Surface acoustic wave concentration of particle and bioparticle suspensions

A rapid particle concentration method in a sessile droplet has been developed using asymmetric surface acoustic wave (SAW) propagation on a substrate upon which the droplet is placed. Due to the asymmetry in the SAW propagation, azimuthal bulk liquid recirculation (acoustic streaming) is generated. Once the local particle concentration is sufficiently high within a particular streamline of the acoustic streaming convective flow, shear-induced migration gives rise to an inward radial force that concentrates the particles at the centre of the droplet. In this paper, a SAW device consists of a 0.75-mm thick, 127.68° Y–X-axis-rotated cut, X-propagating LiNbO₃ for a substrate and an interdigital transducer electrode (IDT) with 25 straight finger pairs in a simple repeating pattern, 12 mm aperture, and a wavelength of λ = 440 μm was patterned on the substrate. The IDT was then driven with a sinusoidal signal at the resonance frequency f ₀ of 8.611 MHz. To investigate the effect of particle type and size on the concentration process, three types of particles were used in this study, including fluorescent particles (1 μm), polystyrene microspheres (3, 6, 20, 45 μm), and living yeast cells (10–20 μm). Different RF powers were applied ranging from 120 to 510 mW. The concentration processes occurs within 2 to 20 s, depending on the particle size, type and input radio frequency (RF) power, much faster than currently available particle concentration mechanisms due to the large convective velocities achieved using the SAW device. Moreover, this concentration method is efficient, concentrating the particles into an aggregate one-tenth the size of the original droplet. Most importantly, bioparticles can also be concentrated by this method; we have verified that yeast cells are not lysed by the SAW radiation during concentration. By using the rapid concentration process described in this work, the breadth of applications and measurement sensitivity of SAW biosensor systems should be greatly enhanced.     

The use of microfluorometric method for activity-guided isolation of antiplasmodial compound from plant extracts

In vitro antiplasmodial activity of methanolic extracts of 16 medicinal plants was evaluated by fluorometric assay using PicoGreen. The IC50s, as determined by parasite DNA concentration, ranged from <11 to >200 and <13 to >200 μg/ml for Plasmodium falciparum 3D7 and K1, respectively; and the most active extracts were those from Anogeissus leiocarpus and Terminalia avicennoides (<11–≥14 μg/ml). Aqueous, butanolic, ethyl acetate, and methanolic fractions of these two extracts revealed butanolic fraction to have a relatively better activity (IC₅₀, 10–12 μg/ml). Activity-guided chromatographic separation of the butanolic fraction on Sephadex LH-20 followed by nuclear magnetic resonance and correlation high-performance liquid chromatography revealed the presence of known hydrolysable tannins and some related compounds—castalagin, ellagic acid, flavogallonic acid, punicalagin, terchebulin, and two other fractions. The IC₅₀s of all these compounds ranged between 8–21 μg/ml (8–40 μM) against both the strains. Toxicity assay with mouse fibroblasts showed all the extracts and isolated compounds to have IC₅₀ ≥ 1500 μg/ml, except for Momordica balsamina with <1500 μg/l. All the extracts and isolated compounds did not affect the integrity of human erythrocyte membrane at the observed IC₅₀s. However, adverse effects manifest in a concentration-dependent fashion (from IC₅₀ ≥ 500 μg/ml).     

