Decreased PU.1 expression in mature B cells induces lymphomagenesis.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma, accounting for 30% of non-Hodgkin lymphomas. Although comprehensive analysis of genetic abnormalities has led to the classification of lymphomas, the exact mechanism of lymphomagenesis remains elusive. The Ets family transcription factor, PU.1, encoded by Spi1, is essential for the development of myeloid and lymphoid cells. Our previous research illustrated the tumor suppressor function of PU.1 in classical Hodgkin lymphoma and myeloma cells. In the current study, we found that patients with DLBCL exhibited notably reduced PU.1 expression in their lymphoma cells, particularly in the non-germinal center B-cell-like (GCB) subtype. This observation suggests that downregulation of PU.1 may be implicated in DLBCL tumor growth. To further assess PU.1's role in mature B cells in vivo, we generated conditional Spi1 knockout mice using Cγ1-Cre mice. Remarkably, 13 of the 23 knockout mice (56%) showed splenomegaly, lymphadenopathy, or masses, with some having histologically confirmed B-cell lymphomas. In contrast, no wild-type mice developed B-cell lymphoma. In addition, RNA-seq analysis of lymphoma cells from Cγ1-Cre Spi1F/F mice showed high frequency of each monoclonal CDR3 sequence, indicating that these lymphoma cells were monoclonal tumor cells. When these B lymphoma cells were transplanted into immunodeficient recipient mice, all mice died within 3 weeks. Lentiviral-transduced Spi1 rescued 60% of the recipient mice, suggesting that PU.1 has a tumor suppressor function in vivo. Collectively, PU.1 is a tumor suppressor in mature B cells, and decreased PU.1 results in mature B-cell lymphoma development.

Single-incision laparoscopic ileocecal resection in a 10-year-old child with appendiceal neuroendocrine tumor.

BACKGROUND: In Japan, the majority of gastrointestinal tract neuroendocrine tumors (NETs) have been reported to originate from the rectum, and appendiceal NETs are relatively rare. Preoperative diagnosis is very difficult and it is diagnosed after appendectomy. Pediatric appendiceal NET is a disease with a good prognosis. However, in rare cases, lymph node metastasis could occur and additional resection is required. CASE PRESENTATION: A 10-year-old boy complained of right lower quadrant abdominal pain and underwent an appendectomy under a diagnosis of acute appendicitis in previous hospital. The final diagnosis was appendiceal NET, so he was referred to our department for additional resection. The tumor was found in the base of the appendix and invasively reached the subserosal layer with obvious vascular invasion. His Ki-67 index was 1 to 2%, so we classified it as appendiceal NET G1 according to the WHO 2015 classification. We considered the possibility of a tumor remnant or lymph node metastasis, so we performed single-incision laparoscopy with D3 lymph node dissection. The pathological diagnosis revealed no tumor remnant but metastasis to one lymph node. He was discharged on the 9th postoperative day. There has been no recurrence at 3 years and 7 months after surgery. CONCLUSION: When the tumor size is 10-20 mm, the frequency of lymph node metastasis in some reports is variable, and there is no consensus yet on the indications for additional resection. However, there are definitely a certain number of cases with lymph node metastasis that require additional resection. In the present patient, long-term survival can be obtained by additional resection. At present, factors such as the presence of vascular or lymph node invasion and the malignancy grade and tumor's location must be considered on a case-by-case basis. Although the incidence rate of appendiceal NET is rare, the diagnosis can be made only during postoperative pathological examination; thus, reliable histopathological examination is required.

A xenograft model reveals that PU.1 functions as a tumor suppressor for multiple myeloma in vivo.

