The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.

Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specific beta subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both the in vitro and in vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles, in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greater in vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with both alpha 2,3 and alpha 2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greater in vitro biological potency compared to those of the pituitary human follicle stimulating hormone.

Pentoxifylline modifies three-dimensional collagen lattice model contraction and expression of collagen types I and III by human fibroblasts derived from post-burn hypertrophic scars and from normal skin

Fibroblasts are thought to be partially responsible for the persisting contractile forces that result in burn contractures. Using a monolayer cell culture and fibroblast populated collagen lattice (FPCL) three-dimensional model we subjected hypertrophic scar and non-cicatricial fibroblasts to the antifibrogenic agent pentoxifylline (PTF - 1 mg/mL) in order to reduce proliferation, collagen types I and III synthesis and model contraction. Fibroblasts were isolated from post-burn hypertrophic scars (HSHF) and non-scarred skin (NHF). Cells were grown in monolayers or incorporated into FPCL`s and exposed to PTF. In monolayer, cell number proliferation was reduced (46.35% in HSHF group and 37.73% in NHF group, p < 0.0001). PTF selectively inhibited collagen III synthesis in the HSHF group while inhibition was more evident to type I collagen synthesis in the NHF group. PTF also reduced contraction in both (HSHF and NHF) FPCL. (C) 2009 Elsevier Ltd and ISBI. All rights reserved.

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on hormone secretion from sheep pituitary cells in vitro

Although vasoactive intestinal polypeptide (VIP) is thought to be a prolactin releasing factor, in vivo studies on sheep suggest that it is inactive in this species. Recent studies, based primarily on the rat, suggest that the related pituitary adenylate cyclase-activating polypeptide (PACAP) is also a hypophysiotrophic factor but again in sheep, this peptide has no in vivo effects on hormone secretion despite being a potent activator of adenylate cyclase in vitro. This lack of response to either peptide in vivo in sheep could be due to the low concentration of peptide that reaches the pituitary gland following peripheral injection. In the present study we therefore adopted an alternative approach of evaluating in vitro effects of these peptides on GH, FSH, LH or prolactin secretion from dispersed sheep pituitary cells. In a time-course study, PACAP (1 mu mol/l) increased GH concentrations in the culture medium between 1 and 4 h and again at 12 h but had no effect in the 6 and 24 h incubations. Prolactin, LH and FSH were not affected by PACAP. The response to various concentrations of PACAP (1 nmol/l-1 mu mol/l) were then evaluated using a 3 h incubation. Again prolactin and LH were not affected by PACAP and there was a small increase in GH concentrations but only at high concentrations of PACAP (0.1 and 1 mu mol/l; P<0.05), PACAP also stimulated FSH secretion in cells from some animals although this effect was small, The GH response to PACAP was inhibited by PACAP(6-38), a putative PACAP antagonist; but not by (N-Ac-Tyr(1), D-Arg(2))-GHRH(1-29)-NH2, a GH-releasing hormone (GHRH) antagonist. The cAMP antagonist Rp-cAMPS was unable to block the GH response to PACAP suggesting that cAMP does not mediate the secretory response to this peptide. At incubation times from 1-24 h, VIP (1 mu mol/l) had no effects on prolactin, LH or GH secretion and, in a further experiment based on a 3 h incubation, concentrations of VIP from 1 nmol/l-1 mu mol/l were again without effect on prolactin concentrations. Interactions between PACAP and gonadotrophin releasing hormone (GnRH), GHRH and dopamine were also investigated. PACAP (1 nmol/l-1 mu mol/l) did not affect the gonadotrophin or prolactin responses to GnRH or dopamine respectively. However, at a high concentration (1 mu mol/l), PACAP inhibited the GH response to GHRH. In summary, these results show that PACAP causes a modest increase in FSH and GH secretion from sheep pituitary cells but only at concentrations of PACAP that are unlikely to be in the physiological range. The present study confirms that VIP is not a prolactin releasing factor in sheep.

Melphalan dosing regimens for management of recurrent melanoma by isolated limb perfusion: Application of a physiological pharmacokinetic model based on melphalan distribution in the isolated perfused rat hindlimb

