In vitro and in vivo measurement of red cell velocity with epi- and transillumination

Measurement of red cell velocity with the dual-slit cross-correlation method in glass capillary tubes during transillumination indicates that the measured velocity must be divided by a correction factor of approximately 1.6 to equal the average velocity calculated from a known flow and inner diameter. Whether the same correction factor exists when red cell velocity is measured during epiillumination is questionable. Red cell velocity was measured with the dual-slit correlation method nearly simultaneously using epi- (EL) and transillumination (TL) while glass tubes (40–100 μm, i.d.) were pump perfused with whole human blood (hemotocrit 39–42%). With TL, the measured velocity is 1.58 ± 0.07 (SEM) times the calculated average velocity, whereas a factor of 2.04 ± 0.04 (SEM) was obtained with epiillumination. When intestinal arterioles with approximately the same inner diameters and flow velocities as the glass tubes were used, the ratio of velocities measured with TL to EL was 1.21 ± 0.02 (SEM) as compared to 1.31 ± 0.09 (SEM) for glass tubes using TL and EL of the tube at the same pump flow. This similarity of TL to EL velocity ratios for glass tubes and microvessels may be fortuitous or indicate that comparable flow properties and measurement conditions exist for in vitro and in vivo situations. The major finding of the study is, however, that different velocity correction factors exist for EL and TL measurements when the dual-slit correlation method is used to estimate red cell velocities in tubes of an internal diameter of 40–100 μm at normal hematocrits.     

Perfluorochemical perfusion of the rat heart: in vivo and in vitro

Perfluorochemical (PFC) perfusates were evaluated in this study for inherent differences obtained by perfusion in vivo and of the heart in vitro. The sources of the PFC's were commercial products: Fluosol DA 20%, Fluosol DA 35%, and Fluosol-43. The first two contain both perfluorodecalin (PD) and perfluorotripropylamine (PTPA), while the third utilizes only perfluorotributylamine (PTBA). The hearts studied were excised from control animals or from animals that were first exchange perfused in vivo with Fluosol-43. For studies in vitro hearts perfused in the presence of PTBA beat at a normal rate for 10 to 12 h and then gradually slowed. By comparison, hearts perfused with Krebs-Henseleit solution (KHS) or PD-PTPA containing perfusates maintained a normal heart rate only briefly and ceased beating within 5 to 10 h. However, excised hearts from animals perfused previously in vivo with the PD-PTPA (35%) mixture showed an initial increase followed by a rapid drop in cAMP and cGMP concentrations of the left ventricle. When the other two PFC-containing perfusates were used, no significant changes were found. Only the PTBA-containing mixture maintained normal levels of the two nucleotides and Na⁺, K⁺ and Ca²⁺ levels of the left ventricle in vitro. Also, for studies in vitro when linoleic (0.156 μm) and palmitic (0.086 μm) acids were added simultaneously to all PFC preparations the PTBA-containing perfusate maintained a normal heart rate for over 10 h. Beating hearts were maintained most effectively when hydroxyethylstarch, an oncotic component, was present at 3% (w/v). These studies demonstrate that suitable PFC-containing perfusates can maintain beating rat hearts for many hours at 37°C.     

Uric acid suppresses 1 alpha hydroxylase in vitro and in vivo

Objective

Patients with gout have lower calcitriol levels that improve when uric acid is lowered. The mechanism of these observations is unknown. We hypothesized that uric acid inhibits 1-αhydroxylase.

Materials and methods

In vivo, Sprague Dawley rats were randomized to control (n = 5), allantoxanamide (n = 8), febuxostat (n = 5), or allantoxanamide + febuxostat (n = 7). Vitamin D, PTH, and 1-αhydroxylase protein were evaluated. In order to directly evaluate the effect of uric acid on 1-αhydroxylase, we conducted a series of dose response and time course experiments in vitro. Nuclear factor κ-B (NFκB) was inhibited pharmacologically. Finally, to evaluate the potential implications of these findings in humans, the association between uric acid and PTH in humans was evaluated in a cross-sectional analysis of data from the NHANES (2003–2006); n = 9773.

