7,8-Dihydroxyflavone facilitates the action exercise to restore plasticity and functionality: Implications for early brain trauma recovery.

Metabolic dysfunction accompanying traumatic brain injury (TBI) severely impairs the ability of injured neurons to comply with functional demands. This limits the success of rehabilitative strategies by compromising brain plasticity and function, and highlights the need for early interventions to promote energy homeostasis. We sought to examine whether the TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF) normalizes brain energy deficits and reestablishes more normal patterns of functional connectivity, while enhancing the effects of exercise during post-TBI period. Moderate fluid percussion injury (FPI) was performed and 7,8-DHF (5mg/kg, i.p.) was administered in animals subjected to FPI that either had access to voluntary wheel running for 7days after injury or were sedentary. Compared to sham-injured controls, TBI resulted in reduced hippocampal activation of the BDNF receptor TrkB and associated CREB, reduced levels of plasticity markers GAP-43 and Syn I, as well as impaired memory as indicated by the Barnes maze task. While 7,8-DHF treatment and exercise individually mitigated TBI-induced effects, administration of 7,8-DHF concurrently with exercise facilitated memory performance and augmented levels of markers of cell energy metabolism viz., PGC-1α, COII and AMPK. In parallel to these findings, resting-state functional MRI (fMRI) acquired at 2weeks after injury showed that 7,8-DHF with exercise enhanced hippocampal functional connectivity, and suggests 7,8-DHF and exercise to promote increases in functional connectivity. Together, these findings indicate that post-injury 7,8-DHF treatment promotes enhanced levels of cell metabolism, synaptic plasticity in combination with exercise increases in brain circuit function that facilitates greater physical rehabilitation after TBI.

Curcumin boosts DHA in the brain: Implications for the prevention of anxiety disorders.

Dietary deficiency of docosahexaenoic acid (C22:6 n-3; DHA) is linked to the neuropathology of several cognitive disorders, including anxiety. DHA, which is essential for brain development and protection, is primarily obtained through the diet or synthesized from dietary precursors, however the conversion efficiency is low. Curcumin (diferuloylmethane), which is a principal component of the spice turmeric, complements the action of DHA in the brain, and this study was performed to determine molecular mechanisms involved. We report that curcumin enhances the synthesis of DHA from its precursor, α-linolenic acid (C18:3 n-3; ALA) and elevates levels of enzymes involved in the synthesis of DHA such as FADS2 and elongase 2 in both liver and brain tissues. Furthermore, in vivo treatment with curcumin and ALA reduced anxiety-like behavior in rodents. Taken together, these data suggest that curcumin enhances DHA synthesis, resulting in elevated brain DHA content. These findings have important implications for human health and the prevention of cognitive disease, particularly for populations eating a plant-based diet or who do not consume fish, a primary source of DHA, since DHA is essential for brain function and its deficiency is implicated in many types of neurological disorders.

Flavonoid derivative 7,8-DHF attenuates TBI pathology via TrkB activation.

Traumatic brain injury (TBI) is followed by a state of metabolic dysfunction, affecting the ability of neurons to use energy and support brain plasticity; there is no effective therapy to counteract the TBI pathology. Brain-derived neurotrophic factor (BDNF) has an exceptional capacity to support metabolism and plasticity, which highly contrasts with its poor pharmacological profile. We evaluated the action of a flavonoid derivative 7,8-dihydroxyflavone (7,8-DHF), a BDNF receptor (TrkB) agonist with the pharmacological profile congruent for potential human therapies. Treatment with 7,8-DHF (5mg/kg, ip, daily for 7 days) was effective to ameliorate the effects of TBI on plasticity markers (CREB phosphorylation, GAP-43 and syntaxin-3 levels) and memory function in Barnes maze test. Treatment with 7,8-DHF restored the decrease in protein and phenotypic expression of TrkB phosphorylation after TBI. In turn, intrahippocampal injections of K252a, a TrkB antagonist, counteracted the 7,8-DHF induced TrkB signaling activation and memory improvement in TBI, suggesting the pivotal role of TrkB signaling in cognitive performance after brain injury. A potential action of 7,8-DHF on cell energy homeostasis was corroborated by the normalization in levels of PGC-1α, TFAM, COII, AMPK and SIRT1 in animals subjected to TBI. Results suggest a potential mechanism by which 7,8-DHF counteracts TBI pathology via activation of the TrkB receptor and engaging the interplay between cell energy management and synaptic plasticity. Since metabolic dysfunction is an important risk factor for the development of neurological and psychiatric disorders, these results set a precedent for the therapeutic use of 7,8-DHF in a larger context.

