Innate immune response in bovine neutrophils stimulated with Mycoplasma bovis.

Mycoplasma bovis (M. bovis) is a significant worldwide pathogen of cattle. Neutrophils have an important role in the innate immune response during infection with M. bovis. However, even though neutrophils accumulate in M. bovis infection, the interaction of M. bovis and neutrophils has not been fully elucidated. We attempted to elucidate the innate immune response of neutrophils stimulated with M. bovis and evaluate the transcriptome and functional analysis of bovine neutrophils stimulated with M. bovis. Proinflammatory cytokines, such as inducible nitric oxide (iNOS), which was the most increased gene in transcriptome analysis, were increased in quantitative polymerase chain reaction analysis of bovine neutrophils stimulated with live or heat-killed M. bovis. Nitric oxide and intracellular reactive oxygen species production of neutrophils stimulated with M. bovis was significantly increased. Neutrophils stimulated with M. bovis showed an increased ratio of nonapoptotic cell death compared to unstimulated controls. We demonstrated that neutrophil extracellular traps (NETs) formation was not recognized in neutrophils stimulated with live M. bovis. However, heat-killed M. bovis induced NETs formation. We also showed the interaction with M. bovis and bovine neutrophils regarding proinflammatory cytokine gene expression and functional expression related to NETs formation. Live and killed M. bovis induced innate immune responses in neutrophils and had the potential to induce NETs formation, but live M. bovis escaped NETs.

Immunosuppression in Cows following Intramammary Infusion of Mycoplasma bovis.

Mycoplasma bovis is a destructive pathogen that causes large economic losses in rearing cattle for beef and dairy worldwide. M. bovis causes suppression of and evades the host immune response; however, the mechanisms of host immune function involved in M. bovis mastitis have not been elucidated. The purpose of this study was to elucidate the characteristics of the bovine immune response to mycoplasmal mastitis. We evaluated the responsiveness of the bovine mammary gland following infusion of M. bovis Somatic cell counts and bacterial counts in milk from the infected quarter were increased. However, the proliferation of peripheral blood mononuclear cells (blood MNCs) and mononuclear cells isolated from M. bovis-stimulated mammary lymph nodes (lymph node MNCs) did not differ from that in the unstimulated cells. Transcriptome analysis revealed that the mRNA levels of innate immune system-related genes in blood MNCs, complement factor D (CFD), ficolin 1 (FCN1), and tumor necrosis factor superfamily member 13 (TNFSF13) decreased following intramammary infusion of M. bovis The mRNA levels of immune exhaustion-related genes, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), lymphocyte activation gene 3 (LAG3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) of milk mononuclear cells (milk MNCs) in the infected quarter were increased compared with those before infusion. Increase in immune exhaustion-related gene expression and decrease in innate immune response-related genes of MNCs in quarters from cows were newly characterized by M. bovis-induced mastitis. These results suggested that M. bovis-induced mastitis affected the immune function of bovine MNCs, which is associated with prolonged duration of infection with M. bovis.

Diversity changes of microbial communities into hospital surface environments.

Previous works have demonstrated considerable variability in hospital cleanliness in Japan, suggesting that contamination is driven by factors that are currently poorly controlled. We undertook 16S rRNA sequence analysis to study population structures of hospital environmental microbiomes to see which factor(s) impacted contamination. One hundred forty-four samples were collected from surfaces of three hospitals with distinct sizes ("A": >500 beds, "B": 100-500 beds, "C": <100 beds). Sample locations of two ward types (Surgical and Internal) included patient room bed table (multiple) (4BT), patient overbed table (multiple) (4OT), patient room sink (multiple) (4S), patient room bed table (single) (SBT), patient overbed table (single) (SOT), patient room sink (single) (SS), nurse desk (ND), and nurse wagon (NW). Total DNA was extracted from each sample, and the 50 samples that yielded sufficient DNA were used for further 16S rRNA sequencing of hospital microbiome populations with cluster analysis. The number of assigned bacterial OTU populations was significantly decreased in hospital "C" compared to the other hospitals. Cluster analysis of sampling locations revealed that the population structure in almost all locations of hospital "C" and some locations in the other hospitals was very similar and unusually skewed with a family, Enterobacteriaceae. Interestingly, locations included patient area (4OT, 4BT, SBT) and nurse area (ND), with a device (NW) bridging the two and a place (4S and SS) shared between patients or visitors. We demonstrated diversity changes of hospital environmental microbiomes with a skewed population, presumably by medical staff pushing NWs or sinks shared by patients or visitors.

