Significance of platelet-derived microparticles and activated platelets in diabetic nephropathy

We measured levels of platelet-derived microparticles (PMP), which have coagulative activity and are produced by platelet activation or physical stimulation, and CD62P/CD63-positive platelets in patients with diabetes mellitus to determine their clinical significance and effects on complications of diabetes including diabetic nephropathy. We also compared these levels before and after administration of the antiplatelet drug cilostazol. Plasma PMP and CD62P/CD63-positive platelet levels were significantly higher in patients with diabetes mellitus than normal controls. CD62P-positive platelet levels were significantly higher in patients with nephropathy than in patients without complications. After administration of cilostazol, PMP and CD62P/CD63-positive platelet levels were significantly decreased. The increases in platelet activity and its related procoagulant activity appear to account in part for the hypercoagulability observed in diabetes mellitus. Our findings suggest that activated platelets might play a role in the development of diabetic nephropathy. Furthermore, antiplatelet therapy with cilostazol for diabetic patients may be useful as antithrombin therapy including antiplatelet therapy, since it suppresses the production of intrinsic coagulants produced by platelet activation.     

<Emphasis Type="Italic">KRAS</Emphasis> and <Emphasis Type="Italic">BRAF</Emphasis> Mutations in 203 Esophageal Squamous Cell Carcinomas: Pyrosequencing Technology and Literature Review

Background

Epidermal growth factor receptor (EGFR) signaling is one of the most promising targets for molecular-targeted therapies in esophageal squamous cell carcinoma (ESCC). Thus, the molecular diagnosis of KRAS and BRAF mutations is clinically important in therapeutic decision making. However, the frequency of KRAS and BRAF mutations in ESCCs remains inconclusive because of the limited sample sizes of previous studies (all N ≤ 80). Pyrosequencing is a nonelectrophoretic nucleotide extension sequencing technology that can be used for mutation testing.

Methods

The frequency of KRAS and BRAF mutations was examined using a nonbiased database of 203 resected ESCCs and a high-throughput pyrosequencing assay.

Results

The validity of the KRAS pyrosequencing method was initially demonstrated by detection of all 4 types of KRAS mutations [c.35G>T (codon 12 GGT>GTT), c.35G>A (codon 12 GGT>GAT), c.34G>T (codon 12 GGT>TGT), c.38G>A mutation (codon 13 GGC>GAC)], which had been previously diagnosed using Scorpion-ARMS technology, in 9 colon cancer tissues (9 of 9; 100 %). Similar results were demonstrated for BRAF mutational status in 3 colon cancer cell lines (HCT116, Colo201, and HT29), which were validated by Sanger dideoxy sequencing. Subsequently, the KRAS mutation was found to be extremely rare (1 of 203; 0.5 %), and the BRAF mutation was absent (0 of 203; 0 %), in the dataset of 203 ESCCs.

Conclusions

These results suggest that KRAS and BRAF mutations play a limited role in the development of ESCC and that mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy in ESCC.

     

Primary liposarcoma of the anterior mediastinum —Case report and review of literature—

A 76-year-old male with anterior mediastinal tumor was admitted to our hospital. He had undergone mediastinal lipoma surgery 3 years earier. The tumor was excised surgically. Microscopic sections of the tumor showed liposarcoma composed of myxoid tissue. Further examination of prior specimens taken from this patient proved this case to be a recurrence of liposarcoma. Poorly differentiated tumors, which pathologically tend to be more cellular with less fat per cell component, are likely to have high CT numbers. But CT number is not sufficient to distinguish well-differentiated liposarcoma from benign lipoma.     

Limited value of perineural invasion in radical prostatectomy specimens as a predictor of biochemical recurrence in Japanese men with clinically localized prostate cancer

The objective of this study was to determine whether the presence of perineural invasion (PNI) in radical prostatectomy specimens could be a useful prognostic parameter in Japanese men with prostate cancer. Between January 1995 and September 2003, 202 Japanese men underwent radical retropubic prostatectomy for prostate cancer without any neoadjuvant therapies prior to surgery. We retrospectively analyzed the relationship between PNI in radical prostatectomy specimens and other prognostic factors, and also assessed the significance of PNI in biochemical recurrence after radical prostatectomy. The presence of PNI was significantly related to clinical stage, pathological stage, Gleason score, seminal vesicle invasion, lymph node metastasis and tumor volume, but not pretreatment serum prostate specific antigen value. During the observation period, biochemical recurrence occurred in 20 patients (3 in patients without PNI and 17 in those with PNI), and the biochemical recurrence-free survival rate in patients with PNI was significantly lower than that in patients without PNI. In addition to-PNI, pathological stage, seminal vesicle invasion, lymph node metastasis and tumor volume were significantly associated with the biochemical recurrence-free survival rate; however, among these five factors, only seminal vesicle invasion was an independent predictor of biochemical recurrence on multivariate analysis. Despite a significant association between several prognostic parameters, PNI was not an independent predictor of biochemical recurrence; therefore, it may not provide an additive effect to consider the presence of PNI in predicting the prognosis of Japanese men who underwent radical prostatectomy if there are other conventional parameters available.     

