Assessment of clinical outcomes of cervical epidural neuroplasty using a Racz-catheter and predictive factors of efficacy in patients with cervical spinal pain.

BACKGROUND: Epidural neuroplasty using the Racz catheter has a therapeutic effect in patients with cervical disc herniation and central stenosis who do not respond to fluoroscopically guided epidural injections. OBJECTIVE: To evaluate the clinical outcomes of cervical epidural neuroplasty in patients with posterior neck and upper extremity pain and to demonstrate correlations between predictive factors and unsuccessful results of cervical epidural neuroplasty. STUDY DESIGN: Observational study. SETTING: An interventional pain management practice in a university hospital. METHODS: Outcome measures were obtained using the numeric rating scale (NRS) for total pain, neck pain, arm pain, and sleep disturbance; and the neck pain and disability scale (NPDS); as well as opioid consumption at preprocedure, one month, 3, 6, and 12 months after procedure. Successful epidural neuroplasty was defined as 50% or greater reduction from the preprocedure NRS for total pain, and at least a 40% reduction in the NPDS. We obtained clinical data and radiologic findings to evaluate correlations between predictive factors and efficacy of epidural neuroplasty. RESULTS: Of the 169 patients, successful outcomes were observed in 108 patients (63.9%) at one month following the procedure, in 109 patients (64.5%) at 3 months, in 96 patients (56.8%) at 6 months, and in 89 patients (52.7%) at 12 months. Previous surgery, spondylolisthesis, and ossification of the posterior longitudinal ligament were significantly associated with unsuccessful outcomes as measured by NRS and NPDS (P < 0.05). LIMITATIONS: Limitations of this evaluation include the lack of a placebo group. CONCLUSIONS: Cervical epidural neuroplasty may be an effective treatment for pain reduction and functional improvement in patients with cervical spinal pain who did not respond to conservative treatment, and may decrease surgical demand. Previous surgery, spondylolisthesis, and ossification of the posterior longitudinal ligament are associated with unsuccessful outcomes of epidural neuroplasty.

Comparison of Serum Adipocytokine Levels according to Metabolic Health and Obesity Status.

BACKGROUND: Metabolic health is an emerging concept that is highly correlated with various metabolic complications, and adipocytokines have been causally linked to a wide range of metabolic diseases. Thus, this study compared serum adipocytokine levels according to metabolic health and obesity status. METHODS: Four hundred and fifty-six nondiabetic subjects (mean age, 40.5 years) were categorized into four groups according to metabolic health and obesity status: metabolically healthy nonobese (MHNO), metabolically healthy obese (MHO), metabolically unhealthy nonobese (MUHNO), and metabolically unhealthy obese (MUHO). Being metabolically healthy was defined as the presence of fewer than two of the following five metabolic abnormalities: high blood pressure, high fasting blood glucose, high triglyceride, low high density lipoprotein cholesterol, and being in the highest decile of the homeostatic model assessment of insulin resistance index. Obesity status was assessed using body mass index (BMI), with obesity defined as a BMI higher than 25 kg/m². Levels of serum interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor α (TNF-α), and adipocyte fatty acid binding protein (A-FABP) were also evaluated. RESULTS: Of the 456 subjects, 247 (54.2%) were in the MHNO group, 66 (14.5%) were in the MHO group, 66 (14.5%) were in the MUHNO group, and 77 (16.9%) were in the MUHO group. There were no significant differences in IL-6 or MCP-1 levels among the groups, but levels of TNF-α and A-FABP were significantly higher in the MUHNO group compared to the MHNO group. CONCLUSION: High TNF-α and A-FABP levels are significantly associated with metabolically unhealthiness in nonobese Korean individuals.

Increased risk of diabetes development in subjects with the hypertriglyceridemic waist phenotype: a 4-year longitudinal study.