[Analysis of prognostic factors in transplant-eligeble newly diagnosed myeloma patients treated with bortezomib plus dexamethasone as induction therapy].

We retrospectively analyzed, and herein discuss, the outcomes of and prognostic factors for 35 untreated multiple myeloma patients less than 65 years of age who received induction therapies with bortezomib (Bor) and dexamethasone (BD) for the purpose of up-front autologous peripheral blood stem cell transplantation (SCT). The overall response rate was 77% (27 cases, including 4 [11%] complete response and 13 [37%] very good partial response cases). The rate of SCT accomplishment was 51% (18 cases). The 3 year-progression free survival (PFS) rate for the SCT group was significantly higher than that of the non-SCT group (41% vs 0%, P=0.0037). This result reflects the significantly more severe adverse effects of induction therapy for the non-SCT than the SCT group. Among reasons for SCT drop-out, 29% of cases suffered severe peripheral neuropathy with features such as irreversible numbness and pain. The analysis of PFS revealed a cytogenetic factor, favorable chromosomal type at diagnosis, to predict a better outcome (P values on univariate and multivariate analyses were 0.0004 and 0.0405, respectively). Our observations suggest establishment of induction therapy, aimed at reducing adverse effects and overcoming unfavorable cytogenetic abnormalities, to be necessary for improving the outcomes of patients with multiple myeloma.

Peripheral blood absolute lymphocyte/monocyte ratio as a useful prognostic factor in diffuse large B-cell lymphoma in the rituximab era.

OBJECTIVES: The tumor microenvironment, including tumor-infiltrating lymphocytes and myeloid-derived cells, is an important factor in the pathogenesis and clinical behavior of malignant lymphoma. However, the prognostic significance of peripheral lymphocytes and monocytes in lymphoma remains unclear. METHODS: We evaluated the prognostic impact of the absolute lymphocyte count (ALC), absolute monocyte count (AMC), and lymphocyte/monocyte ratio (LMR) in 359 diffuse large B-cell lymphoma (DLBCL) patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). RESULTS: The median follow-up time of the surviving patients was 58 months. Low ALC and an elevated AMC were both associated with poor survival rates. Receiver operating characteristic curve analysis showed that LMR was the best predictor of survival, with 4.0 as the cutoff point. Patients with LMR ≤4.0 were more likely to have an aggressive tumor, and this was associated with poor treatment responses. Patients with LMR ≤4.0 at diagnosis had significantly poorer overall survival (OS) and progression-free survival (PFS) than those with LMR >4.0. Multivariate analysis, which included prognostic factors of the International Prognostic Index, showed LMR ≤4.0 to be an independent predictor for the OS (hazard ratio [HR], 2.507; 95% confidence interval [CI], 1.255-5.007; P = 0.009) and PFS (HR, 2.063; 95% CI, 1.249-3.408; P = 0.005). CONCLUSIONS: The LMR at diagnosis, as a simple index which reflects host systemic immunity, predicts clinical outcomes in DLBCL patients treated with R-CHOP.

Central nervous system event in patients with diffuse large B-cell lymphoma in the rituximab era.