We previously demonstrated that PU.1 expression is down-regulated in the majority of myeloma cell lines and primary myeloma cells from patients. We introduced the tet-off system into the human myeloma cell lines U266 and KMS12PE that conditionally express PU.1 and demonstrated that PU.1 induces cell cycle arrest and apoptosis in myeloma cells in vitro. Here, we established a mouse xenograft model of myeloma using these cell lines to analyze the effects of PU.1 on the phenotype of myeloma cells in vivo. When doxycycline was added to the drinking water of mice engrafted with these myeloma cells, all mice had continuous growth of subcutaneous tumors and could not survived more than 65 days. In contrast, mice that were not exposed to doxycycline did not develop subcutaneous tumors and survived for at least 100 days. We next generated mice engrafted with subcutaneous tumors 5-10 mm in diameter that were induced by exposure to doxycycline. Half of the mice stopped taking doxycycline-containing water, whereas the other half kept taking the water. Although the tumors in the mice taking doxycycline continued to grow, tumor growth in the mice not taking doxycycline was significantly suppressed. The myeloma cells in the tumors of the mice not taking doxycycline expressed PU.1 and TRAIL and many of such cells were apoptotic. Moreover, the expression of a cell proliferation marker Ki67 was significantly decreased in tumors from the mice not taking doxycycline, compared with that of tumors from the mice continuously taking doxycycline. The present data strongly suggest that PU.1 functions as a tumor suppressor of myeloma cells in vivo.

[A Case Report of Neuroendocrine Carcinoma of the Esophagus Treated with Multidisciplinary Therapy].

A 72-year-old man was referred to our department because of esophageal tumor. Immunohistochemical findings were CD56-positive, synaptophysin-positive, chromogranin A-positive, Ki-67(labeling index)≥90%. The diagnosis was esophageal neuroendocrine carcinoma, categorized as cT4b(106recR-main bronchus), cN1(106recR), cM0, cStage III C. We had initiated irinotecan plus cisplatin(IP)as neoadjuvant chemotherapy(NAC). Biopsy specimens of primary lesion after 1 course chemotherapy showed a change to squamous cell carcinoma(SCC). The target lesion exhibited partial response(PR)after 2 courses of chemotherapy, and the primary lesion was reduced, but was still present. We performed subtotal esophagectomy and subtotal stomach reconstruction with lymphadenectomy(R0, Cur A). The histopathological findings showed the primary lesion was SCC, metastatic lymph nodes(106recR)was NEC. The final diagnosis was SCC plus NEC, categorized as CT-pT1a (MM), pN1(106recR), M0, fStage II B. After that, we selected treatment regimen considering tissue type, and performed surgery and chemotherapy for 2 times of recurrences. At a follow-up examination 1 year and 2 months after the start of first chemotherapy, the patient is alive without recurrence. Esophageal neuroendocrine carcinoma is relatively rare and the prognosis is poor, but there is as yet no standard therapy. We experienced a case of neuroendocrine carcinoma of the esophagus treated with multidisciplinary therapy.

Immunomodulatory drugs act as inhibitors of DNA methyltransferases and induce PU.1 up-regulation in myeloma cells.

Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide are efficacious in the treatment of multiple myeloma and significantly prolong their survival. However, the mechanisms of such effects of IMiDs have not been fully elucidated. Recently, cereblon has been identified as a target binding protein of thalidomide. Lenalidomide-resistant myeloma cell lines often lose the expression of cereblon, suggesting that IMiDs act as an anti-myeloma agent through interacting with cereblon. Cereblon binds to damaged DNA-binding protein and functions as a ubiquitin ligase, inducing degradation of IKZF1 and IKZF3 that are essential transcription factors for B and T cell development. Degradation of both IKZF1 and IKZF3 reportedly suppresses myeloma cell growth. Here, we found that IMiDs act as inhibitors of DNA methyltransferases (DMNTs). We previously reported that PU.1, which is an ETS family transcription factor and essential for myeloid and lymphoid development, functions as a tumor suppressor in myeloma cells. PU.1 induces growth arrest and apoptosis of myeloma cell lines. In this study, we found that low-dose lenalidomide and pomalidomide up-regulate PU.1 expression through inducing demethylation of the PU.1 promoter. In addition, IMiDs inhibited DNMT1, DNMT3a, and DNMT3b activities in vitro. Furthermore, lenalidomide and pomalidomide decreased the methylation status of the whole genome in myeloma cells. Collectively, IMiDs exert demethylation activity through inhibiting DNMT1, 3a, and 3b, and up-regulating PU.1 expression, which may be one of the mechanisms of the anti-myeloma activity of IMiDs.

Experimental myositis inducible with transfer of dendritic cells presenting a skeletal muscle C protein-derived CD8 epitope peptide.