BACKGROUND: The hypertriglyceridemic waist (HTGW) phenotype is a simple and inexpensive screening parameter to identify people at increased risk of cardiovascular disease. We evaluated whether the HTGW phenotype predicts diabetes in urban Korean adults. METHODS: A total of 2,900 nondiabetic subjects (mean age 44.3 years), comprising 2,078 males (71.7%) and 822 females (28.3%) who underwent annual medical check-ups at our center between January 2005 and December 2009, were recruited. The subjects were divided into four groups according to baseline serum triglyceride (TG) level and waist circumference (WC): normal WC-normal TG (NWNT) level, normal WC-high TG level, enlarged WC-normal TG level, and enlarged WC-high TG (EWHT) level. High serum TG level was defined as ≥150 mg/dL and enlarged WC was defined as ≥90 cm for men and ≥85 cm for women. New cases of diabetes were determined according to questionnaires filled in by participants and the diagnostic criteria of the American Diabetes Association. Cox proportional hazards model analysis was used to assess the association of HTGW phenotype with the incidence of diabetes. RESULTS: A total of 101 (3.5%) new diabetes cases were diagnosed during the study period. The EWHT group had a higher incidence of diabetes (8.3%) compared with the NWNT group (2.2%). The adjusted hazard ratio for diabetes for subjects with the EWHT phenotype at baseline was 4.113 (95% confidence interval [CI], 2.397 to 7.059) after adjustment for age, and 2.429 (95% CI, 1.370 to 4.307) after adjustment for age, sex, total cholesterol, systolic blood pressure, and alcohol drinking history. It was attenuated by inclusion of baseline fasting glucose level in the model. CONCLUSION: Subjects with the HTGW phenotype showed the highest risk of incident diabetes. This tool could be useful for identifying individuals at high risk of diabetes.

Age Is the Strongest Effector for the Relationship between Estimated Glomerular Filtration Rate and Coronary Artery Calcification in Apparently Healthy Korean Adults.

BACKGROUND: Chronic kidney disease (CKD) is considered one of the most common risk factors for cardiovascular disease. Coronary artery calcification (CAC) is a potential mechanism that explains the association between renal function and cardiovascular mortality. We aimed to evaluate the association between renal function and CAC in apparently healthy Korean subjects. METHODS: A total of 23,617 participants in a health-screening program at Kangbuk Samsung Hospital were included in the study. Estimated glomerular filtration rate (eGFR) was assessed using the Cockcroft-Gault equation. Coronary artery calcium score (CACS) was measured via multidetector computed tomography. Subjects were divided into three groups according to the CKD Staging system with eGFR grade: stage 1, eGFR ≥90 mL/min/1.73 m(2); stage 2, eGFR 60 to 89 mL/min/1.73 m(2); and stage 3, eGFR 30 to 59 mL/min/1.73 m(2). RESULTS: The mean age of the participants was 41.4 years and the mean eGFR was 103.6±21.7 mL/min/1.73 m(2). Hypertension and diabetes were noted in 43.7% and 5.5% of the participants, respectively. eGFR showed a weakly negative but significant association with CACS in bivariate correlation analysis (r=-0.076, P<0.01). Mean CACS significantly increased from CKD stage 1 to 3. The proportion of subjects who had CAC significantly increased from CKD stage 1 to 3. Although the odds ratio for CAC significantly increased from stage 1 to 3 after adjustment for confounding factors, this significance was reversed when age was included in the model. CONCLUSION: In early CKD, renal function negatively correlated with the degree of CAC in Korean subjects. Age was the strongest effector for this association.

Intranasal adenovirus-vectored vaccine for induction of long-lasting humoral immunity-mediated broad protection against influenza in mice.

UNLABELLED: Influenza vaccines aimed at inducing antibody (Ab) responses against viral surface hemagglutinin (HA) and neuraminidase (NA) provide sterile immunity to infection with the same subtypes. Vaccines targeting viral conserved determinants shared by the influenza A viruses (IAV) offer heterosubtypic immunity (HSI), a broad protection against different subtypes. We proposed that vaccines targeting both HA and the conserved ectodomain of matrix protein 2 (M2e) would provide protection against infection with the same subtype and also HSI against other subtypes. We report here that single intranasal immunization with a recombinant adenovirus (rAd) vector encoding both HA of H5 virus and M2e (rAdH5/M2e) induced significant HA- and M2e-specific Ab responses, along with protection against heterosubtypic challenge in mice. The protection is superior compared to that induced by rAd vector encoding either HA (rAdH5), or M2e (rAdM2e). While protection against homotypic H5 virus is primarily mediated by virus-neutralizing Abs, the cross-protection is associated with Abs directed to conserved stalk HA and M2e that seem to have an additive effect. Consistently, adoptive transfer of antisera induced by rAdH5/M2e provided the best protection against heterosubtypic challenge compared to that provided by antisera derived from mice immunized with rAdH5 or rAdM2e. These results support the development of rAd-vectored vaccines encoding both H5 and M2e as universal vaccines against different IAV subtypes. IMPORTANCE: Current licensed influenza vaccines provide protection limited to the infection with same virus strains; therefore, the composition of influenza vaccines has to be revised every year. We have developed a new universal influenza vaccine that is highly efficient in induction of long-lasting cross-protection against different influenza virus strains. The cross-protection is associated with a high level of vaccine-induced antibodies against the conserved stalk domain of influenza virus hemagglutinin and the ectodomain of matrix protein. The vaccine could be used to stimulate cross-protective antibodies for the prevention and treatment of influenza with immediate effect for individuals who fail to respond to or receive the vaccine in due time. The vaccine offers a new tool to control influenza outbreaks, including pandemics.

