Russell Higbee

Assessing the immunopotency of Toll‐like receptor agonists in an in vitro tissue‐engineered immunological model

The in vitro Peripheral Tissue Equivalent (PTE) module is a three‐dimensional tissue‐engineered endothelial cell/collagen matrix culture system, which has been reported to reproduce in vivo physiological conditions and which generates dendritic cells (DC) autonomously. In the present study, we used the PTE module to investigate the immunopotency of Toll‐like receptor (TLR) agonists, including polyinosine‐polycytidylic acid, Gardiquimod, CpG 2006 and lipopolysaccharide. Application of TLR agonists in the PTE module induced a wide range of cytokines, including interleukins 1α/β, 6, 8 and 10 and tumour necrosis factor‐α. Compared with traditional peripheral blood mononuclear cell (PBMC) cultures, the PTE module produced twofold to 100‐fold higher levels of cytokine secretion, indicating that it can be a highly sensitive assay system. This increased sensitivity is the result of the natural synergy between the leucocytes and the endothelium. Furthermore, the application of TLR agonists, such as lipopolysaccharide and Gardiquimod, to the PTE module enhanced DC differentiation and promoted DC maturation, as indicated by up‐regulated expression of CD83, CD86 and CCR7(CD197). In addition, functional assays indicated PTE‐derived DC treated with Gardiquimod, a TLR‐7 agonist, significantly augmented anti‐tetanus toxoid antibody production. Interestingly, replacing PBMC with purified myeloid cells (CD33+) significantly reduced the responsiveness of the PTE module to TLR stimulation. The reduced sensitivity was partly the result of the removal of plasmacytoid DC that participated in the response to TLR stimulation and sensitization of the PTE module. Overall, the in vitro PTE module clearly demonstrated the effects of TLR agonists on DC generation, maturation and antigen‐presenting capacity, and may serve as a sensitive and predictive test bed for the evaluation of adjuvant candidates.

A predictive biomimetic model of cytokine release induced by TGN1412 and other therapeutic monoclonal antibodies

Human peripheral blood mononuclear cells (PBMC) are routinely used in vitro to detect cytokine secretion as part of preclinical screens to delineate agonistic and antagonistic action of therapeutic monoclonal antibodies (mAbs). Preclinical value of standard human PBMC assays to detect cytokine release syndrome (CRS) has been questioned, as they did not predict the “cytokine storm” that occurred when healthy human volunteers were given a CD28-specific super-agonist mAb, TGN1412. In this article, we describe a three-dimensional biomimetic vascular test-bed that can be used as a more physiologically relevant assay for testing therapeutic Abs. For developing such a system, we used TGN1412 as a model mAb. We tested soluble TGN1412 on various combinations of human blood components in a module containing endothelial cells grown on a collagen scaffold and measured cytokine release using multiplex array. Our system, consisting of whole leukocytes, endothelial cells, and 100% autologous platelet-poor plasma (PPP) consistently produced proinflammatory cytokines in response to soluble TGN1412. In addition, other mAb therapeutics known to induce CRS or first infusion reactions, such as OKT3, Campath-1H, or Herceptin, generated cytokine profiles in our model system consistent with their in vivo responses. As a negative control we tested the non-CRS mAbs Avastin and Remicade and found little difference between these mAbs and the placebo control. Our data indicate that this novel assay may have preclinical value for predicting the potential of CRS for mAb therapeutics.

Femtosecond laser ablation of porcine intestinal mucosa: potential autologous transplant for segmental cystectomy

Nearly 80% of patients with newly diagnosed bladder cancer present with superficial bladder tumors (confined to the bladder lining such as transitional cell carcinoma [90%], squamous cell carcinoma [6-8%], and adenocarcinoma[2%]) in stages Ta, Tis, or T1. Segmental cystectomy is one surgical treatment for patients who have a low-grade invasive tumor. Transposition of small intestine is a viable surgical treatment option. Success of the transplantation is also dependent upon removal of the entire SI mucosal layer. A Clark Spitfire Ti:Sapphire laser operating at 775 nm and 1 kHz repetition rate, was used to investigate the damage induced to fresh cadaveric porcine small intestinal mucosal epithelium. The laser was held constant at a focal spot diameter of 100 μm using a 200 mm focal point lens, with a power output maximum of 257 mW. A high resolution motorized X-Y-Z stage translated the SI tissue through the beam at 500 μm/sec with a line spacing of 50 μm. This produced a 50% overlap in the laser etching for each pass over a 1 cm x 1.5 cm grid. To determine if the mucosal lining of the SI was adequately removed, the targeted area was covered with 1% fluorescein solution for 30 seconds and then rinsed with phosphate buffered saline. Fluorescein staining was examined under UV illumination, to determine the initial degree of mucosal removal. Tissues were fixed and processed for light and scanning electron microscopy by standard protocols. Brightfield light microscopy of hematoxylin and eosin stained 4 μm thick cross sections, scanning electron microscopy were examined to determine the degree of mucosal tissue removal. Clear delineation of the submucosal layer by fluorescein staining was also observed. The Ti:Sapphire laser demonstrated precise, efficient removal of the mucosal epithelium with minimal submucosal damage.

