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.

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

SUMMARY: 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 1alpha/beta, 6, 8 and 10 and tumour necrosis factor-alpha. 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.

An immunologic model for rapid vaccine assessment -- a clinical trial in a test tube.

While the duration and size of human clinical trials may be difficult to reduce, there are several parameters in pre-clinical vaccine development that may be possible to further optimise. By increasing the accuracy of the models used for pre-clinical vaccine testing, it should be possible to increase the probability that any particular vaccine candidate will be successful in human trials. In addition, an improved model will allow the collection of increasingly more-informative data in pre-clinical tests, thus aiding the rational design and formulation of candidates entered into clinical evaluation. An acceleration and increase in sophistication of pre-clinical vaccine development will thus require the advent of more physiologically-accurate models of the human immune system, coupled with substantial advances in the mechanistic understanding of vaccine efficacy, achieved by using this model. We believe the best viable option available is to use human cells and/or tissues in a functional in vitro model of human physiology. Not only will this more accurately model human diseases, it will also eliminate any ethical, moral and scientific issues involved with use of live humans and animals. An in vitro model, termed "MIMIC" (Modular IMmune In vitro Construct), was designed and developed to reflect the human immune system in a well-based format. The MIMIC System is a laboratory-based methodology that replicates the human immune system response. It is highly automated, and can be used to simulate a clinical trial for a diverse population, without putting human subjects at risk. The MIMIC System uses the circulating immune cells of individual donors to recapitulate each individual human immune response by maintaining the autonomy of the donor. Thus, an in vitro test system has been created that is functionally equivalent to the donor's own immune system and is designed to respond in a similar manner to the in vivo response.

Effect of wavelength on low-intensity laser irradiation-stimulated cell proliferation in vitro.

BACKGROUND AND OBJECTIVES: There exist contradictory reports about low-intensity laser light-stimulated cell proliferation. The purpose of this study was to determine the effect of wavelength on proliferation of cultured murine cells. STUDY DESIGN/MATERIALS AND METHODS: Proliferation of primary cell cultures was measured after irradiation with varying laser wavelengths. RESULTS: Fibroblasts proliferated faster than endothelial cells in response to laser irradiation. Maximum cell proliferation occurred with 665 and 675 nm light, whereas 810 nm light was inhibitory to fibroblasts. CONCLUSIONS: These observations suggest that both wavelength and cell type influence the cell proliferation response to low-intensity laser irradiation.

Histologic comparison of skin biopsy specimens collected by use of carbon dioxide or 810-nm diode lasers from dogs.

OBJECTIVE: To compare histologic artifacts caused by carbon dioxide (CO2) or 810-nm diode surgical lasers used to obtain small biopsy specimens of skin from healthy dogs. DESIGN: Prospective study. ANIMALS: 4 dogs. PROCEDURE: 21 skin biopsy specimens were collected from each dog. Three biopsy specimens were obtained with a CO2 or an 810-nm diode laser at 3 operating settings each, and 3 biopsy specimens were obtained with a 6-mm biopsy punch instrument (controls). After processing, biopsy specimens were examined for artifacts related to laser-tissue interactions. Microscopically visible char was measured from the lateral edge of each specimen obtained with a laser. RESULTS: There were no significant differences among mean char distances in biopsy specimens obtained with the CO2 laser at various settings. Mean char distance was significantly greater in all skin biopsy specimens obtained with the diode laser, compared with those obtained with the CO2 laser. Mean char distance was significantly greater in biopsy specimens obtained with the 810-nm diode laser at high power, compared with biopsy specimens obtained with the 810-nm diode laser at low power. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the CO2 laser caused less thermal injury at margins of skin biopsy specimens; therefore, if a surgical laser is used for removal of cutaneous masses or to obtain skin biopsy specimens, use of the CO2 laser is recommended. Veterinarians performing a biopsy by using a surgical laser should be aware that laser-induced artifacts may render small biopsy specimens useless for providing accurate histologic diagnosis.

Laser lithotripsy for treatment of canine uroliths.

