High-Fat Diet-Induced Diabetic Conditions Exacerbate Cognitive Impairment in a Mouse Model of Alzheimer's Disease Via a Specific Tau Phosphorylation Pattern.

BACKGROUND: Epidemiological evidence has demonstrated a clear association between diabetes mellitus and increased risk of Alzheimer's disease (AD). Cerebral accumulation of phosphorylated tau aggregates, a cardinal neuropathological feature of AD, is associated with neurodegeneration and cognitive decline. Clinical and experimental studies indicate that diabetes mellitus affects the development of tau pathology; however, the underlying molecular mechanisms remain unknown. OBJECTIVE: In the present study, we used a unique diabetic AD mouse model to investigate the changes in tau phosphorylation patterns occurring in the diabetic brain. DESIGN: Tau-transgenic mice were fed a high-fat diet (n = 24) to model diabetes mellitus. These mice developed prominent obesity, severe insulin resistance, and mild hyperglycemia, which led to early-onset neurodegeneration and behavioral impairment associated with the accumulation of hyperphosphorylated tau aggregates. RESULTS: Comprehensive phosphoproteomic analysis revealed a unique tau phosphorylation signature in the brains of mice with diabetic AD. Bioinformatic analysis of the phosphoproteomics data revealed putative tau-related kinases and cell signaling pathways involved in the interaction between diabetes mellitus and AD. CONCLUSION: These findings offer potential novel targets that can be used to develop tau-based therapies and biomarkers for use in AD.

Studies on expression of aldehyde dehydrogenase in normal and cancerous tissues of thyroids.

Recently published articles have reported the controversial data regarding expression of aldehyde dehydrogenase isozyme 1A1 (ALDH1A1), a potential candidate marker for normal and cancer stem cells (CSCs), in thyroid tissues. These data prompted us to re-evaluate expression of ALDH1A1 in normal and cancerous thyroid tissues by 2 different means. The first method was immunohistochemistry with 2 different anti-ALDH1A1 antibodies from distinct companies. Following validating the integrity of these 2 antibodies by Western blotting with ALDH-expressing and nonexpressing cancer cell lines and immunohistochemistry with breast and colon tissues, we report here significant and comparable expression of ALDH1A1 in both normal and cancerous thyroid tissues with both antibodies. Next, relative expression levels of ALDH isozymes were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), revealing that ALDH1A1 was the most highly expressed isozyme followed by ALDH9A1 and relative expression patterns of isozymes were very similar in normal and cancerous tissues. All these data demonstrate that thyroid cells of normal and cancer origins do express ALDH1A1 and to a lesser extent 9A1. Further study will be necessary to study functional significance of ALDH1A1 in the function and behaviors of thyroid normal and cancer stem cells.

Therapeutic potential of cholesteryl O-acyl α-glucoside found in Helicobacter pylori.

Steryl glycosides are derivatives of sterols where the 3ß-hydroxy group is glycosylated. Some of them are further converted to steryl O-acyl glycosides. Steryl glycosides and their derivatives are widely distributed in plants, algae, and fungi, but are relatively rarely distributed in bacteria and animals. Accumulating evidence suggests that glycosylation of sterols not only modifies physicochemical properties of cell membranes but also alters immunogenicity of the cells. Helicobacter pylori, that colonizes the stomach and causes gastric diseases, is auxotrophic for cholesterol, so that it extracts this lipid from plasma membranes of epithelial cells of the host stomach. Since incorporation of cholesterol promotes immune responses of the host, Helicobacter pylori converts cholesterol to cholesteryl glucoside (ChG) and then to cholesteryl 6'-O-acyl glucoside (ChAcG) to evade the immune surveillance. We have found that ChAcG thus produced is specifically recognized by invariant Vα14-Jα18 TCR(+) (Vα14) NKT cells in a CD1-dependent manner. We have also found that activation of Vα14 NKT cells by administration of ChAcG retains homeostasis of immunity upon exposure to allergens and reduces the incidence of allergy. In this article, overview of immunological functions of steryl glycosides with an emphasis on the immunoregulatory functions of ChAcG, is demonstrated.

Prophylactic and therapeutic efficacies of a selective inhibitor of the immunoproteasome for Hashimoto's thyroiditis, but not for Graves' hyperthyroidism, in mice.