Anemia and iron status in young fertile non-professional female athletes

We evaluated the effects of regular physical exercise on anemia and iron status in young non-professional female athletes. A total of 191 healthy white Italian women (23.5 ± 4.68 years) were analyzed; 70 were non-professional athletes performing 11.1 ± 2.63 h week⁻¹ exercise and 121 were sedentary controls. Blood markers of anemia and iron status—hemoglobin (Hb), hematocrit (Hct), red blood cells (RBC), serum ferritin, iron, transferrin (Tf), transferrin saturation (TfS), soluble transferrin receptor (sTfR), and the sTfR/log ferritin ratio (sTfR-F index)—were evaluated. Anemia threshold was Hb < 120 g l^–1. Ferritin concentrations < 12 μg l^–1 were considered as iron deficiency (ID). Frequency of anemia (15.7 versus 10.7%, P = 0.32), ID (27.1 versus 29.8%, P = 0.70), and ID anemia (8.6 versus 5.8%, P = 0.46) was not different in athletes and controls. However, athletes were threefold more likely than controls (17.1 versus 5.8%) to have serum iron < 50 μg dl^–1 [odds ratio (OR) 3.37, P = 0.012]. Low-TfS (<15%) was found in 25.7% of athletes and in 13.2% of controls, OR 2.27, P = 0.030. Elevated-sTfR (>1.76 mg l^–1) was found in 24.3% of athletes and in 12.4% of controls, OR 2.27, P = 0.034. Regular non-professional sport activity does not cause an increased rate of anemia or of iron deficiency in fertile women. However, physical exercise has an impact on iron status as it reduces serum iron and transferrin saturation, and elevates sTfR. Nearly one fifth of recreational athletes have anemia and a third have iron deficit, these conditions can decrease their physical performance.     

The role of exocytosis in the activation of the chloride conductance in Chinese hamster ovary cells (CHO) stably expressing CFTR

 The aim of this study was to examine the question of whether activation of wt-CFTR (wild-type cystic fibrosis transmembrane conductance regulator) by cAMP and the opening of a Cl^–conductance is paralleled by exocytosis and corresponding increases in membrane capacitance. To this end three types of Chinese hamster ovary (CHO) cells were examined: a control group of CHO cells; a group of CHO cells stably expressing wt-CFTR at high levels (also called BQ2-CHO); and a group of CHO cells stably expressing the frequent mutation ΔF508-CFTR. Whole-cell patch-clamp studies were performed to measure the membrane voltage (V _m), the membrane conductance (G _m) and the membrane capacitance (C _m). C _m was assessed by a two-frequency lock-in amplifier method. Forskolin (Fsk, 0.1 μmol/l) and isobutylmethylxanthine (IBMX, 0.1 mmol/l) were used to increase cytosolic cAMP. It is shown that Fsk and IBMX had no effect on V _m and G _m in control CHO and ΔF508-CFTR-CHO cells. Fsk and IBMX depolarized wt-CFTR-expressing CHO cells significantly (from –40 ± 1.5 to –32 ± 1.6 mV, n = 41) and enhanced G _m strongly from 5.0 ± 0.9 to 36 ± 3.9 nS (n = 65). The conductance increase was mostly for Cl^–, because under stimulated conditions a reduction in bath Cl^–concentration depolarized these cells further and significantly from –26 ± 1.8 to –10 ± 1.2 mV (n = 16). This conductance had the characteristic wt-CFTR selectivity of Br^– > Cl^– > I^–(n = 16). Despite this large increase in the Fsk- and IBMX-induced conductance C _m was not altered significantly (15.5 versus 15.7 pF, n = 50). These data indicate that stable overexpression of wt-CFTR but not of ΔF508-CFTR in CHO cells induces a cAMP-activated Cl^–conductance. The activation of this large conductance obviously proceeds with little if any exocytosis. Received: 13 May 1997 / Received after revision: 1 July 1997 / Accepted: 7 July 1997     

The anti-amoebic activity of some medicinal plants used by AIDS patients in southern Thailand

The anti-amoebic activities of chloroform, methanol and water extracts from 12 Thai medicinal plants (39 extracts) commonly used by AIDS patients in southern Thailand were screened, at a concentration of 1,000 μg/ml, against Entamoeba histolytica strain HTH-56:MUTM and strain HM1:IMSS growing in vitro. The extracts were incubated with 2×10⁵ E. histolytica trophozoites/ml of medium at 37°C under anaerobic conditions for 24 h. The cultures were examined with an inverted microscope and scored (1–4) according to the appearance and numbers of the trophozoites. The extracts that caused inhibition were selected and retested using the same conditions but with concentrations that ranged from 31.25 to 1,000 μg/ml using E. histolytica strain HM1:IMSS, and the IC₅₀ values for each extract were calculated. The chloroform extracts from Alpinia galanga (IC₅₀ 55.2 μg/ml), Barleria lupulina (IC₅₀ 78.5 μg/ml), Boesenbergia pandurata (IC₅₀ 45.8 μg/ml), Piper betle (IC₅₀ 91.1 μg/ml) and Piper chaba (IC₅₀ 71.4 μg/ml) and the methanol extract from B. pandurata (IC₅₀ 57.6 μg/ml) were all classified as “active”, i.e. with an IC₅₀ of less than 100 μg/ml, whereas those from Murraya paniculata (IC₅₀ 116.5 μg/ml) and Zingiber zerumbet (IC₅₀ 196.9 μg/ml) were classified as being “moderately active”. The IC₅₀ of a standard drug, metronidazole, was 1.1 μg/ml.     