OBJECTIVE: To confirm in vivo optimal laser energy settings previously determined in vitro for lithotripsy, to develop a technique for Ho:YAG laser lithotripsy, and to evaluate acute and chronic tissue effects of laser lithotripsy. STUDY DESIGN: Prospective, randomized preclinical trial. ANIMALS OR SAMPLE POPULATION: Nineteen intact, adult male dogs. METHODS: Via cystotomy, a single urolith was inserted into the urethra to the level of the base of the os penis to simulate obstruction. Uroliths (calcium oxalate, urate, or magnesium ammonium phosphate) were fragmented by Ho:YAG laser, in contact mode through a 320 micro optic fiber, passed through the operating channel of a 2.8 mm flexible endoscope. The time and total energy to fragmentation were recorded. Dogs were euthanatized immediately after lithotripsy (3 dogs) or at 3 days (7 dogs), and urethral lesions and any stone remnants were evaluated. Urethral integrity was also evaluated in 9 other dogs by endoscopy on day 10; these were also monitored clinically for 30 days. RESULTS: The mean time for adequate fragmentation was 166.7 seconds (range, 47-494.5 seconds). The mean+/-SD energy used was 1418+/-851.2 J. In part 1, 2 dogs were obstructed with urolith fragments at necropsy. Eight dogs had minimal (<30 mg) or no urolith material evident within the urethra. Four dogs had gross focal or circumferential erosion, ulceration, or hemorrhage of the urethral mucosa. Lesions were not associated with the site of laser irradiation in 2 dogs. In dogs observed for 30 days, hematuria, pollakiuria, and stranguria that were observed after lithotripsy, resolved in all affected dogs by day 5. No mucosal lesions were observed by endoscopy and none of the dogs became obstructed. CONCLUSION: Laser lithotripsy with the Ho:YAG laser in contact mode successfully fragmented obstructive uroliths in male dogs. CLINICAL RELEVANCE: Laser lithotripsy may be a clinically relevant technique for treatment of urolithiasis in male dogs; clinical studies to evaluate long-term effects on urethral mucosa and the role of repeat treatment for recurrence are indicated.

In vitro effects of pulsed holmium laser energy on canine uroliths and porcine cadaveric urethra.

BACKGROUND AND OBJECTIVES: To assess the effect of holmium laser energy on canine uroliths and porcine urethra. STUDY DESIGN/MATERIALS AND METHODS: Uroliths of known composition and fresh cadaveric urethra were exposed to holmium laser energy. Urolith fragmentation times and depth of urethral lesions were determined. RESULTS: Overall mean fragmentation time was 11.8 +/- 8.01 seconds. Magnesium ammonium phosphate (MAP) and urate uroliths had significantly shorter fragmentation times compared to other uroliths. Fragmentation time of MAP uroliths irradiated with 1.2 J/pulse was significantly longer than the fragmentation time of MAP uroliths irradiated with 0.3 J/pulse. Overall mean lesion depth for urethral specimens treated with 90 degrees contact mode irradiation was significantly greater than overall mean lesion depth for specimens treated with 30 degrees non-contact mode. CONCLUSIONS: Holmium laser energy effectively fragmented canine uroliths and caused minimal urethral damage in vitro. Dogs with urolithiasis may represent a useful animal model for developing human lithotripsy procedures.

Photodynamic therapy for the treatment of intranasal tumors in 3 dogs and 1 cat.

Three dogs and 1 cat with intranasal tumors were treated with pyropheophorbide-a-hexyl ether-based photodynamic therapy (PDT). PDT was well tolerated by all the animals, and no adverse effects from photosensitizer injection, such as cutaneous photosensitization, were observed. Facial swelling was observed in all animals after each PDT treatment but resolved spontaneously within 72 hours after treatment. All animals had a decrease in severity of epistaxis, frequency of sneezing, and amount of nasal discharge after PDT. Clinical signs were controlled for variable time, although long-term responses were comparable with radiation therapy in 2 animals. This small case series demonstrates another application for PDT in veterinary medicine. On the basis of these findings. further studies are warranted to define the role of PDT in the management of intranasal tumors in dogs and cats.