Combined rTMS to the auditory cortex and prefrontal cortex for tinnitus control in patients with depression: a pilot study.

CONCLUSION: The study showed that combined repetitive transcranial magnetic stimulation (rTMS) on the auditory cortex and prefrontal cortex has more benefit than rTMS on the auditory cortex alone for tinnitus control in patients with depression. Further studies for the most optimal combination of stimulation on both areas are needed. OBJECTIVE: Recent studies suggest that the neuronal network changes of chronic tinnitus are beyond the auditory pathway. There is increasing evidences for the application of rTMS on multiple brain cortices in addition to the auditory cortex for the treatment of tinnitus. Sequential rTMS was performed on the auditory cortex alone as well as the auditory cortex combined with prefrontal cortex in patients with both chronic tinnitus and depression. METHODS: Patients who presented with chronic tinnitus of more than 1 year were enrolled in the present study (seven males, four females; mean age 54 years). To select the site for the rTMS, PET CT was performed. Patients received the first rTMS on the primary auditory cortex for 5 days and on the primary auditory cortex and prefrontal cortex in the second application after tinnitus relapse. The Tinnitus Handicap Inventory (THI), visual analog scale (VAS), and Beck Depression Inventory (BDI) were evaluated before and after rTMS. RESULTS: The mean THI score of the eight patients with depression changed from 77.5 ± 15 to 61.8 ± 20.1 after the second rTMS. There was statistical significance only for the second rTMS. The VAS score changed from 8.6 ± 1.6 to 6.3 ± 1.8 after the first rTMS and from 7.6 ± 2.4 to 4.6 ± 2.7 after the second rTMS, showing statistically significant changes both times. The THI changes after the second rTMS were greater than after the first rTMS, and the changes in VAS score showed a similar pattern. The changes in BDI score, which indicates the severity of depression, showed a variable pattern after rTMS. Patients with mild depression (10≤ BDI score <16, n = 4) showed significant improvement of THI with the second combined rTMS (ΔTHI = 24.5) as compared with the first rTMS on the auditory area (ΔTHI = 6). In contrast, combined rTMS did not show any better improvement on THI (ΔTHI = 6.5) than the first rTMS on the auditory cortex (ΔTHI = 7) in patients without depression (BDI <10, n = 3) and patients with moderate to severe depression (BDI ≥16, n = 4).

Sublingual immunization with a live attenuated influenza a virus lacking the nonstructural protein 1 induces broad protective immunity in mice.

The nonstructural protein 1 (NS1) of influenza A virus (IAV) enables the virus to disarm the host cell type 1 IFN defense system. Mutation or deletion of the NS1 gene leads to attenuation of the virus and enhances host antiviral response making such live-attenuated influenza viruses attractive vaccine candidates. Sublingual (SL) immunization with live influenza virus has been found to be safe and effective for inducing protective immune responses in mucosal and systemic compartments. Here we demonstrate that SL immunization with NS1 deleted IAV (DeltaNS1 H1N1 or DeltaNS1 H5N1) induced protection against challenge with homologous as well as heterosubtypic influenza viruses. Protection was comparable with that induced by intranasal (IN) immunization and was associated with high levels of virus-specific antibodies (Abs). SL immunization with DeltaNS1 virus induced broad Ab responses in mucosal and systemic compartments and stimulated immune cells in mucosa-associated and systemic lymphoid organs. Thus, SL immunization with DeltaNS1 offers a novel potential vaccination strategy for the control of influenza outbreaks including pandemics.

Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza.

BACKGROUND: The ectodomain of matrix protein 2 (M2e) of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs) have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. METHODS AND RESULTS: A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. CONCLUSIONS: The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.

Combinatorial therapy for liver metastatic colon cancer: dendritic cell vaccine and low-dose agonistic anti-4-1BB antibody co-stimulatory signal.

BACKGROUND: The combination of dendritic cell (DC) vaccine and 4-1BB ligation may be a suitable choice of immunotherapy for incurable cancer. However, at anti-tumor effector doses over 100 µg, 4-1BB Ab ligation is toxic to CD4(+) T cells, thus limiting its therapeutic use. MATERIALS AND METHODS: A liver metastatic colon cancer model was established by hepatic injection of CT26 cells into Balb/c mice. Intraperitoneal administration of 1 × 10(6)/200 µL/mouse therapeutic-DCs (tumor lysate pulsed-DCs, P-DCs) began on d 7 after tumor cell inoculation. A P-DC injection was performed twice within a 1-wk interval. Agonistic anti 4-1BB Ab was intraperitoneally injected on d 7, 9, and 11 after tumor cell inoculation. Animals were sacrificed on d 21, and tumor growth was determined by weighing the liver with the tumor. RESULTS: In the 20 µg 4-1BB ligation group, significant induction of CD3(+)CD8(+) T cells was observed without toxicity to CD3(+)CD4(+) T cells. DC vaccine treatment induced tumor antigen-specific Th1 cytokine (IL-2 and IFN-γ) secretion from the splenic lymphocytes. Ligation of 4-1BB reduced the DC vaccine-related IL-10 secretion and regulatory T cell population. Compared with anti-tumor effect of DC vaccine or 20 µg 4-1BB Ab alone, the combination therapy significantly increased the tumor rejection power to the level observed with higher doses of 4-1BB Ab alone. The combination therapy did not induce high-dose 4-1BB-related toxicity with CD4(+) T cell reduction, but did significantly induce tumor antigen-specific IFN-γ secreting effector CD8(+) cytotoxic T cells. CONCLUSIONS: The data from our study reveal the value of using a DC vaccine combined with as little as 20 µg 4-1BB Ab as an improved immunotherapeutic for cancer.

Interleukin 7 receptor gene polymorphisms and haplotypes are associated with susceptibility to IgA nephropathy in Korean children.

An abnormal T-cell response is involved in the pathogenesis of various renal diseases. Survival of naïve T cells is dependent on interleukin 7 (IL7) and its receptor (IL7R). Thus, we investigated the association between IL7R single nucleotide polymorphisms (SNPs) and childhood IgA nephropathy (IgAN). We analyzed the genotypic distributions of two missense SNPs of IL7R, rs1494558 (Ile66Thr) and rs1494555 (Val138Ile), among 198 pediatric IgAN patients and 288 healthy controls. Haplotype analysis and measurement of pair-wise linkage disequilibrium were performed. In addition, the genotypes of patient subgroups, determined by the presence of nephrotic range proteinuria (>40 mg/m(2)/h) and pathological advancement, were analyzed. The genotyping data of IgAN patients and controls showed significant differences in rs1494558 (codominant, P=0.0003; dominant, P=0.0003) and rs1494555 (codominant, P=0.0038; dominant, P=0.0099). In the haplotype analysis, AC (codominant, P=0.0066) and GT (codominant, P=0.0005; dominant, P=0.0006) were significantly associated with susceptibility to IgAN. Furthermore, in the analysis of clinical subgroups of IgAN patients, rs1494558 was associated with nephrotic range proteinuria (codominant, P=0.027; recessive, P=0.023). Our results suggest that IL7R may be associated with disease susceptibility and proteinuria in childhood IgAN.

CD40-targeted recombinant adenovirus significantly enhances the efficacy of antitumor vaccines based on dendritic cells and B cells.

