Assessment of diagnostic accuracy for cryptorchidism and risk factors for delayed orchidopexy.

OBJECTIVES: Cryptorchidism (CO) diagnosis by palpation is challenging. Patients with suspected CO are primarily referred to pediatric urologists by general pediatricians and urologists. Currently, surgical treatment for CO is recommended earlier than in previous guidelines. In this study, we evaluated factors that lead to diagnosis discordance and delayed orchidopexy in patients referred with suspected CO in addition to timing of initial screening. METHODS: In total, 731 patients (1052 testes) with suspected CO were included. Risk factors for diagnostic discrepancy in CO diagnosis by pediatric urologists and risk of delayed orchiopexy were evaluated. RESULTS: Herein, 659 (90%) patients were diagnosed during routine public health checkups for infants and young children, and 419 (57%) patients were referred by pediatric practitioners. Of 1052 testes, 374 (36%) were diagnosed with CO by pediatric urologists. In multivariate analysis, risk factors of diagnostic discrepancy for CO diagnosis by pediatric urologists were bilateral testis (odds ratio [OR] = 9.17, p < 0.0001), >6 months old at initial diagnosis (OR = 1.036, p < 0.0001), and pediatric referral (OR = 4.60, p < 0.0001). In total, 296 patients underwent orchiopexy for CO. In multivariate analysis, risk factors for delayed orchiopexy were presence of comorbidities (OR = 3.43, p = 0.003) and >10 months old at referral (OR = 12.62, p < 0.0001). CONCLUSIONS: Pediatric referral is a risk factor for discordant CO diagnostics, and late age at referral brings a risk of delayed orchiopexy. It is necessary to enlighten pediatricians, who are mainly responsible for routine health checkups, in teaching CO diagnostic techniques to ensure early referral.

Long-term follow-up of congenital hydronephrosis in a single-center study.

OBJECTIVES: Many congenital hydronephroses spontaneously resolve. This study evaluated a long-term follow-up of more than 4 years of patients with congenital hydronephrosis at a single center. METHODS: In total, 215 patients (286 kidneys) with congenital hydronephrosis were included. Hydronephrosis outcomes (resolution, improvement, and persistence) and time-to-outcome were evaluated. RESULTS: Fourteen patients underwent early surgical intervention until the age of 2 years. A total of 189 congenital hydronephrosis cases (66%) showed resolution at a median of 16 months (interquartile range: 7-21 months) and 169 (80%) of 210 kidneys with grade I to II hydronephrosis showed resolution at a median of 14 months (interquartile range: 6-23 months). Of 76 kidneys with grade III to IV hydronephrosis, 24 (32%) showed resolution at a median of 29 months (interquartile range: 24-41 months), and 56 (74%) showed improvement to grade II or less at a median of 12 months (interquartile range: 5-23 months). Of the 76 kidneys with grade III to IV hydronephrosis, five required delayed pyeloplasty at a median of 66 months (interquartile range: 42-89 months). One patient was asymptomatic, with a marked worsening of hydronephrosis and decreased renal function 6 years after the resolution of hydronephrosis. CONCLUSIONS: None of the patients with grade I to II hydronephrosis required surgical treatment, and a shorter follow-up may be sufficient. Grade III to IV severe hydronephrosis should be considered for a longer and more careful follow-up, given the possibility of asymptomatic exacerbation of hydronephrosis.

First-line pembrolizumab for patients with early relapsing urothelial carcinoma after perioperative chemotherapy: a retrospective analysis of bladder cancer and upper urinary tract cancer.