Results

1,25(OH)₂D and 1-αhydroxylase protein were reduced in hyperuricemic rats and improved with febuxostat treatment. Uric acid suppressed 1-αhydroxylase protein and mRNA expression in proximal tubular cells. This was prevented by NFκB inhibition. In humans, for every 1 mg/dL increase in uric acid, the adjusted odds ratio for an elevated PTH (> 65 pg/mL) was 1.21 (95% C.I. 1.14, 1.28; P < 0.0001), 1.15 (95% C.I. 1.08, 1.22; P < 0.0001), and 1.16 (95% C.I. 1.03, 1.31; P = 0.02) for all subjects, subjects with estimated GFR ≥ 60, and subjects with estimated GFR < 60 mL/min/1.73 m² respectively.

Conclusion

Hyperuricemia suppresses 1-αhydroxylase leading to lower 1,25(OH)₂D and higher PTH in rats. Our results suggest this is mediated by NFκB. The association between uric acid and PTH in NHANES suggests potential implications for human disease.     

In vivo but not in vitro leptin enhances lymphocyte proliferation in Siberian hamsters (Phodopus sungorus)

Mounting an immune response requires a relatively substantial investment of energy and marked reductions in energy availability can suppress immune function and presumably increase disease susceptibility. We have previously demonstrated that a moderate reduction in energy stores by partial surgical lipectomy impairs humoral immunity of Siberian hamsters (Phodopus sungorus) and is mediated, in part, by changes in the adipose tissue hormone leptin. The goals of the present study were to assess the role of leptin in cell-mediated immunity and to determine if the potential effects of leptin on immunity are via the direct actions of this hormone on lymphocytes, or indirect, via the sympathetic nervous system (SNS). In Experiment 1, hamsters received osmotic minipumps containing either murine leptin (0.5 μl/h) or vehicle alone for 10 days and splenocyte proliferation in response to the T-cell mitogen Concanavalin A (Con A) was determined. In Experiment 2, Con A-induced splenocyte proliferation was tested in the presence or absence of leptin in vitro. In Experiment 3, exogenous leptin was administered to intact or sympathetically denervated hamsters. Hamsters treated with in vivo leptin displayed increased splenocyte proliferation compared with control hamsters receiving vehicle. In contrast, in vitro leptin had no effect on splenocyte proliferation. Sympathetic denervation attenuated, but did not block, leptin-induced increases in immunity. Taken together, these results are consistent with the idea that leptin can enhance cell-mediated immunity; the SNS appears to contribute, least in part, to leptin-induced increases in immunity. Importantly, these findings confirm previous studies that leptin serves as an important endocrine link between energy balance and immunity.     

Diarylheptanoid compounds from Alnus nepalensis express in vitro and in vivo antifilarial activity

A large number of medicinal plants remain to be explored for antifilarial compounds. In the present study a crude methanolic extract of leaves of Alnus nepalensis, chloroform- and n-butanol-partitioned fractions from the crude extract and 6 bioactivity-guided isolated compounds including two new diarylheptanoid from the fractions were assayed for microfilaricidal, macrofilaricidal and female worm sterilizing activity using the lymphatic filariid Brugia malayi in in vitro and in vivo systems. In vitro, the crude methanolic extract exerted better microfilaricidal (LC100: 15.63 μg/ml, IC50: 6.00 μg/ml) than macrofilaricidal (LC100: >250; IC50: 88 μg/ml) activity whereas chloroform and n-butanol fractions were more macrofilaricidal (LC100: 125 and 31.25 μg/ml; IC50: 13.14 and 11.84, respectively) than microfilaricidal (LC100: 250–500 μg/ml, IC50: 44.16 μg/ml). In addition, n-butanol fraction also caused 74% inhibition in MTT reduction potential of the adult worms. In vivo (doses: crude: 100–200 mg/kg; fractions: 100 mg/kg, i.p. × 5 days) the chloroform fraction exerted >50% macrofilaricidal activity whereas methanolic extract and n-butanol fraction produced 38–40% macrofilaricidal action along with some female sterilizing efficacy. Of the 5 diarylheptanoid compounds isolated, alnus dimer, and (5S)-5-hydroxy-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-3-heptanone were found to show the most potent with both macrofilaricidal (LC100: 15.63 μg/ml, IC50: 6.57–10.31 μg/ml) and microfilaricidal (LC100: 31.25–62.5 μg/ml, IC50: 11.05–22.10 μg/ml) activity in vitro. These findings indicate that the active diarylheptanoid compounds may provide valuable lead for design and development of new antifilarial agent(s).     