Central nervous system (CNS) events, including CNS relapse and progression to CNS, are known to be serious complications in the clinical course of patients with lymphoma. This study aimed to evaluate the risk of CNS events in patients with diffuse large B-cell lymphoma in the rituximab era. We performed a retrospective survey of Japanese patients diagnosed with diffuse large B-cell lymphoma who underwent primary therapy with R-CHOP chemoimmunotherapy between September 2003 and December 2006. Patients who had received any prophylactic CNS treatment were excluded. Clinical data from 1221 patients were collected from 47 institutions. The median age of patients was 64 years (range, 15-91 years). We noted 82 CNS events (6.7%) and the cumulative 5-year probability of CNS events was 8.4%. Patients with a CNS event demonstrated significantly worse overall survival (P < 0.001). The 2-year overall survival rate after a CNS event was 27.1%. In a multivariate analysis, involvement of breast (relative risk [RR] 10.5), adrenal gland (RR 4.6) and bone (RR 2.0) were identified as independent risk factors for CNS events. We conclude that patients with these risk factors, in addition to patients with testicular involvement in whom CNS prophylaxis has been already justified, are at high risk for CNS events in the rituximab era. The efficacy and manner of CNS prophylaxis in patients for each involvement site should be evaluated further.

Extranodal metastasis predicts poor survival in advanced colorectal cancer.

BACKGROUND/AIMS: The prognostic significance of extranodal metastasis (ENM) in colorectal cancer (CRC) is disregarded by the TNM classification system. The influence of ENM on survival among locally advanced CRC patients was examined. METHODOLOGY: We reviewed retrospectively the clinical course of 263 patients who underwent surgical resection of locally advanced CRC at our Department between 2005 and 2009. We analyzed the prognostic factors with special reference to the clinicopathological factors of primary tumors. RESULTS: Thirty-eight cases of ENM were detected among patients with CRC. Compared with ENM negative cancers, ENM-positive cancers were associated with poorer tumor differentiation grade (p=0.026) and higher prevalence of TNM-stage (p<0.0001), T-status (p=0.024), N-status (p<0.0001) and postoperative recurrence (p<0.0001). In univariate analysis, TNM-stage (p<0.0035), T-status (p=0.002), N-status (p<0.0024) and positive ENM (p<0.0001) were significant predictors of poor survival. Multivariate analyses showed a positive ENM to be a highly significant independent predictor of mortality (HR=1.98, 95% CI=1.23- 3.23, p=0.0053). Survival analyses using Kaplan-Meier curves demonstrated that patients with ENM-positive cancers had significantly poorer survival than patients with ENM-negative cancers. Patients with ENM-negative cancers did not show significantly different survival from patients with node-negative cancers (p=0.272, data not shown). CONCLUSIONS: ENM appears to be a strong independent negative prognostic factor of poor survival in locally advanced CRC.

Feasibility of solo laparoscopic colorectal resection.

BACKGROUND/AIMS: To demonstrate the feasibility of solo laparoscopic colorectal resection (SLCR) is performed by the laparoscopist only. This study is an evaluation of the feasibility of SLCR for patients with colorectal cancer. METHODOLOGY: Fifty-one consecutive patients received SLCR from 2008 to 2009. The procedure was performed with four trocars and one laparoscopist. The short-term outcomes and complications were investigated retrospectively. RESULTS: The median age of the patients was 67 years (range 42-81). The median operating time for SLCR was 168 minutes (range 90-268). For one patient (1.96%) conversion to open surgery was required. Anastomotic leakage developed in 1 (1.96%) patient and ileus developed in 2 (3.9%). The median postoperative hospital stay was 8 (range 6-60) days without in-hospital deaths. CONCLUSIONS: In our experience, SLCR for patients with colorectal cancer is feasible and compares favorably with the standard technique. The diminished need for human operative assistance provides significant economic and organizational benefits.

Effects of intra-articular inoculation with Mycoplasma bovis on immunological responses in calf joints.