Deterioration of plasticity and metabolic homeostasis in the brain of the UCD-T2DM rat model of naturally occurring type-2 diabetes.

The rising prevalence of type-2 diabetes is becoming a pressing issue based on emerging reports that T2DM can also adversely impact mental health. We have utilized the UCD-T2DM rat model in which the onset of T2DM develops spontaneously across time and can serve to understand the pathophysiology of diabetes in humans. An increased insulin resistance index and plasma glucose levels manifested the onset of T2DM. There was a decrease in hippocampal insulin receptor signaling in the hippocampus, which correlated with peripheral insulin resistance index along the course of diabetes onset (r=-0.56, p<0.01). T2DM increased the hippocampal levels of 4-hydroxynonenal (4-HNE; a marker of lipid peroxidation) in inverse proportion to the changes in the mitochondrial regulator PGC-1α. Disrupted energy homeostasis was further manifested by a concurrent reduction in energy metabolic markers, including TFAM, SIRT1, and AMPK phosphorylation. In addition, T2DM influenced brain plasticity as evidenced by a significant reduction of BDNF-TrkB signaling. These results suggest that the pathology of T2DM in the brain involves a progressive and coordinated disruption of insulin signaling, and energy homeostasis, with profound consequences for brain function and plasticity. All the described consequences of T2DM were attenuated by treatment with the glucagon-like peptide-1 receptor agonist, liraglutide. Similar results to those of liraglutide were obtained by exposing T2DM rats to a food energy restricted diet, which suggest that normalization of brain energy metabolism is a crucial factor to counteract central insulin sensitivity and synaptic plasticity associated with T2DM.

Interactive actions of Bdnf methylation and cell metabolism for building neural resilience under the influence of diet.

Quality nutrition during the period of brain formation is a predictor of brain functional capacity and plasticity during adulthood; however it is not clear how this conferred plasticity imparts long-term neural resilience. Here we report that early exposure to dietary omega-3 fatty acids orchestrates key interactions between metabolic signals and Bdnf methylation creating a reservoir of neuroplasticity that can protect the brain against the deleterious effects of switching to a Western diet (WD). We observed that the switch to a WD increased Bdnf methylation specific to exon IV, in proportion to anxiety-like behavior, in Sprague Dawley rats reared in low omega-3 fatty acid diet, and these effects were abolished by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. Blocking methylation also counteracted the reducing action of WD on the transcription regulator CTCF binding to Bdnf promoter IV. In vitro studies confirmed that CTCF binding to Bdnf promoter IV is essential for the action of DHA on BDNF regulation. Diet is also intrinsically associated to cell metabolism, and here we show that the switch to WD downregulated cell metabolism (NAD/NADH ratio and SIRT1). The fact that DNA methyltransferase inhibitor did not alter these parameters suggests they occur upstream to methylation. In turn, the methylation inhibitor counteracted the action of WD on PGC-1α, a mitochondrial transcription co-activator and BDNF regulator, suggesting that PGC-1α is an effector of Bdnf methylation. Results support a model in which diet can build an "epigenetic memory" during brain formation that confers resilience to metabolic perturbations occurring in adulthood.

Single cell molecular alterations reveal target cells and pathways of concussive brain injury.

