Measurement of the vascular input function in mice for DCE-MRI

DCE-MRI is an important technique in the study of small animal cancer models because its sensitivity to vascular changes opens the possibility of quantitative assessment of early therapeutic response. However, extraction of physiologically descriptive parameters from DCE-MRI data relies upon measurement of the vascular input function (VIF), which represents the contrast agent concentration time course in the blood plasma. This is difficult in small animal models due to artifacts associated with partial volume, inflow enhancement, and the limited temporal resolution achievable with MR imaging. In this work, the development of a suite of techniques for high temporal resolution, artifact resistant measurement of the VIF in mice is described. One obstacle in VIF measurement is inflow enhancement, which decreases the sensitivity of the MR signal to the presence of contrast agent. Because the traditional techniques used to suppress inflow enhancement degrade the achievable spatiotemporal resolution of the pulse sequence, improvements can be achieved by reducing the time required for the suppression. Thus, a novel RF pulse which provides spatial presaturation contemporaneously with the RF excitation was implemented and evaluated. This maximizes the achievable temporal resolution by removing the additional RF and gradient pulses typically required for suppression of inflow enhancement. A second challenge is achieving the temporal resolution required for accurate characterization of the VIF, which exceeds what can be achieved with conventional imaging techniques while maintaining adequate spatial resolution and tumor coverage. Thus, an anatomically constrained reconstruction strategy was developed that allows for sampling of the VIF at extremely high acceleration factors, permitting capture of the initial pass of the contrast agent in mice. Simulation, phantom, and in vivo validation of all components were performed. Finally, the two components were used to perform VIF measurement in the murine heart. An in vivo study of the VIF reproducibility was performed, and an improvement in the measured injection-to-injection variation was observed. This will lead to improvements in the reliability of quantitative DCE-MRI measurements and increase their sensitivity.

Molecular analysis of TNF sensitivity in normal and Ha-{\it ras\/} transformed murine fibroblasts

Tumor necrosis factor (TNF) is known to have antiproliferative effects on a wide variety of tumor cells but proliferative effects on normal cells. However, the molecular basis for such differences in the action of TNF are unknown. The overall objectives of my research are to investigate the role of oncogenes in TNF sensitivity and delineate some of the molecular mechanisms involved in TNF sensitivity and resistance. To accomplish these objectives, I transfected TNF-resistant C3H mouse embryo fibroblasts (10T1/2) with an activated Ha-ras oncogene and determined whether these cells exhibit altered sensitivity to TNF. The results indicated that 10T1/2 cells transfected with an activated Ha-ras oncogene (10T-EJ) not only produced tumors in nude mice but also exhibited extreme sensitivity to cytolysis by TNF. In contrast, 10T1/2 cells transfected with the pSV2-neo gene alone were resistant to the cytotoxic effects of TNF. I also found that TNF-induced cell death was mediated through apoptosis. The differential sensitivity of 10T1/2 and 10T-EJ cell lines to TNF was not due to differences in the number of TNF receptors on their cell surface. In addition, TNF-resistant revertants isolated from Ha-ras-transformed, TNF-sensitive cells still expressed the same amount of p21 as TNF-sensitive cells and were still tumorigenic, suggesting that Ha-ras-induced transformation and TNF sensitivity may follow different pathways. Interestingly, TNF-resistant but not sensitive cells expressed higher levels of bcl-2, c-myc, and manganese superoxide dismutase (MnSOD) mRNA following exposure to TNF. However, TNF treatment resulted in a marginal induction of p53 mRNA in both TNF-sensitive and resistant cells. Based on these results I can conclude that (i) Ha-ras oncogene induces both transformation and TNF sensitivity, (ii) TNF-induced cytotoxicity involves apoptosis, and (iii) TNF-induced upregulation of bcl-2, c-myc, and MnSOD genes is associated with TNF resistance in C3H mouse embryo fibroblasts. ^

