Genetic analysis of adult leukoencephalopathy patients using a custom-designed gene panel.

Leukoencephalopathies encompass all clinical syndromes that predominantly affect brain white matter. Genetic diagnosis informs clinical management of these patients, but a large part of the genetic contribution to adult leukoencephalopathy remains unresolved. To examine this genetic contribution, we analyzed genomic DNA from 60 Japanese patients with adult leukoencephalopathy of unknown cause by next generation sequencing using a custom-designed gene panel. We selected 55 leukoencephalopathy-related genes for the gene panel. We identified pathogenic mutations in 8 of the 60 adult leukoencephalopathy patients (13.3%): NOTCH3 mutations were detected in 5 patients, and EIF2B2, CSF1R, and POLR3A mutations were found independently in 1 patient each. These results indicate that cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 mutations is the most frequent adult leukoencephalopathy in our cohort. Moreover, brain imaging analysis indicates that CADASIL patients who do not present typical phenotypes may be underdiagnosed if not examined genetically.

Preferential changes of skeletal muscle echogenicity in myotonic dystrophy type 1.

BACKGROUND AND PURPOSE: In myotonic dystrophy type 1 (DM1), weakness of distal limb muscles affects quality of life. Non-invasive evaluation of muscular involvement by muscle sonography could be useful for characterizing muscle-specific involvement. METHODS: Sonography of the lower leg and forearm was performed in 19 patients with DM1 and 10 control subjects. The mean echo intensities (EIs) of seven limb muscles were obtained by computer-assisted histogram analysis and compared within DM1 according to the overall clinical severity. RESULTS: The EIs of the muscles were significantly higher in DM1 than in the controls (P < 0.01), except for the soleus (P = 0.4). Comparison of adjacent muscles showed the following: (i) greater EIs in flexor digitorum profundus than flexor carpi ulnaris (P < 0.01) and flexor digitorum superficialis (P = 0.02), and (ii) greater EIs in the medial head of the gastrocnemius than the soleus (P < 0.00001). In a subgroup analysis of DM1 according to the modified Rankin Scale (mRS), the more severe subgroup (mRS = 4-5) had lower mean EIs than the less severe subgroup (mRS from 1-3) (P = 0.01) in the flexor digitorum superficialis but not in other muscles. CONCLUSIONS: Preferential high echogenicity in the medial gastrocnemius and deep finger flexors is suggestive of DM1. Muscle echogenicity is not generally related to functional dysfunction in DM1.

Intra- and postoperative low-dose ketamine for adolescent idiopathic scoliosis surgery: a randomized controlled trial.

BACKGROUND: In this randomized controlled trial, we examined whether intra- and postoperative infusion of low-dose ketamine decreased postoperative morphine requirement and morphine-related adverse effects as nausea and vomiting after scoliosis surgery. METHODS: After IRB approval and informed consent, 36 patients, aged 10-19 years, undergoing posterior correction surgery for adolescent idiopathic scoliosis, were randomly allocated into two groups: intra- and postoperative ketamine infusion at a rate of 2 µg/kg/min until 48 h after surgery (ketamine group, n = 17) or infusion of an equal volume of saline (placebo group, n = 19). All patients were administered total intravenous anesthesia with propofol and remifentanil during surgery and intravenous morphine using a patient-controlled analgesia device after surgery. The primary outcome was cumulative morphine consumption in the initial 48 h after surgery. Pain scores (Numerical Rating Scale, NRS, 0-10), sedation scales, incidence of postoperative nausea and vomiting (PONV), and antiemetic consumption were recorded by nurses blinded to the study protocol for 48 h after surgery. RESULTS: Patient characteristics did not differ between the two groups. Cumulative morphine consumption for 48 h after surgery was significantly lower in the ketamine group compared to the placebo group (0.89 ± 0.08 mg/kg vs. 1.16 ± 0.07 mg/kg, 95% confidence interval for difference between the means, 0.03-0.48 mg/kg, P = 0.019). NRS pain, sedation scales, and incidence of PONV did not differ between the two groups. Antiemetic consumption was significantly smaller in ketamine group. CONCLUSIONS: Intra- and postoperative infusion of low-dose ketamine reduced cumulative morphine consumption and antiemetic requirement for 48 h after surgery.

