Carboxymethyl cellulose-based rotigotine nanocrystals-loaded hydrogel for increased transdermal delivery with alleviated skin irritation.

Transdermal rotigotine (RTG) therapy is prescribed to manage Parkinson's disease (Neupro® patch). However, its use is suffered from application site reactions. Herein, drug nanocrystalline suspension (NS)-loaded hydrogel (NS-HG) employing polysaccharides simultaneously as suspending agent and hydrogel matrix was constructed for transdermal delivery, with alleviated skin irritation. RTG-loaded NS-HG was prepared using a bead-milling technique, employing sodium carboxylmethyl cellulose (Na.CMC) as nano-suspending agent (molecular weight 90,000 g/mol) and hydrogel matrix (700,000 g/mol), respectively. NS-HG was embodied as follows: drug loading: ≤100 mg/mL; shape: rectangular crystalline; crystal size: <286.7 nm; zeta potential: -61 mV; viscosity: <2.16 Pa·s; and dissolution rate: >90 % within 15 min. Nuclear magnetic resonance analysis revealed that the anionic polymers bind to RTG nanocrystals via charge interaction, affording uniform dispersion in the matrix. Rodent transdermal absorption of RTG from NS-HG was comparable to that from microemulsions, and proportional to drug loading. Moreover, NS-HG was skin-friendly; erythema and epidermal swelling were absent after repeated application. Further, NS-HG was chemically stable; >95 % of the drug was preserved up to 4 weeks under long term (25 °C/RH60%), accelerated (40 °C/RH75%), and stress (50 °C) storage conditions. Therefore, this novel cellulose derivative-based nanoformulation presents a promising approach for effective transdermal RTG delivery with improved tolerability.

Design of Montelukast Nanocrystalline Suspension for Parenteral Prolonged Delivery.

Background: Montelukast (MTK), a representative leukotriene receptor antagonist, is currently being investigated as a potential candidate for treating Alzheimer's disease. For potent and effective dosing in elderly patients, a parenteral prolonged delivery system is favored, with improved medication adherence with reduced dosage frequency. Purpose: This study aimed to design a nanocrystalline suspension (NS)-based MTK prolonged delivery system and evaluate its pharmacokinetics profile and local tolerability following subcutaneous administration. Methods: To decelerate the dissolution rate, the amorphous MTK raw material was transformed into a crystalline state using a solvent-mediated transformation method and subsequently formulated into NS using a bead-milling technique. The MTK NSs were characterized by morphology, particle size, crystallinity, and in vitro dissolution profiles. The pharmacokinetic profile and local tolerability at the injection site following subcutaneous injection of MTK suspension were evaluated in rats. Results: Microscopic and physical characterization revealed that the amorphous MTK powder was lucratively transformed into a crystalline form in acidic media (pH 4). MTK crystalline suspensions with different diameters (200 nm, 500 nm, and 3 µm) were uniformly prepared using bead-milling technology, employing polysorbate 80 as suspending agent. Prepared crystalline suspensions exhibited analogous crystallinity (melting point, 150°C) and size-dependent in vitro dissolution profiles. MTK NSs with particle sizes of 200 nm and 500 nm provided a protracted pharmacokinetic profile for up to 4 weeks in rats, with a higher maximum drug concentration in plasma than the 3 µm-sized injectable suspensions. Histopathological examination revealed that MTK NS caused chronic granulomatous inflammation at the injection site, which resolved after 4 weeks. Conclusion: The MTK parenteral NS delivery system is expected to be a valuable tool for treating Alzheimer's disease with extended dose intervals.

High-Payload Nanosuspension of Centella asiatica Extract for Improved Skin Delivery with No Irritation.