It is suggested that polymyositis, an autoimmune inflammatory myopathy, is mediated by autoaggressive CD8 T cells. Skeletal muscle C protein is a self-antigen that induces C protein-induced myositis, a murine model of polymyositis. To establish a new murine model of myositis inducible with a single CD8 T-cell epitope peptide that derives from the C protein, three internet-based prediction systems were employed to identify 24 candidate peptides of the immunogenic fragment of the C protein and bind theoretically to major histocompatibility complex class I molecules of C57BL/6 (B6) mice. RMA-S cell assay revealed that a HILIYSDV peptide, amino acid position 399-406 of the C protein, had the highest affinity to the H2-K(b) molecules. Transfer of mature bone marrow-derived dendritic cells pulsed with HILIYSDV induced myositis in naive B6 mice. This myositis was suppressed by anti-CD8-depleting antibodies but not by anti-CD4-depleting antibodies. Because this myositis model is mediated by CD8 T cells independently of CD4 T cells, it should be a useful tool to investigate pathology of polymyositis and develop therapies targeting CD8 T cells.

Abatacept may be effective and safe in patients with amyloid A amyloidosis secondary to rheumatoid arthritis.

OBJECTIVES: To examine the efficacy and safety of abatacept (ABT) in patients with amyloid A (AA) amyloidosis secondary to rheumatoid arthritis (RA), and to speculate about the immunologic association of ABT with AA amyloid deposit regression. METHODS: We administered ABT to 70- and 65-year-old Japanese women with RA and AA amyloidosis. We quantified serum cytokine concentrations and analysed regulatory T lymphocytes (Treg cells) via flow cytometry. We also studied AA amyloid deposits via histopathology and immunohistochemistry. RESULTS: ABT improved rheumatoid inflammation and AA amyloidosis, one case showing clinical remission and the other demonstrating incomplete recovery of nephrosis but stable kidney function. Serum levels of interleukin-6 and tumour necrosis factor α decreased to baseline in the first 6 months of treatment, but serum interleukin-2 concentrations did not change. CD4+CD25++FoxP3+ Treg cells gated on T lymphocytes and CD4+ T lymphocytes decreased to baseline in the first 3 treatment months. One case showed complete regression of AA amyloid fibrils in serial upper gastrointestinal biopsies, but the other case still had AA amyloid deposits despite ABT-induced normalised rheumatoid inflammation, with polymorphonuclear leukocytes and macrophages infiltrating tissues containing AA amyloid. CONCLUSIONS: ABT demonstrated efficacy and safety in AA amyloidosis secondary to RA and affected Treg cells and inflammatory cytokines. Because the gradual decrease in Treg cells population coincided with AA amyloid deposit regression during ABT therapy, AA amyloid fibril turnover in these patients may involve an immunologic mechanism. Phagocytes seemed to have an important role in AA amyloid fibril regression, which suggests an immunologic interaction.

Usefulness of platelet count to predict concomitant valvular heart disease in patients with systemic lupus erythematosus.

Background: Although the prevalence rate of valvular heart disease (VHD) is high in patients with systemic lupus erythematosus (SLE), the predictive factors of concomitant VHD have not been fully evaluated. Methods and results: Among 288 patients with SLE who underwent transthoracic echocardiography at Kumamoto University Hospital from 2016 to 2021, 241 patients with sufficient echocardiographic data were retrospectively analysed. Among them, 22 (9 %) had VHD (10 had mitral regurgitation, 3 had aortic regurgitation, 6 had tricuspid regurgitation, 1 had mitral regurgitation and tricuspid regurgitation, and 2 had a prosthetic cardiac valve). After excluding the two patients with a prosthetic cardiac valve, we divided the remaining patients into two groups: the VHD group and non-VHD group. Multivariate logistic regression analysis revealed that age and the platelet count were significantly and independently associated with having VHD (age: odds ratio, 1.06; 95 % confidence interval, 1.02-1.10; p < 0.01) (platelet count: odds ratio, 0.99; 95 % confidence interval, 0.98-1.00; p < 0.05). After excluding 95 patients aged < 40 years, receiver operating characteristic analysis revealed that the area under the curve of the platelet count for prediction of VHD was 0.73 with an optimal cut-off value of 166.5 × 103/µL (sensitivity: 76.6 %, specificity: 60.0 %). Among patients with a low platelet count (<166.5 × 103/µL), the rate of having VHD was 29 % (12/41 patients). However, among those with a high platelet count (≥166.5 × 103/µL), this rate was only 8 % (8/103 patients). Conclusion: The platelet count is useful to predict concomitant VHD in middle-aged and older patients with SLE.

