Richard H. Smith

A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation

This study examines the intranasal instillation of lipopolysaccharide (LPS) into BALB/c mice causing acute pulmonary damage, due to neutrophil infiltration and sepsis. A dose response with LPS showed that an intranasal instillation of 167 microg/ml (10 microg/mouse) caused acute lung injury within 2-4 h and reached maximal damage at 24-48 h. We found the method of LPS administration for induction of acute pulmonary damage to be crucial. After 24 h post-LPS injection, a comparison showed a substantial increase in pulmonary damage with intranasal instillation of LPS. As for intravenous injection, it showed a baseline effect. This study indicates that LPS administered intranasally causes acute pulmonary damage, whereas with intravenous and intraperitoneal endotoxin administration a tissue-specific or similar degree of pulmonary injury may not develop.

Induction of T-cell immunity to oligosaccharide antigens immobilized on crystalline bacterial surface layers (S-layers)

Immunization of Balb/c mice with conjugates of oligosaccharide haptens and crystalline bacterial surface-layer proteins (S-layers) primed the mice for a strong, hapten-specific, delayed-type hypersensitivity (DTH) response. Conjugates of haptens with bovine serum albumin produced only weak DTH responses but, when mixed with aluminium hydroxide, elicited DTH responses comparable to those against S-layer conjugates. Surface-layer conjugates also elicited strong anti-hapten DTH responses when administered by an oral/nasal route. Apparently, the natural assembly of S-layer proteins into large, two-dimensional arrays endows them with intrinsic adjuvant properties.

Effect of Sulfo Lewis C on smoke inhalation injury in an ovine model

OBJECTIVE To evaluate the effect of Sulfo Lewis C (SO3-3âGal1-3GlcNAc-O(CH2)8-COOMe), a putative ligand of selectins, on smoke inhalation injury. DESIGN Prospective animal study with concurrent controls. SETTING An animal laboratory. SUBJECTS Twelve 1-yr-old female sheep, weighing 24 to 33 kg. INTERVENTIONS Twelve sheep received nine exposure units of smoke generated by thermolysis of pine woodchips (80 g). Group 1 (n = 6) was untreated. Group 2 (n = 6) was treated with an intravenous infusion of Sulfo Lewis C after smoke exposure. Animals were killed 48 hrs after injury. MEASUREMENTS AND MAIN RESULTS Cardiopulmonary variables and blood gases were measured serially. Granulocyte free-radical production was measured before smoke exposure and at 4 and 48 hrs after injury. Ventilation/perfusion distribution (VA/Q) was analyzed using the multiple inert gas elimination technique. Granulocyte free-radical production was increased after smoke exposure in both groups. Oxygenation was significantly improved by the administration of Sulfo Lewis C. VA/Q analysis demonstrated significantly less blood flow to low VA/Q lung segments in treated animals. CONCLUSIONS Selectin blockade attenuated lung injury after smoke exposure. These data support the hypothesis that neutrophils play a pivotal role in smoke inhalation injury.

Crystalline Bacterial Cell Surface Layers (S-Layers) as Combined Carrier/Adjuvants for Conjugate Vaccines

The development of vaccines to prevent human and animal diseases has been a challenging task for generations of physicians, microbiologists and biochemists. Jenner’s fundamental publication on inoculation of susceptible people with cowpox virus was the first documented evidence for the efficacy of vaccination (Jenner 1798). Since then, a number of vaccines against a variety of viral and bacterial infections have been used in man and animals. Other vaccines have been developed to protect against parasitic infections. More recently, experimental immunotherapies against cancers have been developed which utilise conjugate vaccine technology (for review see Bittle and Murphy 1989). Although only a few diseases have been totally eliminated through vaccination, the availability of vaccines has led to dramatic reductions in morbidity and mortality. However, there are still many infectious diseases against which vaccines need to be developed (for reviews see Bell and Torrigiani 1987; Mizrahi 1990). Moreover, problems remain regarding the efficacy of some currently available vaccines in the general population. The development of new carrier materials, capable of enhancing immunogenicity and long term immunological memory, is seen as an important contribution toward solving the remaining problems in vaccination.