BACKGROUND: The titrated extract of Centella asiatica (CA) has received much attention as a cosmeceutical ingredient owing to its anti-wrinkle effect. However, due to the low solubility and high molecular weight of pharmacologically active constituents, including asiatic acid (AA), madecassic acid (MA), and asiaticoside (AS), it is challenging to fabricate high-payload topical preparations of CA with satisfactory skin absorption profiles. PURPOSE: This study aimed to design a high-payload topical preparation of CA using nanocrystallization technique and to evaluate its skin absorption profile and local tolerability. METHODS: High-payload nanocrystal suspensions (NSs) were prepared using lab-scale bead-milling technology, by adjusting the type and amount of suspending agent, CA content, type of vehicle, and milling speed. CA-loaded NSs were characterized in terms of morphology, particle size, crystallinity, and in vitro dissolution pattern. Skin absorption of CA nanocrystals was evaluated using a vertical Franz diffusion cell mounted with porcine skin. In vivo skin irritation following topical application of high-payload NS was assessed in normal rats. RESULTS: The optimized NS system, composed of 10% (w/v) CA, 0.5% polyvinylpyrrolidone (PVP) K30 as steric stabilizer, and 89.5% of distilled water, was characterized as follows: spherical or elliptical in shape, 200 nm in size, with low crystallinity. The in vitro dissolution of AA or MA from NSs was markedly faster compared to raw material, under sink condition. Penetration of AA, MA, and AS in the porcine skin was markedly elevated using the high-payload NS formula, providing 5-, 4-, and 4.5-fold higher accumulation in skin layer, compared to that of the marketed cream formula (CA 1%, Madeca cream). Moreover, topical application of high-payload NS was tolerable, showing neither erythema nor oedema in normal rats. CONCLUSION: The novel NS system is expected to be a virtuous approach for offering a better skin absorption of CA, without using an excess quantity of solubilizers.

Benign meningioma manifesting with acute subdural hematoma and cerebral edema: a case report and review of the literature.

BACKGROUND: Spontaneous subdural hematoma rarely presents with a hypervascular or malignant tumor but even less frequently in a benign tumor like meningioma. We encountered a patient with acute subdural hematoma associated with benign meningioma. Here, we report this case along with a review of previous reports, especially focusing on their clinical features and possible bleeding mechanisms. CASE PRESENTATION: A 53-year-old Asian woman presented with severe headache and progressive neurologic deterioration due to cerebral edema. The patient was submitted to open surgery for evacuation of the subdural hematoma and concurrent tumor removal on the ipsilateral parietal convexity. A hypervascular, encapsulated mass was identified during surgery and completely removed including the adjacent dura mater (Simpson grade 0). The tumor was histologically confirmed as an angiomatous meningioma (World Health Organization grade I). Her clinical course was uneventful after surgery. CONCLUSIONS: Although meningiomas are commonly benign according to their histological traits, they can lead to spontaneous bleeding and cause neurologically unstable condition. Therefore, meningiomas need to be considered as a cause of spontaneous subdural hematoma if radiologically suspicious, which should be reflected by proper management for a positive outcome.

Public Awareness of Stroke and Its Predicting Factors in Korea: a National Public Telephone Survey, 2012 and 2014.

The aim of this study was to investigate time trends in the public awareness of stroke and its predicting factors. The target population was 9,600 community-dwelling adults, aged 19-79 years, in 16 metropolitan cities and provinces in Korea. The survey samples in 2012 and 2014 were selected separately (entirely different sets of subjects) using a proportionate quota sampling method. Information concerning knowledge of stroke and demographics was collected by trained telephone interviewers using random digit dialing. After excluding subjects with a non-response or refusal to answer any question, the analyses included 8,191 subjects in 2012 and 8,127 subjects in 2014. Respondents' awareness of stroke warning signs (numbness or weakness, difficulty speaking or understanding speech, dizziness, visual impairment, and severe headache) was highest for difficulty speaking or understanding speech (80.9% in 2012 and 86.4% in 2014). There were significant increases in the proportion of respondents understanding the appropriate action (i.e., calling an ambulance) at the time of stroke occurrence (59.6% to 67.1%), and in the proportion aware of the general need for prompt treatment (86.7% to 89.8%). In multivariable logistic regression analysis, older age, higher education level, higher household income, current non-smoking, exposure to stroke-related public relations materials, and experience of stroke education were significantly associated with both high knowledge of stroke warning signs and awareness of the need for prompt treatment. Between 2012 and 2014, the public's awareness of stroke increased significantly. More specialized interventions, including public relations materials and education, should focus on subgroups who have lower stroke knowledge.

Effects on the Qualities of Proteolysis to Beef by Non-coating and Coating Protease Treatment.