Salvage surgery for a locally persistent or recurrent tumour in maxillary cancer patients who have undergone radiotherapy and concomitant intra-arterial cisplatin: implications for surgical margin assessment.

Limited information about salvage surgery is available for locally persistent and recurrent maxillary sinus cancers after the completion of chemoradiation therapy. Seventy-six maxillary sinus cancer patients who had undergone chemoradioselection using initial radiotherapy and concomitant intra-arterial cisplatin were screened retrospectively. Twenty-four of these patients who had a locally persistent or recurrent tumour were investigated. The 2-year overall survival rate of patients with maxillary sinus cancer of all types was 39.0% for those who underwent salvage surgery and 10.0% for those who did not. The 2-year overall survival rate of patients with maxillary sinus squamous cell carcinoma was 45.8% for those who underwent salvage surgery and 11.1% for those who did not. Furthermore, the 2-year local control and overall survival rates of patients with positive and negative surgical margins were 14.3% and 83.3% and 14.3% and 66.7%, respectively. There were significant differences in local control (P=0.004) and overall survival (P=0.005) regarding surgical margin status. Although salvage surgery for a locally persistent or recurrent maxillary sinus cancer is a feasible treatment, patients with positive surgical margins are more prone to local relapse. Therefore, surgical safety margins should be assessed thoroughly.

Psychological and endocrine factors and pain after mastectomy.

BACKGROUND: This prospective study was designed to examine the associations of demographic, clinical, psychological and neuroendocrine factors with acute and chronic post-operative pain following partial mastectomy. METHODS: Sixty-four female patients scheduled for partial mastectomy were enrolled. Pre-operative anxiety/depression was assessed, using the Hospital Anxiety and Depression Scale (HADS). Pre-operative 24-h urinary cortisol levels were measured 2 days before surgery. Post-operative pain was examined using a visual analog scale (VAS) for acute pain on 0-2 post-operative day (POD), and a short-form McGill Pain Questionnaire for chronic pain at 6 months after surgery. In the last 29 subjects, post-operative 24-h urinary cortisol levels were also measured on 0 POD and were subjected to correlation analysis. RESULTS: Multivariate logistic regression analysis revealed that lower pre-operative cortisol secretion and greater pre-operative anxiety were significantly associated with an increased risk of moderate to severe acute post-operative pain [Odds Ratio (95% Confidence Interval); 0.96 (0.92-0.98), and 1.24 (1.04-1.54)], and that patients with greater pre-operative anxiety and moderate to severe acute pain were more likely to develop chronic post-operative pain [OR (95% CI); 1.63 (1.23-2.40), and 5.07 (1.30-24.6)]. Correlational analysis demonstrated that the post-operative cortisol level was inversely correlated with pre-operative anxiety and the intensity of acute post-operative pain (r = -0.40, p < 0.05, and r = -0.50, p < 0.01), but not with the intensity of chronic pain. CONCLUSIONS: This study confirms that pre-operative anxiety is associated with both acute and chronic post-operative pain after partial mastectomy. It also suggests that lower perioperative cortisol secretion might be associated with greater acute post-operative pain. SIGNIFICANCE: Although the associations between psychological stress/stress hormone levels and chronic post-operative pain remain to be determined, pre-operative psychological stress and perioperative cortisol levels are correlated with acute post-operative pain.

Development of bone canaliculi during bone repair.