Mycoplasma arthritis that caused by Mycoplasma bovis exhibit severe lameness. This disease is difficult to cure with antibiotics, but the detailed pathological mechanisms have not been fully clarified. In this study, we examined the effects of intra-articular inoculation with M. bovis on immunological responses in calf joints. We inoculated three calves each with M. bovis or phosphate buffer saline (control) into the right stifle joint and dissected them at 15 days postinoculation. Mycoplasma bovis-inoculated calves exhibited swelling of the stifle joint, increases in synovial fluid, fibrin deposition, and cartilage thinning. Intracellular M. bovis was detected in synovial tissues analyzed by immunohistochemistry and transmission electron microscopy. Messenger RNA expressions of interleukin (IL)-1ß, IL-6, IL-8, IL-12p40, and IL-17A in synovial fluid cells and synovial tissues from M. bovis-inoculated calves were significantly higher than those from control calves. Protein levels of these cytokines in synovial fluid from M. bovis-inoculated calves were markedly higher than those from control calves. Our study clarified that inoculation with M. bovis into the stifle joint induced the production of inflammatory cytokines by synovial fluid cells and synovial tissues, causing a severe inflammatory response in joints. Additionally, M. bovis could invade cells in synovial tissues, which may have aided it in evading antibiotics and host immune surveillance.

Scheduled prospective tri-weekly modified FOLFOX6 maintenance chemotherapy in the treatment of metastatic colorectal cancer.

BACKGROUND/AIMS: Oxaliplatin, which is effective for colorectal cancer (CRC) in combination with 5-fluorouracil (5-FU) and leucovorin (LV), is widely used for metastatic CRC. With the increasing use of oxaliplatin, however, serious adverse events have been experienced, including hematologic and neurologic toxicities. The aim of this study was to evaluate whether tri-weekly modified FOLFOX6 (mFOLFOX6) maintenance chemotherapy is associated with a low incidence of severe hematologic and neurologic toxicities in the treatment of patients with metastatic CRC. METHODOLOGY: We developed a new treatment regimen with mFOLFOX6 biweekly for 8-10 consecutive cycles (induction phase) followed by a 3-week rest period, after which treatment was resumed with cycles of tri-weekly mFOLFOX6 at standard doses (maintenance phase). Validity and complications were investigated retrospectively. RESULTS: Twenty-nine patients were enrolled in this study. The median progression-free survival (PFS) and overall survival (OS) times were 9.4 months and 23 months, respectively. All patients had peripheral neuropathy during treatment, but grade 3 neurotoxicity was observed in only 2 patients (6.9%). CONCLUSIONS: mFOLFOX6 maintenance chemotherapy was associated with a very low incidence of grade 3 hematologic and neurologic toxicities. The toxicities associated with PFS and OS were comparable to those reported in the treatment of patients with metastatic CRC. A tri-weekly mFOLFOX maintenance strategy of alternative treatment with a less-toxic regimen may reduce toxicity and maintain efficacy.

Transcriptome analysis of Mycoplasma bovis stimulated bovine peripheral blood mononuclear cells.

Mycoplasma bovis is a pathogenic bacterium in bovines that causes huge global economic losses. Numerous factors play important roles in M. bovis pathogenesis; however, the host immune response involved in M. bovis infection has not been fully elucidated. We aimed to determine the characteristics of the host immune response to Mycoplasma infection. We evaluated the responsiveness of bovine peripheral blood mononuclear cells (PBMCs) stimulated with M. bovis via microarray analysis. The transcriptional abundance of innate immune-related genes IL-36A, IL-27, IFN-γ, and IL-17 in PBMCs increased after M. bovis exposure. Upon M. bovis infection, there was increased expression of the lymphocyte activated genes basic leucine zipper transcription factor (BATF) and signaling lymphocytic activation molecule family members 1 and 7 (SLAMF 1 and SLAMF 7) in PBMCs compared with that in unstimulated cells. The study revealed that the transcriptional abundance of innate immunity genes in PBMCs increased during M. bovis infection. This induced the activation of PBMCs, giving rise to an immune response, which is followed by the development of the inflammatory response. The results from this study could be used as the basis for the development of novel vaccine candidates against M. bovis.

Central nervous system involvement in diffuse large B-cell lymphoma.