This study was performed to improve the techniques used for tenderizing red meat as elderly food. Beef meat was immersed in liposome encapsulated enzyme solution and the effect of protease encapsulation on the beef properties was analyzed. The protease encapsulation properties were analyzed according to the size distribution and enzymatic activity. After enzyme reaction on the beef, the chemical properties of the meat such as pH, water holding capacity, shear rate, lipid oxidation and total volatile basic nitrogen (TVB-N) were analyzed. The pH of the beef increased during the reaction and coating protease (CP) was higher than non-coating protease (NCP). Total color differences were increased remarkably after 36 h and generally, the difference in CP was relatively lower than in NCP. WHC was significantly decreased within 24 h, and no effect from the protease coating was observed. Protease activity was significantly increased within 48 h and no differences in the enzyme coating were observed. The TVB-N value of NCP was increased within 24 h while CP was sustained for up to 36 h. The TVB-N value of protease treated meat increased after 36 h and no effect from the protease coating was detected. Consequently, liposome encapsulated protease was found to have similar properties as non-coated protease. Application of liposome seems to be an interesting option for injecting various functional materials without changing the properties of meat.

Comparison of Dexmedetomidine and Remifentanil on Airway Reflex and Hemodynamic Changes during Recovery after Craniotomy.

PURPOSE: During emergence from anesthesia for a craniotomy, maintenance of hemodynamic stability and prompt evaluation of neurological status is mandatory. The aim of this prospective, randomized, double-blind study was to compare the effects of dexmedetomidine and remifentanil on airway reflex and hemodynamic change in patients undergoing craniotomy. MATERIALS AND METHODS: Seventy-four patients undergoing clipping of unruptured cerebral aneurysm were recruited. In the dexmedetomidine group, patients were administered dexmedetomidine (0.5 µg/kg) for 5 minutes, while the patients of the remifentanil group were administered remifentanil with an effect site concentration of 1.5 ng/mL until endotracheal extubation. The incidence and severity of cough and hemodynamic variables were measured during the recovery period. Hemodynamic variables, respiration rate, and sedation scale were measured after extubation and in the post-anesthetic care unit (PACU). RESULTS: The incidence of grade 2 and 3 cough at the point of extubation was 62.5% in the dexmedetomidine group and 53.1% in the remifentanil group (p=0.39). Mean arterial pressure (p=0.01) at admission to the PACU and heart rate (p=0.04 and 0.01, respectively) at admission and at 10 minutes in the PACU were significantly lower in the dexmedetomidine group. Respiration rate was significantly lower in the remifentanil group at 2 minutes (p<0.01) and 5 minutes (p<0.01) after extubation. CONCLUSION: We concluded that a single bolus of dexmedetomidine (0.5 µg/kg) and remifentanil infusion have equal effectiveness in attenuating coughing and hemodynamic changes in patients undergoing cerebral aneurysm clipping; however, dexmedetomidine leads to better preservation of respiration.

The General Public's Awareness of Early Symptoms of and Emergency Responses to Acute Myocardial Infarction and Related Factors in South Korea: A National Public Telephone Survey.

BACKGROUND: Prompt treatment affects prognosis and survival after acute myocardial infarction (AMI) onset. This study evaluated the awareness of early symptoms of AMI and knowledge of appropriate responses on symptom occurrence, along with related factors. METHODS: Participants' knowledge of the early symptoms of and responses to AMI onset were investigated using a random digit dialing survey. We included 9600 residents of 16 metropolitan cities and provinces in Korea. RESULTS: The proportions of respondents who were aware of early symptoms of AMI ranged from 32.9% (arm or shoulder pain) to 79.1% (chest pain and discomfort). Of the respondents, 67.0% would call an ambulance if someone showed signs of AMI, 88.7% knew ≥1 symptom, 10.9% knew all five symptoms, and 3.1% had excellent knowledge (correct identification of all five AMI symptoms, not answering "Yes" to the trap question, and correctly identifying calling an ambulance as the appropriate response when someone is exhibiting AMI symptoms). The odds ratio (OR) for having excellent knowledge was significantly higher for those who graduated college or higher (OR 3.42; 95% confidence interval [CI], 1.09-10.76) than for those with less than a primary school education, as well as for subjects with AMI advertisement exposure (OR 1.49; 95% CI, 1.10-2.02) and with knowledge of AMI (OR 1.63; 95% CI, 1.16-2.27). The 60- to 79-year-old group had significantly lower OR for excellent knowledge than the 20- to 39-year-old group (OR 0.53; 95% CI, 0.28-0.99). CONCLUSIONS: Awareness of AMI symptoms and the appropriate action to take after symptom onset in South Korea was poor. Therefore, educational and promotional strategies to increase the overall awareness in the general public, especially in the elderly and those with low education levels, are needed.