Antifilarial activity in vitro and in vivo of some flavonoids tested against Brugia malayi

We evaluated the antifilarial activity of 6 flavonoids against the human lymphatic filarial parasite Brugia malayi using an in vitro motility assay with adult worms and microfilariae, a biochemical test for viability (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-reduction assay), and two animal models, Meriones unguiculatus (implanted adult worms) and Mastomys coucha (natural infections). In vitro, naringenin and hesperetin killed the adult worms and inhibited (>60%) MTT-reduction at 7.8 and 31.2 μg/ml concentration, respectively. Microfilariae (mf) were killed at 250-500 μg/ml. The half maximal inhibitory concentration (IC₅₀) of naringenin for motility of adult females was 2.5 μg/ml. Flavone immobilized female adult worms at 31.2 μg/ml (MTT > 80%) and microfilariae at 62.5 μg/ml. Rutin killed microfilariae at 125 μg/ml and inhibited MTT-reduction in female worms for >65% at 500 μg/ml. Naringin had adulticidal effects at 125 μg/ml while chrysin killed microfilariae at 250 μg/ml. In vivo, 50 mg/kg of naringenin elimiated 73% of transplanted adult worms in the Meriones model, but had no effect on the microfilariae in their peritoneal cavity. In Mastomys, the same drug was less effective, killing only 31% of the naturally acquired adult worms, but 51%, when the dose was doubled. Still, effects on the microfilariae in the blood were hardly detectable, even at the highest dose. In summary, all 6 flavonoids showed antifilarial activity in vitro, which can be classed, in a decreasing order: naringenin > flavone = hesperetin > rutin > naringin > chrysin. In jirds, naringenin and flavone killed or sterilized adult worms at 50 mg/kg dose, but in Mastomys, where the parasite produces a patent infection, only naringenin was filaricidal. Thus naringenin and flavone may provide a lead for design and development of new antifilarial agent(s). This is the first report on antifilarial efficacy of flavonoids.     

Pentamidine exerts in vitro and in vivo anti Trypanosoma cruzi activity and inhibits the polyamine transport in Trypanosoma cruzi

Pentamidine is an antiprotozoal and fungicide drug used in the treatment of leishmaniasis and African trypanosomiasis. Despite its extensive use as antiparasitic drug, little evidence exists about the effect of pentamidine in Trypanosoma cruzi, the etiological agent of Chagas’ disease. Recent studies have shown that pentamidine blocks a polyamine transporter present in Leishmania major; consequently, its might also block these transporters in T. cruzi. Considering that T. cruzi lacks the ability to synthesize putrescine de novo, the inhibition of polyamine transport can bring a new therapeutic target against the parasite. In this work, we show that pentamidine decreases, not only the viability of T. cruzi trypomastigotes, but also the parasite burden of infected cells. In T. cruzi-infected mice pentamidine decreases the inflammation and parasite burden in hearts from infected mice. The treatment also decreases parasitemia, resulting in an increased survival rate. In addition, pentamidine strongly inhibits the putrescine and spermidine transport in T. cruzi epimastigotes and amastigotes. Thus, this study points to reevaluate the utility of pentamidine and introduce evidence of a potential new action mechanism. In the quest of new therapeutic strategies against Chagas disease, the extensive use of pentamidine in human has led to a well-known clinical profile, which could be an advantage over newly synthesized molecules that require more comprehensive trials prior to their clinical use.     