Despite the advantages of using adenoviral vectors for specific antigenic gene delivery in the development of antigen-presenting cell (APC)-based vaccines, the lack of the coxsackievirus-adenovirus receptor (CAR) on APCs limits the use of adenoviral vectors for in vitro gene delivery. In this study, we used a recombinant adapter protein, CFm40L, which consists of the ectodomain of CAR genetically fused to the ectodomain of CD40 ligand (CD40L) via a trimerization motif, to target Her-2/neu- or human papillomavirus 16 (HPV16) E6/E7-encoding adenoviruses to CD40 on dendritic cells (DCs) and B cells. Targeting CD40 enabled the enhancement of tumor antigen delivery and simultaneous activation of APCs via the CD40-CD40L interaction. We found that these transduced DCs and B cells substantially enhanced the CTL response against human Her-2/neu- and HPV16 E6/E7-expressing tumors, resulting in significant inhibition of tumor growth in a murine tumor model. In addition, the use of the CFm40L adapter protein in combination with gemcitabine treatment allowed for a successful immune response against a self-tumor antigen, murine Her-2/neu. Our results suggest that targeting adenovirus to APCs via CD40, using CFm40L, represents a great improvement in anticancer cellular vaccines.

Prophylactic and therapeutic efficacy of avian antibodies against influenza virus H5N1 and H1N1 in mice.

BACKGROUND: Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY) found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1. METHODS AND FINDINGS: We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection. CONCLUSIONS: The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the current H1N1 pandemic.

Mycophenolate mofetil promotes down-regulation of expanded B cells and production of TNF-alpha in an experimental murine model of colitis.

In this study, we used a murine intestinal inflammation model that mimics immunologic characteristics of human Crohn's disease (CD) to investigate the anti-inflammatory effects of mycophenolate mofetil (MMF) on intestinal injury and tissue inflammation. When these colitic mice were pretreated with MMF, we observed a significant decrease in mortality rates and body weight loss as well as an improvement in both wasting and histopathologic signs of colonic inflammation, relative to untreated colitic mice. To determine the mechanisms of action of MMF, we compared various immunological characteristics of the untreated and MMF-pretreated colitic mice. MMF-pretreated colitic mice showed an 18% decrease in the proportion of CD19+ B cells compared with untreated colitic mice 3 days. As a result, MMF pretreatment increases proportion of apoptotic T and B cells, especially CD19+ B cells. Also, down-regulation of Th1 cytokines (TNF-alpha, IFN-gamma) and augmentation of CD4+CD45RB(low) regulatory T (Treg) cells were observed in MMF-pretreated colitic mice compared with untreated colitic mice. Furthermore, mycophenolic acid (MPA) reduced TNF-alpha-stimulated NF-kappaB activation in HT-29 colon epithelial cells. Also, MMF-pretreated colitic mice significantly reduced expression of MD-1 compared with untreated colitic mice on B cells and dendritic cells (DCs). These studies show that MMF pretreatment can improve experimental colitis by down-regulation of expanded B cells population through apoptosis and augmentation of Treg cells. Through these mechanisms, MMF might also be an effective agent for the treatment of other diseases characterized by mucosal inflammation.

15-deoxyspergualin prevents mucosal injury by inhibiting production of TNF-alpha and down-regulating expression of MD-1 in a murine model of TNBS-induced colitis.

The immunosuppressive drug 15-deoxyspergualin (DSG) is currently being used in clinical trials to prolong graft survival and reverse graft rejection. Here we evaluated whether DSG has a potential for ameliorating diseases characterized by mucosal inflammation. Using a murine model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, we were able to demonstrate that DSG reduced the severity of colitis. Therefore, colitic mice pretreated with DSG showed a striking improvement of the wasting disease compared with colitic mice, as assessed by weight loss as well as clinical, macroscopic and microscopic analysis. Also, we observed the significant change occurred in the CD19(+) B cell subset, which was decreased 15% in DSG pretreated colitic mice compared with colitic mice. However, DSG pretreatment does not influence the apoptotic population of T and B cells. Compared with colitic mice, down-regulation of TNF-alpha production was observed in DSG pretreated colitic mice. In addition, DSG pretreated colitic mice significantly reduced expression of MD-1 compared with colitic mice on B cells and dendritic cells (DCs). Therefore, pretreatment with DSG resulted in a significant histologic improvement, protecting against mucosal ulcerations and reduced inflammatory response by modulating expression of MD-1, which plays a very important role in immune response on B cells and DCs. Also, this improvement was paralleled by a reduction in TNF-alpha levels. Collectively, current results demonstrate that DSG may be an effective agent for the treatment of diseases characterized by mucosal inflammation.