Major histocompatibility complex (MHC) class I-restricted T cell epitopes are generated mainly by the immunoproteasome in antigen-presenting cells. Therefore, inhibition of activity of this proteolytic complex molecule is thought to be a potential treatment for cell-mediated autoimmune diseases. We therefore studied the efficacy of an immunoproteasome inhibitor, ONX 0914 (formerly PR-957), for the treatment of autoimmune thyroid diseases, including cell-mediated Hashimoto's thyroiditis and autoantibody-mediated Graves' hyperthyroidism using mouse models. Our data show that ONX 0914 was effective prophylactically and therapeutically at suppressing the degree of intrathyroidal lymphocyte infiltration and, to a lesser degree, the titres of anti-thyroglobulin autoantibodies in non-obese diabetic (NOD)-H2(h4) mice, an iodine-induced autoimmune thyroiditis model. It also inhibited differentiation of T cells to T helper type 1 (Th1) and Th17 cells, effector T cell subsets critical for development of thyroiditis in this mouse strain. In contrast, its effect on the Graves' model was negligible. Although ONX 0914 exerts its immune-suppressive effect through not only suppression of immune proteasome but also other mechanism(s), such as inhibition of T cell differentiation, the present results suggest that the immunoproteasome is a novel drug target in treatment of Hashimoto's thyroiditis in particular and cell-mediated autoimmune diseases in general.

Glycolipid stimulators for NKT cells bearing invariant V alpha 19-J alpha 33 TCR alpha chains.

Attempts have been made to find specific antigens for a novel NKT cell subset bearing invariant V alpha 19-J alpha 33 TCR alpha chains (V alpha 19 NKT cell). Comprehensive examinations revealed substantial antigenic activity in synthetic alpha-mannosylceramide (ManCer) that was presented by MR1. Structural modification of the sphingosine moiety of alpha-ManCer improved antigenic activity to enhance either Th1 or Th2 -promoting cytokine production by V alpha 19 NKT cells. Such alpha-ManCer analogues will be useful for developing new therapies as immunomodulators.

Role of interleukin-1beta and tumor necrosis factor-alpha-dependent expression of cyclooxygenase-2 mRNA in thermal hyperalgesia induced by chronic inflammation in mice.

The present study investigated whether the endogenous pro-inflammatory cytokines [interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha)]-dependent expression of cyclooxygenase-2 (COX-2) mRNA within the spinal cord could be involved in the development of chronic inflammatory pain-like behaviors in mice. We demonstrated that the expression of COX-2 mRNA on the ipsilateral side of the spinal cord was significantly increased 6 h and 3 days after intraplantar injection of complete Freund's adjuvant (CFA), compared with the expression in saline-treated mice. In addition, the chronic pain-like behaviors following CFA injection were markedly suppressed by repeated intrathecal (i.t.) pre-treatment with the COX-2 inhibitor etodolac, but not with the COX-1 inhibitor mofezolac. The cytosolic level of the activated form of nuclear factor-kappa B (NF-kappaB), which is a major contributor to the induction of COX-2, on the ipsilateral side of the mouse spinal cord was also increased compared with that in the saline-treated mice. The key finding in the present study was that a single i.t. injection with either IL-1beta or TNF-alpha induced a marked increase in spinal COX-2 mRNA and persistent thermal hyperalgesia in mice. Furthermore, CFA-induced hypersensitivity to inflammatory pain was significantly reduced by repeated i.t. pre-injection of the recombinant Fc chimera of IL-1 receptor I or soluble TNF receptor I, which sequesters endogenous IL-1beta or TNF-alpha, respectively. In contrast, the expression of spinal COX-2 mRNA in CFA-treated mice was similar to that in saline-treated mice at 7 days after CFA injection. The present findings strongly indicate the early intrathecal use of the COX-2 inhibitor for the relief of chronic inflammatory pain. Furthermore, together with the result in a previous study that pro-inflammatory cytokines lead to stimulation of a NF-kappaB-dependent transcriptional pathway, these findings suggest that a spinal cytokine/NF-kappaB/COX-2 pathway may play an important role in the development, but not maintenance, of chronic pain following peripheral tissue inflammation.

Alleviation of Abeta-induced cognitive impairment by ultrasound-mediated gene transfer of HGF in a mouse model.

