Functional recovery after peripheral nerve injury is dependent on the pro-inflammatory cytokines IL-1ß and TNF: implications for neuropathic pain.

IL-1ß and TNF are potential targets in the management of neuropathic pain after injury. However, the importance of the IL-1 and TNF systems for peripheral nerve regeneration and the mechanisms by which these cytokines mediate effects are to be fully elucidated. Here, we demonstrate that mRNA and protein levels of IL-1ß and TNF are rapidly upregulated in the injured mouse sciatic nerve. Mice lacking both IL-1ß and TNF, or both IL-1 type 1 receptor (IL-1R1) and TNF type 1 receptor (TNFR1), showed reduced nociceptive sensitivity (mechanical allodynia) compared with wild-type littermates after injury. Microinjecting recombinant IL-1ß or TNF at the site of sciatic nerve injury in IL-1ß- and TNF-knock-out mice restored mechanical pain thresholds back to levels observed in injured wild-type mice. Importantly, recovery of sciatic nerve function was impaired in IL-1ß-, TNF-, and IL-1ß/TNF-knock-out mice. Notably, the infiltration of neutrophils was almost completely prevented in the sciatic nerve distal stump of mice lacking both IL-1R1 and TNFR1. Systemic treatment of mice with an anti-Ly6G antibody to deplete neutrophils, cells that play an essential role in the genesis of neuropathic pain, did not affect recovery of neurological function and peripheral axon regeneration. Together, these results suggest that targeting specific IL-1ß/TNF-dependent responses, such as neutrophil infiltration, is a better therapeutic strategy for treatment of neuropathic pain after peripheral nerve injury than complete blockage of cytokine production.

Development of a novel noncompetitive antagonist of IL-1 receptor.

IL-1 is a major proinflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein subunits. Currently available strategies to counter pathological IL-1 signaling rely on a recombinant IL-1 receptor antagonist, which directly competes with IL-1 for its binding site. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1R accessory protein, which are putative interactive sites with the IL-1RI subunit. In this study, we substantiate the merits of one of these peptides, rytvela (we termed "101.10"), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC(50) approximately 1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed noncompetitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. Findings describe the discovery of a potent and specific small (peptide) antagonist of IL-1RI, with properties in line with an allosteric negative modulator.

Clinical outcome of adjuvant treatment of endometrial cancer using aperture-based intensity-modulated radiotherapy.

PURPOSE: To assess disease control and acute and chronic toxicity with aperture-based intensity-modulated radiotherapy (AB-IMRT) for postoperative pelvic irradiation of endometrial cancer. METHODS AND MATERIALS: Between January and July 2005, after hysterectomy for endometrial cancer, 15 patients received 45 Gy to the pelvis using AB-IMRT. The AB-IMRT plans were generated by an in-house treatment planning system (Ballista). The AB-IMRT plans were used for treatment and were dosimetrically compared with three other approaches: conventional four-field, enlarged four-field, and beamlet-based IMRT (BB-IMRT). Disease control and toxicity were prospectively recorded and compared with retrospective data from 30 patients treated with a conventional four-field technique. RESULTS: At a median follow-up of 27 months (range, 23-30), no relapse was noted among the AB-IMRT group compared with five relapses in the control group (p = 0.1). The characteristics of each group were similar, except for the mean body mass index, timing of brachytherapy, and applicator type used. Patients treated with AB-IMRT experienced more frequent Grade 2 or greater gastrointestinal acute toxicity (87% vs. 53%, p = 0.02). No statistically significant difference was noted between the two groups regarding the incidence or severity of chronic toxicities. AB-IMRT plans significantly improved target coverage (93% vs. 76% of planning target volume receiving 45 Gy for AB-IMRT vs. conventional four-field technique, respectively). The sparing of organs at risk was similar to that of BB-IMRT. CONCLUSION: The results of our study have shown that AB-IMRT provides excellent disease control with equivalent late toxicity compared with the conventional four-field technique. AB-IMRT provided treatment delivery and quality assurance advantages compared with BB-IMRT and could reduce the risk of second malignancy compared with BB-IMRT.

Postoperative irradiation of gynecologic malignancies: improving treatment delivery using aperture-based intensity-modulated radiotherapy.