The complex neuropathology of traumatic brain injury (TBI) is difficult to dissect, given the convoluted cytoarchitecture of affected brain regions such as the hippocampus. Hippocampal dysfunction during TBI results in cognitive decline that may escalate to other neurological disorders, the molecular basis of which is hidden in the genomic programs of individual cells. Using the unbiased single cell sequencing method Drop-seq, we report that concussive TBI affects previously undefined cell populations, in addition to classical hippocampal cell types. TBI also impacts cell type-specific genes and pathways and alters gene co-expression across cell types, suggesting hidden pathogenic mechanisms and therapeutic target pathways. Modulating the thyroid hormone pathway as informed by the T4 transporter transthyretin Ttr mitigates TBI-associated genomic and behavioral abnormalities. Thus, single cell genomics provides unique information about how TBI impacts diverse hippocampal cell types, adding new insights into the pathogenic pathways amenable to therapeutics in TBI and related disorders.

Traumatic Brain Injury Induces Genome-Wide Transcriptomic, Methylomic, and Network Perturbations in Brain and Blood Predicting Neurological Disorders.

The complexity of the traumatic brain injury (TBI) pathology, particularly concussive injury, is a serious obstacle for diagnosis, treatment, and long-term prognosis. Here we utilize modern systems biology in a rodent model of concussive injury to gain a thorough view of the impact of TBI on fundamental aspects of gene regulation, which have the potential to drive or alter the course of the TBI pathology. TBI perturbed epigenomic programming, transcriptional activities (expression level and alternative splicing), and the organization of genes in networks centered around genes such as Anax2, Ogn, and Fmod. Transcriptomic signatures in the hippocampus are involved in neuronal signaling, metabolism, inflammation, and blood function, and they overlap with those in leukocytes from peripheral blood. The homology between genomic signatures from blood and brain elicited by TBI provides proof of concept information for development of biomarkers of TBI based on composite genomic patterns. By intersecting with human genome-wide association studies, many TBI signature genes and network regulators identified in our rodent model were causally associated with brain disorders with relevant link to TBI. The overall results show that concussive brain injury reprograms genes which could lead to predisposition to neurological and psychiatric disorders, and that genomic information from peripheral leukocytes has the potential to predict TBI pathogenesis in the brain.

Systems Nutrigenomics Reveals Brain Gene Networks Linking Metabolic and Brain Disorders.

Nutrition plays a significant role in the increasing prevalence of metabolic and brain disorders. Here we employ systems nutrigenomics to scrutinize the genomic bases of nutrient-host interaction underlying disease predisposition or therapeutic potential. We conducted transcriptome and epigenome sequencing of hypothalamus (metabolic control) and hippocampus (cognitive processing) from a rodent model of fructose consumption, and identified significant reprogramming of DNA methylation, transcript abundance, alternative splicing, and gene networks governing cell metabolism, cell communication, inflammation, and neuronal signaling. These signals converged with genetic causal risks of metabolic, neurological, and psychiatric disorders revealed in humans. Gene network modeling uncovered the extracellular matrix genes Bgn and Fmod as main orchestrators of the effects of fructose, as validated using two knockout mouse models. We further demonstrate that an omega-3 fatty acid, DHA, reverses the genomic and network perturbations elicited by fructose, providing molecular support for nutritional interventions to counteract diet-induced metabolic and brain disorders. Our integrative approach complementing rodent and human studies supports the applicability of nutrigenomics principles to predict disease susceptibility and to guide personalized medicine.

Methamphetamine blocks exercise effects on Bdnf and Drd2 gene expression in frontal cortex and striatum.