A new therapeutic approach to treat Alzheimer's disease (AD) is needed, and the use of growth factors is considered to be a candidate. Hepatocyte growth factor (HGF) is a unique multifunctional growth factor, which has the potential effect to exert neurotrophic action and induce angiogenesis. In this study, we examined the effects of overexpression of human HGF plasmid DNA using ultrasound-mediated gene transfer into the brain in an Abeta-infused cognitive dysfunction mouse model. We demonstrated that HGF gene transfer significantly alleviated Abeta-induced cognitive impairment in mice in behavioral tests. These beneficial effects of HGF might be due to (1) significant recovery of the vessel density in the dentate gyrus of the hippocampus, (2) upregulation of BDNF, (3) a significant decrease in oxidative stress and (4) synaptic enhancement. A pharmacological approach including gene therapy to increase the HGF level in combination with anti-Abeta therapy might be a new therapeutic option for the treatment of AD.

Coexpression of an unusual form of the EWS-WT1 fusion transcript and interleukin 2/15 receptor betamRNA in a desmoplastic small round cell tumour.

BACKGROUND: The beta chain of the interleukin 2/15 receptor (IL-2/15Rbeta) is induced by the expression of the EWS-WT1. A case of desmoplastic small round cell tumour (DSRCT) expressing only an unusual EWS-WT1 treated by us is reported here. AIM: To characterise an unusual form of EWS-WT1. METHODS: Frozen tissue sections of the axillary tumour were examined using a laser-assisted microdissection technique and reverse transcriptase polymerase chain reaction. RESULTS: The novel fusion of exon 8 of EWS and the defective exon 10 of WT1 (-KTS) was detected. Although it was an unusual form, the coexpression of the present EWS-WT1, IL-2/15Rbeta and Janus kinase (JAK1) mRNA was detected in the tumour cells. IL-2 and signal transducers and activators of transcription (STAT5) mRNA were detected in both tumour and stromal cells. CONCLUSION: The induction of the IL-2/15 receptor signalling pathway may contribute to tumorigenesis in DSRCT through a paracrine or an autocrine system, even though the EWS-WT1 was an unusual form.

Prevention of onset of Parkinson's disease by in vivo gene transfer of human hepatocyte growth factor in rodent model: a model of gene therapy for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SNi). As neurotrophic factors support the survival and enhance the function of dopaminergic neurons, gene therapy using neurotrophic factors has become the center of interest. Thus, we focused on hepatocyte growth factor (HGF) as a neurotrophic and angiogenic growth factor. At 7 days before injection of 6-hydroxydopamine into the SNi, stereotaxic transfection of human HGF or lacZ plasmid was performed into the unilateral striatum of rats. Expression of human HGF in the injected sites could be detected in rats transfected with HGF plasmid DNA, using immunohistochemical staining. Consistently, human immunoreactive HGF protein could be detected at least up to 12 days after transfection. Interestingly, PD rats transfected with lacZ demonstrated amphetamine-induced rotational asymmetry. However, transfection of HGF plasmid DNA resulted in significant inhibition of abnormal rotation up to 24 weeks in a dose-dependent manner. Over 90% of dopaminergic neurons were lost in PD rats transfected with lacZ, whereas over 70% survived in rats transfected with HGF, as assessed by immunohistochemical staining. Overall, the present study demonstrated that overexpression of HGF prevented neuronal death in a PD rat model, providing a potential novel therapy for PD.

The validity of a hyperventilation test for an investigation of autonomic failure: assessment in patients with multiple system atrophy and Parkinson's disease.

Although heart rate (HR) responses to hyperventilation (HV) have been used as a cardiovascular autonomic function test, autonomic involvement during HV remains uncertain. To clarify the relationship between autonomic activity and cardiovascular changes during HV, we compared cardiovascular responses during HV among subjects with different autonomic function, namely 16 patients with probable multiple system atrophy (MSA), 16 with possible MSA, 28 with Parkinson's disease (PD) and 28 healthy controls. Abnormalities of cardiovascular responses to head-up postural change and the Valsalva maneuver were definitely present in the order of probable MSA, possible MSA and PD, and abnormal HR and blood pressure (BP) responses during HV were observed in probable MSA and possible MSA, but not in PD. Unlike the significant difference in standard cardiovascular autonomic function tests, the HR and BP responses during HV were equivalent between probable and possible MSA. These findings suggest that cardiovascular control during HV may be affected not only by autonomic activity but also by other factors.

L-3,4-dihydroxyphenylalanine-induced c-Fos expression in the CNS under inhibition of central aromatic L-amino acid decarboxylase.