BACKGROUND: Malignant lymphoma with central nervous system (CNS) involvement has an extremely poor prognosis. We retrospectively studied the risk factors for CNS involvement in patients with diffuse large B-cell lymphoma (DLBCL) treated by cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or rituximab (R) -CHOP chemotherapy. PATIENTS AND METHODS: We studied 375 consecutive patients who were newly diagnosed with DLBCL between 1996 and 2006. Patients with primary CNS involvement and patients who received CNS prophylaxis were excluded. All the patients received CHOP (n = 172) or R-CHOP (n = 203) chemotherapy. The following variables were assessed for their potential to predict CNS involvement: gender, age, serum lactate dehydrogenase (LDH) level, performance status, clinical stage, number of extranodal involvements, International Prognostic Index (IPI), bone marrow involvement, presence of a bulky mass, presence of B symptom, and treatment. RESULTS: CNS involvement was observed in 13 cases (3.5%). In univariate analysis, LDH more than normal range, LDH more than twice as normal range, high IPI, bone marrow involvement, and systemic relapse were the predictors for CNS involvement. In multivariate analysis, no risk factors were detected for CNS involvement. The use of rituximab did not have an impact on CNS involvement. CONCLUSIONS: The incidence of CNS involvement does not decrease in rituximab-era.

Mycoplasma bovis induces matrix metalloproteinase-3 expression in bovine synovial cells via up-regulation of interleukin-1ß expression in mononuclear cells.

Mycoplasma bovis causes chronic arthritis in calves, presenting as osteolysis in affected joints. Matrix metalloproteinase-3 (MMP-3), an enzyme involved in cartilage degradation, is produced by synovial cells. Production of this proteinase is regulated by interleukin (IL)-1ß, which is produced by mononuclear cells. Both factors are known to play important roles in osteolysis in human autoimmune and bacterial arthritis. However, the pathophysiology of Mycoplasma arthritis (MA) has not been elucidated. In this study, we evaluated the levels of MMP-3 and IL-1ß in synovial fluid (SF) from MA calves and examined the effect of IL-1ß on MMP-3 expression in bovine synovial cells in vitro. Levels of MMP-3 and IL-1ß in SF from MA calves were significantly higher than those of clinically healthy calves. Mycoplasma bovis induced significant increases in the expression of IL-1ß mRNA and protein in mononuclear cells, compared with cells not exposed to M. bovis. Interestingly, the supernatant of mononuclear cells stimulated with M. bovis contained high levels of IL-1ß, which induced higher expression of MMP-3 mRNA and protein in synovial cells than direct stimulation by M. bovis. Recombinant bovine IL-1ß also induced increased MMP-3 mRNA and protein expression in synovial cells. Our results indicate that M. bovis induces IL-1ß expression by bovine mononuclear cells, and this cytokine then promotes MMP-3 production by synovial cells. These findings suggest that MMP-3 and IL-1ß are key factors in the development of osteolysis in MA calves.

Adolescent wilms tumor with intraspinal and bone metastases: a case report and the review of literature.

A 14-year-old girl was referred for a large tumor of the left kidney, with intraspinal and vertebral metastases. Left nephrectomy and intraspinal tumor resection were performed. The histology of both tumors was nephroblastoma with no anaplasia and favorable histology, and they were diagnosed as stage IV. The tumor bed and vertebras were irradiated. We started chemotherapy according to the DD-4A regimen of Japanese Wilms' Tumor Study Group. The vertebral metastasis was additionally irradiated. The patient has remained in disease-free remission for 45 months after the surgical resection. Intensive multimodality therapy including DD-4A regimen of National Wilms' Tumor Study can result in long-term disease-free remission.

The standard international prognostic index for predicting the risk of CNS involvement in DLBCL without specific prophylaxis.

Central nervous system (CNS) involvement is a serious complication in patients with diffuse large B-cell lymphoma (DLBCL) and evaluating CNS risk is an important issue. Using the standard international prognostic index (IPI) and CNS-IPI, a recently proposed model including IPI risk factors and adrenal/kidney involvement, we assessed CNS risk in 1220 untreated DLBCL patients who received R-CHOP without prophylaxis. According to the standard IPI, the cumulative incidences of CNS involvement at 2 years were 1.3, 4.6, 8.8, and 12.7% in the low-, low-intermediate-, high-intermediate-, and high-risk groups, respectively (p <.001). This result is comparable with that of the CNS-IPI. Patients with breast involvement tended to have lower risk according to the standard IPI but showed frequent CNS involvement, similar to patients with testis involvement. The standard IPI is also a useful predictor of CNS involvement. Patients with breast/testis involvement would be candidates for prophylaxis regardless of the standard IPI risk.