BACKGROUND: First-line pembrolizumab is available for recurrent disease within 12 months after the receipt of platinum-based perioperative chemotherapy. However, the benefit of first-line pembrolizumab is unclear. This study evaluated the oncological outcome of patients treated with pembrolizumab compared with chemotherapy as first-line therapy for early relapsing disease after the receipt of platinum-based perioperative chemotherapy. METHODS: Data from a multicenter study included 454 patients diagnosed with unresectable or metastatic UC from November 2006 to July 2021. We identified patients with early and non-early relapsing disease. Oncological outcomes were evaluated using progression-free survival, overall survival, and survival with disease control. RESULTS: Fifty-three patients with early relapsing disease and 15 patients with non-early relapsing disease were identified. Of 53 patients with early relapsing disease, 26 (49.1%) were treated with pembrolizumab and 27 (50.9%) were treated with chemotherapy as first-line therapy. Fifteen patients with non-early relapsing disease were treated with chemotherapy. Early relapsing disease was associated with shorter progression-free survival and overall survival than non-early relapsing disease. Pembrolizumab was associated with longer progression-free survival and survival with disease control than chemotherapy in patients with early relapsing disease. There was no significant difference in overall survival between pembrolizumab and chemotherapy, but overall survival plateau with a long tail was observed in pembrolizumab. CONCLUSIONS: First-line pembrolizumab in earlier clinical settings for highly malignant tumors might improve the prognosis of patients with early relapsing disease after the receipt of platinum-based perioperative chemotherapy.

[A Case of Refractory Intermittent Hematuria that Occurred during Paclitaxel/Ramucirumab Combination Therapy for Metastatic Gastric Cancer].

A 76-year-old male patient developed right hydronephrosis due to peritoneal and retroperitoneal dissemination after surgery for gastric cancer. A ureteral stent was inserted, and systemic chemotherapy was introduced for metastatic gastric cancer. Disease progression was observed, and paclitaxel/ramucirumab combination therapy was started as the second-line treatment. After seven courses, severe gross hematuria appeared intermittently, and refractory epistaxis was observed concurrently. No hemorrhagic lesion was found in the imaging test and urethrocystoscopy. The patient received conservative treatment, such as blood transfusion, and further examination was planned. However, hematuria and epistaxis resolved spontaneously during the course of treatment. From the clinical course, it was thought to be a hemorrhagic adverse event due to ramucirumab, and the patient's treatment was therefore changed to another drug. The patient recovered without recurrence of gross hematuria.

Clinical benefit of early treatment with bone-modifying agents for preventing skeletal-related events in patients with genitourinary cancer with bone metastasis: A multi-institutional retrospective study.

OBJECTIVES: To evaluate the clinical benefit of bone-modifying agents and identify the risk factors of skeletal-related events in patients with genitourinary cancer with newly diagnosed bone metastasis. METHODS: This was a multicenter retrospective study including a total of 650 patients with bone metastasis of the following cancer types: hormone-sensitive prostate cancer (n = 443), castration-resistant prostate cancer (n = 50), renal cell carcinoma (n = 80) and urothelial carcinoma (n = 77). Clinical factors at the time of diagnosis of bone metastasis were analyzed. Early treatment with bone-modifying agents was defined as follows: administration of bone-modifying agents before the development of skeletal-related events and within 6 months from the diagnosis of bone metastasis. RESULTS: During the follow-up period (median 19.0 months, interquartile range 6.0-43.8 months), skeletal-related events were reported in 88 (20%) patients with hormone-sensitive prostate cancer, 17 (34%) patients with castration-resistant prostate cancer, 58 (73%) patients with renal cell carcinoma and 34 (44%) patients with urothelial carcinoma. Early treatment with bone-modifying agents significantly prolonged the time to the first skeletal-related event in castration-resistant prostate cancer, renal cell carcinoma and urothelial carcinoma, but not in hormone-sensitive prostate cancer. Bone pain and elevated alkaline phosphatase levels were independent predictive risk factors of the first skeletal-related event. The subgroup analysis showed that early treatment with bone-modifying agents was associated with prolonged time to the first skeletal-related events in patients with bone pain or elevated alkaline phosphatase levels. CONCLUSIONS: Early treatment with bone-modifying agents should be considered, especially for patients with bone pain and elevated alkaline phosphatase levels, to prevent skeletal-related events in patients with genitourinary cancer with bone metastasis.

Modified feedback-control task for examining the relationship between sense of agency and motor control: a pilot study.