Optimal hemoglobin level for anemia treatment in a cohort of hemodialysis patients.

BACKGROUND: Anemia is a major risk factor that contributes to mortality in patients with chronic kidney disease. There is controversy over the optimal hemoglobin (Hb) target in these patients. This study investigated the association between Hb level and mortality in a cohort of hemodialysis (HD) patients in Korea. METHODS: This study was a multicenter prospective observational study of maintenance HD patients that was performed for 5 years in western Seoul, Korea. Three hundred and sixty-two participants were enrolled. Laboratory values and mortality were accessed every 6 months. Repeated measures of laboratory values in each interval were averaged to obtain one semiannual mean value. The Hb values were divided into six groups: (1) Hb<9 g/dL; (2) 9 g/dL≤Hb<10 g/dL; (3) 10 g/dL≤Hb<11 g/dL; (4) 11 g/dL≤Hb<12 g/dL; (5) 12 g/dL≤Hb<13 g/dL; and (6) Hb≥13 g/dL. We analyzed the odds ratio for all-cause mortality, based on the Hb group, and adjusted for demographics and various laboratory values. Statistics were performed with SAS, version 9.1 software (SAS Institute Inc., Cary, NC, USA). RESULTS: Mortality odds ratios relative to the reference group (10-11 g/dL) in the fully adjusted model were 3.61 for<9 g/dL; 3.17 for 9-10 g/dL(⁎); 4.65 for 11-12 g/dL(⁎); 5.50 for 12-13 g/dL(⁎); and 2.05 for≥13 g/dL ((⁎) indicates P<0.05). CONCLUSION: In this study, a Hb level of 10-11 g/dL was associated with the lowest mortality among the groups with Hb level<13 g/dL. Larger interventional trials are warranted to determine the optimal Hb target for Korean HD patients.

The Prognostic Value of the Tumor Shrinkage Rate for Progression-Free Survival in Patients with Non-Small Cell Lung Cancer Receiving Gefitinib.

BACKGROUND: The efficacy of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy can be measured based on the rate of treatment response, based on the Response Evaluation Criteria in Solid Tumors (RECIST) criteria or progression-free survival (PFS). However, there are some patients harboring sensitive EGFR mutations who responded poorly to EGFR-TKI therapy. In addition, there is variability in the PFS after EGFR-TKI treatment. METHODS: We performed a retrospective analysis of the medical records of 85 patients with non-small cell lung cancer, who had achieved a stable disease or better response at the first evaluation of treatment response, after receiving a 2-month course of gefitinib. We calculated the tumor shrinkage rate (TSR) by measuring the longest and perpendicular diameter of the main mass on computed tomography before, and 2 months after, gefitinib therapy. RESULTS: There was a significant positive correlation between the TSR and PFS (R=0.373, p=0.010). In addition, a simple linear regression analysis showed that the TSR might be an indicator for the PFS (B±standard error, 244.54±66.79; p=0.001). On univariate analysis, the sex, histologic type, smoking history and the number of prior chemotherapy regimens, were significant prognostic factors. On multivariate regression analysis, both the TSR (ß=0.257, p=0.029) and adenocarcinoma (ß=0.323, p=0.005) were independent prognostic factors for PFS. CONCLUSION: Our results showed that the TSR might be an early prognostic indicator for PFS in patients receiving EGFR-TKI therapy.

Malignant rhabdoid tumor of the kidney and spine in an infant.

Rhabdoid tumor of the kidney (RTK) is a rare malignancy in infancy. Central nervous system involvement in RTK is already known. However, solitary spinal metastasis in RTK has been hardly reported. The authors report a case of metastatic RTK to spine causing paraplegia in an 8-month-old girl. Since the patient was young, the diagnosis of spine metastasis was delayed until paraplegia was seen after radical nephrectomy. Thorough neurological examination should be performed for early diagnosis of spinal metastasis in young patients with RTK. If there are any abnormal signs in neurologic examination, magnetic resonance images of brain and spine are recommended.

Colonic mucormycosis presented with ischemic colitis in a liver transplant recipient.