The optimal dosing schedule for melphalan therapy of recurrent malignant melanoma in isolated limb perfusions has been examined using a physiological pharmacokinetic model with data from isolated rat hindlimb perfusions (IRHP), The study included a comparison of melphalan distribution in IRHP under hyperthermia and normothermia conditions. Rat hindlimbs were perfused with Krebs-Henseleit buffer containing 4.7% bovine serum albumin at 37 or 41.5 degrees C at a flow rate of 4 ml/min. Concentrations of melphalan in perfusate and tissues were determined by high performance liquid chromatography with fluorescence detection, The concentration of melphalan in perfusate and tissues was linearly related to the input concentration. The rate and amount of melphalan uptake into the different tissues was higher at 41.5 degrees C than at 37 degrees C. A physiological pharmacokinetic model was validated from the tissue and perfusate time course of melphalan after melphalan perfusion. Application of the model involved the amount of melphalan exposure in the muscle, skin and fat in a recirculation system was related to the method of melphalan administration: single bolus > divided bolus > infusion, The peak concentration of melphalan in the perfusate was also related to the method of administration in the same order, Infusing the total dose of melphalan over 20 min during a 60 min perfusion optimized the exposure of tissues to melphalan whilst minimizing the peak perfusate concentration of melphalan. It is suggested that this method of melphalan administration may be preferable to other methods in terms of optimizing the efficacy of melphalan whilst minimizing the limb toxicity associated with its use in isolated limb perfusion.

The effect of the aged garlic extract, 'Kyolic', on the development of experimental atherosclerosis

The aged garlic extract 'Kyolic' lowers serum cholesterol levels in humans and experimental animals and thus is presumed to have a protective effect against atherosclerosis. However, to date no studies have examined the effect of this substance on the actual development of the disease. In the present study, the right carotid artery of 24 rabbits was de-endothelialized by balloon catheterisation in order to produce a myointimal thickening. After 2 weeks the rabbits were randomly assigned to four groups: Group I received a standard diet; Group II received the standard diet supplemented with 800 mu 1/kg body weight/day 'Kyolic'; Group III received a 1% cholesterol supplemented standard diet; and Group IV received a 1% cholesterol supplemented standard diet plus 'Kyolic'. After 6 weeks, the cholesterol diet caused a 6-fold increase in serum cholesterol level (Group III; 6.4 +/- 0.6 mmol/1) compared to normal diet (Group I; 1.2 +/- 0.4 mmol/1) (P < 0.05) with only a minor, non-significant reduction seen by the addition of 'Kyolic' (Group IV; 6.2 +/- 0.7 mmol/l). Group III rabbits developed fatty streak lesions covering approximately 70 +/- 8% of the surface area of the thoracic aorta, which was significantly reduced to 25 +/- 3% in the 'Kyolic'-treated Group IV. No lesions were present in Groups I and II. The hypercholesterolaemic diet caused an increase in aortic arch cholesterol (2.1 +/- 0.1 mg cholesterol/g tissue) which was significantly reduced by 'Kyolic' supplementation (1.7 +/- 0.2 mg cholesterol/g tissue) (P < 0.05). 'Kyolic' significantly inhibited the development of thickened, lipid-filled lesions in the pre-formed neointimas produced by balloon-catheter injury of the right carotid artery in cholesterol-fed rabbits (intima as percent of artery wall, Group III 42.6 +/- 6.5% versus Group IV 23.8 +/- 2.3%, P < 0.01), but had little effect in rabbits on a standard diet (Group II 18.4 +/- 5.0% versus Group I 16.7 +/- 2.0%). In vitro studies showed that 'Kyolic' has a direct effect on inhibition of smooth muscle proliferation. In conclusion,'Kyolic' treatment reduces fatty streak development, vessel wall cholesterol accumulation and the development of fibro fatty plaques in neointimas of cholesterol-fed rabbits, thus providing protection against the onset of atherosclerosis. (C) 1997 Elsevier Science Ireland Ltd.

A comparison of the expressive communication skills of triplet, twin and singleton children

Few studies have focused on the language acquisition of higher multiple birth sets. In this study, the communication skills of 51 triplet children are described. The measures used were: mean length of utterance; type-token ratio; conversational nets; phoneme repertoire; and number of different types of phonological processes used. The data gained were used to compare the communication skills of triplets with those of twins, singletons and normative data available in the literature. Siblings within triplet sets were also compared using language samples obtained from adult-child interactions and when the three children were playing together. The results indicated that the triplets' early communication skills were different from those of both singletons and twins. The triplets' difficulties included delayed syntactic development, limited use of different language functions and delayed phonological development. In contrast, twins' communication profile is characterised by disordered phonological development.

In vitro infectivity of species of Leishmania (Viannia) responsible for American cutaneous leishmaniasis

There is little available information regarding the infectivity of New World Leishmania species, particularly those from the Amazonian Brazil, where there are six species of the subgenus Viannia causing American cutaneous leishmaniasis (ACL). The aim of this study was to compare, in vitro, the potential infectivity of the following Leishmania (Viannia) spp.: L. (V.) braziliensis from localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL) patients, L. (V.) guyanensis, L. (V.) shawi, L. (V.) lainsoni and L. (V.) naiffi from LCL patients only, in cultured BALB/c mice peritoneal macrophage, as well as the production of NO by the infected cells. The infectivity of parasites was expressed by the infection index and, the nitric oxide (NO) production in the macrophage culture supernatant was measured by the Griess method. It was found that L. (V.) braziliensis from MCL, the more severe form of disease, showed the highest (p <= 0.05) infection index (397), as well as the lowest NO production (2.15 mu M) compared with those of other species. In contrast, L. (V.) naiffi which is less pathogenic for the human showed the lowest infection index (301) and the highest NO production (4.11 mu M). These results demonstrated a negative correlation between the infectivity and the ability of these parasites to escape from the microbicidal activity of the host cell.