The expression of platelet-derived growth factor and connective tissue growth factor in different types of abdominal aortic aneurysms

AIM: An atherosclerotic abdominal aortic aneurysms (AAAA) differ from inflammatory abdominal aortic aneurysms (IAAA), which are characterized by a non specific inflammatory reaction leading to considerable aneurysmal wall thickness from the media to adventitia and retroperitoneal fibrosis in the surrounding tissue. Platelet-derived growth factor (PDGF) and its receptor have been localized to specific cell types within atherosclerotic plaques. Human connective tissue growth factor (CTGF) is a cysteine rich polypeptide that has similar structures to PDGF and has been implicated in connective tissue formation. PDGF and CTGF may play a role in the development of aneurysmal walls in both AAAA and IAAA. METHODS: Using in situ hybridization technique with DIG-labeled RNA probes and immunostaining, we investigated CTGF gene expression, and expression of PDGF and its receptor protein, in human aneurysmal walls. RESULTS: Expression of CTGF mRNA was found on vascular smooth muscle cells (VSMC) in specimens from AAAA and IAAA. Strong CTGF expression was localized in VSMC around calcification in AAAA. In IAAA, strong expression of CTGF was observed around inflammatory cells. In the aneurysmal walls of AAAA, PDGF A and B chains were strongly stained on small vessels, and the PDGF beta receptor was also strongly stained on VSMC around calcification. In the aneurysmal wall of IAAA, weak expressions of PDGF A and B chains were observed in endothelial cells of vessel walls around the inflammatory cells, but the intensity of expression was much weaker than that on the vessel walls in AAAA. CONCLUSIONS: Such differences in fibrogenic cytokine expression may be involved in characteristic aneurysmal formation.     

CT‐Guided Wire Localization for Involved Axillary Lymph Nodes After Neo‐adjuvant Chemotherapy in Patients With Initially Node‐Positive Breast Cancer

Resection of biopsy‐proven involved axillary lymph nodes (iALNs) is important to reduce the false‐negative rates of sentinel lymph node (SLN) biopsy after neo‐adjuvant chemotherapy (NAC) in patients with initially node‐positive breast cancer. Preoperative wire localization for iALNs marked with clips placed during biopsy is a technique that may help the removal of iALNs after NAC. However, ultrasound (US)‐guided localization is often difficult because the clips cannot always be reliably visible on US. Computed tomography (CT)‐guided wire localization can be used; however, to date there have been no reports on CT‐guided wire localization for iALNs. The aim of this study was to describe a series of patients who received CT‐guided wire localization for iALN removal after NAC and to evaluate the feasibility of this technique. We retrospectively analyzed five women with initially node‐positive breast cancer (age, 41–52 years) who were scheduled for SLN biopsy after NAC and received preoperative CT‐guided wire localization for iALNs. CT visualized all the clips that were not identified on post‐NAC US. The wire tip was deployed beyond or at the target, with the shortest distance between the wire and the index clip ranging from 0 to 2.5 mm. The total procedure time was 21–38 minutes with good patient tolerance and no complications. In four of five cases, CT wire localization aided in identification and resection of iALNs that were not identified with lymphatic mapping. Residual nodal disease was confirmed in two cases: both had residual disease in wire‐localized lymph nodes in addition to SLNs. Although further studies with more cases are required, our results suggest that CT‐guided wire localization for iALNs is a feasible technique that facilitates identification and removal of the iALNs as part of SLN biopsy after NAC in situations where US localization is unsuccessful.     