We recently found that silver impregnation staining with protargol (silver protein), that is, a modified Bodian method, is useful for histologically identifying the details of bone canaliculi structure, using thin sections of decalcified bone tissues. With this staining method, we conducted the present study to assess the development of bone canaliculi during the process of intramembranous ossification using a fracture-like stimulation model of the rat femur. After making a drill-hole in the cortex of the rat femur, decalcified thin sections were obtained after 3, 5, 7, and 14 days by the standard paraffin-embedding procedure. Silver staining for bone canaliculi was performed using our previously reported technique. The results showed that woven bone covered the fracture surface of the cortex after 5 days, then immature lamellar bone attached to the woven bone after 7 days, and finally the lamellar bone matured and became thick with appositional growth after 14 days. The osteocytes in the woven bone appeared at an early stage of bone repair and developed a few canaliculi that were short and irregularly distributed in the osteoid matrix, while the osteocytes in the lamellar bone at a late stage formed many bone canaliculi that were long and regularly distributed in mature bone matrix. Therefore, we concluded that woven bone osteocytes may be necessary for induction of the lamellar bone osteocytes followed by active appositional growth of the lamellar bone at the early stage of bone repair, and also that both bone tissues could be clearly distinguished from one another based on the pattern of development of bone canaliculi by the osteocytes, as seen with the use of our sensitive staining method.

Vascular smooth muscle cell outgrowth, proliferation, and apoptosis in young and old rats.

We attempted to identify the effects of aging on the response of vascular smooth muscle cells (VSMCs) to injury. Rat aortas were injured by ballooning, and cell outgrowth of the aortic explants, as well as cell proliferation and apoptosis induced by 7-ketocholesterol in the culture system were compared in young (6-7 weeks) and old (40-50 weeks) rats. Explant outgrowth in uninjured rats did not differ between young and old rats. However, 14 days after balloon injury, the number of explants with outgrowth increased by a factor of four (P<0.01) in young rats, while that of the old rats did not change. Cell proliferation in cultured VSMCs also did not differ between young and old uninjured rats, but, in the injured group, proliferation doubled in young rats (P<0.01), but did not change in the older group. The rate of unadhered cells in the presence of 7-ketocholesterol (30 microM) did not differ between young and old uninjured rats. In the injured rats, however, that of young rats was lower (P<0.01) while that of older rats was higher (P<0.01). The extent of DNA fragmentation (%) after the addition of 7-ketocholesterol (30 microM) did not differ between young and old uninjured rats. In the injured groups, DNA fragmentation was lower in the young rats (P<0.01), and higher in the old ones (P<0.01), when compared to their respective controls. These results indicate that the VSMC injury responses of cell outgrowth and proliferation were more pronounced in the young, and that 7-ketocholesterol-induced apoptosis was more extensive in old rats.

Cytofluorometric DNA ploidy analysis in giant cell tumor of bone: histologic and prognostic value.

DNA ploidy analysis by DNA cytofluorometry was performed on 41 tumors obtained from 37 patients with primary giant cell tumor of bone (GCT). Histologically, 26 of the tumors from primary or recurrent lesions were evaluated as grade I, and 13 tumors as grade II. Among the 33 primary GCT patients, 4 patients had local recurrence or pulmonary metastasis. The DNA ploidy pattern and the percentage of hyperdiploid cells showing a greater DNA content than diploid cells, were obtained from DNA cytofluorometry. All of the 33 primary tumors were diploid. Of 6 recurrent tumors, 4 were diploid and 2 were euploid-polyploid. One of the two pulmonary metastatic tumors was diploid, but another that demonstrated a malignant transformation to malignant fibrous histiocytoma was aneuploid. The percentage of hyperdiploid cells was significantly different between primary and recurrent tumors (P = 0.0188) and between grade I and grade II tumors (P = 0.0052), while there was no difference between primary tumors in the cases that recurred or metastasized and those that did not. Thus, these data indicate that cell proliferative activity is closely correlated with biological aggressiveness and histological grading, although DNA ploidy is not useful for predicting prognosis.

Response of DNA ploidy to chemotherapy in primary and metastatic lesions in human osteosarcomas.

Primary and pulmonary metastatic and pulmonary metastatic tumors (two synchronous and seven metachronous metastases) in nine patients with osteosarcomas were studied by DNA cytofluorometry. All patients were treated with both pre and postoperative chemotherapy. The results showed that all five diploid osteosarcomas and three of the four aneuploid tumors did not markedly change their ploidy pattern after preoperative chemotherapy, and had almost the same ploidy patterns as the pulmonary metastatic lesions. Those eight tumors showed poor histologic response and chemoresistance by the doxorubicin binding assay. Only one aneuploid osteosarcoma showing good histologic response and chemosensitivity changed its ploidy pattern to diploid, with the disappearance of aneuploid tumor cells and its synchronous pulmonary metastatic tumor also showed conversion to a diploid pattern with massive tumor necrosis. It is evident that those tumors showing no change in their ploidy pattern after chemotherapy were resistant to the chemotherapy. Therefore, we conclude that regardless of whether the pulmonary metastatic tumors were synchronous or metachronous, they showed the same change in their ploidy pattern as well as their chemosensitivity as the primary human osteosarcoma from which they were derived.