Intratumoral delivery of IL-18 naked DNA induces T-cell activation and Th1 response in a mouse hepatic cancer model.

BACKGROUND: The novel cytokine, interleukin (IL)-18, is a strong interferon-gamma inducer and costimulatory factor in Th1 cell activation. IL-18 triggers IFN-gamma production and enhances cytolytic activity in both T and NK cells. However, the exact mechanism of antitumor action of IL-18 remains to be clarified. To determine the effects of IL-18 plasmid DNA on hepatic cancer in mice, CT26 murine colon adenocarcinoma cells were established in mouse liver. METHODS: Plasmid vectors encoding IL-18 were transferred directly into the liver 7 days after tumor injection to restrict IL-18 expression within the tumor site. The IL-18 protein level was increased in the liver 4 days after plasmid injection, and a marked antitumoral effect was observed at day 7. Antitumor effects were evaluated by measuring tumor regression, immune cell population, and IFN-gamma production. RESULTS: The IL-18 plasmid controlled the growth of hepatic tumors and proliferation of splenic immune cells. Moreover, treatment of CT26 tumors with the IL-18 plasmid significantly enhanced the population of the effector T and NK cells in the spleen and peripheral blood. In spleen, the population of CD4+CD62Low cells was augmented in response to IL-18 on day 7. These results are consistent with the increase in CD4+ T cells secreting IFN-gamma, but not CD8+ T cells. The marked reduction of tumor growth in tumor-bearing mice was associated with the maintenance of IFN-gamma production in spleen in response to IL-18. These antitumoral effects were maintained until 14 days after plasmid injection. CONCLUSION: Our results suggest that direct plasmid DNA transfer of IL-18 with no accompanying reagents to augment transfection efficiency may be useful in tumor immunotherapy.

Two-signal blockade with anti-CD45RB and anti-CD154 monoclonal antibodies inhibits graft rejection via CD4-dependent mechanisms in allogeneic skin transplantation.

Blockade of signal 1 or 2 for T-cell activation by the use of anti-CD45RB and anti-CD154 monoclonal antibodies (mAb) (two-signal blockade) has been proven effective in preventing or delaying graft rejection. However, the mechanisms of its immunomodulatory effects are clearly unknown and the present studies were performed to determine how the two-signal blockade modulate allogeneic immune responses, especially T-cell mediated cellular immunity, in a murine skin allograft model. We now report on the profound inhibition of alloreactive T cells by two-signal blockade via CD4-dependent mechanisms. C57BL/6 mice of BALB/c skin allograft were treated with anti-CD45RB, anti-CD154, CTLA4-Ig, or their combinations. For depletion of CD4 or CD8 T cells, the recipients received CD4-depleting or CD8-depleting mAb. We confirmed that survival of skin allograft was markedly prolongated in the two-signal blockade-treated group. In depletion study, anti-CD45RB, anti-CD154 and CD4-depleting mAb-treated group showed acute rejection of skin allograft in contrast to CD8-depleting group treated with the two-signal blockade. In the group treated with the two-signal blockade, the proportions of CD4+CD45RB(low) and CD8+CTLA-4 regulatory T cells were increased while effector CD8+ T cells, including IFN-gamma-secreting and CD8+CD62L(low) T cells, were decreased when compared with non-treated group. In contrast, the CD4-depleted group treated with the two-signal blockade resulted in recovery from immunoregulatory effects of two-signal blockade. In addition, results of IL-4 and IL-10 production were also showed CD4-dependence. Therefore, the two- signal blockade is accompanied by CD4-dependent mechanisms in allogeneic skin transplantation.

Tolerance induction through simultaneous double bone marrow transplantation with two-signal blockade.