[Purpose] A sense of agency and feedback control may be related when the sensory feedback is attributed to the self; however, the relationship between sense of agency and movement disorders remains unclear. Although a feedback-control task might enable the examination of this relationship, it may be difficult for patients with movement disorders to complete this task. The present study modified the feedback-control task for future clinical research. [Participants and Methods] Twenty-four healthy adults participated in the study. The basic procedure followed that of a previous study in which participants traced a target line while receiving visual feedback of their actual or fake movement. The task was modified to reduce the width of the movement area, change the shape of the line from sinusoidal to horizontal, and reduce the number of trials from 45 to 15. [Results] When participants received the visual feedback of their actual movement, the movement error significantly decreased, whereas when participants received the fake movement that represented pre-recordings of their previous own movements, the movement error significantly increased. [Conclusion] The results partially agreed with those of the previous study. This modified task might help in examining the relationship between sense of agency and movement disorders in terms of motor control.

Insignificant role of bacillus Calmette-Guérin maintenance therapy after complete transurethral resection of bladder tumor for intermediate- and high-risk non-muscle-invasive bladder cancer: Results from a randomized trial.

OBJECTIVES: To investigate the effect of bacillus Calmette-Guérin maintenance therapy on patients with intermediate- and high-risk non-muscle-invasive bladder cancer receiving aggressive complete transurethral resection of bladder tumors standardized by well-trained surgeons. METHODS: A total of 95 patients were prospectively enrolled. Patients were diagnosed with multiple or recurrent non-muscle-invasive bladder cancer (Ta and T1), or with carcinoma in situ after complete transurethral resection of bladder tumors. Patients with Ta or T1 tumors without carcinoma in situ received six bacillus Calmette-Guérin instillations as induction therapy. Those with carcinoma in situ underwent eight bacillus Calmette-Guérin instillations as induction therapy. The patients were randomized into maintenance and non-maintenance groups. The maintenance group received intravesical bacillus Calmette-Guérin instillations once a week for 3 weeks at 3, 6, 12 and 18 months after bacillus Calmette-Guérin instillation. The primary end-point was recurrence-free survival. RESULTS: A total of 88 patients were evaluated. The average follow-up period was 48.3 ± 19.0 months. Five-year recurrence-free survival rates for the maintenance and non-maintenance groups were 80.1% and 79.3%, respectively. Five-year progression-free survival rates of the maintenance and non-maintenance groups were 92.4% and 85.3%, respectively. Recurrence- and progression-free survival rates did not significantly increase in the maintenance group compared with that in the non-maintenance group. CONCLUSIONS: Bacillus Calmette-Guérin maintenance therapy did not improve recurrence- and progression-free survival rates after the initial complete transurethral resection of bladder tumors compared with that after bacillus Calmette-Guérin induction therapy alone.

[A Case of Muscle Invasive Bladder Cancer in a Patient with Systemic Lupus Erythematosus].

Systemic lupus erythematosus (SLE) is an autoimmune disease with various symptoms. We present a case of muscle invasive bladder cancer with lymph node swelling caused by SLE. A 60-year-old man was referred to our hospital with high fever and pollakisuria, micro hematuria, proteinuria. We detecteda papillary tumor located behind the left ureteral orifice. Magnetic resonance imaging showed invasion of the tumor to the fat around the bladder. Computed tomography (CT) showed the swelling of left common iliac lymph node and bilateral inguinal lymph nodes. According to cystoscopy, imaging examination and transurethral resection of bladder tumor, we diagnosed it as a bladder cancer (cT3aN3M1). In addition, a close inspection of proteinuria was performed, and SLE was diagnosed. We started steroid therapy under the influence of neutropenia and thrombopenia caused by SLE. The swelling of lymph nodes disappeared on the CT three months later. After the therapy with gemcitabine andcisplatin, radical cystectomy and cutaneous ureterostomy were performed. Pathological examination showed invasive urothelial carcinoma and no lymph node metastasis. He now shows no evidence of disease 18 months after the operation.

[Burned-out testicular tumor diagnosed triggered by paraneoplastic neurological syndrome: a case report].

