Mechanical characterization of collagen fibers and scaffolds for tissue engineering

Engineered tissues must utilize scaffolding biomaterials that support desired cellular functions and possess or can develop appropriate mechanical characteristics. This study assessed properties of collagen as a scaffolding biomaterial for ligament replacements. Mechanical properties of extruded bovine achilles tendon collagen fibers were significantly affected by fiber diameter, with smaller fibers displaying higher tangent moduli and peak stresses. Mechanical properties of 125 micrometer-diameter extruded fibers (tangent modulus of 359.6+/-28.4MPa; peak stress of 36.0+/-5.4MPa) were similar to properties reported for human ligaments. Scaffolds of extruded fibers did not exhibit viscoelastic creep properties similar to natural ligaments. Collagen fibers from rat tail tendon (a well-studied comparison material) displayed characteristic strain-softening behavior, and scaffolds of rat tail fibers demonstrated a non-intuitive relationship between tangent modulus and specimen length. Composite scaffolds (extruded collagen fibers cast within a gel of Type I rat tail tendon collagen) were maintained with and without fibroblasts under standard culture conditions for 25 days; cell-incorporated scaffolds displayed significantly higher tangent moduli and peak stresses than those without cells. Because tissue-engineered products must possess appropriate mechanical as well as biological/chemical properties, data from this study should help enable the development of improved tissue analogues.     

Autogeny in Ochlerotatus vigilax (Diptera: Culicidae) from southeast Queensland, Australia

Field and laboratory investigations were undertaken to determine the level of expression of autogeny in the mosquito Ochlerotatus vigilax (Skuse) from southeast Queensland, Australia, and whether there was evidence of seasonal variation. At two field sites in southeast Queensland, Wellington Point and Donnybrook, autogeny rates were determined on six occasions between January 2001 and January 2002. The autogeny rate varied between 71 and 100% at Wellington Point and between 63 and 100% at Donnybrook. Autogenous fecundity ranged from 17 to 63 eggs per female at Wellington Point and from 13 to 88 eggs per female at Donnybrook. Positive relationships were found between adult body size (indicated by wing length), autogeny rate, and fecundity. A laboratory study was conducted to investigate the influence of larval nutrition and adult diet (water versus sucrose) on the expression of autogeny. The autogeny rate at a low-diet treatment was between 73 and 90% when sucrose was withheld from females and 100% when sucrose was provided. All high-diet females were autogenous. Autogenous egg development required 80 +/- 6 h from emergence at 27 degrees C. We conclude that autogeny rates are consistently high in Oc. vigilax from the southeast Queensland region.     

Immunization with a polyprotein vaccine consisting of the T-Cell antigens thiol-specific antioxidant, Leishmania major stress-inducible protein 1, and Leishmania elongation initiation factor protects against leishmaniasis

Development of an effective vaccine against Leishmania infection is a priority of tropical disease research. We have recently demonstrated protection against Leishmania major in the murine and nonhuman primate models with individual or combinations of purified leishmanial recombinant antigens delivered as plasmid DNA constructs or formulated with recombinant interleukin-12 (IL-12) as adjuvant. In the present study, we immunized BALB/c mice with a recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant, L. major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF) delivered with adjuvants suitable for human use. Aspects of the safety, immunogenicity, and vaccine efficacy of formulations with each individual component, as well as the polyprotein referred to as Leish-111f, were assessed by using the L. major challenge model with BALB/c mice. No adverse reactions were observed when three subcutaneous injections of the Leish-111f polyprotein formulated with either MPL-squalene (SE) or Ribi 529-SE were given to BALB/c mice. A predominant Th1 immune response characterized by in vitro lymphocyte proliferation, gamma interferon production, and immunoglobulin G2A antibodies was observed with little, if any, IL-4. Moreover, Leish-111f formulated with MPL-SE conferred immunity to leishmaniasis for at least 3 months. These data demonstrate success at designing and developing a prophylactic leishmaniasis vaccine that proved effective in a preclinical model using multiple leishmanial antigens produced as a single protein delivered with a powerful Th1 adjuvant suitable for human use.     

Cloning and characterization of a gene encoding an immunoglobulin-binding receptor on the cell surface of some members of the family Trypanosomatidae

