An immediate consequence of traumatic brain injury (TBI) is the induction of an inflammatory response. Mounting data suggest that inflammation is a major contributor to TBI-induced brain damage. However, much remains unknown regarding the induction and regulation of the inflammatory response to TBI. In this study we compared the TBI-induced inflammatory response to severe parenchymal injury (controlled cortical impact) vs. mild brain injury (craniotomy) over a 21-day period. Our data show that both severe and mild brain injury induce a qualitatively similar inflammatory response, involving highly overlapping sets of effector molecules. However, kinetic analysis revealed that the inflammatory response to mild brain injury is of much shorter duration than the response to severe TBI. Specifically, the inflammatory response to severe brain injury persists for at least 21 days, whereas the response to mild brain injury returns to near baseline values within 10 days post-injury. Our data therefore imply that the development of accurate diagnostic tests of TBI severity that are based on imaging or biomarker analysis of the inflammatory response may require repeated measures over at least a 10-day period, post-injury.

Mice lacking the gene for the IL-1R antagonist (IL-1ra) show abnormal development and homeostasis as well as altered responses to infectious and inflammatory stimuli. A reduction in the level of IL-1 signaling, either by deletion of the receptor or increased expression of IL-1ra, does not affect development or homeostasis, but does alter immune responses. In this study we use genetic epistasis to investigate the interdependence of selected genes in the IL-1 family in the regulation of these developmental and immunological processes. Deletion of the gene encoding the type I IL-1R (IL-1RI) is epistatic to deletion of the IL-1ra gene. Therefore, all functions of IL-1ra depend upon the presence of a functional receptor; there is no other target. Similarly, overexpression of the mRNA encoding the secreted form of IL-1ra is epistatic to deletion of the receptor antagonist, leaving the role of the intracellular splice variants of IL-1ra unknown. The abnormal development of IL-1ra-deficient mice is probably due to chronic overstimulation of the proinflammatory pathway via IL-1, but a clear single pathological defect is not apparent. These results support the model that the only essential function of IL-1ra in both health and disease is competitive inhibition of the IL-1RI.

Imreg (Tyr-Gly-Gly) is a well-known immunostimulant. However, it possesses a short half-life. Stabilized analogues of Imreg were prepared by a regioselective insertion in which peptide bonds at position 1,2 or 2,3 were replaced by thioamide linkages. This was achieved by using new thioacylating agents based on thioacyl-fluoro-N-benzimidazolone. The synthesis and properties of these reagents are described herein. This peptide modification enhanced significantly the half-life of the thioanalogues relative to Imreg in blood. The thioanalogues and Imreg were tested in vitro in T and B cell proliferation assays and for their ability to stimulate cytotoxic T-lymphocytes (CTLs). Only thiotyrosyl glycyl glycine 11 displayed some activity as evidenced by a weak stimulation of CTLs. On the basis of this activity and the increased stability, an in vivo immunological evaluation was undertaken. Immunophenotyping of 11 revealed a significant increase in activated CTL and NK cell populations in the spleen. This expansion was also accompanied by a significant stimulation of NK cells and the B cell proliferative response. Thioanalogues of Imreg were generally nontoxic, as exemplified by 11. The latter is a promising immunostimulant which may be targeted for cancer and viral infections, where CTLs and NK cells play an important role, or as a vaccine adjuvant where stimulation of antibody-producing B cells is important.

Neuroinflammation plays a critical role in the pathogenesis of traumatic brain injury (TBI). TBI induces rapid activation of astrocytes and microglia, infiltration of peripheral leukocytes, and secretion of inflammatory cytokines. In the context of modest or severe TBI, such inflammation contributes to tissue destruction and permanent brain damage. However, it is clear that the inflammatory response is also necessary to promote post-injury healing. To date, anti-inflammatory therapies, including the broad class of non-steroidal anti-inflammatory drugs (NSAIDs), have met with little success in treatment of TBI, perhaps because these drugs have inhibited both the tissue-damaging and repair-promoting aspects of the inflammatory response, or because inhibition of inflammation alone is insufficient to yield therapeutic benefit. Salsalate is an unacetylated salicylate with long history of use in limiting inflammation. This drug is known to block activation of NF-κB, and recent data suggest that salsalate has a number of additional biological activities, which may also contribute to its efficacy in treatment of human disease. Here, we show that salsalate potently blocks pro-inflammatory gene expression and nitrite secretion by microglia in vitro. Using the controlled cortical impact (CCI) model in mice, we find that salsalate has a broad anti-inflammatory effect on in vivo TBI-induced gene expression, when administered post-injury. Interestingly, salsalate also elevates expression of genes associated with neuroprotection and neurogenesis, including the neuropeptides, oxytocin and thyrotropin releasing hormone. Histological analysis reveals salsalate-dependent decreases in numbers and activation-associated morphological changes in microglia/macrophages, proximal to the injury site. Flow cytometry data show that salsalate changes the kinetics of CCI-induced accumulation of various populations of CD11b-positive myeloid cells in the injured brain. Behavioral assays demonstrate that salsalate treatment promotes significant recovery of function following CCI. These pre-clinical data suggest that salsalate may show promise as a TBI therapy with a multifactorial mechanism of action to enhance functional recovery.

Water soluble analogues of the lipophilic immunostimulant, octadecyl D-alanyl-L-glutamine, BCH-527, were synthesized and evaluated for the ability to stimulate natural killer (NK) cells. One of these compounds in which the octadecyl chain of BCH-527 was replaced with a shorter chain alcohol, 6-(D-alanyl-L-glutaminylamino)hexan-1-ol, 9, displayed an in vitro stimulation of NK cells comparable to that of interleukin 2 (IL 2). However, when the hydroxyl of 9 was linked to L-fucose to yield 1-beta-[6-(D-alanyl-L-glutaminylamino)hex-1-yl]-L- fucopyranose (BCH-2537, 1), the observed stimulation of NK cells was greater than that observed with IL 2. Further evaluation of these compounds revealed that the improved in vitro activity of BCH-2537 was more pronounced in vivo. That is, while both compounds significantly increased splenic NK cells, only BCH-2537 significantly increased the activity of these cells in vivo. In terms of a structure-activity relationship, NK cell activity was sensitive to minor structural modifications. It was influenced by conservative substitutions within the dipeptide, the length of the hydrocarbon chain, and the functionality at the end of the chain. No other compound enhanced NK cell activity to the extent exhibited by BCH-2537, although a few were equipotent to 9.