Bortezomib modulates TRAIL sensitivity in human bladder and prostate cancer

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a member of the TNF superfamily of cytokines that can induce cell death through engagement of cognate death receptors. Unlike other death receptor ligands, it selectively kills tumor cells while sparing normal cells. Preclinical studies in non-human primates have generated much enthusiasm regarding its therapeutic potential. However, many human cancer cell lines exhibit significant resistance to TRAIL-induced apoptosis, and the molecular mechanisms underling this are controversial. Possible explanations are typically cell-type dependent, but include alterations of receptor expression, enhancement of pro-apoptotic intracellular signaling molecules, and reductions in anti-apoptotic proteins. We show here that the proteasome inhibitor bortezomib (Velcade, PS-341) produces synergistic apoptosis in both bladder and prostate cancer cell lines within 4-6 hours when co-treated with recombinant human TRAIL which is associated with accumulation of p21 and cdk1/2 inhibition. Our data suggest that bortezomib's mechanism of action involves a p21-dependent enhancement of caspase maturation. Furthermore, we found enhanced tumor cell death in in vivo models using athymic nude mice. This is associated with increases in caspase-8 and caspase-3 cleavage as well as significant reductions in microvessel density (MVD) and proliferation. Although TRAIL alone had less of an effect, its biological significance as a single agent requires further investigations. Toxicity studies reveal that the combination of bortezomib and rhTRAIL has fatal consequences that can be circumvented by altering treatment schedules. Based on our findings, we conclude that this strategy has significant therapeutic potential as an anti-cancer agent. ^

Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

INTRODUCTION: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. METHODS: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. RESULTS: Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. CONCLUSION: We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.

Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice.

Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.

Skeletal abnormalities in mice lacking extracellular matrix proteins, thrombospondin-1, thrombospondin-3, thrombospondin-5, and type IX collagen.

Thrombospondin-5 (TSP5) is a large extracellular matrix glycoprotein found in musculoskeletal tissues. TSP5 mutations cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia; both show a characteristic growth plate phenotype with retention of TSP5, type IX collagen (Col9), and matrillin-3 in the rough endoplasmic reticulum. Whereas most studies focus on defining the disease process, few functional studies have been performed. TSP5 knockout mice have no obvious skeletal abnormalities, suggesting that TSP5 is not essential in the growth plate and/or that other TSPs may compensate. In contrast, Col9 knockout mice have diminished matrillin-3 levels in the extracellular matrix and early-onset osteoarthritis. To define the roles of TSP1, TSP3, TSP5, and Col9 in the growth plate, all knockout and combinatorial strains were analyzed using histomorphometric techniques. While significant alterations in growth plate organization were found in certain single knockout mouse strains, skeletal growth was only mildly disturbed. In contrast, dramatic changes in growth plate organization in TSP3/5/Col9 knockout mice resulted in a 20% reduction in limb length, corresponding to similar short stature in humans. These studies show that type IX collagen may regulate growth plate width; TSP3, TSP5, and Col9 appear to contribute to growth plate organization; and TSP1 may help define the timing of growth plate closure when other extracellular proteins are absent.

Regulation of hepatic cytochrome P450 expression in mice with intestinal or systemic infections of citrobacter rodentium.

We reported previously that infection of C3H/HeOuJ (HeOu) mice with the murine intestinal pathogen Citrobacter rodentium caused a selective modulation of hepatic cytochrome P450 (P450) gene expression in the liver that was independent of the Toll-like receptor 4. However, HeOu mice are much more sensitive to the pathogenic effects of C. rodentium infection, and the P450 down-regulation was associated with significant morbidity in the animals. Here, we report that oral infection of C57BL/6 mice with C. rodentium, which produced only mild clinical signs and symptoms, produced very similar effects on hepatic P450 expression in this strain. As in HeOu mice, CYP4A mRNAs and proteins were among the most sensitive to down-regulation, whereas CYP4F18 was induced. CYP2D9 mRNA was also induced 8- to 9-fold in the C57BL/6 mice. The time course of P450 regulation followed that of colonic inflammation and bacterial colonization, peaking at 7 to 10 days after infection and returning to normal at 15 to 24 days as the infection resolved. These changes also correlated with the time course of significant elevations in the serum of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, as well as of interferon-gamma and IL-2, with serum levels of IL-6 being markedly higher than those of the other cytokines. Intraperitoneal administration of C. rodentium produced a rapid down-regulation of P450 enzymes that was quantitatively and qualitatively different from that of oral infection, although CYP2D9 was induced in both models, suggesting that the effects of oral infection on the liver are not due to bacterial translocation.