A 37-Year-Old Man With Intellectual Disability Discovered to Have Aspartylglucosaminuria: Implications for the Diagnosis of Genetic Causes.

Objectives: The causes of intellectual disability (ID) are varied, with as many as 1,400 causative genes. We attempted to identify the causative gene in a patient with long-standing undiagnosed ID. Methods: Although this was an isolated case with no family history, we searched for the causative gene using trio-based whole-exome sequencing (trio-WES), because severe ID is often caused by genetic variations, and inherited metabolic disorders (IMDs) are assumed to be the cause when regression and epilepsy occur. Results: We identified homozygous donor splice-site variants in the AGA gene (aspartylglucosaminidase; NM_000027.4) Chr4(GRCh38):g. 177436275C>A, c.698+1G>T. This gene is implicated in aspartylglucosaminuria (AGU; OMIM #208400) and originated from both of the patient's parents. We confirmed the pathogenicity of the variant by detecting the splicing defect in cDNA from the patient's blood and accumulation of aberrant metabolites in the patient's urine. Discussion: We discuss how to more readily achieve an accurate diagnosis for patients with undiagnosed intellectual disabilities. Medical practitioners' awareness of the characteristics of the disease leading to clinical suspicion in patients with matching presentations, and the performance of newborn screening when possible, is important for the diagnosis of ID. In addition, the characteristic symptoms and course of the disease give rise to suspicion of IMDs. Given our results, we consider trio-WES to be a powerful method for identifying the causative genes in cases of ID with genetic causes.

Overlap syndrome involving anti-OJ antibody-positive polymyositis, systemic lupus erythematosus and Sjögren's syndrome: A case report and literature review.

Anti-OJ antibody is relatively rarely detected in patients with the anti-synthetase syndrome, which is polymyositis (PM)/dermatomyositis (DM) with anti-aminoacyl transfer ribonucleic acid (RNA) synthetase antibodies. There have been few case reports of anti-OJ antibody-positive PM/DM complicated by other connective tissue disorders. Herein, we report the case of a 33-year-old woman who was admitted to our hospital with fever, muscle weakness, and dyspnoea on exertion. She was diagnosed with anti-OJ antibody-positive PM, overlapping systemic lupus erythematosus, and Sjögren's syndrome (SS). Her symptoms and clinical findings improved after treatment with prednisolone 1 mg/kg/day without immunosuppressive agents. This is the first case of overlap syndrome with anti-OJ antibody-positive PM, systemic lupus erythematosus, and Sjögren's syndrome.

Possible Association of Mutations in the MEFV Gene with the Intestinal Phenotype of Behçet's Disease and Refractoriness to Treatment.

BACKGROUND: Mediterranean fever (MEFV) gene mutations are responsible for familial Mediterranean fever (FMF) and associated with other inflammatory diseases. However, the effects of MEFV gene mutations on intestinal Behçet's disease (BD) are unknown. In this study, we investigated these mutations and clinical features in patients with intestinal BD. METHODS: MEFV gene analysis was performed in 16 patients with intestinal BD, 10 with BD without intestinal lesions, and 50 healthy controls. Clinical features of patients with intestinal BD were retrospectively assessed. RESULTS: The rates of MEFV gene mutations in patients with intestinal BD, BD without intestinal lesions, and healthy controls were 75%, 50%, and 38%, respectively. Only 2 of 12 patients with intestinal BD harboring MEFV gene mutations (17%) were controlled without immunosuppressive treatment, while 8 patients (67%) required therapy with tumor necrosis factor (TNF) inhibitors. Among patients with intestinal BD without MEFV gene mutations (four patients), three (75%) were controlled by the administration of 5-aminosalicylic acid with or without colchicine, and one (25%) required TNF inhibitors. All patients who underwent intestinal resection had MEFV gene mutations. Immunohistochemical analysis and in situ hybridization with interleukin-1ß (IL-1ß) showed a high expression of IL-1ß only in injured areas, suggesting that IL-1ß may be involved in the formation of ulcers in patients with intestinal BD carrying MEFV gene mutations. CONCLUSION: Mutations in the MEFV gene may be associated with intestinal lesions of BD and refractoriness to treatment.

Dual effects of TRAIL in suppression of autoimmunity: the inhibition of Th1 cells and the promotion of regulatory T cells.