Rapid decrease of free vancomycin in dense staphylococcal cultures

Bacterial numbers in broth cultures were determined by bioluminescence assay of intracellular bacterial ATP. Broth MICs for strains of Staphylococcus epidermidis (ATCC 14990 and 35984) and Staphylococcus aureus (ATCC 25923, 29213 and 6538) were determined for cultures with different inocula (10⁵–10⁸ bacteria/ml) after 24 h of incubation in supplemented Mueller–Hinton broth containing vancomycin. All of the tested strains except one were susceptible to methicillin, and all of the strains were susceptible to vancomycin. Free vancomycin concentrations in the broth cultures of all strains were determined with an agar well bioassay after 24 h of incubation. Free vancomycin concentrations and bacterial numbers of ATCC 35984 and ATCC 29213 were also determined after 0.5, 2, 4, and 8 h. In a low inoculum (10⁵ bacteria/ml), the broth MICs were 1–4 μg/ml. In a high inoculum (∼10⁸ bacteria/ml), the broth MICs increased two- to fourfold to 4–8 μg/ml. In dense inocula (∼10⁹–10¹⁰ bacteria/ml), the concentrations of free vancomycin in the broth were reduced, in most cases below the detection limit of the bioassay (≤0.5 μg/ml). This reduction of free vancomycin was fast, occurring in initially dense inocula in less than 30 min. No emergence of resistance was seen. These results show a rapid reduction of free vancomycin in the broth and a simultaneous increase in broth MICs in high inocula, without development of resistance. This indicates that the dosing regimen of vancomycin is of particular importance in staphylococcal infections with dense inocula, e.g. infective endocarditis.     

Zschokkella helmii n. sp. (Myxozoa: Myxosporea), a new parasite of marbled spinefoot Siganus rivulatus (Forsskal 1775), Red Sea, Egypt: light and transmission electron microscopy

Zschokkella helmii n. sp., a new parasite of Siganus rivulatus from the Red Sea, Egypt, was described using light and transmission electron microscopy. However, the infection was severe; single “histozoic” plasmodium was encountered in the gallbladder wall. Spores are ellipsoid with 9–11 valvar striations. Spore mean length is 10.8 μm (10.0–11.0), while the spore mean width is 7.5 μm (7.0–8.0). Polar capsules are nearly round with a diameter of 2.2 μm (2.0–3.0) and have five filaments. Ultrastructure of the plasmodial wall and sporogenesis of the present species followed the usual pattern valid for most studied myxosporean species.     

Effect of zoledronic acid and an antibody against bone sialoprotein II on MDA-MB-231GFP breast cancer cells in vitro and on osteolytic lesions induced in vivo by this cell line in nude rats