L-3,4-dihydroxyphenylalanine (DOPA) is a neurotransmitter candidate. To map the DOPAergic system functionally, DOPA-induced c-Fos expression was detected under inhibition of central aromatic L-amino acid decarboxylase (AADC). In rats treated with a central AADC inhibitor, DOPA significantly increased the number of c-Fos-positive nuclei in the paraventricular nuclei (PVN) and the nucleus tractus solitarii (NTS), and showed a tendency to increase in the supraoptic nuclei (SON), but not in the striatum. On the other hand, DOPA with a peripheral AADC inhibitor elevated the level of c-Fos-positive nuclei in the four regions, suggesting that DOPA itself induces c-Fos expression in the SON, PVN and NTS. In rats treated with 6-hydroxydopamine (6-OHDA) to lesion the nigrostriatal dopamine (DA) pathway, DOPA significantly induced c-Fos expression in the four regions under the inhibition of peripheral AADC. However, under the inhibition of central AADC, DOPA did not significantly increase the number of c-Fos-positive nuclei in the four regions, suggesting that DOPA at least in part induces c-Fos expression through its conversion to DA. It was likely that the 6-OHDA lesion enhanced the response to DA, but attenuated that to DOPA itself. In conclusion, we proposed that the SON, PVN and NTS include target sites for DOPA itself.

Suppression of PI3K/mTOR pathway rescues LLC cells from cell death induced by hypoxia.

Cancer cells in solid tumors are challenged by various microenvironmental stresses, including hypoxia, and cancer cells in hypoxic regions are resistant to current cancer therapies. To investigate the mechanism of resistance to hypoxia in cancer cells, we examined mouse Lewis lung carcinoma (LLC) cells, which died due to necrosis at high density under hypoxic but not under normoxic conditions. Levels of mammalian target of rapamycin (mTOR), a central regulator of cellular energy, are reported to be suppressed in hypoxia. We found that phosphorylation of two molecules downstream to it, ribosomal p70 S6 kinase (S6K) and ribosomal protein S6, was markedly suppressed by hypoxia. Overexpression of the active form of S6K increased the sensitivity of LLC cells to hypoxia. On the other hand, inhibition of PI3K or mTOR dramatically reduced hypoxia-induced cell death under hypoxic conditions. Under hypoxic conditions, blockade of the PI3K or mTOR pathway increased levels of intracellular ATP and delayed decreases in pH and glucose level in culture medium, without affecting the cell cycle.

Analysis of long-term gene expression in neurons of the hippocampal subfields following traumatic brain injury in rats.

After experimental traumatic brain injury (TBI), widespread neuronal loss is progressive and continues in selectively vulnerable brain regions, such as the hippocampus, for months to years after the initial insult. To clarify the molecular mechanisms underlying secondary or delayed cell death in hippocampal neurons after TBI, we compared long-term changes in gene expression in the CA1, CA3 and dentate gyrus (DG) subfields of the rat hippocampus at 24 h and 3, 6, and 12 months after TBI with changes in gene expression in sham-operated rats. We used laser capture microdissection to collect several hundred hippocampal neurons from the CA1, CA3, and DG subfields and linearly amplified the nanogram samples of neuronal RNA with T7 RNA polymerase. Subsequent quantitative analysis of gene expression using ribonuclease protection assay revealed that mRNA expression of the anti-apoptotic gene, Bcl-2, and the chaperone heat shock protein 70 was significantly downregulated at 3, 6 (Bcl-2 only), and 12 months after TBI. Interestingly, the expression of the pro-apoptotic genes caspase-3 and caspase-9 was also significantly decreased at 3, 6 (caspase-9 only), and 12 months after TBI, suggesting that long-term neuronal loss after TBI is not mediated by increased expression of pro-apoptotic genes. The expression of two aging-related genes, p21 and integrin beta3 (ITbeta3), transiently increased 24 h after TBI, returned to baseline levels at 3 months and significantly decreased below sham levels at 12 months (ITbeta3 only). Expression of the gene for the antioxidant glutathione peroxidase-1 also significantly increased 6 months after TBI. These results suggest that decreased levels of neuroprotective genes may contribute to long-term neurodegeneration in animals and human patients after TBI. Conversely, long-term increases in antioxidant gene expression after TBI may be an endogenous neuroprotective response that compensates for the decrease in expression of other neuroprotective genes.

Development of efficient plasmid DNA transfer into adult rat central nervous system using microbubble-enhanced ultrasound.