Therapeutic evaluation of free and liposome-loaded furazolidone in experimental visceral leishmaniasis

Drug delivery systems are promising pharmaceutical formulations used to improve the therapeutic index of drugs. In this study, we developed a liposomal formulation of furazolidone that targets Leishmania (Leishmania) chagasi amastigotes in a hamster model. Using laser scanning confocal microscopy, it was demonstrated that the liposomal drug co-localised with L. (L.) chagasi amastigotes within macrophages. Liposomal furazolidone administered intraperitoneally at 0.5 mg/kg for 12 consecutive days reduced spleen (74%) and liver (32%) parasite burden at a 100-fold lower dose than the free drug. Free furazolidone (50 mg/kg) also effectively reduced spleen (82.5%) and liver (85%) parasites; its in vitro activity against promastigotes and intracellular amastigotes demonstrated a high degree of parasite selectivity. Thus, furazolidone, both in the free and liposome-loaded formulation, is an effective inhibitor of L. (L.) chagasi, representing a possible cost-effective drug candidate for the treatment of visceral leishmaniasis. (C) 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

CD1a and Factor XIIIa Immunohistochemistry in Leprosy: A Possible Role of Dendritic Cells in the Pathogenesis of Mycobacterium leprae Infection

Leprosy is a curable chronic granulomatous infectious disease caused by the bacillus Mycobacterium leprae. This organism has a high affinity for skin and peripheral nerve cells. In the evolution of infections, the immune status of patients determines the disease expression. Dendritic cells are antigen-presenting cells that phagocytose particles and microorganisms. In skin, dendritic cells are represented by epidermal Langerhans cells and dermal dendrocytes, which can be identified by expression of CD1a and factor XIIIa (FXIIIa). In the present study, 29 skin samples from patients with tuberculoid (13 biopsies) and lepromatous (16 biopsies) leprosy were analyzed by immunohistochemistry using antibodies to CD1a and FXIIIa. Quantitative analysis of labeling pattern showed a clear predominance of dendritic cells in tuberculoid leprosy. Difference between the number of positive cells of immunohistochemistry for the CD1a and FXIIIa staining observed in this study indicates a role for dendritic cells in the cutaneous response to leprosy. Dendritic cells may be a determinant of the course and clinical expression of the disease.

Salbutamol improves markers of epithelial function in mice with chronic allergic pulmonary inflammation

We investigated the effects of salbutamol on the markers of epithelial function in a murine model of chronic allergic pulmonary inflammation by recording the ciliary beat frequency (CBF) and the transepithelial potential difference (PD) in vivo. Mice were sensitized and received four challenges of ovalbumin (OVA group) or 0.9% saline (control group). Forty-eight hours after the 4th inhalation, we observed eosinophilia in the bronchoalveolar lavage and epithelium remodeling with stored acid mucus in the OVA group (P < 0.001). No difference in the baseline CBF was noticed between the groups; however, the OVA group had a significantly lower baseline PD (P = 0.013). Salbutamol increased the CBF in all groups studied, and the dose response curve to salbutamol increased the PD in the OVA group from 10(-4) M to 10(-2) M. We suggest that salbutamol affects the CBF and the depth of the periciliary layer, which, in great part, determines the ability of the cilia to propel the mucus layer. This effect may have a positive impact on airway mucociliary transport in asthma and may have clinical implications. (C) 2011 Elsevier B.V. All rights reserved.

Bovine Bone Screws: Metrology and Effects of Chemical Processing and Ethylene Oxide Sterilization on Bone Surface and Mechanical Properties

We assess the effects of chemical processing, ethylene oxide sterilization, and threading on bone surface and mechanical properties of bovine undecalcified bone screws. In addition, we evaluate the possibility of manufacturing bone screws with predefined dimensions. Scanning electronic microscopic images show that chemical processing and ethylene oxide treatment causes collagen fiber amalgamation on the bone surface. Processed screws hold higher ultimate loads under bending and torsion than the in natura bone group, with no change in pull-out strength between groups. Threading significantly reduces deformation and bone strength under torsion. Metrological data demonstrate the possibility of manufacturing bone screws with standardized dimensions.