TRAIL is known to play a pivotal role in the inhibition of autoimmune disease. We previously demonstrated that administration of dendritic cells engineered to express TRAIL and myelin-oligodendrocyte glycoprotein reduced the severity of experimental autoimmune encephalomyelitis and suggested that CD4(+)CD25(+) regulatory T cells (Tregs) were involved in mediating this preventive effect. In the current study, we investigated the effect of TRAIL on Tregs, as well as conventional T cells, using TRAIL-deficient mice. Upon induction of experimental autoimmune encephalomyelitis, TRAIL-deficient mice showed more severe clinical symptoms, a greater frequency of IFN-γ-producing CD4(+) T (Th1) cells, and a lower frequency of CD4(+)Foxp3(+) Tregs than did wild-type mice. In vitro, conventional T cells stimulated by bone marrow-derived dendritic cells (BM-DCs) from TRAIL-deficient mice showed a greater magnitude of proliferation than did those stimulated by BM-DCs from wild-type mice. In contrast, TRAIL expressed on the stimulator BM-DCs enhanced the proliferative response of CD4(+)CD25(+) Tregs in the culture. The functional TRAILR, mouse death receptor 5 (mDR5), was expressed in conventional T cells and Tregs upon stimulation. In contrast, the decoy receptor, mDc-TRAILR1, was slightly expressed only on CD4(+)CD25(+) Tregs. Therefore, the distinct effects of TRAIL may be due to differences in the mDc-TRAILR1 expression or the signaling pathways downstream of mouse death receptor 5 between the two T cell subsets. Our data suggest that TRAIL suppresses autoimmunity by two mechanisms: the inhibition of Th1 cells and the promotion of Tregs.

Easy preparation of a liposome-mediated protein delivery system by freeze-thawing a liposome-protein complex.

Homeostasis can be achieved by adding a protein supplement; however, an appropriate vector is required to deliver the protein into the cell because of the low stability of proteins in the blood and low cell membrane permeability. Here we report an easy one-step method of encapsulating proteins into liposomes for delivery. We used negatively charged superoxide dismutase (SOD) and a polycation liposome as protein and liposome models, respectively. Liposome-encapsulated SOD was prepared by freeze-thawing the SOD-liposome complex (lipoplexes). The amount of immobilized SOD within the lipoplex significantly increased on freeze-thawing. Surprisingly, subjecting the single-layered lipoplexes to freeze-thawing produced multilayered liposomes with SOD localized between the lipid layers. The amount of SOD delivered intracellularly significantly increased by freeze-thawing compared with that delivered by lipoplexes without freeze-thawing. SOD, liposomes, and endosomes were separately localized in the cells. The freeze-thawed lipoplex-encapsulated SOD samples were intravenously injected in mice. The SOD biodistribution was dramatically changed compared with the injection of free SOD or lipoplex. SOD was detached from the lipoplex in the bloodstream after the injection of non-freeze-thawed lipoplex, whereas the encapsulation of SOD in the liposomes upon freeze-thawing enabled the stable circulation of SOD with the liposomes in the bloodstream. This work paves the way for the application of the freeze-thawing technology for the easy one-step encapsulation of proteins into liposomes for protein delivery.

Peptides derived from human insulin-like growth factor-II mRNA binding protein 3 can induce human leukocyte antigen-A2-restricted cytotoxic T lymphocytes reactive to cancer cells.

Insulin-like growth factor-II mRNA binding protein 3 (IMP-3) is an oncofetal protein expressed in various malignancies including lung cancer. This study aimed to identify immunogenic peptides derived from IMP-3 that can induce tumor-reactive and human leukocyte antigen (HLA)-A2 (A*02:01)-restricted cytotoxic T lymphocytes (CTL) for lung cancer immunotherapy. Forty human IMP-3-derived peptides predicted to bind to HLA-A2 were analyzed to determine their capacity to induce HLA-A2-restricted T cells in HLA-A2.1 (HHD) transgenic mice (Tgm). We found that three IMP-3 peptides primed HLA-A2-restricted CTL in the HLA-A2.1 Tgm. Among them, human CTL lines reactive to IMP-3 (515) NLSSAEVVV(523) were reproducibly established from HLA-A2-positive healthy donors and lung cancer patients. On the other hand, IMP-3 (199) RLLVPTQFV(207) reproducibly induced IMP-3-specific and HLA-A2-restricted CTL from healthy donors, but did not sensitize CTL in the HLA-A2.1 Tgm. Importantly, these two IMP-3 peptide-specific CTL generated from healthy donors and cancer patients effectively killed the cancer cells naturally expressing both IMP-3 and HLA-A2. Cytotoxicity was significantly inhibited by anti-HLA class I and anti-HLA-A2 monoclonal antibodies, but not by the anti-HLA-class II monoclonal antibody. In addition, natural processing of these two epitopes derived from the IMP-3 protein was confirmed by specific killing of HLA-A2-positive IMP-3-transfectants but not the parental IMP-negative cell line by peptide-induced CTL. This suggests that these two IMP-3-derived peptides represent highly immunogenic CTL epitopes that may be attractive targets for lung cancer immunotherapy.