Exposure to drugs of abuse can produce many neurobiological changes which may lead to increased valuation of rewards and decreased sensitivity to their costs. Many of these behavioral alterations are associated with activity of D2-expressing medium spiny neurons in the striatum. Additionally, Bdnf in the striatum has been shown to play a role in flexible reward-seeking behavior. Given that voluntary aerobic exercise can affect the expression of these proteins in healthy subjects, and that exercise has shown promise as an anti-addictive therapy, we set out to quantify changes in D2 and Bdnf expression in methamphetamine-exposed rats given access to running wheels. Sixty-four rats were treated for two weeks with an escalating dose of methamphetamine or saline, then either sacrificed, housed in standard cages, or given free access to a running wheel for 6 weeks prior to sacrifice. Rats treated with methamphetamine ran significantly greater distances than saline-treated rats, suggesting an augmentation in the reinforcement value of voluntary wheel running. Transcription of Drd2 and Bdnf was assessed via RT-qPCR. Protein expression levels of D2 and phosphorylation of the TrkB receptor were measured via western blot. Drd2 and Bdnf mRNA levels were impacted independently by exercise and methamphetamine, but exposure to methamphetamine prior to the initiation of exercise blocked the exercise-induced changes seen in rats treated with saline. Expression levels of both proteins were elevated immediately after methamphetamine, but returned to baseline after six weeks, regardless of exercise status.

Cross-linking of CD40 using anti-CD40 antibody, 5C11, has different effects on XG2 multiple myeloma cells.

A multiple myeloma (MM) cell line, XG2, has high-level expression of CD40, a tumor necrosis factor receptor (TNFR) family member. CD40 is present on the surfaces of a large variety of cells, including B cells, endothelial cells, dendritic cells and some carcinoma cells, and delivers signals regulating diverse cellular responses, such as proliferation, differentiation, growth suppression, cell death. In this research, we study the effects of cross-linking of CD40 on myeloma cells using different concentrations of anti-CD40 monoclonal antibody (mAb), 5C11. We found that low concentrations of 5C11 induced proliferation of XG2, while high concentrations of 5C11 resulted in homotypic aggregation of XG2, and strongly suppression of its proliferation and apoptosis after 24 h of treatment. These dose-dependent effects of 5C11 were verified by flow cytometry, Western blotting and immunoprecipitation. Autocrine or paracrine induction of IL-6, and up-regulation of membrane TNF and phosphorylation of TNFR1 may partially explain the contradictory biological effects of CD40 cross-linking on XG2 by anti-CD40 mAb.

Vulnerability imposed by diet and brain trauma for anxiety-like phenotype: implications for post-traumatic stress disorders.

Mild traumatic brain injury (mTBI, cerebral concussion) is a risk factor for the development of psychiatric illness such as posttraumatic stress disorder (PTSD). We sought to evaluate how omega-3 fatty acids during brain maturation can influence challenges incurred during adulthood (transitioning to unhealthy diet and mTBI) and predispose the brain to a PTSD-like pathobiology. Rats exposed to diets enriched or deficient in omega-3 fatty acids (n-3) during their brain maturation period, were transitioned to a western diet (WD) when becoming adult and then subjected to mTBI. TBI resulted in an increase in anxiety-like behavior and its molecular counterpart NPY1R, a hallmark of PTSD, but these effects were more pronounced in the animals exposed to n-3 deficient diet and switched to WD. The n-3 deficiency followed by WD disrupted BDNF signaling and the activation of elements of BDNF signaling pathway (TrkB, CaMKII, Akt and CREB) in frontal cortex. TBI worsened these effects and more prominently in combination with the n-3 deficiency condition. Moreover, the n-3 deficiency primed the immune system to the challenges imposed by the WD and brain trauma as evidenced by results showing that the WD or mTBI affected brain IL1ß levels and peripheral Th17 and Treg subsets only in animals previously conditioned to the n-3 deficient diet. These results provide novel evidence for the capacity of maladaptive dietary habits to lower the threshold for neurological disorders in response to challenges.

Brain and spinal cord interaction: protective effects of exercise prior to spinal cord injury.