Temporal effects of femtosecond-to-nanosecond laser ablation on fresh porcine liver

Using a Ti:Sapphire laser operating at 800nm and a repetition rate of 1 kHz, we investigated the damage induced to fresh cadaveric porcine liver after laser irradiation for pulse widths of 120-fs, 8ps, and 7-ns. The laser was held constant at a focal spot diameter of 100μm yielding a maximum fluence of 9J/cm2. Then, using polarization optics, the energy per pulse was controlled to well below ablation threshold fluences. The tissue samples were translated under the laser via 0.1μm resolution encoded X-Y-Z motorized stages. After irradiation and fixation, we evaluated the tissues using brightfield light microscopy of Hematoxylin and Eosin stained 4 μm thick cross sections, scanning electron microscopy, and transmission electron microscopy. The tissue samples were examined for both removal rates of material, thermal damage to surrounding tissue, and cell disruption for equivalent fluence levels across the temporal range. We found an increase in removal rate along with a decrease in thermal damage as the pulse widths approached the femtosecond regime for a constant fluence. With femtosecond pulses, ablation still occurred below fluences of 2J/cm2. However, for nanosecond pulses, ablation no longer occurred, showing a decrease in ablation threshold as the pulse width decreases. Because of the reduced thermal effects compared to nanosecond pulses, ultrafast lasers may offer a solution to more precise tissue removal with less damage to surrounding cells.

Ultrafast pulsed lasers: surgical wave of the future?

A Ti:Sapphire laser operating at 800 nm and 1 kHz repetition rate, was used to investigate the damage induced to fresh cadaveric porcine tissues. The laser was held constant at a focal spot diameter of 100 μm for pulse widths varying from 120-femtoseconds to 7-nanoseconds yielding a maximum fluence of 12.7 J/cm2 irradiation. Polarization optics were used to reduce the energy per pulse to well below tissue ablation threshold fluences. Hollow silica waveguides with a silver inner coating and bore diameters of 300, 500, 750 and 1000 μm were also used for the Ti:Sapphire laser with output pulses <150 fs duration and energy up to 700 μjoules. A high resolution motorized X-Y-Z stage translated the tissue through the beam at 1 mm/sec. A Luxar Novapulse CO2 surgical laser was used as a standard for comparison. Tissues were processed for light, scanning and transmission electron microscopy by standard protocols. Tissue samples were examined for tissue removal rates, thermal damage to adjacent tissue, and cellular disruption for equivalent fluence levels. The Ti:Sapphire laser demonstrated an increase in removal rate along with a decrease in thermal damage as the pulse widths approached the femtosecond regime for a constant fluence. With femtosecond pulses, ablation still occurred below fluences of 2 J/cm2. However, for nanosecond pulses, ablation no longer occurred, showing a decrease in ablation threshold as the pulse width decreases. Because of the reduced thermal effects compared to nanosecond pulses, ultrafast lasers may offer a solution to more precise tissue removal with less damage to surrounding cells as compared to more conventional surgical laser systems.

Noncoherent light for PDT of spontaneous animal tumors

Cultured 9L cells were incubated with graded doses of pheophorbide-a-hexyl ether (HPPH) and exposed to 665 nm red light from either a noncoherent light source or a KTP-pumped dye laser. Cell death was observed after irradiation using either light source, with the noncoherent light being most effective at the highest HPPH concentrations. To determing the practicality of using the noncoherent light source for clinical PDT, dogs and cats with spontaneous tumors were injected intravenously with 0.15 mg/kg HPPH one hour before their tumors were irradiated with 665 nm noncoherent light (50 mW cm-2, 100 J cm-2). Of the 9 tumors treated, 8 complete responses were observed, all of which occurred in animals with squamous cell carcinoma. After 68 weeks of follow up, the median initial disease free interval had not been reached. These data support the use of noncoherent light sources for PDT of spontaneous tumors in animals, representing a cost-effective alternative to medical lasers in both veterinary and human dermatology and oncology.