Adenosine signaling contributes to ethanol-induced fatty liver in mice.

Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5'-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5'-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.

The effect of ultraviolet radiation on the pathogenesis of {\it Candida albicans} in mice

Ultraviolet (UV) radiation produces immunological alterations in both humans and animals that include a decrease in the delayed type hypersensitivity (DTH) response to complex antigens, and to the induction of the suppressor T cell pathway. Cell-mediated immunity of the type that is altered by UV radiation has been shown to be important in host resistance against microorganisms. My dissertation addresses questions concerning the effects of UV radiation on the pathogenesis of opportunistic fungal pathogens such as Candida albicans.^ The (DTH) response of C3H mice exposed to ultraviolet (UV) radiation before (afferent arm of DTH) or after (efferent arm of DTH) infection with Candida albicans was markedly and systemically suppressed. Although suppression of both the afferent and efferent phases of DTH were caused by similar wavebands within the ultraviolet region, the dose of UV radiation that suppressed the efferent arm of DTH was 10-fold higher than the dose that suppressed the afferent arm of the DTH reaction.^ The DTH response of C57BL/6 mice was also suppressed by UV radiation; however the suppression was accomplished by exposure to significantly lower doses UV radiation compared to C3H mice. In C57BL/6 mice, the dose of UV radiation that suppressed the afferent phase of DTH was 5-fold higher than the dose that suppressed the efferent phase.^ Exposure of C3H mice to UV radiation before sensitization induced splenic suppressor T cells that upon transfer to normal recipients, impaired the induction of DTH to Candida. In contrast, the suppression caused by UV irradiation of mice after sensitization was not transferable. Spleen cells from sensitized mice exhibited altered homing patterns in animals that were exposed to UV radiation shortly before receiving cells, suggesting that UV-induced suppression of the efferent arm of DTH could result from an alteration in the distribution of effector cells.^ UV radiation decreased the survival of Candida-infected mice; however, no correlation was found between suppression of the DTH response and the course of lethal infection. This suggested that DTH was not protective against lethal disease with this organism. UV radiation also changed the persistence of the organism in the internal organs. UV-irradiated, infected animals had increased numbers of Candida in their kidneys compared to non-irradiated mice. Sensitization prior to UV irradiation aided clearance of the organism from the kidneys of UV-irradiated mice.^ These data show that UV radiation suppresses cell-mediated immunity to Candida albicans in mice and increases mortality of Candida-infected mice. Moreover, the data suggest that an increase in environmental UV radiation could increase the severity of pathogenic infections. ^

Functional studies on DNA double-strand break repair proteins (MmRad51, Ku80) in mice

RecA in Escherichia coli and it's homologue, ScRad51 in Saccharomyces cerevisiae, play important roles in recombinational repair. ScRad51 homologues have been discovered in a wide range of organisms including Schizosaccharomyces pombe, lily, chicken, mouse and human. To date there is no direct evidence to describe that mouse Rad51(MmRad51) is involved in DNA double-strand break repair. In order to elucidate the role of MmRad51 in vivo, it was mutated by the embryonic stem (ES) cell/gene targeting technology in mice. The mutant embryos arrested in development shortly after implantation. There was a decrease in cell proliferation followed by programmed cell death, and trophectoderm-derived cells were sensitive to $\gamma$-radiation. Severe chromosome loss was observed in most mitotically dividing cells. The mutant embryos lived longer and developed further in a p53 mutant background; however, double-mutant embryonic fibroblasts failed to proliferate in tissue culture, reflecting the embryos limited life span. Based on these data, MmRad51 repairs DNA damage induced by $\gamma$-radiation, is needed to maintain euplody, and plays an important role in proliferating cells.^ Ku is a heterodimer of 70 and 80 kDs subunit, which binds to DNA ends and other altered DNA structures such as hairpins, nicks, and gaps. In addition, Ku is required for DNA-PK activity through a direct association. Although the biochemical properties of Ku and DNA-PKcs have been characterized in cells, their physiological functions are not clear. In order to understand the function of Ku in vivo, we generated mice homozygous for a mutation of the Ku80 gene. Ku80-deficient mice, like scid mice, showed severe immunodeficiency due to a impairment of V(D)J recombination. Mutant mice were semiviable and runted, cells derived from mutant embryos displayed hypersensitivity to $\gamma$-radiation, a decreased growth rate, a slow entry into S phase, altered colony size distributions, and a short life span. Based on these results, mutant cells and mice appeared to prematurely age. ^