Intraductal papillary mucinous tumor of the pancreas associated with autosomal dominant polycystic kidney disease.

A 43-year-old male with a history of autosomal dominant polycystic kidney disease (ADPKD) was admitted to our center with severe abdominal pain and was diagnosed with acute pancreatitis. CT showed multiple cysts in the liver and both kidneys along with ADPKD and a cystic mass, 4 cm in diameter, in the pancreatic head. The main pancreatic duct was dilated to 1 cm in diameter. The patient was diagnosed with acute pancreatitis due to intraductal papillary mucinous tumor (IPMT), and pancreatoduodenectomy was performed. Histologic examination revealed a multiloculated cystic tumor filled with mucin in the head of the pancreas. Microscopically, the tumor was diagnosed as adenocarcinoma and was found to have invaded the main pancreatic duct. Although, in addition to our case, only seven cases with association between ADPKD and malignant neoplasms have been reported, five of these cases had neoplasms arising from the pancreas. Therefore, we suggest that some genetic interactions may exist between ADPKD and pancreatic carcinogenesis.

Post-treatment PET-CT Findings may Predict the Prognosis of DLBCL with a Bulky Mass.

We retrospectively analyzed the prognosis of patients with diffuse large B cell lymphoma (DLBCL) and a bulky mass at diagnosis. We retrospectively analyzed clinical data for 29 consecutive DLBCL patients with an initial bulky mass receiving R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) therapy from 2004 to 2011. Bulky disease was defined as a measurable tumor mass >10 cm in diameter or a mediastinal mass >1/3 of the chest diameter. Patients with primary mediastinal large B-cell lymphoma were excluded. The median age was 65 years (20-78 years) and the maximum tumor diameter was 11.5 cm (10.0-17.0 cm). Complete response and partial response were achieved in 14 patients each, while 1 patient had progressive disease. The 3-year overall survival (OS) rate and progression-free survival (PFS) rate were 66 and 56 %, respectively. Findings on post-treatment positron emission tomography-computed tomography (PET-CT) were significantly associated with OS (34 % for patients with abnormal uptake vs. 75 % for those without, P = 0.014), and were also associated with PFS (36 vs. 83 %, respectively, P < 0.001). Nine patients with a single site of abnormal uptake on PET-CT underwent radiotherapy and 5 of them subsequently relapsed. An initial bulky mass does not indicate a poor prognosis of DLBCL. However, the post-treatment PET-CT findings may have predictive value in DLBCL patients with a bulky mass.

Analysis of outcomes in patients with supra-diaphragmatic vs infra-diaphragmatic diffuse large B cell lymphoma treated with R-CHOP therapy.

The prognostic implications of infra-diaphragmatic (InD) versus supra-diaphragmatic (SpD) primary lesions in limited-stage diffuse large B-cell lymphoma (DLBCL) remains unknown. This retrospective study aimed to assess the prognostic impact of spD and InD lesions as well as presence of gastrointestinal (GI) involvements in adults with limited-stage DLBCL. We analyzed data from 178 patients with limited-stage DLBCL who were treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone therapy at 7 institutions of the Yokohama City University Hematology Group between 2003 and 2009. The median age was 63 years (range, 18-80 years). The primary sites were SpD in 109 patients, and InD in 69. No statistical differences in progression-free survival (PFS) or overall survival (OS) were observed between patients with SpD lesions and those with InD lesions. However, when patients with SpD lesions, InD lesions with (n=35), and without (n=34) GI involvement were compared, the presence of GI lesions was associated with favorable PFS. The multivariate analysis revealed that SpD or InD localization had no independent effect on PFS or OS, whereas the presence of GI lesions was correlated with favorable PFS (P=0.024, HR 0.09).