The aim of this study was to investigate the combined effect of zoledronic acid and an antibody against bone sialoprotein II (BSPII) on proliferation and osteolytic activity of MDA-MB-231^GFP breast cancer cells. For this purpose, the cells were exposed to zoledronic acid (10–20 μg/ml [25–50 μM]) and an anti-BSPII IgY (10–100 μg/ml) for up to 5 days alone or in combination. The combined treatment showed synergistic antiproliferative effects at the higher dose of zoledronic acid. Following inoculation of 1 × 10⁵ MDA-MB-231 ^GFP breast cancer cells into a branch of the femoral artery of nude rats, lytic lesions developed in the tibia, femur or fibula of the injected hind leg after approximately 30 days. The appearance and development of these lesions were monitored radiographically. Rats with lytic lesions were treated with zoledronic acid (60 μg/kg/week sc × 8; n = 10), zoledronic acid and an anti-BSPII IgY antibody (60 μg/kg/week sc × 8 + 10 mg/kg/week sc × 8; n = 10), or left untreated (n = 20). In addition, rats were treated for 4 weeks (n = 10) with both regimens starting right after tumor cell inoculation. Finally, ten rats were treated with zoledronic acid for 2 weeks before tumor cell inoculation (60 μg/kg/week sc × 2). The antiosteolytic effect of zoledronic acid was high as shown by inhibition of osteolytic growth. Addition of the anti-BSPII IgY further decreased the incidence of femoral osteolytic lesions (40% reduction), indicating remineralization, and reduced periosteal defects of cortical bone (20% reduction). These observations favor using the IgY-antibody in addition to zoledronic acid in order to stimulate osteoblast-induced remineralization.     

Determinants for the Development of Oropharyngeal Colonization or Infection by Fluconazole-Resistant Candida Strains in HIV-Infected Patients

 A point prevalence study to document oral yeast carriage was undertaken. Risk factors for the development of oropharyngeal colonization or infection by fluconazole-resistant Candida strains in HIV-infected patients were investigated with a case-control design. Cases included all patients with fluconazole-resistant strains (MIC≥64 μg/ml), and controls were those with susceptible (MIC≤8 μg/ml) or susceptible-dependent-upon-dose (MIC 16–32 μg/ml) strains. One hundred sixty-eight Candida strains were isolated from 153 (88%) patients, 28 (16%) of whom had oropharyngeal candidiasis. Overall, 19 (12%) of the patients harbored at least one resistant organism (MIC≥64 μg/ml). Among patients with resistant strains, tuberculosis (P<0.001), esophageal candidiasis (P=0.001), clinical thrush (P<0.001), and a CD4+ cell count <200/mm³ (P=0.03) were more frequent. These patients had also been treated more commonly with antituberculous drugs (adjusted odds ratio [OR] 6.13; 95% confidence interval [CI] 2.11–17.80), ciprofloxacin (OR 6.0; 95% CI 1.23–29.26), fluconazole (OR 4.59; 95% CI 1.55–13.52), and steroids (OR 4.13; 95% CI 1.11–15.39). Multivariate analysis showed that the determinants for fluconazole resistance were therapy with antituberculous drugs (OR 3.61; 95% CI 1.08–12.07;P=0.03) and one of the following: previous tuberculosis (OR 3.53; 95% CI 1.08–14.57;P=0.03) or fluconazole exposure (OR 3.41; 95% CI 1.10–10.54). Findings from this study indicate that treatment with antituberculous drugs, previous tuberculosis, and fluconazole exposure are the strongest determinants for development of oropharyngeal colonization or infection by fluconazole-resistant Candida strains in HIV-infected patients.     

A Method to Replicate the Microstructure of Heart Tissue <Emphasis Type="Italic">In Vitro</Emphasis> Using DTMRI-Based Cell Micropatterning

A novel cell culture methodology is described in which diffusion tensor magnetic resonance imaging (DTMRI) and cell micropatterning are combined to fabricate cell monolayers that replicate realistic cross-sectional tissue structure. As a proof-of-principle, neonatal rat ventricular myocyte (NRVM) monolayers were cultured to replicate the tissue microstructure of murine ventricular cross-sections. Specifically, DTMRI-measured in-plane cardiac fiber directions were converted into soft-lithography photomasks. Silicone stamps fabricated from the photomasks deposit fibronectin patterns to guide local cellular alignment. Fibronectin patterns consisted of a matrix of 190 μm² subregions, each comprised of parallel lines 11–20 μm-wide, spaced 2–8.5 μm apart, and angled to match local DTMRI-measured fiber directions. Within 6 days of culture, NRVMs established confluent, electrically coupled monolayers, and for 18 μm-wide, 5 μm-spaced lines, directions of cell alignment in subregions microscopically replicated DTMRI-measurements with a local error of 7.2 ± 4.1°. By adjusting fibronectin line widths and spacings, cell elongation, gap junctional membrane distribution, and local cellular disarray were altered without changing the dominant directions of cell alignment in individual subregions. Changes in the anisotropy of electrical propagation were assessed by optically mapping membrane potentials. This novel methodology is expected to enable systematic studies of intramural structure–function relationships in both healthy and structurally remodeled hearts.     