Mucormycosis is an uncommon opportunistic fungal infection with high mortality in liver transplant recipients. Mucormycosis of the gastrointestinal tract can manifest with features similar to ischemic colitis. Typically signs and symptoms of non-gangrenous ischemic colitis resolve spontaneously within 24-48 h. On the other hand, the clinical course of the mucormycosis is commonly fulminant. We encountered a case of invasive fungal colitis presenting with abdominal pain and hematochezia in a liver transplant recipient. Endoscopic examination showed multiple shallow ulcerations and edema with mucosal friabilities on the sigmoid and distal descending colon, which was consistent with ischemic colitis. However, the histological examination obtained from endoscopic biopsies showed fungal hyphae with surrounding inflammatory cells and mucosal necrosis. The patient was successfully managed with antifungal agent without surgical treatment. Thus, early diagnosis and treatment is essential for improving the prognosis of invasive fungal infection after liver transplantation.

Pattern of hepatitis a incidence according to area characteristics using national health insurance data.

OBJECTIVES: Over the past several years, the incidence of hepatitis A infection has been increasing rapidly in the young-adult population in Korea. We examined the effects of area-level socioeconomic status and environmental hygiene on the incidence of hepatitis A. METHODS: This study is based on the registered national population of Korea and the national health insurance data from 2004 to 2008. A total of 73 459 individuals were confirmed to have had hepatitis A. The standardized incidences of hepatitis A in 232 districts adjusted for sex and age of people were calculated for each year, and the rate ratios of the incidence rates were estimated according to area-level socioeconomic status and environmental hygiene using multiple Poisson regression models. RESULTS: The incidence rates of hepatitis A infection were 15.6 (per 100 000) in 2004, 19.0 (per 100 000) in 2005, 27.2 (per 100 000) in 2006, 25.1 (per 100 000) in 2007, and 61.7 (per 100 000) in 2008. The analysis of the area-level effects showed that residential areas of the less deprived than other regions, areas with higher levels of education, and heavily populated areas were significantly associated with increased risk. CONCLUSIONS: There is a very strong possibility that both area-level socioeconomic status and environmental hygiene play a role in increasing the risk of hepatitis A infection in Korea. Therefore, to reduce hepatitis A infection, we need a nationwide strategy that considers these area-level characteristics.

Role of TNFR-related 2 mediated immune responses in dextran sulfate sodium-induced inflammatory bowel disease.

Previous work has suggested that the LIGHT-TR2 costimulatory pathway plays a role in the acute and chronic stages of dextran sulfate sodium (DSS)-induced colitis [Steinberg et al. (2008); Wang et al. (2005)]. To clarify the role of TNFR-related 2 (TR2) signaling in the maintenance of intestinal homeostasis, we generated a TR2 knock-out (KO) mouse. Using DSS to induce colitis, we compared the colitic symptoms and pathological changes in wild type (WT) and TR2 KO mice, and the production of cytokines by the diseased colons. We also studied the role of TR2 in suppressing innate and adaptive immunity in the DSS model. TR2 deficient mice were characterized by reduced symptoms of intestinal inflammation compared with wild-type mice, and reduced production of cytokines. We therefore generated a monoclonal antibody against mouse TR2 which was specific to TR2 and capable of blocking TR2 signals. With this antibody, we demonstrated that antagonizing TR2 during the development of DSS-induced colitis reduced the symptoms of inflammation. Our findings suggest that TR2 is an important mediator in colitis, and may serve as a therapeutic target in inflammatory bowel disease.

[Analysis for the impact of adulthood and childhood socioeconomic positions and intergenerational social mobility on adulthood health].

OBJECTIVES: There are at least three conceptual models for the effects of the childhood social environment on adult health: the critical period model, the social mobility model, and the cumulative risk model. However, few studies have investigated all three different models within the same setting. This study aims to examine the impact of childhood and adulthood socioeconomic positions and intergenerational social mobility over the life course on the health in adulthood based both on the critical period model and the social mobility model. METHODS: This study was conducted on 9583 adults aged between 25 and 64 years old and they were the respondents to the Korea Welfare Panel Study (2006). A multivariate logistic regression analysis was carried out, using the critical period model and the social mobility model out of the life course approaches, to look into the impact of childhood and adulthood socioeconomic positions and intergenerational social mobility on the health status in adulthood. RESULTS: Household income and occupation out of the adulthood socioeconomic position indicators had an independent influence on the adulthood health status. The childhood socioeconomic position indicators, except for the place of childhood residence, affected the adulthood health status even after adjustment for the adulthood socioeconomic position. The effect of intergenerational social mobility was also statistically significant even after adjusting for the adulthood socioeconomic position, but it became insignificant when the childhood socioeconomic position was additionally adjusted for. CONCLUSIONS: Adulthood health is indeed affected by both the childhood and adulthood socioeconomic positions as well as intergenerational social mobility. This result shows that a life course approach needs to be adopted when dealing with health issues.