Several members of the Trypanosomatidae family, when freshly isolated from their mammalian hosts, have immunoglobulins adsorbed to their cell surfaces. However, a significant portion of these antibody molecules is not parasite specific, i.e., the immunoglobulins are bound to the parasite's cell surface molecules via noncognitive interactions. It has been proposed that this noncognitive adsorption of immunoglobulins to the parasite is mediated by an Fc-like receptor present in several members of the Trypanosomatidae family. However, the molecular identification of this receptor has never been defined. Here, we describe the cloning of a gene encoding a protein that might represent this molecule. The gene, named Lmsp1, was cloned by screening a Leishmania major cDNA expression library using a rabbit antiserum. Lmsp1 is present in both Leishmania and Trypanosoma and is expressed in all developmental stages of these parasites. The predicted protein has a molecular mass of 16.6 kDa and contains an RGD sequence starting at residue 104 and three cysteine residues at positions 55, 74, and 116. The purified recombinant protein strongly binds to normal immunoglobulins of various animal species (humans, rabbits, sheep, goats, guinea pigs, donkeys, rats, and mice) and the binding to human immunoglobulins appears to be immunoglobulin G (IgG) and IgM isotype specific. Moreover, Lmsp1 binds to both purified Fc and Fab fragments of IgG from both humans and rabbits. The mapping of the Lmsp1 epitopes that bind human IgG revealed that different sequences of the molecule bind to Fc or Fab. In addition, fluorescence-activated cell sorter analyses with a specific rabbit anti-Lmsp1 antiserum showed that Lmsp1 is associated with the parasite's cell surface. Finally, inhibition experiments point to an active role of this molecule in the immunoglobulin-mediated attachment and penetration of Trypanosoma cruzi in its macrophage host cells, thus suggesting that Lmsp1 is a putative Trypanosomatidae immunoglobulin receptor.     

Widespread IgE-mediated hypersensitivity in the Sudan to the 'green nimitti' midge, Cladotanytarsus lewisi (Diptera: Chironomidae) II. Identification of a major allergen.

A major allergen has been identified in an aqueous extract of the 'green nimitti' midge, Cladotanytarsus lewisi (Diptera: Chironomidae). Following chromatography on Sephadex G-100 allergenic activity, as assessed by skin ('prick') testing, eluted as two closely related peaks (pools I and II) at about 50% bed volume. When these pools were applied separately to columns of CM-cellulose, activity in each eluted with 0 . 05 M NaCl. Isoelectric focusing of the unfractionated allergen gave a single peak of activity at pI 4 . 3. By SDS--PAGE, biological activity in the whole 'green nimitti' extract and the material eluting from both pools I and II of the Sephadex G-100 column migrated to the same positions and were associated with a molecular size of 15,000--20,000 daltons. Skin test reactivity of the unfractionated material and the Sephadex G-100 pool I and II eluates were all destroyed following incubation with trypsin, chymotrypsin, thermolysin and neuraminidase. These experiments indicate that a major allergen derived from the 'green nimitti' midge, a cause of widespread and severe immediate-type allergy in the Sudan, is an acidic glycoprotein of 15,000--20,000 molecular weight.     

ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan

An intracellular protein, dystrophin, plays an important role in keeping muscle fibers intact by binding at its N-terminal end to the subsarcolemmal cytoskeletal actin network and via its C-terminal end to the transmembraneous protein beta-dystroglycan. Duchenne muscular dystrophy is caused by the loss of dystrophin, which can result from the loss of this binding. The N-terminal part of the latter binding site of dystrophin has been well documented using overlay assay and X-ray diffraction assays. However, the binding site at the C-terminal region of dystrophin has not been examined in detail. In the present work, we report a detailed analysis of the C-terminal binding domain as follows. (1). The full binding activity corresponding to the effective binding in vivo is expressed by the dystrophin fragment spanning amino acids 3026-3345 containing the ZZ domain at the C-terminus. Determination of this binding range is important not only for understanding of the mechanism of dystrophy, but also useful for the design of truncated dystrophin constructs for gene therapy. (2). The ZZ domain binds to EF1 domain in the dystrophin fragment to reinforce the binding activity. (3). The cysteine 3340 in the ZZ domain is essential for the binding of dystrophin to beta-dystroglycan. A reported case of DMD due to missense mutation C3340Y may be caused by inability to fix dystrophin beneath the cell membrane. (4). The binding mode of utrophin is different from that of dystrophin. The difference is conspicuous concerning the cysteine residues present in the ZZ domain.     

Cytologic grade independently predicts survival of patients with pancreatic adenocarcinoma

Our objectives were to devise a cytologic grading system and determine whether it would predict survival of patients with solid-type pancreatic adenocarcinoma. We evaluated 116 consecutive patients from July 2000 to November 2002; they were followed up until September 2003. We scored the following features on rapid Romanowsky-stained endoscopic ultrasound-guided fine-needle aspiration smears: cell group architecture, single cells, nuclear grade, mucus, bizarre cells, and necrosis. A cytologic grade (low vs high) was assigned. The Kaplan-Meier estimate of 6-month survival was 76% (SE, 7%) for patients with low-grade tumors vs 50% (SE, 6%) for patients with high-grade carcinoma. The median survival for patients with low-grade vs high-grade tumors was 1 year vs 6 months, respectively (chi2 = 4.45; P = .035). Cox proportional hazards regression showed tumor stage, cancer-specific treatment, and cytologic grade to be independent predictors of survival (P = .001). No other factors (age, mass location, placement of stent, presence of concomitant chronic pancreatitis, race, sex) predicted survival. We devised a grading system that independently predicted survival in patients with pancreatic adenocarcinoma.