In vitro efficacy of diclofenac against <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>

Chemotherapy is often futile in systemic listeriosis, translating to being a peril to public health. There is, thus, an imperative need for novel antilisterial compounds, possibly acting through mechanisms dissimilar to those of existing drugs. The present study describes one such agent—the non-steroidal anti-inflammatory drug (NSAID) diclofenac sodium (Dc). The National Committee for Clinical Laboratory Standards (NCCLS) minimum inhibitory concentration (MIC), mode of action, and two mechanisms of action, i.e., on bacterial DNA and membrane, have been characterized with respect to Dc. The drug showed noteworthy inhibitory action (MIC₉₀ = 50 μg/ml) against Listeria strains, demonstrated cidal (minimum bactericidal concentration [MBC]=100 μg/ml) activity, inhibited listerial DNA synthesis (45.48%; incorporation of [methyl-3H] thymidine), and possessed bacterial membrane-damaging activity (37.33%; BacLight assay). Dc could be used as a lead compound for the synthesis of new, more active agents perhaps devoid of side effects. Further, quantitative structure–activity relationship (QSAR) studies will contribute to a new generation of promising adjuvants to existing antilisterial drugs.     

Effects of the carcinogen dimethylhydrazine (DMH) on the function of rat colonic crypts

 Rats injected with dimethylhydrazine for 5 weeks (DMH, 40 mg/kg body weight) invariably develop colonic cancer after a latency of some 10–14 weeks. Preliminary studies have suggested that Na⁺ absorption by surface colonic crypt cells is attenuated in the preneoplastic period (8–12 weeks after the first injection of DMH). The present study of glucocorticoid-treated (dexamethasone 6 mg/kg body weight, s.c. 3 days or triamcinolone 30 mg/kg body weight, s.c. 3 days) rats was undertaken to examine the ion transport properties of rat distal colon during this period in more detail. Ussing chamber studies of the distal colon and whole-cell patch-clamp measurements in surface cells, mid-crypt cells and crypt-base cells obtained from isolated crypts were performed. In Ussing chamber studies the equivalent short-circuit current inhibitable by amiloride (10 μmol/l) DMH-treated rats was about 40% of control. In addition, the hyperpolarizing effect of amiloride (10 μmol/l) on membrane voltage (V _m) was strongly attenuated in surface and mid-crypt cells of DMH-treated rats. Carbachol (CCH, 100 μmol/l), which predictably hyperpolarized surface, mid-crypt cells and crypt-base cells of control rats, had no significant effect on V _m in DMH-treated rats, but increased membrane conductance (G _m) significantly. This indicates that CCH probably activates both Cl^–and K⁺ channels in all three colonic crypt compartments in the DMH-treated rats. Forskolin (5 μmol/l), which has the most pronounced effect in crypt-base cells in control rats, depolarized V _m and enhanced G _m in all three compartments in DMH-treated rats. These data indicate that DMH profoundly alters Na⁺ and Cl^–transport in colonic crypts prior to the appearance of colonic adenocarcinoma and that these effects can be summarized as follows: (1) the Na⁺ conductance of surface cells is attenuated; (2) cells along the length of the crypt-lumen axis tend to lose their normal response to CCH and instead show simultaneous and comparable increases in K⁺and Cl^–conductances; (3) the effect of forskolin is enhanced along the entire crypt axis. As a result colonic crypt transport is shifted to predominant Cl^–secretion, findings which are characteristic of colonic carcinoma cell lines such as HT₂₉ and T₈₄ cells. Received: 13 May 1996 / Received after revision: 5 August 1996 / Accepted: 14 October 1996     