Calmodulin-dependent kinase II regulates Dlx5 during osteoblast differentiation.

Calmodulin-dependent kinase II (CaMKII) acts as a key regulator of osteoblast differentiation. CaMKII is a Ca(2+)-activated serine/threonine kinase and it regulates the activity of target proteins by phosphorylation. Dlx5 transcription factor plays crucial roles in osteoblast differentiation. The expression of Dlx5 is regulated by several osteogenic signaling pathways from early stages of osteoblastogenesis. In addition, Dlx5 can be phosphorylated and activated by p38, suggesting that the function of Dlx5 can be also modulated by post-translational modification. Although CaMKII and Dlx5 both play crucial roles during osteoblast differentiation, the interaction between CaMKII and Dlx5 has not been investigated. In the current study, we examined the effects CamKII on the function of Dlx5. We found that CaMKII phosphorylates Dlx5, and that CaMKII increases the protein stability and the osteoblastogenic transactivation activity of Dlx5. Conversely, a CaMKII inhibitor KN-93 decreased the osteogenic and transactivation activities of Dlx5. These results indicate that CaMKII regulates osteoblast differentiation, at least in part, by increasing the protein stability and the transcriptional activity of Dlx5.

Mitotic checkpoint gene MAD1 in hepatocellular carcinoma is associated with tumor recurrence after surgical resection.

OBJECTIVE: Underlying mechanism of mitotic checkpoint gene mitosis arrest deficiency 1 (MAD1) in human hepatocellular carcinoma (HCC) is rarely known. MATERIALS AND METHODS: We studied genetic change of the MAD1 gene as well as protein expression in 44 HCC and their associated non-cancerous surrounding liver tissues. RESULTS: Genotype AG of MAD1 G-1849 A promoter was highly significant in microscopic vascular invasion than other genotypes (P = 0.006). Moreover, the mean tumor size of HCC with genotype AG (7.71 cm) was significantly larger than those of other genotypes (AA, 4.41 cm; GG, 4.59 cm; P = 0.033). After a median follow-up of 22 months, 18 (41%) of the 44 patients relapsed. Eleven (32.4%) of 34 with MAD1 protein expression and 7 (70%) of 10 with no expression of MAD1 protein showed tumor recurrence. The incidence of tumor recurrence in patients with the lost MAD1 expression was significantly higher than in those with the expressed MAD1 protein (P = 0.011). CONCLUSION: These results suggest that MAD1 promoter genotype may be involved in tumor progression. Moreover, the loss of MAD1 protein expression may be related to the tumor recurrence after surgical resection of HCC.

4-1BB (CD137) signals depend upon CD28 signals in alloimmune responses.

Our previous study has demonstrated that there is a significant delay of Balb/c cardiac allograft rejection in the C57BL/6 4-1BB-deficient knockout recipient. In this study, we examined the effect of combined blockade of the 4-1BB and CD28 costimulatory pathways on cardiac allograft rejection in the C57BL/6-->Balb/c model. A long-term cardiac allograft survival was induced in CD28/4-1BB- deficient mice (>100 days survival in 3 of 4 mice), which was comparable with CD28-deficient mice (>100 days survival in 2 of 5 mice; P<0.2026). There was no long-term cardiac allograft survival in either wild-type (WT) or 4-1BB-deficient mice, even though 4-1BB-deficient recipients showed a significant delay of cardiac allograft rejection than WT mice. An in vitro mixed leukocyte reaction (MLR) assay showed that 4-1BB-deficient and WT mouse T cells had a similar responsiveness to allostimulation, whereas CD28- and CD28/4-1BB-deficient mouse T cells had a defective responsiveness to allostimulation. Furthermore, 4-1BB-deficient mice showed a similar CTL but an elevated Ab response against alloantigens as compared to WT mice, and the alloimmune responses of 4-1BB-deficient mice were abrogated in the CD28-deficient background. Overall, these results indicate that the CD28 costimulatory pathway plays a major role in the alloimmune response and that 4-1BB signals are dependent upon CD28 signals.