Biology Lab-on-a Chip for Drug Screening

A microfluidic device was designed and tested for screening anti-inflammatory drug candidates using the cell rolling method. Channels were coated with either E- or P-Selectin (adhesion molecule), and isolated human neutrophils or lymphocytes were allowed to roll on the Selectin coated surface under flow conditions which mimic blood flow in the vascular capillary. Undiluted human blood was successfully tested for cell rolling and inhibition. Anti E- or P- Selectin, Sialyl Lewisx and Fucoidan were tested for the inhibition of cell rolling.

Selectin blockade worsened lipopolysaccharide-induced lung injury in a swine model.

BACKGROUND Polymorphonuclear leukocytes have been reported to play an important role in various acute lung injuries. Neutrophil recruitment into tissues is a multistep process involving sequential engagement of adhesion molecules. The objective of this study was to determine the effect of selectin inactivation with Sulfo Lewis C (SO3-3betaGal1-3betaGlcNAc-O(CH2)8-COOMe) on the pulmonary response to lipopolysaccharide (LPS) infusion. METHODS All animals (n = 11) were pretreated with an intramuscular injection of a priming dose of Escherichia coli LPS (10 microg/kg). Eighteen hours later, animals received an intravenous infusion of LPS (20 microg/kg) over 20 minutes. All animals were resuscitated with a lactated Ringers solution. Group I (G1; n = 5) received no additional treatment. Group II (G2; n = 6) received a bolus injection of Sulfo Lewis C (10 mg/kg) 10 minutes before LPS insult followed by a continuous infusion (1 mg/kg per hour) for the rest of the study. Animals were observed for 5 hours from initiation of the LPS infusion and killed. Cardiopulmonary variables and blood gases were measured serially. The multiple inert gas elimination technique (MIGET) was used to evaluate the matching of air flow and blood flow in the lung 5 hours after LPS infusion. Histologic evaluation of the parenchymal injury was performed by using light microscopy. The number of polymorphonuclear leukocytes and red blood cells in the alveolar spaces per field at 400x magnification were counted in 10 randomly selected fields. RESULTS Hypoxemia, indexed as Pao2/FIO2, was exacerbated by the administration of Sulfo Lewis C (G1:437+/-33 vs. G2: 241+/-63 mm Hg at 5 hours, p<0.03). This finding is supported by the multiple inert gas elimination technique analysis, which demonstrated significantly greater blood flow to true shunt in G2 (G1:4.42+/-1.75 vs. G2:23.2+/-5.69, p<0.02). There was no difference between the two groups in red blood cell counts in the alveolar spaces. However, polymorphonuclear leukocyte counts were significantly greater in G2 (G1:1.8+/-0.58 vs. G2:9.9+/-2.34, p<0.01). CONCLUSION Selectin blockade significantly worsened lung injury induced by LPS infusion, and greater numbers of neutrophils were observed in alveolar spaces in the group treated with Sulfo Lewis C. These findings are supported by the multiple inert gas elimination technique analysis, which demonstrated significantly greater blood flow to the true shunt compartment in treated animals. Further studies are required to determine the role of selectins in sepsis-induced lung injury.

Synthesis and characterization of a Pertussis toxin-biotin conjugate.

We prepared a Pertussis toxin-biotin conjugate and found its biological properties to be similar to those of native Pertussis toxin with respect to the hemagglutination, Chinese hamster ovary cell, and lymphocyte proliferation assays. Direct binding to Chinese hamster ovary and Jurkat cells was observed using fluorescence microscopy. Pertussis toxin-biotin was also found to possess similar glycoconjugate binding specificities as those of 125I-labeled Pertussis toxin.

Single Cell Enzymatic Analysis on a Microchip: Lysing of Single Cells and Identification of Their β-Galactosidase Activity

Microchips are introduced as a platform for performing single cell assays. On-chip lysis and incubation of single HL-60 cells with FDG, a fluorogenic, β-galactosidase specific, substrate, followed by detection of the fluorescence response is demonstrated. The advantages of chip based enzymatic single cell analyses are highlighted.