We report a case of burned-out testicular tumor. A 41-year-old man was referred to our department with swelling of iliac lymph nodes detected by computed tomography screening for cerebellar atrophy. Lymph node biopsy revealed metastasis of seminoma. Ultrasound examination showed an irregular hypoechoic area in his left testis. We diagnosed paraneoplastic neurological syndrome secondary to burned-out testicular tumor. So, we underwent left orchiectomy and chemotherapy. He remains free from disease recurrence 15 months after treatment.

[Case of primary malignant lymphoma of the bladder].

We report a case of primary malignant lymphoma of the bladder. An 87-year-old female visited our hospital for incidental bladder tumor. Cystoscopic examination demonstrated a non-papillary tumor over 10 mm in diameter. We performed transurethral resection of the bladder tumor. Histological examination showed malignant lymphoma, diffuse large B cell type. After further examination, it was diagnosed as primary malignant lymphoma of the bladder, stage IA E(Ann Arbor classification). We performed six courses of R-CHOP regimen (rituximab, cyclophosphamide, vincristine, doxorubicin, predonisolone). We did not find any local or distant recurrences after eight months' follow up.

Impaired Relationship between Sense of Agency and Prediction Error Due to Post-Stroke Sensorimotor Deficits.

Sense of agency refers to the experience of controlling one's actions. Studies on healthy people indicated that their self-other attribution can be realized based on prediction error which is an inconsistency between the internal prediction and sensory feedback of the movements. However, studies on patients with post-stroke sensorimotor deficits hypothesized that their self-other attribution can be based on different attribution strategies. This preliminary study examined this hypothesis by investigating whether post-stroke sensorimotor deficits can diminish the correlation between prediction errors and self-other judgments. Participants performed sinusoidal movements with visual feedback and judged if it represented their or another's movements (i.e., self-other judgment). The results indicated that the patient who had worse upper limb sensorimotor deficits and lesser paretic upper limb activity compared with the other patient made more misattributions and showed a lower correlation between prediction errors and self-other judgments. This finding suggests that post-stroke sensorimotor deficits can impair the relationship between prediction error and self-other attribution, supporting the hypothesis that patients with such deficits can have altered strategies for the registration of agency.

Stromal cells' B7-1 is a key stimulatory molecule for interleukin-10 production by HOZOT, a multifunctional regulatory T-cell line.

We have previously shown that xenogeneic stromal cell stimulation of naïve T cells resulted in the generation of a new type of regulatory T (Treg) cell termed HOZOT, which has multifunctional properties and a CD4/CD8 double-positive phenotype. Even after the establishment of HOZOT, stromal cells can function as an antigen-presenting cell (APC) by inducing these cells to produce interleukin (IL)-10. When compared with other stimuli, stromal cells showed an IL-10-producing ability comparable to anti-CD3 antibody (Ab) stimulation, and much greater than dendritic cell (DC) stimulation. Distinct from professional APCs, stromal cells express only major histocompatibility complex (MHC) class I and B7-1 costimulatory molecules, and not MHC class II or other costimulatory molecules, such as ICOSL (CD275), PD-L1 (CD274), PD-L2 (CD273), CD40, OX40L (CD252) and 4-1BBL (CD137L) in the absence of stimulation. Blocking experiments revealed that, in addition to anti-H-2K(d) Ab and anti-human CD8 Ab, anti-mouse B7-1 Ab could effectively block IL-10 production, indicating a key role of the B7-1/CD28 pathway. Using stromal cells expressing different levels of B7-1, IL-10 production correlated with the levels of B7-1 expression. Distinct from ICOSL or PD-L1 expressed on DCs (which are regarded as IL-10-inducing costimulatory molecules), this study showed that B7-1 on stromal cells is a key molecule regulating IL-10 production by multifunctional Treg cells, HOZOT.

The primary therapy chosen for patients with localized prostate cancer between the university hospital and its affiliated hospitals in Nara Uro-Oncological Research Group registration.