We have investigated the effects of a spinal cord injury on the brain and spinal cord, and whether exercise provided before the injury could organize a protective reaction across the neuroaxis. Animals were exposed to 21 days of voluntary exercise, followed by a full spinal transection (T7-T9) and sacrificed two days later. Here we show that the effects of spinal cord injury go beyond the spinal cord itself and influence the molecular substrates of synaptic plasticity and learning in the brain. The injury reduced BDNF levels in the hippocampus in conjunction with the activated forms of p-synapsin I, p-CREB and p-CaMK II, while exercise prior to injury prevented these reductions. Similar effects of the injury were observed in the lumbar enlargement region of the spinal cord, where exercise prevented the reductions in BDNF, and p-CREB. Furthermore, the response of the hippocampus to the spinal lesion appeared to be coordinated to that of the spinal cord, as evidenced by corresponding injury-related changes in BDNF levels in the brain and spinal cord. These results provide an indication for the increased vulnerability of brain centers after spinal cord injury. These findings also imply that the level of chronic activity prior to a spinal cord injury could determine the level of sensory-motor and cognitive recovery following the injury. In particular, exercise prior to the injury onset appears to foster protective mechanisms in the brain and spinal cord.

High-fat diet transition reduces brain DHA levels associated with altered brain plasticity and behaviour.

To assess how the shift from a healthy diet rich in omega-3 fatty acids to a diet rich in saturated fatty acid affects the substrates for brain plasticity and function, we used pregnant rats fed with omega-3 supplemented diet from their 2nd day of gestation period as well as their male pups for 12 weeks. Afterwards, the animals were randomly assigned to either a group fed on the same diet or a group fed on a high-fat diet (HFD) rich in saturated fats for 3 weeks. We found that the HFD increased vulnerability for anxiety-like behavior, and that these modifications harmonized with changes in the anxiety-related NPY1 receptor and the reduced levels of BDNF, and its signalling receptor pTrkB, as well as the CREB protein. Brain DHA contents were significantly associated with the levels of anxiety-like behavior in these rats.

Dietary omega-3 deficiency from gestation increases spinal cord vulnerability to traumatic brain injury-induced damage.

Although traumatic brain injury (TBI) is often associated with gait deficits, the effects of TBI on spinal cord centers are poorly understood. We seek to determine the influence of TBI on the spinal cord and the potential of dietary omega-3 (n-3) fatty acids to counteract these effects. Male rodents exposed to diets containing adequate or deficient levels of n-3 since gestation received a moderate fluid percussion injury when becoming 14 weeks old. TBI reduced levels of molecular systems important for synaptic plasticity (BDNF, TrkB, and CREB) and plasma membrane homeostasis (4-HNE, iPLA2, syntaxin-3) in the lumbar spinal cord. These effects of TBI were more dramatic in the animals exposed to the n-3 deficient diet. Results emphasize the comprehensive action of TBI across the neuroaxis, and the critical role of dietary n-3 as a means to build resistance against the effects of TBI.

Effects of diet and/or exercise in enhancing spinal cord sensorimotor learning.

Given that the spinal cord is capable of learning sensorimotor tasks and that dietary interventions can influence learning involving supraspinal centers, we asked whether the presence of omega-3 fatty acid docosahexaenoic acid (DHA) and the curry spice curcumin (Cur) by themselves or in combination with voluntary exercise could affect spinal cord learning in adult spinal mice. Using an instrumental learning paradigm to assess spinal learning we observed that mice fed a diet containing DHA/Cur performed better in the spinal learning paradigm than mice fed a diet deficient in DHA/Cur. The enhanced performance was accompanied by increases in the mRNA levels of molecular markers of learning, i.e., BDNF, CREB, CaMKII, and syntaxin 3. Concurrent exposure to exercise was complementary to the dietary treatment effects on spinal learning. The diet containing DHA/Cur resulted in higher levels of DHA and lower levels of omega-6 fatty acid arachidonic acid (AA) in the spinal cord than the diet deficient in DHA/Cur. The level of spinal learning was inversely related to the ratio of AA:DHA. These results emphasize the capacity of select dietary factors and exercise to foster spinal cord learning. Given the non-invasiveness and safety of the modulation of diet and exercise, these interventions should be considered in light of their potential to enhance relearning of sensorimotor tasks during rehabilitative training paradigms after a spinal cord injury.