Analysis of BMP signalling through the receptor type IA in embryogenesis using conditional gene inactivation in mice

Although bone morphogenetic proteins (BMPs) were initially identified for their potent bone-inducing activity, their precise roles in processes of endochondral and intramembranous bone formation are far from being clear. Tissue-specific loss-of-function experiments using the BMP receptor type IA (BMPR-IA) are particularly attractive since this receptor is thought to be essential for signaling by the closely related BMPs -2, 4, and 7. To ablate signaling through this receptor during chondrogenesis, we have generated transgenic mice expressing Cre recombinase under the control of the collagen type II (Col2a1) gene regulatory sequences. Mice lacking BMPR-IA function in chondrocytes display a number of skeletal abnormalities, including defects in bones of the chondrocranium, abnormal dorsal vertebral processes, scapulae with severe hypoplasia of dorsal elements, and shortening of the long bones. Alterations in the growth plate of long bones in mutants suggest that BMPR-IA is not required for early steps of the chondrocyte specification, but is rather important in regulation of terminal differentiation. Molecular analysis revealed noticeable downregulation of the Ihh/Ptch signalling pathway, decreased chondrocyte proliferation rate and deregulation of hypertrophy. ^ In order to elucidate the role of BMP signalling in development of the limb and intramembranous ossification, we have used mice expressing Cre recombinase under control of the Prx1 (MHox) regulatory elements (M. Logan, pers comm.). Cre activity was found in those mice in the developing limb bud mesenchyme, as well as in a subset of cranial neural crest cells. Prx1-Cre-induced conditional mutants display prominent defects in distal limb outgrowth, as well as ossification defects in a number of neural crest-derived calvarial bones. Intriguingly, mutant limbs displayed alterations in patterning along all three axes. Molecular analysis revealed ectopic anterior Shh/Ptch signalling pathway activation and expression of some Hox genes. Observed loss of Msx1 and Msx2 expression in the progress zone correlates with downregulation of Cyclin D1 and decreased distal outgrowth. Abnormal ventral localization of Lmx1b-expressing cells along with observed later morphological abnormalities suggest a novel role for BMP signalling in establishment or maintaining of the dorso-ventral polarity in the limb mesoderm. ^

Importance of sperm transition proteins demonstrated in mice carrying Tnp1- and Tnp2-null alleles

Chromatin condensation within the nucleus of developing spermatids involves replacement of histones by transition proteins, which are in turn replaced by protamines. The importance of transition proteins in the complex process of spermiogenesis has, to date, been only speculative. This study sought to investigate the extent to which transition proteins are essential or have redundant functions by characterizing sperm produced in mice expressing all combinations of Tnp-null alleles. Results from breeding trials of 8 weeks duration revealed that, on average, wildtype males produced about 14 offspring whereas TP2 and TP1 single-knockout males produced about 8 and 1 offspring, respectively, demonstrating their subfertility. Genotypes with less than two Tnp wildtype alleles, as well as double-knockout mutants, were completely infertile. Sperm from males with impaired fertility had poor progressive motility, heterogeneous chromatin condensation, incompletely processed protamine 2 and head and tail abnormalities. Generally, as the number of Tnp-null alleles increased so did the severity of abnormalities. However, specific morphological abnormalities were associated with the absence of an individual TP. Studies which sought to identify possible root causes for abnormalities in thiol-rich sperm structures revealed no differences in thiol content or sulfhydryl oxidation status within the nucleus but nuclei and tails from single-knockout mutants were severely disrupted following thiol reduction. Binding of fluorescent dyes to DNA was normal in sperm recovered from caput but abnormal in cauda epididymal sperm from TP1 knockouts and infertile double mutants. Injection of cauda epididymal sperm from double knockouts into oocytes produced very few offspring; however, after injection with testicular sperm, the efficiency was no different from wildtype. These results suggest DNA structural alterations or degradation during epididymal transport of sperm resulting in a diminished capacity of the paternal DNA of these sperm to produce offspring. The overall importance of transition proteins for normal chromatin condensation and production of fertile sperm has been demonstrated. Furthermore, identification of specific morphological abnormalities associated with the absence of an individual transition protein provides new evidence that the proteins are not completely redundant and each fulfills some unique function. ^