BACKGROUND: We investigated the differences between the preferential primary therapy conceived by the primary doctors and the primary therapy actually conducted for prostate cancer patients in Nara, Japan. METHODS: The distribution of primary therapy and clinical characteristics of 2303 prostate cancer patients - diagnosed between 2004 and 2006 at Nara Medical University and its 23 affiliated hospitals - were assessed. Moreover, the preferential primary therapy for the patients at each clinical stage (cT1-T3bN0M0) conceived by the primary doctors was investigated and compared to the actual therapy. RESULTS: Of all patients, 51% received primary androgen deprivation therapy (PADT), 30% underwent radical prostatectomy (RP), and 14% received radiation therapy (RT). The preferential primary therapy for cT1-2N0M0 was RP (92%) while 38% of the patients actually received PADT (RP: 40%). For cT3aN0M0, the preferential primary therapy was both RP and external beam radiation therapy (EBRT) while 58% of the patients actually received PADT (RP: 16%, EBRT: 24%). For cT3bN0M0, the most preferential primary therapy was EBRT (46%) while 67% of the patients actually received PADT (EBRT: 21%). This trend was more notable in the affiliated hospitals than in the University hospital. The hospitals with lower volume of RP per year significantly conducted PADT compared with those with higher volume of RP. CONCLUSIONS: PADT was commonly used to treat localized prostate cancer as well as locally advanced prostate cancer in Japan. There was a definite discrepancy between the preferential primary therapy conceived by the primary doctors and the actual therapy provided to the patients.

External validation of a genitourinary cancer-specific prognostic scoring system to predict survival for patients with bone metastasis (modified B-FOM scoring model): Comparison with other scoring models in terms of accuracy.

OBJECTIVE: We previously developed genitourinary (GU) cancer-specific scoring system for prediction of survival in patients with bone metastasis (the Bone-Fujimoto-Owari-Miyake [B-FOM] scoring model) based on five prognostic factors: the type of primary tumor (prostate cancer (PCa) vs renal cell carcinoma (RCC) and PCa vs urothelial carcinoma (UC)), poor performance status (PS), visceral metastasis, high Glasgow-prognostic score (GPS), elevated neutrophil-to-lymphocyte ratio (NLR). The aim of this study was to externally validate and further improve the performance of the B-FOM score. METHODS: The external validation cohort comprised 309 patients with GU cancer with bone metastasis from multiple institutions. Clinical factors were analyzed using Kaplan-Meier method and COX regression hazard model. Performance of a modified B-FOM score was compared to that of other scoring models by the Kaplan-Meier method and the area under the curve (AUC) of receiver operating characteristic curves. RESULTS: The median follow-up period of development and validation cohort were 25 and 17 months, respectively. Kaplan-Meier curve demonstrated that the type of primary tumor (RCC and UC vs PCa), poor PS, presence of visceral metastasis, high GPS, elevated NLR were significantly associated with shorter cancer-specific survival. Risk groups were successfully stratified by the modified B-FOM score classification. Moreover, the AUC of the modified B-FOM scoring model for predicting mortality at 6, 12, and 24 months were 0.895, 0.856, and 0.815, respectively, which were the highest among evaluated models. CONCLUSIONS: The B-FOM scoring model is a simple and accurate prediction tool. By using this scoring model at the time of the diagnosis of bone metastasis in patients with GU cancers, an individualized optimal treatment strategy can be selected.

The production of IL-10 by human regulatory T cells is enhanced by IL-2 through a STAT5-responsive intronic enhancer in the IL-10 locus.