[Preparation of a novel functional anti-human CD40 monoclonal antibody and identification of its bioactivity].

AIM: To prepare a novel functional mouse anti-human CD40 monoclonal mAb. METHODS: Female BALB/c mice of 6-8 weeks old were immunized with CD40 transfectant (L929-CD40) as immunogen. The spleen B cells of the mice were fused with Sp2/0. The hybridoma cells were screened with CD40 transfectant (L929-CD40) by FCM. Fast-strip analysis was performed to identify Ig subclass of this mAb. The epitope recognized by this mAb was detected by Bio/5C11 competitive assay. The proliferation and cell cycle of tumor cells in vitro were studied by MTT assay and PI staining respectively. RESULTS: One hybridoma cell line named 2B6 was obtained, which had the property of secreting anti-human CD40 monoclonal antibody continuously and steadily. This mAb specifically recognized human CD40 molecule and promoted the proliferation of tumor cells in vitro. CONCLUSION: One hybridoma cell line which can secret a novel functional mouse anti-human CD40 mAb has been developed successfully. This mAb can specifically recognize human CD40 and influence the growth of tumor cells in vitro.

[Serum levels and clinical significance of soluble CD40 in liver disease].

AIM: To measure the level of serum soluble CD40 (sCD40) in patients with acute hepatitis, hepatitis gravis and primary carcinoma of the liver, and to evaluate the relationship of sCD40 with biochemical marks and disease prognosis. METHODS: Patients with acute hepatitis (n=49) hepatitis gravis (n=22) and primary carcinoma of the liver (n=13) were studied, and serum sCD40 was determined in these patients and compared with that of healthy controls (n=44) by enzyme linked immunosorbent assay (ELISA). The binding capacity of serum sCD40 to its ligand CD40L was detected by flow cytometry (FCM) in vitro. RESULTS: Concentration of sCD40 was significantly higher in patients with liver disease than that in healthy controls (P<0.001), but no significant difference was found between the three types of liver disease (P=0.475). In the hepatitis gravis group, sCD40 concentration in dead patients was higher compared with that in the survivals (P<0.05). Level of sCD40 in patients with acute hepatitis was correlated with serum alanine transaminase (ALT) and aspartic transaminase (AST). The serum sCD40 could bind CD40L in vitro. CONCLUSION: These data suggest that sCD40 is an important serological marker in liver disease to evaluate acute injury of hepatocytes, and it shows a relevance with the prognosis of hepatitis gravis. The highly elevated level of sCD40 suggest the involvement of CD40 and its ligand CD40L in liver disease.

[One-step purification of monoclonal antibody against gp130 by cation exchange liquid chromatography].

AIM: To develop a one-step purification method of anti-gp130 monoclonal antibody (mAb) B-S12 from mouse ascites. METHODS: After filtrated by centrifugation, the ascites sample was loaded on a cation exchange column and purified by using ion-strength gradient elution buffer. The effects of pH of the loading buffer and ion strength gradients of the elution buffer on the purity of antibody obtained were investigated. The antibody's biological activity was tested by MTT colorimetry. RESULTS: It was shown that the mAb B-S12 with a purity of over 90% could be achieved by using 20 mmol/L HEPES buffer (pH 4.0) as loading buffer and 0-1.0 mol/L NaCl as elution buffer. The total recovery rate of the mAb was 52%. The purified antibody could stimulate the proliferation of XG-2 cell line. CONCLUSION: The established one-step purification method was simple and suitable for purification of mAb B-S12.

Sensitization of multiple myeloma and B lymphoma lines to dexamethasone and gamma-radiation-induced apoptosis by CD40 activation.