Assessment of cone beam computed tomography techniques for imaging lung damage in mice in vivo

Lung damage is a common side effect of chemotherapeutic drugs such as bleomycin. This study used a bleomycin mouse model which simulates the lung damage observed in humans. Noninvasive, in vivo cone-beam computed tomography (CBCT) was used to visualize and quantify fibrotic and inflammatory damage over the entire lung volume of mice. Bleomycin was used to induce pulmonary damage in vivo and the results from two CBCT systems, a micro-CT and flat panel CT (fpCT), were compared to histologic measurements, the standard method of murine lung damage quantification. Twenty C57BL/6 mice were given either 3 U/kg of bleomycin or saline intratracheally. The mice were scanned at baseline, before the administration of bleomycin, and then 10, 14, and 21 days afterward. At each time point, a subset of mice was sacrificed for histologic analysis. The resulting CT images were used to assess lung volume. Percent lung damage (PLD) was calculated for each mouse on both the fpCT (PLDfpcT) and the micro-CT (PLDμCT). Histologic PLD (PLDH) was calculated for each histologic section at each time point (day 10, n = 4; day 14, n = 4; day 21, n = 5; control group, n = 5). A linear regression was applied to the PLDfpCT vs. PLDH, PLDμCT vs. PLDH and PLDfpCT vs. PLDμCT distributions. This study did not demonstrate strong correlations between PLDCT and PLDH. The coefficient of determination, R, was 0.68 for PLDμCT vs. PLDH and 0.75 for the PLD fpCT vs. PLDH. The experimental issues identified from this study were: (1) inconsistent inflation of the lungs from scan to scan, (2) variable distribution of damage (one histologic section not representative of overall lung damage), (3) control mice not scanned with each group of bleomycin mice, (4) two CT systems caused long anesthesia time for the mice, and (5) respiratory gating did not hold the volume of lung constant throughout the scan. Addressing these issues might allow for further improvement of the correlation between PLDCT and PLDH. ^

Regulation of T cell function by complement C5A in mice during mycobacterial infection

Tuberculosis is the leading cause of death in the world due to a single infectious agent, making it critical to investigate all aspects of the immune response mounted against the causative agent, Mycobacterium tuberculosis , in order to better treat and prevent disease. Previous observations show a disparity in the ability to control mycobacterial growth between mouse strains sufficient in C5, such as C57BL/6 and B10.D2/nSnJ, and those naturally deficient in C5, such as A/J and B10.D2/nSnJ, with C5 deficient mice being more susceptible. It has been shown that during M. tuberculosis infection, C5 deficient macrophages have a defect in production of interleukin (IL)-12, a cytokine involved in the cyclical activation between infected macrophages and effector T cells. T cells stimulated by IL-12 produce interferon (IFN)-γ, the signature cytokine of T helper type 1 (Th1) cells. It is known that a cell-mediated Th1 response is crucial for control of M. tuberculosis in the lungs of humans and mice. This study demonstrates that murine T cells express detectable levels of CD88, a receptor for C5a (C5aR), following antigen presentation by macrophages infected with mycobacteria. T cells from C5 deficient mice infected with M. tuberculosis were found to secrete less IFN-γ and had a reduced Th1 phenotype associated with fewer cells expressing the transcription factor, T-box expressed in T cells (T-bet). The altered Th1 phenotype in M. tuberculosis infected C5 deficient mice coincided with a rise in IL-4 and IL-10 secretion from Th2 cells and inducible regulatory T cells, respectively. It was found that the ineffective T cell response to mycobacteria in C5 deficient mice was due indirectly to a lack of C5a via poor priming by infected macrophages and possibly by a direct interaction between T cells and C5a peptide. Therefore, these studies show a link between the cells of the innate and adaptive arms of the immune system, macrophages and T cells respectively, that was mediated by C5a using a mouse model of M. tuberculosis infection. ^