Development and characterization of a scalable microperforated device capable of long-term zero order drug release

A drug delivery system that consists of microperforated polyimide microtubes was developed and characterized. Two groups of polyimide tubes were used. One set consisted of microtubes (I.D. = 125 μm) with 32.9 ± 1.7 μm size holes. The second set consisted of larger tubes (I.D. = 1000 μm) with 362–542 μm holes. The number of holes was varied between 1 and 3. The small tubes were loaded with crystal violet (CV) and ethinyl estradiol (EE) and the drug release studies were performed in 0.01 M phosphate buffered saline (PBS) (pH 7.1–7.4) at 37.0 ± 1.0°C for upto 4 weeks. The large tubes were loaded with CV and the drug release was studied in vitro in PBS and also ex vivo in rabbit’s vitreous humor. Linear release rates with R² > 0.9900 were obtained for all groups with CV and EE. Release rates of 7.8 ± 2.5, 16.2 ± 5.5, and 22.5 ± 6.0 ng/day for CV and 30.1 ± 5.8 ng/day for EE were obtained for small tubes. For large tubes, a release rate of 10.8 ± 4.1, 15.8 ± 4.8 and 22.1 ± 6.7 μg/day was observed in vitro in PBS and a release rate of 5.8 ± 1.8 μg/day was observed ex vivo in vitreous humor.     

Effect of tuftsin on the functional activity and intracellular pH of murine peritoneal macrophages

The effect of tuftsin and its tripeptide analog in various concentrations (from 0.001 to 10.0 μg/ml) on phagocytosis and on the intracellular pH is studied in murine peritoneal macrophages. Tuftsin causes a uniform dose-dependent increase of these two parameters in the cells. This effect is maximally pronounced at concentrations of the peptide close to its physiological level (about 0.3 μg/ml) and gradually decreases as its content in the incubation medium is lowered or raised. On the other hand, the tripeptide analog of tuftsin does not exhibit such an effect on the cells and under the same conditions suppresses phagocytosis and acidifies their intracellular medium. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 3, pp. 265–267, March 1994 Presented by I. P. Ashmarin, Member of the Russian Academy of Medical Sciences     

Pervanadate inhibits volume-sensitive chloride current in bovine chromaffin cells

 The role of protein tyrosine phosphorylation in the activation of the volume-sensitive Cl^– current in bovine chromaffin cells was investigated by studying the effects of inhibitors of protein tyrosine kinases (PTKs) and phosphatases (PTPs). The whole-cell current was induced by intracellular guanosine-5’-0-(3-thiotriphosphate) (GTP-[γ-S], 100–250 μM), the nonhydrolysable GTP analogue, or by cell inflation through the patch pipette under voltage-clamp conditions. PTK inhibitors tyrphostin B46 (5–50 μM) and genistein (200 μM) did not inhibit the volume-sensitive Cl^– current nor did they induce it in the absence of other stimuli. In contrast, the PTP inhibitor pervanadate (200 μM) applied intracellularly prevented activation of the current. Voltage-activated Na⁺ and Ca²⁺ currents were unaffected by pervanadate. Neither sodium orthovanadate nor hydrogen peroxide alone mimicked the action of pervanadate. Other PTP inhibitors tested, i.e. ammonium molybdate (10–100 μM), phenylarsine oxide (10 μM), and ZnCl₂ (500 μM), as well as the serine/threonine protein phosphatases inhibitor okadaic acid (200 nM) failed to inhibit the volume-sensitive Cl^– current. It is suggested that the inhibitory action of pervanadate indicates the involvement of protein tyrosine phosphorylation in the regulation of volume-sensitive Cl^– channels in bovine chromaffin cells. The possibility of pervanadate acting via a pathway unrelated to protein phosphorylation is also discussed. Received: 27 June / Received after revision: 15 September / Accepted: 16 September     