We examined the effects of CD40 activation with dexamethasone (Dex) or 60Co-gamma-irradiation on the growth of malignant B cells in vitro, using the human multiple myeloma (MM) cell line, XG2, and the B lymphoma Daudi cell line as models. Both lines are resistant to Dex and irradiation; 10(-7)M Dex or 10 Gy of gamma-irradiation induced only minimal growth arrest and apoptosis of the cells. Treatment of the cells with the agonistic anti-CD40 monoclonal antibody 5C11 partially inhibited the proliferation of the Daudi cells; XG2 underwent apoptosis. XG2 is an Interleukin-6 (IL-6)-dependent myeloma cell line and CD40 activation blocked XG2 in the G1 phase of the cell cycle, in a manner similar to the effect of IL-6 deprivation. Daudi was blocked in the G2/M phase after treatment with the agonistic CD40 mAb 5C11. Furthermore, the activation of CD40 on Daudi and XG2 enhanced their sensitivity to dexamethasone-and gamma-irradiation -induced growth arrest and apoptosis. CD40 activation stimulated both anti-apoptotic Bcl-XL and pro-apoptotic Bax mRNA synthesis in the Daudi cell line; CD40 activation increased the Bax mRNA level but had no effect on the Bcl-XL mRNA level in the XG2 cell line. Apoptosis in both cell lines was associated with an increasing ratio of Bax-to-Bcl-XL both in mRNA and in protein levels. It is concluded that use of the anti-CD40 mAb 5C11 either by itself or in combination with chemotherapy and/or radiotherapy may have significant therapeutic potential.

[Effect of CD(40) on the in vitro biological behavior of malignant B lymphocytes].

OBJECTIVE: To explore the effect of recombinant human soluble CD(40) ligand (rhsCD(40)L) and CD(40)L cDNA transfected cell (CD(40)L-TC) on the behavior of malignant B lymphocytes, and investigate the possibility of using rhsCD(40)L as a new bio-factor in tumor immunotherapy. METHOD: rhsCD(40)L and CD(40)L-TC were obtained by gene recombinant techniques. Multiple myeloma cell lines, XG2, XG7, U266 and 8226, B-lymphoma cell lines, Raji and Daudi were selected to detect responses to rhsCD(40)L and CD(40)L-TC stimulation. Cell growth curve, cell cycle, early apoptosis as well as membrane surface molecules on these cell lines were analyzed. RESULTS: (1) The expression levels of CD(40) molecule on malignant B lymphocytes showed heterogeneity. High level of CD(40) on XG2, moderate on 8266, Raji, and Daudi, and no expression on U266 and XG7 were detected. The rhsCD(40)L stimulation gave rise to a typical homo-type cell aggregation of XG2 and Daudi. Meanwhile, at least 10 to 20 of CD(40)(+) XG2 or CD(40)(+) Daudi cells were found adherent to one pre-treat ed CD(40)L-TC. (2) Co-incubation with rhsCD(40)L (5 micro g/ml), or CD(40)L-TC (tumor cell: CD(40) = 5:1) resulted in a significant inhibition of in vitro cell growth of XG2, Raji and Daudi, with G(1)-phase arrest for XG2 and G(2)-phase for Raji and Daudi. These two kinds of CD(40) stimulators induced XG2, Raji and Daudi cells to apoptosis in vitro. The apoptotic rate for XG2 was 23.3% (rhsCD(40)L) and 18.8% (CD(40)L-TC), for Daudi 14.2% and 15.9%, and for Raji 11.6% and 8.9% respectively. (3) Phenotype analysis showed that CD(95) expression levels were significantly up-regulated on XG2, Raji and Daudi after stimulation with rhsCD(40)L or CD(40)L-TC, and CD(80) and CD(18) expression levels on Raji were respectively enhanced and decreased. CONCLUSION: The abilities to directly inhibit XG2, Daudi and Raji cell proliferation, to induce themapoptosis, as well as to up-regulate immune co-stimulator molecule CD(80) expression on Raji cells would make rhsCD(40)L a potential bio-factor for tumor immuno-therapy.