Maximizing Fibroblast Adhesion on Protein-Coated Surfaces Using Microfluidic Cell Printing.

translation of in vitro cell based assays to in vivo cellular response is imprecise at best. The advent of three-dimensional cell cultures in addition to bioreactor type microfluidics has improved the situation. However, these technical advances cannot be easily combined due to practical limitations. Development of a vertical microfluidic cell printer overcomes this obstacle, providing the ability to more closely recapitulate complex cellular environments and responses. As a proof of concept, we investigated the adhesion of fibroblasts under flow on protein-coated surfaces using a novel vertical microfluidic print head to isolate and manipulate both mechanical and biological factors as a model of fibroblast behavior during the foreign body response following implant insertion. A low flow rate with larger microfluidic channels onto a serum-coated surface has been determined to allow the highest density of viable fibroblasts to attach to the surface. While these insights into fibroblast surface attachment may lead to better material designs, the methods developed herein will certainly be useful as a biomaterials testing platform.

An inexpensive system for evaluating the tussive and anti-tussive properties of chemicals in conscious, unrestrained guinea pigs.

INTRODUCTION: Commercial whole-body plethysmography systems used to evaluate the anti-tussive potential of drugs employ sophisticated technology, but these systems may be cost prohibitive for some laboratories. The present study describes an alternative, inexpensive system for evaluating the tussive and anti-tussive potential of drugs in conscious, unrestrained guinea pigs. METHODS: The system is composed of a transparent small animal anesthesia induction box fitted with a microphone, a camera and a pneumotachometer to simultaneously capture audio, video, air flow and air pressure in real time. Data acquisition and analysis was performed using free software for audio and video, and a research pneumotach system for flow and pressure. System suitability testing was performed by exposing conscious, unrestrained guinea pigs to nebulized aqueous solutions of a selective agonist for TRPV1 (citric acid) or a selective agonist for TRPA1 (AITC), with or without pre-treatment with a selective antagonist for TRPV1 (BCTC) or a selective antagonist for TRPA1 (HC-030031). RESULTS: The system easily discerned coughs from other respiratory events like sneezes. System suitability test results are as follows: AITC caused 10.7 (SEM=1.4592) coughs vs. 5.8 (SEM=1.6553) when pre-treated with HC-030031 (P<0.05). Citric acid caused 12.4 (SEM=1.4697) coughs vs. 3.2 (SEM=1.3928) when pre-treated with BCTC (P<0.002). DISCUSSION: We have described in detail an inexpensive system for evaluating the tussive and anti-tussive potential of chemicals in conscious, unrestrained guinea pigs. Suitability testing indicates that the system is comparable to a commercial whole-body plethysmography system for detecting and differentiating between coughs and sneezes. This system may provide some investigators a cost-conscious alternative to more expensive commercial whole-body plethysmography systems.

Pancreatic galanin: molecular forms and anatomical locations.

Galanin-like immunoreactivity (GalLI) was found to be present in extracts taken from human, canine, and porcine pancreata. The dominant gel filtration peak in each species co-elutes with standard synthetic porcine galanin (Gal), but an apparently smaller molecular weight Gal form was present in human pancreas and intestine and in dog intestine. Reverse-phase HPLC demonstrated identity of porcine pancreatic Gal immunoreactivity with synthetic intestinal Gal. Heterogeneity was seen on reverse-phase HPLC: Human pancreas and intestine had three peaks of immunoreactivity; the retention times were identical between the pancreas and intestinal extracts; and human Gal elutes at an earlier retention time than porcine Gal. Similarly, dog pancreatic GalLI eluted earlier than porcine Gal on reverse-phase HPLC. Immunohistochemical studies revealed the presence of specific staining for GalLI in varicose nerve fibers in the pancreas of the three species. In the dog pancreas an association between Gal-containing nerve fibers and islet cells was readily demonstrable. This was not the case with pig or human pancreas. We conclude that pancreatic Gal is present in the pancreas of the three species and that molecular heterogeneity is similar between intestinal and pancreatic forms. In the dog, a distinct anatomical relationship is demonstrable between Gal-containing nerves and islet cells.

Distribution and function of enteric GAL-IR nerves in dogs: comparison with VIP.