Histamine H1-receptor antagonists against Leishmania (L.) infantum: an in vitro and in vivo evaluation using phosphatidylserine-liposomes

Considering the limited and toxic therapeutic arsenal available for visceral leishmaniasis (VL), the drug repositioning approach could represent a promising tool to the introduction of alternative therapies. Histamine H1-receptor antagonists are drugs belonging to different therapeutic classes, including antiallergics and anxyolitics. In this work, we described for the first time the activity of H1-antagonists against L. (L.) infantum and their potential effectiveness in an experimental hamster model. The evaluation against promastigotes demonstrated that chlorpheniramine, cinnarizine, hydroxyzine, ketotifen, loratadine, quetiapine and risperidone exerted a leishmanicidal effect against promastigotes, with IC₅₀ values in the range of 13–84 μM. The antihistaminic drug cinnarizine demonstrated effectiveness against the intracellular amastigotes, with an IC₅₀ value of 21 μM. The mammalian cytotoxicity was investigated in NCTC cells, resulting in IC₅₀ values in the range of 57–229 μM. Cinnarizine was in vivo studied as a free formulation and entrapped into phosphatidylserine-liposomes. The free drug was administered for eight consecutive days at 50 mg/kg by intraperitoneal route (i.p.) and at 100 mg/kg by oral route to L. infantum-infected hamsters, but showed lack of effectiveness in both regimens, as detected by real time PCR. The liposomal formulation was administered by i.p. route at 3 mg/kg for eight days and reduced the parasite burden to 54% in liver when compared to untreated group; no improvement was observed in the spleen of infected hamsters. Cinnarizine is the first antihistaminic drug with antileishmanial activity and could be used as scaffold for drug design studies for VL.     

Aqueous leaf extracts display endocrine activities in vitro and disrupt sexual differentiation of male Xenopus laevis tadpoles in vivo

The occurrence of natural substances acting as endocrine disrupting compounds (EDC) in the environment is to date poorly understood. Therefore, (anti)androgenic and (anti)estrogenic activities of three different aqueous leaf extracts (beech, reed and oak) were analyzed in vitro using yeast androgen and estrogen screen. The most potent extract was selected for in vivo exposure of Xenopus laevis tadpoles to analyze the potential effects on development and reproductive biology of amphibians. Tadpoles were exposed from stage 48 to stage 66 (end of metamorphosis) to aqueous oak leaf extracts covering natural occurring environmental concentrations of dissolved organic carbon. Gene expression analyses of selected genes of the hypothalamus-pituitary-gonad and of the hypothalamus-pituitary-thyroid axis as well as histological investigation of gonads and thyroid glands were used to evaluate endocrine disrupting effects on the reproductive biology and development. Female tadpoles remained unaffected by the exposure whereas males showed severe significant histological alterations of testes at the two highest oak leaf extract concentrations demonstrated by the occurrence of lacunae and oogonia. In addition, a significant elevation of luteinizing hormone β mRNA expression with increasing extract concentration in male tadpoles indicates an involvement of hypothalamus-pituitary-gonad axis mainly via antiandrogenic activity. These results suggest that antiandrogenic EDC of oak leaf extract are responsible for inducing the observed effects in male tadpoles. The present study demonstrates for the first time that in surface waters, natural occurring oak leaf compounds at environmentally relevant concentrations display antiandrogenic activities and have considerable effects on the endocrine system of anurans affecting sexual differentiation of male tadpoles.     

Disparity between in vivo and in vitro synthesis of yolk protein by fat bodies of vitellogenic Locusta migratoria after allatectomy

Allatetomy of female locusts at an early stage of oocyte maturation halts subsequent development of terminal oocytes, but this is not due to lack of vitellogenin in the hemolymph. The fat body ceases to secrete vitellogenin in vivo shortly after allatectomy. When excised and incubated in vitro such fat bodies do actively synthesize vitellogenin even 3 days after allatectomy. These results suggest a negative control of vitellogenesis, released as a consequence of allatectomy, and only operative on the fat body in vivo.     

