4-hydroperoxycyclophosphamide and had an 1.4-fold increased glutathione (GSH) level, a 1.5-fold increased glutathione S-transferase (GST) activity, and a 1.4-fold increased GST pi expression compared to Tera. Tera-CP was cross-resistant to 5-fluorouracil, but thymidylate synthase activity was not increased. Topoisomerase I and II activities and c-myc RNA and protein expression were the same in both lines. Platinum accumulation was equal in both lines, and platinum-DNA binding was lower in Tera-CP than in Tera. Both cell lines were xenografted into nude mice and tumors showed marked differentiation. Tera-CP tumors were 2.8-fold resistant to CDDP compared to Tera tumors. In new cell lines derived from xenografts of Tera and Tera-CP CDDP sensitivity, GST activity and GSH level corresponded with their sensitivity and resistant origin. Tera-CP is a model of in vitro and in vivo CDDP resistance with the GSH/GST detoxifying system as an important mechanism. CDDP resistance could be induced without a concomitant increase in differentiation.     

In vitro and in vivo activity of 3-(1-methyl 1-5-nitro-2-imidazolylmethylideneamino)-2-oxazolidinone against Pasteurella

Furazolidone was more active than 3-(1-methyl)-5-nitro-2-imidazolyl-methylideneamino)-2-oxazolidinone (MABN) against a series of 34 isolates of Pasteurella and 11 of Yersinia (formerly designated Pasteurella). However, the nitroimidazole was superior to furazolidone by both subcutaneous and oral routes against a series of mouse infections incited by strains of Pasteurella. It also was superior to furazolidone and sulfaquinoxaline when administered in the diet against two Pasteurella strains in a fowl cholera model infection in chickens. The good in vitro activity of MABN plus its low toxicity suggest its further study as an agent for fowl cholera and the shipping fever complex of cattle.