Albendazole-praziquantel interaction in healthy volunteers: kinetic disposition, metabolism and enantioselectivity

center dot Pharmacokinetic interactions between albendazole and praziquantel are based on plasma concentrations of the enantiomeric mixture of both drugs with contradictory data, although the antiparasitic activity arises from (-)-(R)-praziquantel and (+)-albendazole sulfoxide. WHAT THIS STUDY ADDS center dot The pharmacokinetic interaction between albendazole and praziquantel is enantioselective. Praziquantel increased the plasma concentrations of (+)-albendazole sulfoxide more than those of (-)-albendazole sulfoxide and the administration of albendazole did not change the kinetic disposition of (+)-(S)-praziquantel, but increased the plasma concentration of (-)-(R)-praziquantel. AIM This study investigated the kinetic disposition, metabolism and enantioselectivity of albendazole (ABZ) and praziquantel (PZQ) administered alone and in combination to healthy volunteers. METHODS A randomized crossover study was carried out in three phases (n = 9), in which some volunteers started in phase 1 (400 mg ABZ), others in phase 2 (1500 mg PZQ), and the remaining volunteers in phase 3 (400 mg ABZ + 1500 mg PZQ). Serial blood samples were collected from 0-48 h after drug administration. Pharmacokinetic parameters were calculated using a monocompartmental model with lag time and were analyzed using the Wilcoxon test; P < 0.05. RESULTS The administration of PZQ increased the plasma concentrations of (+)-ASOX (albendazole sulphoxide) by 264% (AUC 0.99 vs. 2.59 mu g ml-1 h), (-)-ASOX by 358% (0.14 vs. 0.50 mu g ml-1 h) and albendazole sulfone (ASON) by 187% (0.17 vs. 0.32 mu g ml-1 h). The administration of ABZ did not change the kinetic disposition of (+)-(S)-PZQ (-)-(R)-4-OHPZQ or (+)-(S)-4-OHPZQ, but increased the plasma concentration of (-)-(R)-PZQ by 64.77% (AUC 0.52 vs. 0.86 mu g ml-1 h). CONCLUSIONS The pharmacokinetic interaction between ABZ and PZQ in healthy volunteers was demonstrated by the observation of increased plasma concentrations of ASON, both ASOX enantiomers and (-)-(R)-PZQ. Clinically, the combination of ABZ and PZQ may improve the therapeutic efficacy as a consequence of higher concentration of both active drugs. On the other hand, the magnitude of this elevation may represent an increased risk of side effects, requiring, certainly, reduction of the dosage. However, further studies are necessary to evaluate the efficacy and safety of this combination.

Anti-mullerian hormone is the best predictor of poor response in ICSI cycles of patients with endometriosis

Purpose: To correlate ovarian reserve (OR) markers with response in assisted reproduction techniques (ART) and determine their ability to predict poor response among patients with endometriosis (EDT). Methods: We evaluated ART cycles of 27 women with EDT and 50 with exclusive male factor. Basal follicle stimulating hormone (FSH) and anti-mullerian hormone (AMH) levels were determined. Ovarian response to gonadotropin stimulation was assessed and correlation coefficients calculated between the variables and reserve markers. Areas under the curve (AUC) determined ability of tests to predict poor response. Results: AMH was significantly correlated with response in both groups and it was the only marker with significant discriminative capacity to predict poor response among EDT (AUC = 0.842; 95% CI: 0.651-0.952) and control group (AUC = 0.869; 95% CI: 0.743-0.947). Conclusion: Infertile patients with endometriosis can benefit from the pre-therapeutic assessment of OR markers. However, regardless of disease presence, only AMH predicts poor response to stimulus.

Increased basal FSH levels as predictors of low-quality follicles in infertile women with endometriosis

Objective: To determine whether basal levels of follicle-stimulating hormone (FSH) and anti-mullerian hormone (AMH), antral follicle count (AFC), and the numbers of dominant follicles, oocytes, and mature oocytes retrieved after ovarian stimulation differed between infertile women with endometriosis and healthy women undergoing assisted reproduction techniques (ART). Method: Of 77 consecutive ART candidates, 27 were infertile and had endometriosis. A male factor caused the infertility of the other 50, who acted as controls. Results: The AMH and AFC levels were similar in the 2 groups. The FSH levels were higher (8.28 mIU/ML [range, 5.25-24.1 mIU/ML] vs 5.91 mIU/mL [range, 2.47-18.7 mIU/ML]; P<0.01) in the study group. And the numbers of retrieved (n = 5 [range, 0-12] vs n = 9 [range, 0-27]; P<0.05) and mature oocytes (n = 4 [range, 0-11] vs n = 5 [range, 0-16]; P<0.05) were less in the study group. Conclusion: Because AMH levels were unchanged, endometriosis seems not to damage the primordial pool of follicles and oocytes, but to lessen the quality of the ovarian response to the hCG injection. Basal FSH levels may be of value in predicting ART success in women with the disease. (C) 2010 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.