STAT5 molecules are key components of the IL-2 signaling pathway, the deficiency of which often results in autoimmune pathology due to a reduced number of CD4(+)CD25(+) naturally occurring regulatory T (Treg) cells. One of the consequences of the IL-2-STAT5 signaling axis is up-regulation of FOXP3, a master control gene for naturally occurring Treg cells. However, the roles of STAT5 in other Treg subsets have not yet been elucidated. We recently demonstrated that IL-2 enhanced IL-10 production through STAT5 activation. This occurred in two types of human Treg cells: a novel type of umbilical cord blood-derived Treg cell, termed HOZOT, and Tr1-like Treg cells, IL-10-Treg. In this study, we examined the regulatory mechanisms of IL-10 production in these Treg cells, focusing specifically on the roles of STAT5. By performing bioinformatic analysis on the IL-10 locus, we identified one STAT-responsive element within intron 4, designated I-SRE-4, as an interspecies-conserved sequence. We found that I-SRE-4 acted as an enhancer element, and clustered CpGs around the I-SRE-4 were hypomethylated in IL-10-producing Treg cells, but not in other T cells. A gel-shift analysis using a nuclear extract from IL-2-stimulated HOZOT confirmed that CpG DNA methylation around I-SRE-4 reduced STAT5 binding to the element. Chromatin immunoprecipitation analysis revealed the in situ binding of IL-2-activated STAT5 to I-SRE-4. Thus, we provide molecular evidence for the involvement of an IL-2-STAT5 signaling axis in the expression of IL-10 by human Treg cells, an axis that is regulated by the intronic enhancer, I-SRE-4, and epigenetic modification of this element.

[A case report of toxic shock syndrome after high orchiectomy].

We report our experience of toxic shock syndrome (TSS) in a 54-year-old male patient after high orchiectomy for testicular cancer. Four days after the surgery, he began to have diarrhea, high fever, and diffuse erythroderma followed by severe hypotension. There were no signs of postsurgical wound infection, so serious drug eruption was suspected. Methicillin-resistant Staphylococcus aureus (MRSA) was detected in the culture of the drain and result of skin biopsy, leading to the diagnosis of TSS. Following treatment with intravenous fluids, antibiotics, human immunoglobulin and corticosteroids, the symptoms went into remission. TSS progresses rapidly and becomes life-threatening if treatment is delayed. Therefore, when TSS is suspected in postsurgical patients who experience fever of 39 degrees C or higher, dermal erythema, shock, treatment should be started promptly.

Agency judgments in post-stroke patients with sensorimotor deficits.

Sense of agency refers to the feeling of being in control of one's actions. Previous research has demonstrated that sense of agency is produced through the sensorimotor system, which is involved in comparing internal predictions with sensory feedback in motor control. Therefore, sensorimotor deficits might impair agency through a sensorimotor system malfunction. The present study examined this hypothesis by investigating post-stroke patients who had suffered a subcortical stroke that damaged regions associated with sensorimotor function. To examine agency judgments with respect to motor control, we adopted a self-other attribution task and applied it to post-stroke patients. Participants traced a horizontal straight line and received visual feedback through a cursor on a monitor. The cursor movement reflected either the participants' actual movement or the movement of an "other" that had been previously recorded. Participants judged whether the cursor movement reflected their own movement (self) or an other's movement while they engaged in four cycles of the horizontal tracing movement. After each trial, participants reported their self-other judgment on a nine-point scale. Post-stroke patients completed the experiment with their paretic as well as their non-paralyzed upper limbs. Compared to healthy controls, patients made significantly more self-attributions of others' movements. Interestingly, such misattributions were observed in the patients' performance using both paretic and non-paralyzed upper limbs. These results suggest that post-stroke patients with sensorimotor deficits form misattributions that cannot be explained solely by the sensorimotor system's role in motor control. We discuss these misattributions in post-stroke patients in terms of cue integration theory.

Dynamic Relationship between Sense of Agency and Post-Stroke Sensorimotor Deficits: A Longitudinal Case Study.

Post-stroke sensorimotor deficits impair voluntary movements. This impairment may alter a person's sense of agency, which is the awareness of controlling one's actions. A previous study showed that post-stroke patients incorrectly aligned themselves with others' movements and proposed that their misattributions might be associated with their sensorimotor deficits. To investigate this hypothesis, the present study compared the agency dynamics in a post-stroke patient A (PA) with sensorimotor deficits, who rarely used her paretic upper limbs in her daily life to patient B (PB), who had a paretic upper limb with almost normal functions and activity. At the second, fourth, and eighth weeks following their strokes, PA and PB completed experiments where they performed horizontal movements while receiving visual feedback, and analyzed if the visual feedback represented their own or another's movements. Consequently, PB made no misattributions each week; whereas, PA made incorrect self-attributions of other's movements at the fourth week. Interestingly, this misattribution noticeably decreased at the eighth week, where PA, with an improved paretic upper limb, used her limb almost as much as before her stroke. These results suggest that the sense of agency alters according to the sensorimotor deficit severity and paretic upper limb activity.