<Emphasis Type="Italic">In-Situ</Emphasis> Visualization and Quantification of Mineralization of Cultured Osteogenetic Cells

An osteoblastic cell line (HOS cells) produces a prominent osteoid matrix with mineralization. Fibroblasts, on the other hand, do not exhibit this mineralization. To evaluate the degree of mineralization, we added calcein to the culture medium and then observed the culture wells by using an image analyzer. The calcein uptake into the cell/matrix layer was detected in the HOS cells but not in the fibroblasts. The calcein uptake was also quantified in situ by using an image analyzer, which revealed high levels in the HOS cells, which correlated well with the calcium content of the mineralized matrix. Rat marrow cells were also cultured in media containing calcein, fetal bovine serum, -glycerophosphate, L-ascorbic acid 2-phosphate, and with or without dexamethasone. With the dexamethasone, the cells exhibited osteogenic differentiation that resulted in mineralized matrix formation after about 10 days. The matrix formation coincided with the appearance of calcein uptake into the cell/matrix layer, with the amount of calcein uptake increasing with time. By contrast, the culture without the dexamethasone did not exhibit matrix formation and the calcein uptake was negligible. In the case of both HOS cell and rat marrow cell cultures in vitro, calcein did not affect expressions of their alkaline phosphatase activity or osteocalcin production. Furthermore, histologic observation revealed that rat marrow cells subcultured with calcein could show osteogenic ability after in vivo implantation. These results suggest that the current method of detecting calcein uptake in a culture allows the monitoring of the osteogenic capacity of cultured cells, as well as the measurement of the amount of mineralization produced by the osteogenic cells. Given that osteogenic cultured cells/mineralized matrices are used in bone reconstruction surgery, the in situ monitoring method is invaluable in that it allows us to evaluate the osteogenic capacity of in vitro constructs.     

Changes in bone turnover markers during 14-day 6° head-down bed rest

Osteoporosis caused by exposure to microgravity represents a serious clinical concern, but the mechanisms have yet to be fully elucidated. The present research aimed to elucidate the effects of microgravity environments on bone turnover, with a specific focus on changes in bone resorption markers such as type I collagen cross-linked N-telopeptides (NTx) and deoxypyridinoline (Dpyr), for which scant data are available regarding detailed time course. Methods using 6° head-down bed rest were utilized to simulate a microgravity environment. Eleven adult male volunteers underwent 6° head-down bed rest for 14 days; measurements were made of serum and urine Ca concentrations, in addition to osteocalcin (OC), bone alkaline phosphatase (ALP), NTx, and Dpyr as bone turnover markers. By the end of bed rest, concentrations of bone ALP had significantly increased, but OC displayed a tendency toward decrease. Concentrations of Dpyr significantly increased from day 6, remaining elevated until the end of bed rest. Concentrations of NTx significantly increased on day 13 and at the end of bed rest. Serum and urinary concentrations of Ca increased significantly at the end of bed rest. Bone ALP represents a relatively early marker of osteoblast differentiation at the matrix maturation phase and OC is a late marker in osteoblast differentiation at the calcification phase. The present results therefore suggest an absolute increase in bone resorption and normal or reduced bone formation, together causing prominent uncoupling and rapid bone loss after simulated microgravity. Moreover, the present results suggest that bone resorption is enhanced at an early stage of exposure to microgravity environments.     

Oxygen dynamics at paraspinal muscles during exertion using near-infrared spectroscopy in patients with degenerative lumbar scoliosis

 Oxygen dynamics were measured in both sides of the paraspinal muscles of patients with degenerative lumbar scoliosis. The objective was to investigate the extent of stress by measuring the changes in oxygenation and blood flow volume using near-infrared spectroscopy. There were 44 patients with degenerative lumbar scoliosis (7 men, 37 women) and 11 controls with no scoliosis (2 men, 9 women). The measured recovery time for deoxyhemoglobin indicated the recovery from energy deficit after exercise. The average recovery time of the 11 control subjects was 2.4 ± 0.7 s on the right and 2.4 ± 0.8 s on the left. Among the 44 patients the average recovery time was 3.8 ± 1.8 s on the convex side and 2.1 ± 1.1 s on the concave side. There was a significant difference between the two groups. The recovery time on the concave side in patients was faster than that on the convex side. The results of the current study support the theory that stretching the muscles at the convex side results in chronic increases in the intramuscular compartment and reduced muscular blood flow. Received: May 23, 2002 / Accepted: October 29, 2002 Offprint requests to: M. Miyake, Department of Orthopaedic Surgery, Eiko Hospital, 1381 Takuma, Takuma-cho, Mitoyo-gun, Kagawa 769-1101, Japan