Lmcd1/Dyxin, a novel Z-disc associated LIM protein, mediates cardiac hypertrophy in vitro and in vivo

To identify new mediators of cardiac hypertrophy, we performed a genome-wide mRNA screen of stretched neonatal rat cardiomyocytes (NRCMs). In addition to known members of the hypertrophic gene program, we found the novel sarcomeric Z-disc LIM protein Lmcd1/Dyxin markedly upregulated. Consistently, Lmcd1 was also induced in several mouse models of myocardial hypertrophy suggesting a causal role in cardiac hypertrophy. We overexpressed Lmcd1 in NRCM, which led to cardiomyocyte hypertrophy and induction of the hypertrophic gene program. Likewise, the calcineurin-responsive gene RCAN1-4 was found significantly upregulated. Conversely, knockdown of Lmcd1 blunted the response to hypertrophic stimuli such as stretch and phenylephrine (PE), suggesting that Lmcd1 is required for the hypertrophic response. Furthermore, PE-mediated activation of calcineurin was completely blocked by knockdown of Lmcd1. To confirm these results in vivo, we generated transgenic mice with cardiac-restricted overexpression of Lmcd1. Despite normal cardiac function, adult transgenic mice displayed significant cardiac hypertrophy, again accompanied by induction of hypertrophic marker genes such as ANF and α-skeletal actin. Likewise, Rcan1-4 was found upregulated. Moreover, when crossed with transgenic mice overexpressing constitutionally active calcineurin, Lmcd1 transgenic mice revealed an exacerbated cardiomyopathic phenotype with depressed contractile function and further increased cardiomyocyte hypertrophy. We show that the novel z-disc protein Lmcd1/Dyxin is significantly upregulated in several models of cardiac hypertrophy. Lmcd1/Dyxin potently induces cardiomyocyte hypertrophy both in vitro and in vivo, while knockdown of this molecule prevents hypertrophy. Mechanistically, Lmcd1/Dyxin appears to signal through the calcineurin pathway. Lmcd1/Dyxin may thus represent an attractive target for novel antihypertrophic strategies.     

Testosterone-dependent transformation of nephronic tubule cells into serous and mucous gland cells in stickleback kidneys in vivo and in vitro

When male sticklebacks come into breeding condition, the cells of the second proximal segments and collecting tubules of the nephrons are transformed into cells producing secretions used for nest building. This investigation is aimed at determining whether this transformation of renal tubule cells is induced directly by androgens. To this end, renal tissue is cultured with androgen supplement, either methyltestosterone or 11-ketotestosterone. The results are compared with the effects of methyltestosterone on the tubule cells in castrated males. In the nephronic tubules of methyltestosterone-treated fish, the basal labyrinth—and thus most of the ion reabsorbing capacity of the tubules—disappears within 4 days, probably as a result of autophagous digestion. During this period the cells become considerably enlarged and an extensive granular endoplasmic reticulum develops. The Golgi apparatus proliferates and starts to form secretory granules. In renal tissue cultured for 10 days with methyltestosterone, secretory granules are observed in the second proximal tubule cells, but no secretory activity can be detected in the collecting tubules. After 5 days in cultures supplemented with 11-ketotestosterone, however, signs of secretory activity are apparent already in the second proximal tubules, and to a lesser extent, in the collecting tubules. After 10 days of incubation with 11-ketotestosterone, glandular transformation is almost completed. The results provide strong evidence that the transformation of nephronic tubule cells into mucus-secreting cells in sexually mature sticklebacks is due to a direct effect of androgen. It can be concluded then that, at least in vitro, 11-ketotestosterone may be considered more androgenic than methyltestosterone.     

Extravascular protein measurements in vivo and in situ by ultramicrospectrophotometry

By means of ultramicrospectrophotometry the amount of extravascular plasma protein in the perivasal connective tissue was determined in vivo and in situ, using as an object the mesenterial plate of an everted intestinal loop of the rat. As with the circulating blood plasma, the protein content in the perivascular space lies in the range of 7–52%. We calculated the mean protein amount in 1 g of interstitial tissue as 3.4 mg. Outside the wall of venules the protein content was significantly higher than around capillaries or arterioles. Further from the vascular wall, the protein content decreases in the interstitial tissue. These concentration gradients were significantly higher around the venules than around the arterioles. The extravascular protein in steady state conditions of permeability is, therefore, not equally distributed within the interstitial space.     