Neodymium:Yttrium-aluminum-Garnet (Nd:YAG) laser ablation of an obstructive urethral polyp in a dog.

A miniature schnauzer presented for evaluation of a persistent lower urinary tract obstruction. Further examination revealed that the dog had developed an obstructive, inflammatory polyp secondary to a long-standing urinary tract infection. The polyp was located within the proximal urethra and interfered with normal voiding. The polyp was visualized using flexible endoscopy and then was successfully ablated using the neodymium:yttrium-aluminum-garnet (Nd:YAG) laser. The Nd:YAG laser is a potentially useful tool for treating various lesions of the lower urinary tract.

Outcome of and complications associated with prophylactic percutaneous laser disk ablation in dogs with thoracolumbar disk disease: 277 cases (1992-2001).

OBJECTIVE: To determine outcome of and complications associated with prophylactic percutaneous laser disk ablation in dogs with thoracolumbar disk disease. DESIGN: Retrospective study. ANIMALS: 277 dogs. PROCEDURE: Medical records of dogs with a history of thoracolumbar disk disease in which the 7 intervertebral disks from T10-11 through L3-4 were ablated with a holmium-yttrium-aluminum-garnet laser inserted through percutaneously placed needles were reviewed. Complications and episodes of a recurrence of neurologic signs (eg, paresis or paralysis) were recorded. Owners were contacted by telephone for follow-up information. RESULTS: Nine of 262 (3.4%) dogs for which follow-up information was available had a recurrence of paresis or paralysis. Follow-up time ranged from 1 to 85 months (mean, 15 months); signs recurred between 3 and 52 months (mean, 15.1 months) after laser disk ablation. Acute complications occurred in 5 dogs and included mild pneumothorax in 1 dog, an abscess at a needle insertion site in 1 dog, and proprioceptive deficits in 3 dogs, 1 of which required hemilaminectomy within 1 week because of progression and severity of neurologic signs. One dog developed diskospondylitis. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that prophylactic percutaneous laser disk ablation is associated with few complications and may reduce the risk of recurrence of signs of intervertebral disk disease in dogs.

Use of a neodymium:yttrium-aluminum-garnet laser to remove exuberant granulation tissue from the esophagus of a horse.

Use of a neodymium:yttrium-aluminum-garnet laser to remove exuberant granulation tissue from the esophagus of a horse A 4-year-old Quarter Horse mare was evaluated because of recurrent esophageal obstruction. Endoscopic examination revealed a proliferative mass in the esophagus approximately 55 cm aborad to the nares. Histologic examination of biopsy specimens revealed diffuse suppurative esophagitis and granulation tissue with no evidence of neoplasia. The mass was excisedand ablated transendoscopically with a neodymium:yttrium-aluminum-garnet laser. During a follow-up examination 2 weeks after the final laser procedure, the mass was no longer apparent, and the esophagus appeared healed. Six months after the final examination, there had been no recurrence of the mass. In this horse, transendoscopic laser excision was efficacious and avoided problems associated with an esophagotomy.

Selective laser-induced hyperthermia for the treatment of spontaneous tumors in dogs.

Indocyanine green (ICG), with spectral absorption at 800-810 nm, is a chromophore that strongly absorbs and converts near-infrared laser energy into thermal energy. By directly injecting ICG into a tumor, selective tumor hyperthermia during laser irradiation occurs. In this study, 4 dogs with spontaneous tumors were injected in situ with a 0.25% wt/vol ICG solution and immediately irradiated with 810 nm laser energy. Thermal data were collected from multiple thermocouples placed within the tumor and tumor response was recorded. Tissue heating to ≥ 42°C was observed in all tumors. One dog had a complete remission, one dog had stable disease for 12 weeks, and the other 2 dogs developed progressive disease shortly after treatment. Results from this study demonstrate that chromophore-enhanced laser-induced hyperthermia can effectively and selectively heat canine tumors. However, tumor size and pigmentation of overlying skin are potentially limiting factors to the success of this novel c ancer treatment.