IL-2 activation of STAT5 enhances production of IL-10 from human cytotoxic regulatory T cells, HOZOT.

OBJECTIVE: Interleukin (IL)-10 is an immunosuppressive cytokine produced by many cell types, including T cells. We previously reported that a novel type of regulatory T (Treg) cells, termed HOZOT, which possesses a FOXP3+CD4+CD8+CD25+ phenotype and dual suppressor/cytotoxic activities, produced high levels of IL-10. In this study, we examined the mechanisms of high IL-10 production by HOZOT, focusing on Janus activating kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway. MATERIALS AND METHODS: We prepared five different types of T cells, including HOZOT from human umbilical cord blood. Cytokine productions of IL-10, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) were compared among these T cells after anti-CD3/CD28 antibody stimulation in the presence or absence of IL-2. Specific inhibitors for JAK/STAT, nuclear factor-kappaB (NF-kappaB), and nuclear factor for activated T cell (NFAT) were used to analyze signal transduction mechanisms. RESULTS: IL-10 production by HOZOTs was greatly enhanced by the addition of IL-2. Little or no enhancement of IFN-gamma and TNF-alpha production was observed under the same conditions. The enhancing effect of IL-2 was specific for both HOZOT and IL-10-secreting Treg cells. T helper type 2 cells, whose IL-10 production mechanisms involve GATA-3, failed to show IL-2-mediated enhancement of IL-10. Similar enhancing effects of IL-15 and IFN-alpha suggested a major role of JAK/STAT activation pathway for high IL-10 production. Further inhibitor experiments demonstrated that STAT5 rather than STAT3 was critically involved in this mechanism. CONCLUSION: Our results demonstrated that IL-2 selectively enhanced production of IL-10 in HOZOT primarily through activation of STAT5, which synergistically acts with NF-kappaB/NFAT activation, implying a novel regulatory mechanism of IL-10 production in Treg cells.

IL-2-independent generation of FOXP3(+)CD4(+)CD8(+)CD25(+) cytotoxic regulatory T cell lines from human umbilical cord blood.

OBJECTIVE: Since the existence of mouse naturally occurring CD4(+)CD25(+) T regulatory (Treg) cells was demonstrated, a variety of human Treg subsets have been identified as distinct T cell populations. Here we show the establishment of novel Treg cell lines possessing unique characteristics. METHODS: Novel Treg cell lines, designated HOZOT, were generated by coculturing human umbilical cord blood cells with mouse stromal cell lines in the absence of exogenous IL-2 or other cytokines. HOZOT were characterized and compared with CD4(+)CD25(+) Treg cells in terms of the CD phenotype, FOXP3 expression, suppressor activity against allogeneic MLR, anergy property, and IL-10 production. RESULTS: HOZOT were generated and expanded as normal lymphoblastoid cells with cytotoxic activity against the cocultured stromal cells. HOZOT consisted of three subpopulations as defined by phenotype: CD4(+)CD8(+), CD4(+)CD8(dim), and CD4(-)CD8(+). All three subpopulations showed both suppressor and cytotoxic activities. While HOZOT's expression of FOXP3, CD25, GITR, and cytoplasmic CTLA-4 implied a similarity to naturally occurring CD4(+)CD25(+) Treg cells, these two Treg cells differed in IL-2 responsiveness and IL-10 production. CONCLUSIONS: Our studies introduce a new method of generating Treg cells in an IL-2-independent manner and highlight a unique Treg cell type with cytotoxic activity and a phenotype of FOXP3(+)CD4(+)CD8(+)CD25(+).