The non-synonymous polymorphism at position 114 of the WRN protein affects cholesterol efflux in vitro and correlates with cholesterol levels in vivo

Werner syndrome (WS) is a recessive disorder characterized by the premature onset of a number of age-related diseases. The objective of the present study was to examine the degree of associations between non-synonymous coding Single Nucleotide Polymorphisms (SNPs) in the WRN gene and markers of obesity, diabetes, and hypertension using meta-analyses publically available and to test their effect in WS fibroblasts. The P-value, after genomic control correction, for each non-synonymous coding SNP present in the WRN gene was retrieved from the International Consortium for Blood Pressure Genome-Wide Association Study, the Genome Wide Associations Scans for Total Cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides, and the Meta-Analyses of Glucose and Insulin-related traits Consortium. For SNPs significantly associated with cholesterol traits, we generated expression vectors containing the amino acid changes and measured cholesterol uptake and efflux in transfected WS fibroblasts. One SNP (rs2230009) changing a valine for an isoleucine at position 114 of the WRN protein was nominally associated with cholesterol and LDL-cholesterol measurements (P-values < 0.05). Interestingly, a WRN cDNA expression vector bearing a valine at position 114 instead of isoleucine significantly affected cholesterol efflux in WS fibroblasts. These results implicate a functional effect of this WRN polymorphism on cholesterol metabolism.     

Prolactin release in vitro and in vivo in the pigeon and the domestic fowl following administration of synthetic thyrotrophin-releasing factor (TRF)

Chicken and pigeon pituitaries were incubated in vitro in the presence of different concentrations of hypothalamic extract and of synthetic thyrotrophin-releasing factor (TRF). It was found that prolactin, measured by pigeon crop-sac bioassay, was released in both species. The amount of prolactin released from the chicken pituitaries was increased at the higher doses of TRF, the increase occurring at a dose which corresponded to the decrease in the biphasic TSH secretion. In the pigeon, prolactin and growth hormone release in vitro were measured by an electrophoretic-densitometric method. Both prolactin synthesis and release showed a biphasic response to the dose level of TRF, as did growth hormone synthesis.Thyrotrophin-releasing factor administered in vivo in pigeons resulted in a significant cropsac response, which was biphasic with respect to dose of TRF. There were also corresponding changes in pituitary prolactin content.     

Enhancement of ultrasonic thrombus imaging using novel liposomal bubbles targeting activated platelet glycoprotein IIb/IIIa complex—in vitro and in vivo study

Background

We developed perfluorocarbon gas-containing bubble liposomes (BL) with Arg-Gly-Asp (RGD) sequence-containing peptides, which bind to activated platelet glycoprotein IIb/IIIa complexes. The aim of this study was to examine the enhancing effects in ultrasonic thrombus imaging using these targeted BL in vitro and in vivo.

Methods

Liposomes composed of phosphatidylcholine and cholesterol were manufactured, and RGD peptide was attached by a covalent coupling reaction. Sonication was used to conjugate liposomes and perfluorocarbon gas, which formed targeted BL. In vitro, targeted BL were mixed with whole blood, which was allowed to coagulate while being shaken and rotated. In vivo, we administered targeted BL to 10 rabbits with acute thrombotic occlusions in the ilio-femoral artery. Thrombi were imaged using a 7.5-9 MHz linear transducer and a conventional ultrasound machine, and by scanning electron microscopy. Ultrasound images were digitized, and mean pixel gray-scale level (black = 0, white = 255) was measured.

Results

In vitro, mean pixel gray-scale level of the thrombi in targeted BL group was significantly higher than in control and non-targeted BL groups (93 ± 26 vs. 58 ± 16, 48 ± 9, p = 0.002, n = 10). Scanning electron microscopy revealed large amounts of targeted BL attached to the thrombi. In vivo, mean pixel gray-scale level of the thrombi with targeted BL was significantly higher (33.2 ± 6.4 vs. 24.8 ± 8.5, p = 0.0051, n = 10) than that before targeted BL administration.

Conclusions

Perfluorocarbon gas-containing BL with RGD peptide represent a novel echo contrast agent, which can markedly enhance ultrasonic thrombus imaging in vitro and in vivo, and may be useful for noninvasively diagnosing acute thrombotic vessel occlusion.