Cotransplantation of donor bone marrow cells (BMCs) in allograft recipients is currently the most promising concept for clinical tolerance induction; however, it still has many difficulties in its successful performance due to the toxicity of the required host conditioning, the risk of engraftment failure, and the problem of graft-versus-host disease (GVHD), as well as the limited accessibility of donor bone marrow cells. Therefore, we performed the studies to determine whether BMCs from multi-donors are simultaneously engrafted and lead to induction of chimerism-based tolerance through the tolerogenic protocol of whom effectiveness we have shown in a previous study. Using a murine model, it was demonstrated that grafted BMCs from BALB/c (H-2(d)) and CBA mice (H-2(k)) establish mixed type and multi-lineage double chimerism and induce immunological donor-specific tolerance to fully MHC-mismatched skin allografts in host C57BL/6 mice (H-2(b)) receiving conditioning with Busulfan and treatment with the two-signal blockade comprised of anti-CD45RB and anti-CD154 monoclonal antibodies.

Tolerance induction through megadose bone marrow transplantation with two-signal blockade.

BACKGROUND: Induction of mixed chimerism is currently the most promising concept for clinical tolerance induction; however, the toxicity of the required host conditioning for allogeneic bone marrow transplantation (BMT) should be overcome. Therefore, we explored tolerogenic effectiveness of megadose BMT with anti-CD45RB and anti-CD154 mAb (two-signal blockade) in murine recipients without conditioning. MATERIALS AND METHODS: Recipient B6 mice of BALB/c skin allograft received conditioning and an optimal dose (2x10(7) cells) of BMT. For a megadose BMT model, the conditioning was not performed; instead, megadose (2x10(8) cells) of BM was transplanted. The recipients were then treated with anti-CD45RB mAb and anti-CD154 mAb alone or their combination. Flow cytometry was performed to analyze the degree and distribution of donor-derived cells, peripheral deletion of Vbeta5 or Vbeta11 T cells and intrathymic presence of donor MHC class II+ cells. Induction of chimerism-based tolerance to skin allograft was further determined. RESULTS: High levels ( approximately 23.7%) of mixed and multi-lineage chimerism-based tolerance to skin allograft were induced in the recipients (91%) treated with the optimal-dose BMT and the two-signal blockade. The megadose BMT could replace the recipient conditioning and establish low (approximately 10%) and stable multilineage chimerism. Donor-specific tolerance to skin allograft was induced in these chimeras through clonal deletion of donor-reactive cells. CONCLUSIONS: The megadose BMT with the two-signal blockade could effectively establish mixed and multi-lineage chimerism and induce donor-specific tolerance, suggesting its potential for clinical application.

Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of inflammatory bowel disease.

4-1BB (CDw 137), a member of the tumor necrosis factor receptor (TNFR) superfamily, is a costimulatory receptor primarily expressed on activated T cells. It has been shown that the administration of agonistic anti-4-1BB monoclonal antibody (mAb) enhances tumor immunity and allogenic immune responses. Paradoxically, we found that the administration of anti-4-1BB mAb reduced the incidence and severity of inflammatory bowel disease. In this study, we investigated the effects of anti-4-1BB mAb in a murine intestinal inflammation model, which induced by the hapten reagent, 2,4,6-trinitrobenzene sulfonic acid (TNBS) and mimics immunologic characteristics of human Crohn's disease (CD). Colitis was induced by rectal administration of 2mg of TNBS in 35% ethanol using a vinyl catheter positioned 4cm from the anus. All mice were sacrificed 3 and 10 days after the TNBS administration. The disease activity index (DAI), histological changes of the colon and production of cytokines (IL-2, IL-4, IL-10 and IFN-gamma) were evaluated. The surface molecules of T cells in peripheral blood, spleen and mesenteric lymph nodes were analyzed by flow cytometry. When mice were treated with anti-4-1BB mAb, improvement in both wasting and histopathologic signs of colonic inflammation was observed. The increase a number of splenic CD4(+)CD25(+) T cells and decreased synthesis of the Th1 cytokine IL-2 also occurred. Interestingly, increased production of Th1 cytokine IFN-gamma and proportion of CD8(+) T cells were observed in mice treated with anti-4-1BB mAb in comparison to the colitic mice. These studies show, for the first time, that agonistic anti-4-1BB mAb can improve experimental colitis by reduction of IL-2 and augmentation of CD4(+)CD25(+) regulatory T cells. TNBS colitis is Th1-mediated and has similar histologic features and distribution of inflammation to CD. This study suggests that anti-4-1BB mAb therapy could be effective in the treatment of patients with CD.