Although gene therapy might become a promising approach for central nervous system diseases, the safety issue is a serious consideration in human gene therapy. To overcome this problem, we developed an efficient gene transfer method into the adult rat brain based on plasmid DNA using a microbubble-enhanced ultrasound method, since microbubble-enhanced ultrasound has shown promise for transfecting genes into other tissues such as blood vessels. Using the microbubble-enhanced ultrasound method, luciferase expression was increased approximately 10-fold as compared to injection of naked plasmid DNA alone. Interestingly, the site of gene expression was limited to the site of insonation with intracisternal injection, in contrast to previous studies using viruses. Expression of the reporter gene, Venus, was readily detected in the central nervous system. The transfected cells were mainly detected in meningeal cells with intracisternal injection, and in glial cells with intrastriatal injection. There was no obvious evidence of tissue damage by microbubble-enhanced ultrasound. Overall, the present study demonstrated the feasibility of efficient plasmid DNA transfer into the central nervous system, providing a new option for treating various diseases such as tumors.

[Complications of video-assisted thoracoscopic surgery].

We introduced video-assisted thoracoscopic surgery (VATS) for chest disorders in our institution in March, 1992. At first, many of the subjects' disorders were non-malignant diseases such as spontaneous pneumothorax, but later we started to perform this procedure for lung cancer and mediastinum neoplasm, with improved result over thoracoscopic surgical procedures. Now most of the chest disorders at our institution are treated with VATS. However, many kinds of complications due to manual techniques and instrument troubles surfaced during this period. Therefore, in this article we would like to describe the complications that we have experienced in our institution using VATS and discuss how we have attempted to deal with these complications.

A novel hypoxia-dependent 2-nitroimidazole KIN-841 inhibits tumour-specific angiogenesis by blocking production of angiogenic factors.

Tumour angiogenesis is initiated by angiogenic factors that are produced in large amounts by hypoxic tumour cells. The inhibition of this step may lead to tumour-specific antiangiogenesis because normal tissues are not usually hypoxic. On the other hand, blocking a biological function of endothelial cells is known to result in angiogenic inhibition. To produce a tumour-specific and powerful antiangiogenesis, we determined whether potent angiogenic inhibition could be achieved by inhibiting the production of angiogenic factors by hypoxic tumour cells and simultaneously blocking certain angiogenic steps in endothelial cells under normoxia. We focused on the 2-nitroimidazole moiety, which is easily incorporated into hypoxic cells and exhibits its cytotoxicity as hypoxic cytotoxin. We designed and synthesised 2-nitroimidazole derivatives designated as KIN compounds, and investigated their antiangiogenic activities under normoxia using a chick embryo chorioallantoic membrane. KIN-841 (2-nitroimidazole 1-acetylhydroxamate) showed a potent angiogenic inhibition in a dose-dependent manner. This compound inhibited the proliferation of bovine pulmonary arterial endothelial (BPAE) cells more strongly than that of tumour cells, such as Lewis lung carcinoma (3LL) cells, under normoxia. The inhibition of cell proliferation by KIN-841 under hypoxia increased about five-fold compared to that under normoxia. Moreover, under hypoxia, KIN-841 significantly decreased the excessive production of vascular endothelial cell growth factors induced by 3LL cells as determined by tritium-labelled thymidine ([(3)H]thymidine) incorporation into BPAE cells and by ELISA. Intraperitoneal administration of KIN-841 suppressed 3LL-cell-induced in vivo angiogenesis in the mouse dorsal air sac system. These results indicate that the regulation of the production of angiogenic factors by hypoxic tumour cells is a useful target for tumour-specific angiogenesis inhibition, and that KIN-841, which causes simultaneous direct inhibition of endothelial cell function and production of angiogenic factors by hypoxic tumour cells, is a very potent inhibitor of tumour-specific angiogenesis. Thus, the potential for clinical use of KIN-841 as an antitumour drug is very high.

Magnetic resonance imaging analysis on the relationship between anterior disc displacement and balancing-side occlusal contact.