Microsporidian parasites: a danger facing marine fishes of the Red Sea

Out of 600 marine fish from the Red Sea belonging to three different species that were collected and examined for microsporidian parasites, 87 (14.5%) fish were found to be infected. The infection was recorded as cysts or xenomas embedded in the gut epithelium and the peritoneal cavity of the three fish species. The highest percent of infection with microsporidian parasites was recorded in Saurida tumbil 19.5% (39/200) followed by Pagrus pagrus 15% (45/300) and the lowest percent of infection was recorded in Epinephelus chlorostigma 3% (three out of 100). After rupture of the cysts, the spores were released and examined by light microscopy. Each spore was elongated to ellipsoidal in shape and possessed a posterior vacuole which is characteristic to phylum Microspora. They measure 1.6 ± 0.5 μm (1.5–2.4 μm) × 1.3 ± 0.1 μm (1.3–2.0 μm) in Saurida tumbil and Pagrus pagrus, respectively. The spores of Pleistophora sp recorded from E. chlorostigma were ovoid to pyriform in shape and measure 1.9 ± 0.5 μm (1.8–2.7 μm) × 1.6 ± 0.4 μm (1.5–2.4 μm).     

Inhibition of platelet aggregation by monoclonal antibodies against glycoprotein IIb–IIIa complex

Monoclonal antibodies CRC64 are obtained against Ca²⁺-dependent glycoprotein IIb–IIIa complex of the platelet membrane which possess the ability to inhibit completely fibrinogen-dependent platelet aggregation. CRC64 is directed against the epitope formed by the glycoprotein IIb–IIIa complex and does not interact with proteins isolated after platelets are treated with ethylenediamine tetraacetate. Complete, reproducible blockade of platelet aggregation caused by 5 μM adenosine diphosphate is noted in an MCA concentration of 3 μg/ml, while in the case of a stronger inductor, namely 1 U/ml thrombin, platelet aggregation is inhibited in a concentration of 5 μg/ml. F(ab′)₂ fragments are also able to inhibit platelet aggregation completely and are usually effective in concentrations lower than native monoclonal antibodies. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 10, pp. 402–405, October, 1994 Presented by V. N. Smirnov, Member of the Russian Academy of Medical Sciences     

Thermal Injury Prediction During Cryoplasty Through <Emphasis Type="Italic">In Vitro</Emphasis> Characterization of Smooth Muscle Cell Biophysics and Viability

Restenosis in peripheral arteries is a major health care problem in the United States. Typically, 30–40% of angioplasties result in restenosis and hence alternative treatment techniques are being actively investigated. Cryoplasty, a novel technique involving simultaneous stretching and freezing of the peripheral arteries (e.g., femoral, iliac, popliteal) using a cryogen-filled balloon catheter, has shown the potential to combat restenosis. However, evaluation of the thermal and biophysical mechanisms that affect cellular survival during cryoplasty is lacking. To achieve this, the thermal history in arteries was predicted for different balloon temperatures using a thermal model. Cellular biophysical responses (water transport (WT) and intracellular ice formation (IIF)) were then characterized, using in vitro model systems, based on the thermal model predictions. The thermal and biophysical effects on cell survival were eventually determined. For this study, smooth muscle cells (SMC) isolated from porcine femoral arteries were used in suspensions and attached in vitro systems (monolayer and fibrin gel). Results showed that for different balloon temperatures, the thermal model predicted cooling rates from 2200 to 5 °C/min in the artery. Biophysical parameters (WT & IIF) were higher for SMCs in attached systems as compared to suspensions. The “combined” fit WT parameters for SMCs in suspension (at 5, 10, and 25 °C/min) are L _pg = 0.12 μm/(min atm) and E _Lp = 24.1 kcal/mol. Individual WT parameters for SMCs in attached cell systems at higher cooling rates are approximately an order of magnitude higher compared to suspensions (e.g., at 130 °C/min, WT parameters in monolayer and fibrin TE systems are L _pg = 18.6, 19.4 μm/(min atm) and E _Lp = 112, 127 kcal/mol, respectively). Similarly, IIF parameters assessed at 130 °C/min are higher for SMCs in attached systems than suspensions ( = 1.1, 354, 378 (× 10⁸ (1/m² s)) and κ_o = 1.6, 1.8, 2.1 (× 10⁹ K⁵) for suspensions, monolayer, and fibrin TE, respectively). One possible reason for the differences in IIF kinetics was verified to be the presence of gap junctions, which facilitate cell–cell connections through which ice can propagate. This is reflected by the change in the predicted IIF parameters when a gap junction inhibitor was added and tested in monolayer ( (1/m² s)); κ_o = 2.1 × 10⁹ K⁵). SMC viability was affected by the model system (lower viability in attached systems), the thermal conditions and the biophysics. For e.g., IIF is lethal to cells and SMC viability was verified to be the least in fibrin TE (most % IIF) and the most in suspensions (least % IIF) at all cooling rates. Using the results from the fibrin TE (suggested as the best in vitro system to mimic a restenosis environment), conservative estimates of injury regimes in the artery during cryoplasty is predicted. The results can be used to suggest future optimizations and modifications during cryoplasty and also to design future in vivo studies.