The distribution of nerves containing galanin-immunoreactive (GAL-IR) material was compared to the distribution of neurons containing vasoactive intestinal polypeptide (VIP) immunoreactivity in the canine gastrointestinal tract. The actions of intra-arterially administered galanin and VIP on motility in the gastric antrum and corpus and the intestines were also studied. All sphincter muscles contained galanin- and VIP-immunoreactive nerve profiles. VIP-immunoreactive nerve profiles were present in all layers of the stomach, small intestine, and colon. GAL-IR nerve somata were common in the submucous plexus of ileum and colon and in the myenteric plexus of the terminal antrum, as were nerve processes in various layers. In the small intestine, galanin inhibited contractile responses to field stimulation of intrinsic nerves and also reduced the contractions after nerve blockade with tetrodotoxin (TTX). VIP often enhanced field-stimulated contractions at low doses but inhibited these and the contractions after TTX at higher doses. In the stomach and colon, both peptides inhibited responses to field stimulation; whether these effects were due to actions on smooth muscle was not tested. The distribution and actions of galanin in gut are consistent with the hypothesis that it acts at smooth muscle sites and possibly at prejunctional sites.

Phase I trial of adjuvant chemotherapy with cyclophosphamide, epirubicin and 5-fluorouracil (CEF) for stage II breast cancer.

Epirubicin is a new anthracycline with a potentially more favorable toxicity profile than the parent compound, doxorubicin. Accordingly, the feasibility and toxicity of 6 courses of adjuvant chemotherapy with cyclophosphamide (C), epirubicin (E), and 5-fluorouracil (F) were assessed in 10 patients with Stage 2 (node positive) breast cancer. Doses of C and F were 600 mg/m2 and E was 75 mg/m2. Moderate granulocytopenia (median count = 610/mm3) occurred on day 14 of the first 21 day treatment course and was the main toxicity encountered with treatment, although there were no episodes of granulocytopenic fever. Grade 3 or 4 vomiting occurred in 40% and significant alopecia in 30% of patients. Four patients experienced transient asymptomatic decreases in calculated radionuclide cardiac ejection fraction of greater than or equal to 10% but no signs or symptoms of cardiac failure were observed. If epirubicin proves to be less cardiotoxic than doxorubicin, this combination would merit further evaluation as potential adjuvant therapy for early breast cancer.

Characterization of content and chromatographic forms of neuropeptides in purified nerve varicosities prepared from guinea pig myenteric plexus.

Partially purified nerve varicosities (PV) prepared from guinea pig ileal myenteric plexus were found to contain, by radioimmunoassay, gastrin-releasing polypeptide (GRP), substance P (SP), galanin, Leu-enkephalin (LE), Met-enkephalin (ME), and vasoactive intestinal polypeptide (VIP). SP was present in the highest concentration followed by, in descending order, ME, LE, VIP, GRP and galanin. On reverse-phase HPLC, SP-, LE- and ME-like immunoreactivity in the PV preparation eluted at retention times similar to their synthetic analogues, galanin-like immunoreactivity eluted at a retention time different from that of synthetic porcine galanin and VIP-like immunoreactivity eluted at the retention time of synthetic guinea pig VIP. GRP-like immunoreactivity, on reverse-phase HPLC, eluted at retention times close to that of synthetic porcine GRP-(1-27) and its major oxidized form. Evidence was obtained for the presence of an alpha-neurokinin-like immunoreactive entity and an unidentified SP-like immunoreactive entity in guinea pig myenteric plexus.

Therapeutic plasma exchange in the immune hemolytic anemias and immunologic thrombocytopenic purpura.

Thus, plasma exchange appears to have a potential but limited role in the care of antibody mediated injury of red cells or platelets. Its clearest indication appears to be in those uncommon instances of warm antibody mediated hemolysis with a highly fulminant course where the classical therapeutic measures do not have adequate time for effect. Similarly, such therapy appears to have significant potential in cases of acute immunologic thrombocytopenic purpura where some clinical contraindication to steroids, immunosuppressive agents or splenectomy exists. Acute ITP of pregnancy may provide another important setting for such a therapeutic approach. Finally, plasma exchange has been shown to provide temporary clinical stability in cases of chronic cold agglutinin disease.