Intracranial and extracranial multiple arterial dissecting aneurysms in rheumatoid arthritis: A case report.

OBJECTIVE: We describe a case of intracranial and extracranial multiple arterial dissecting aneurysms in rheumatoid arthritis (RA). CASE PRESENTATION: A 29-year-old man with a medical history of RA since 18 years of age was admitted to our hospital for vomiting, dysarthria, and conscious disturbance. At 23, he underwent ligation of the left internal carotid artery (ICA) with superficial temporal artery to middle cerebral artery anastomosis because of acute infarct of the left hemisphere caused by arterial dissection of the left ICA. During the current admission, computed tomography (CT) revealed subarachnoid hemorrhage, and digital subtraction angiography (DSA) demonstrated dissecting aneurysms of the left intracranial vertebral artery (VA) and right extracranial VA. We diagnosed him with a ruptured dissecting aneurysm of the left intracranial VA and performed endovascular parent artery occlusion on the left VA. For the right unruptured VA aneurysm, we performed coil embolization simultaneously. At 2 weeks after the endovascular treatment, follow-up DSA revealed that multiple de novo dissecting aneurysms developed on the origin of the left VA and left and right internal thoracic arteries. Those aneurysms were treated with coil embolization. Other remaining aneurysms on the left thyrocervical trunk, right transverse cervical artery, and both common iliac arteries were treated by conservative therapy. While continuing medical treatment for RA, the patient recovered and was discharged to a rehabilitation hospital. CONCLUSION: Considering that RA-induced vasculitis can be a potential risk of vascular complications including multiple arterial dissections, physicians should carefully perform endovascular interventional procedures for patients with long-term RA.

Identification of SPARC as a candidate target antigen for immunotherapy of various cancers.

To establish efficient anticancer immunotherary, it is important to identify tumor-associated antigens (TAAs) directing the immune system to attack cancer. A genome-wide cDNA microarray analysis identified that secreted protein acidic and rich in cysteine (SPARC) gene is overexpressed in the gastric, pancreatic and colorectal cancer tissues but not in their noncancerous counterparts. This study attempted to identify HLA-A24 (A*2402)-restricted and SPARC-derived CTL epitopes. We previously identified H-2K(d)-restricted and SPARC-derived CTL epitope peptides in BALB/c mice, of which H-2K(d)-binding peptide motif is comparable with that of HLA-A24 binding peptides. By using these peptides, we tried to induce HLA-A24 (A*2402)-restricted and SPARC-reactive human CTLs and demonstrated an antitumor immune response. The SPARC-A24-1(143-151) (DYIGPCKYI) and SPARC-A24-4(225-234) (MYIFPVHWQF) peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A24 (A*2402) positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both SPARC and HLA-A24 (A*2402). Furthermore, the adoptive transfer of the SPARC-specific CTLs could inhibit the tumor growth in nonobese diabetic/severe combined immunodeficient mice bearing human cancer cells expressing both HLA-A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy.

Activation of antigen-specific cytotoxic T lymphocytes by beta 2-microglobulin or TAP1 gene disruption and the introduction of recipient-matched MHC class I gene in allogeneic embryonic stem cell-derived dendritic cells.