Schistosomal granuloma modulation

Recurrent experimental evidence indicates that schistosomal egg granuloma formation – at least in the murine model – results from a host response generated against both egg- and worm-derived antigens. Further experiments aimed at identifying the existence in vivo of cross-sensitization between Schistosoma haematobium worms and S. mansoni-derived egg antigens were performed with respect to S. mansoni egg antigen-induced granuloma formation and fibrogenesis in the liver. Male OF1 mice bisexually infected with S. haematobium or S. mansoni were hepatically challenged (cecal vein injection) with S. mansoni SEA (soluble egg antigen)-coupled Sepharose beads at the end of prepatent infection (8–10 days prior to the start of egg deposition). The mean granuloma volume (MGV) of in-vivo-generated synchronized hepatic granulomas (8 days old) and the fibrotic response were estimated. Just like S. mansoni-infected rodents, mice carrying an S. haematobium infection generated an accelerated hepatic granulomogenesis [respective MGVs 4.72 ± 0.56 and 5.41 ± 0.75 × 10⁶ μm³; P < 0.0001 versus unsensitized (MGV 3.00 ± 0.40 × 10⁶ μm³) mice] and an enhanced fibrotic response against S. mansoni SEA. They also had significantly enlarged spleens (P < 0.0001) and moderately enlarged livers (P = 0.02) as compared with S. haematobium-infected mice that were not challenged with SEA. From these observations we infer that in vivo, S. haematobium worms can positively modulate S. mansoni egg antigen-induced granuloma formation and hepatic fibrogenesis, resulting in more severe liver pathology. Received: 10 May 1999 / Accepted: 20 May 1999     

The effects of training intensity on muscle buffer capacity in females

We examined changes in muscle buffer capacity (βm_{in vitro}), and the lactate threshold (LT) after 5 weeks of high-intensity interval training (INT) above the LT or moderate-intensity continuous training (CON) just below the LT. Prior to and immediately after training, 16 female subjects performed a graded exercise test to determine and the LT, followed 2 days later by a resting muscle biopsy from the vastus lateralis muscle to determine βm_{in vitro}. Following baseline testing, the subjects were randomly placed into the INT (n=8) or CON training group (n=8). Subjects then performed 5 weeks of cycle training (3 days per week), performing either high-intensity INT (6–10×2 min at 120–140% LT with 1 min rest) or moderate-intensity CON (80–95% LT) training. Total training volume was matched between the two groups. After the training period, both groups had significant improvements in (12–14%; P<0.05) and the LT (7–10%; P<0.05), with no significant differences between groups. The INT group, however, had significantly greater improvements in βm_{in vitro} (25%; 123±5–153±7 μmol H⁺·g muscle dm⁻¹·pH⁻¹; P<0.05) than the CON group (2%; 130±12–133±7 μmol H⁺·g muscle dm⁻¹·pH⁻¹, P>0.05). Our results show that when matched for training volume, high-intensity interval training above the LT results in similar improvements in and the LT, but greater improvements in βm_{in vitro} than moderate-intensity continuous training below the LT. This suggests that training intensity is an important determinant of changes to βm_{in vitro}.