The purpose of this study was to evaluate the relationship between balancing-side occlusal contact patterns and the prevalence of the internal derangement of the temporomandibular joint (TMJ). Forty-one patients were used for the magnetic resonance image (MRI) analysis of TMJ and occlusal examination. Balancing-side occlusal contact patterns observed during mandibular lateral excursive movements were classified into the three following categories: group A, simultaneous balancing-side contact, group B, balancing-side contact (with clenching only) and Group C, no balancing-side contact (with or without clenching). By the occlusal examination of 57 sides, 31.6% showed group A, 8.8% showed group B and 59.6% showed group C contact. Group A could not be observed in the patient group with normal disc position. In the disc displacement group, the prevalence of group A, group B and group C were 40.9, 6.8 and 52.3%, respectively. The higher prevalence of simultaneous balancing-side contact was revealed to be associated with articular disc dislocation.

Spinorphin as an endogenous inhibitor of enkephalin-degrading enzymes: roles in pain and inflammation.

It is possible that enkephalins are involved in the pain-modulating mechanism in the spinal cord. Enkephalins, however, are short-lived, being rapidly degraded by various endogenous enzymes. Many substances that inhibit enkephalin-degradation have been investigated and it has been reported that some inhibitors (e.g. kelatorphan and RB101) alone showed anti-nociceptive activity. We found an endogenous factor that modulated enkephalin-degrading activity and purified it from bovine spinal cord based on its inhibitory activity toward enkephalin-degrading enzymes. Structural analysis revealed the factor to be Leu-Val-Val-Tyr-Pro-Trp-Thr and it was named spinorphin. It has been found that spinorphin inhibited the activity toward various enkephalin-degrading enzymes from monkey brain, especially dipeptidyl peptidase III (DPPIII, Ki=5.1 x 10(-7) M). Recently we reported that this inhibitor significantly inhibited bradykinin (BK)-induced nociceptive flexor responses. Importantly, the mode of inhibition to BK-responses by spinorphin was different from the case with morphine. The morphine-induced blockade of BK-response was attenuated by pertussis toxin treatment, whereas that of spinorphin was not. We also have reported roles for spinorphin in inflammation. Spinorphin significantly inhibited the functions of polymorphonuclear neutrophils (PMNs) by suppressing the binding of fMLF to its receptor on PMNs. Further, this inhibitor suppressed the carrageenan-induced accumulation of PMN in mouse air pouches after intravenous administration. These results indicate that spinorphin may be an endogenous anti-inflammatory regulator. The possible role of spinorphin and its analog as regulators in pain and inflammation will be discussed.

Evolution of CHR-2 SINEs in cetartiodactyl genomes: possible evidence for the monophyletic origin of toothed whales.

Short interspersed repetitive elements (SINEs) are a kind of retroposons dispersed among the eukaryotic genomes. Previously, we isolated and characterized a new SINE family, named CHR-2, members of which are distributed in the genomes of cetaceans, hippopotamuses, and ruminants. We analyzed systematically more than a hundred members of the CHR-2 SINEs, which were isolated from the genomes of cetaceans and cow, together with the additional data available in the DNA databases, and showed that these SINEs are divided into at least five distinct subfamilies that share diagnostic nucleotides and/or deletions. A hybridization analysis clearly demonstrated that, among these five subfamilies, two subfamilies, named CD and CDO, are specific to cetaceans and toothed whales, respectively. We reconstruct the evolutionary history of the CHR-2 SINEs during evolution of cetartiodactyl genomes.

Insight into the mechanism of trypanosome lytic factor-1 killing of Trypanosoma brucei brucei.

It has been known for almost a century that normal human serum can lyse the extracellular blood parasite Trypanosoma brucei brucei. This process is a result of a non-immune killing factor in human sera known as trypanosome lytic factor (TLF). In this work, we demonstrate that killing of T. b. brucei by trypanosome lytic factor-1 (TLF-1) in vitro is inhibited by the lipophyllic iron chelator, LI, the lipophyllic antioxidant DPPD, and the protease inhibitors antipain and E64. Thus TLF-1 killing likely requires iron, oxidants, and serine and cysteine proteases. Furthermore, we demonstrate that TLF-1 mediated lysis causes measurable peroxidation in T. brucei lipids via a reaction that is inhibited by DPPD, weak bases, and human haptoglobin. We hypothesize that TLF-1 lysis requires intracellular factors within the trypanosome including high intracellular H2O2 and high polyenoic lipid concentrations, lysosomal acidification and proteases, and intracellular iron sources. The data presented supports the hypothesis that the combination of these factors with TLF-1 inside the lysosome results in lysosomal membrane breakdown, release of the lysosomal contents, and subsequent autodigestion of the cell.