A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or beta(2)-microglobulin (beta(2)m) gene was disrupted in 129 (H-2(b))-derived ES cells and then expression vectors for the H-2K(d) or beta(2)m-linked form of K(d) (beta2m-K(d)) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1(-/-)/K(d) ES-DC and beta(2)m(-/-)/beta(2)m-K(d) ES-DC. As intended, both of the transfectant ES-DC expressed K(d) but not the intrinsic H-2(b) haplotype-derived MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) and TAP1(-/-)/K(d) ES-DC were not recognized by pre-activated H-2(b)-reactive CTL and did not prime H-2(b) reactive CTL in vitro or in vivo. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC and TAP1(-/-)/K(d) ES-DC had a survival advantage in comparison to beta(2)m(+/-)/beta(2)m-K(d) ES-DC and TAP1(+/+)/K(d) ES-DC, when transferred into BALB/c mice. K(d)-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2(b) haplotype-encoded MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than beta(2)m(+/-)/beta(2)m-K(d) ES-DC. The modification of the beta(2)m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.

An open-label, randomized controlled trial of sulfamethoxazole-trimethoprim for Pneumocystis prophylaxis: results of 52-week follow-up.

OBJECTIVES: The aim was to investigate the long-term prophylactic efficacy, drug retention and safety of low-dose sulfamethoxazole-trimethoprim (SMX/TMP) prophylaxis against Pneumocystis pneumonia (PCP). METHODS: Adult patients with rheumatic diseases receiving prednisolone ≥0.6 mg/kg/day were randomized into the single-strength group (SS; SMX/TMP 400/80 mg daily), the half-strength group (HS; 200/40 mg daily) or the escalation group (ES; starting at 40/8 mg and increasing incrementally to 200/40 mg daily) and treated for 24 weeks, then observed for 52 weeks. The primary endpoint, the PCP non-incidence rate (non-IR) at week 24, has been reported previously. The secondary endpoints were the PCP non-IR at week 52, treatment discontinuation rate and adverse events. RESULTS: Fifty-eight, 59 and 55 patients in the SS, HS and ES, respectively, received SMX/TMP. PCP did not develop in any of the patients by week 52. The estimated PCP non-IR in patients receiving SMX/TMP 200/40 mg daily (HS and ES) was 96.8-100%. Throughout the 52-week observation period, the overall discontinuation rate was significantly lower in HS than in SS (22.7 vs 47.2%, P = 0.004). The discontinuation rates attributable to adverse events were significantly lower in HS (19.1%, P = 0.007) and ES (20.3%, P = 0.007) than in SS (41.8%). The IRs of adverse events requiring SMX/TMP dose reduction before week 52 differed among the three groups, with a significantly higher IR in SS than in HS or ES (P = 0.007). CONCLUSION: SMX/TMP 200/40 mg had a high PCP prevention rate and was superior to SMX/TMP 400/80 mg in terms of drug retention and safety. TRIAL REGISTRATION: University Hospital Medical Information Network Clinical Trials Registry, UMIN000007727.

Identification of the H2-Kd-restricted cytotoxic T lymphocyte epitopes of a tumor-associated antigen, SPARC, which can stimulate antitumor immunity without causing autoimmune disease in mice.

We previously reported that the secreted protein acidic and rich in cystein (SPARC) was overexpressed in melanoma in humans, and the serum SPARC level was useful as a novel tumor marker for melanoma. SPARC was also reported to be overexpressed in various human cancers. In this study, we asked whether SPARC-specific cytotoxic T lymphocytes (CTL) could induce antitumor immunity to SPARC-expressing tumor in mice or not as a preclinical study of SPARC-directed anticancer immunotherapy. Because of similarities in the structural motifs of major histocompatibility complex-binding peptides between H2-Kd and HLA-A24 (A*2402), the most common human leukocyte antigen class I allele in the Japanese population, we attempted to identify the H2-Kd-restricted SPARC epitope for CTL in BALB/c mice and we found that the mouse SPARC143-151 (DYIGPCKYI) and SPARC225-234 (MYIFPVHWQF) peptides could induce peptide-reactive CTL in BALB/c mice without causing autoimmune diseases. The immunization of mice with SPARC225-234 peptide-pulsed bone marrow-derived dendritic cells (BMDC) inhibited the growth of s.c. inoculated mouse mammary cancer cell line, N2C, expressing SPARC and these mice lived longer than the mice immunized with peptide-unpulsed BMDC. In conclusion, our study indicated that SPARC peptide-based cancer immunotherapy was effective and safe at least in a mouse tumor prevention model.