PURPOSE: To evaluate dosimetric and treatment delivery advantages of aperture-based intensity-modulated radiotherapy (AB-IMRT) for the treatment of patients receiving whole pelvic radiotherapy for gynecologic malignancies. METHODS AND MATERIALS: Nineteen patients undergoing pelvic radiotherapy after resection of endometrial cancers were selected. A 45-Gy dose was prescribed to the target volume delineated on a planning CT scan. An in-house inverse planning system, Ballista, was used to develop a treatment plan using aperture-based multileaf collimator segments. This approach was compared with conventional four-field, enlarged four-field, and static beamlet-based IMRT (BB-IMRT) techniques in terms of target coverage, dose-volume histogram statistics for surrounding normal tissues, and numbers of segments and monitor units (MU). RESULTS: Three quarters (76.4%) of the planning target volume received the prescription dose with conventional four-field plans. With adequate target coverage, the Ballista plans significantly reduced the volume of bowel and bladder irradiated at the prescribed dose (p < 0.001), whereas the two approaches provided equivalent results for the rectum (p = 0.5). On the other hand, AB-IMRT and BB-IMRT plans showed only small differences in dose-volume histogram statistics of unknown clinical impact, whereas Ballista plan delivery required on average 73% and 59% fewer segments and MU, respectively. CONCLUSION: With respect to conventional techniques, AB-IMRT for the treatment of gynecologic malignancies provides dosimetric advantages similar to those with BB-IMRT but with clear treatment delivery improvements.

A transcriptional role for C/EBP beta in the neuronal response to axonal injury.

The molecular mechanisms responsible for inducing gene expression following neuronal injury are not well understood. Here, we address this issue by focusing upon C/EBPbeta, a transcription factor implicated in cellular injury and regeneration. We show that C/EBPbeta mRNA is expressed in neurons throughout the mature brain and that levels of both C/EBPbeta mRNA and phosphoprotein are increased in facial motor neurons following axonal injury. To determine the importance of these increases, we examined the regeneration-associated Talpha1 alpha-tubulin gene which contains functional C/EBP binding sites in its promoter. In transgenic mice, expression of a minimal 176 nucleotide Talpha1 alpha-tubulin promoter:nlacZ reporter gene was upregulated in injured facial motor neurons. This injury-induced transcriptional increase was inhibited in C/EBPbeta -/- mice. A similar inhibition was observed in C/EBPbeta -/- mice that carried a larger 1.1-kb promoter Talpha1:nlacZ reporter construct. Moreover, in situ hybridization revealed that the injury-induced upregulation of the endogenous mouse alpha1 alpha-tubulin mRNA, and of a second regeneration-associated mRNA, GAP-43, was inhibited in C/EBPbeta -/- mice. Thus, C/EBPbeta is essential for the neuronal injury response, acting to transcriptionally activate regeneration-associated gene expression.

Glucocorticoids play a fundamental role in protecting the brain during innate immune response.

The innate immune system plays a crucial role in protecting the host against infectious microorganisms. An inappropriate control of this system may have profound consequences, because of the maintained production of specific proinflammatory molecules. Glucocorticoids are the most efficient endogenous molecules that provide negative feedback on proinflammatory signaling and gene expression. Here we show that activation of this system is not detrimental for the brain but a profound neurodegeneration takes place in animals treated with the glucocorticoid receptor inhibitor Mifepristone (RU486). This drug increased the inflammatory reaction induced by a single intracerebral bolus of lipopolysaccharide (LPS). Inhibition of tumor necrosis factor alpha (TNF-alpha) totally abolished the neurotoxic effect of the endotoxin, and chronic infusion of the cytokine mimicked the treatment combining RU486 and LPS. The neuronal damage caused by TNF-alpha is dependent on both nitric oxide and caspase pathways. In controlling the cerebral innate immunity and microglial TNF-alpha production, glucocorticoids play a major role in protecting the brain against bacterial cell wall components.

Endotoxemia prevents the cerebral inflammatory wave induced by intraparenchymal lipopolysaccharide injection: role of glucocorticoids and CD14.

There is a robust and transient innate immune response in the brain during endotoxemia, which is associated with a cascade of NF-kappaB signaling events and transcriptional activation of genes that encode TNF-alpha and the LPS receptor CD14. The present study investigated whether circulating LPS has the ability to modulate the cerebral innate immune response caused by an intrastriatal (IS) injection of the endotoxin. We also tested the possibility that CD14 plays a role in these effects and male rats received an intracerebroventricular injection with an anti-CD14 before the IS LPS administration. The single LPS bolus into the striatum caused a strong and time-dependent transcriptional activation of TNF-alpha, IkappaBalpha, CD14, and monocyte chemoattractant protein-1 mRNA in microglial cells ipsilateral to the site of injection. Surprisingly, this wave of induced transcripts was essentially abolished by the systemic endotoxin pretreatment. Such anti-inflammatory properties of circulating LPS are mediated via plasma corticosterone, because exogenous corticoids mimicked while glucocorticoid receptor antagonist RU486 prevented the effects of systemic endotoxin challenge. Of interest is the partial involvement of CD14 in LPS-induced neuroinflammation; the anti-CD14 significantly abolished the microglial activity at day 3, but not at times earlier. The inflammatory response provoked by an acute intraparenchymal LPS bolus was not associated with convincing neurodegenerative processes. These data provide compelling evidence that systemic inflammation, through the increase in circulating glucocorticoids, has the ability to prevent the cerebral innate immune reaction triggered by an IS endotoxin injection. This study also further consolidates the existence of such system in the brain, which is finely regulated and its transient activation is not harmful for the neuronal elements.