Relationship between P-glycoprotein positivity, doxorubicin binding ability and histologic response to chemotherapy in osteosarcomas.

We previously reported that the doxorubicin binding ability detected by the doxorubicin (adriamycin) binding assay was closely correlated with the chemosensitivity of human osteosarcomas. In this study, we undertook to clarify the relationship between P-glycoprotein positivity (%PPG) and doxorubicin binding ability (%DB) in human osteosarcomas in order to determine which is a more sensitive index of histologic response to chemotherapy. Ten primary osteosarcomas were analyzed by the doxorubicin binding assay and by immunofluorescence to detect cellular P-glycoprotein positivity. Three good responders to chemotherapy containing doxorubicin showed a %DB greater than 90% (average: 96.43%), whereas the seven poor responders had values less than 80% (average: 35.31%). The difference between the two groups was statistically significant (P = 0.0167). However, the average %PPG of the three good responders was 6.73%, whereas the %PPG of the seven poor responders was 14.27%. There was no significant difference in %PPG between the two groups (P = 0.3051). No negative correlation between the %DB and the %PPG of all osteosarcomas (r = 0.536, P = 0.1104) was found, although there was a trend that those tumors with a high %PPG showed a low %DB. These results suggest that osteosarcomas showing a low %DB and %PPG with poor response to chemotherapy, may have multidrug resistance mechanisms other than P-glycoprotein. Therefore, we conclude that doxorubicin binding ability, which reflects all of the doxorubicin-resistant mechanisms, was more sensitive than P-glycoprotein positivity in predicting the chemosensitivity of human osteosarcoma.

Prognostic significance of DNA ploidy pattern in osteosarcomas in association with chemotherapy.

In this study, we analysed the DNA ploidy of osteosarcomas at biopsy and attempted to clarify the relationship between DNA ploidy pattern and prognosis. Thirty patients with non-metastatic osteosarcoma of an extremity were studied. All underwent intensive chemotherapy with doxorubicin, cisplatin and methotrexate, in addition to wide tumor resection. DNA ploidy was detected by DNA cytofluorometry, using isolated and smeared cells of biopsied tumor tissue. Twelve tumors showed a diploid ploidy pattern and 18 showed a non-diploid pattern such as aneuploidy (15 tumors) and euploid-polyploidy (3 tumors). The event-free survival rate at 9 years was 63.5% in non-diploid osteosarcoma patients and 13.3% in diploid osteosarcoma patients. There was a statistically significant difference between the two groups (P = 0.0278). These results lead us to conclude that a non-diploid osteosarcoma may be more sensitive to chemotherapy than a diploid tumor.

Prognostic value of DNA ploidy response to chemotherapy in human osteosarcomas.

We analyzed the DNA ploidy alterations after preoperative chemotherapy in 30 patients with non-metastatic osteosarcomas of the extremities. All of the patients received intensive chemotherapy with doxorubicin, cisplatin and methotrexate as well as wide tumor resection. DNA ploidy was determined by DNA cytofluorometry using isolated and smeared cells from biopsied and resected tumors after preoperative chemotherapy. The results showed that 12 diploid and nine non-diploid osteosarcomas did not change their ploidy pattern, but nine non-diploid tumors changed to a diploid pattern with the disappearance of the aneuploid cells. The nine patients with altered ploidy tumors had a better histologic response to chemotherapy and a better prognosis than the patients with non-altered tumors especially diploid tumors (P = 0.0138). Therefore, we conclude that a decrease in aneuploid cells after chemotherapy is closely correlated with a good prognosis in half of the cases of aneuploid osteosarcoma. These results also suggest that aneuploid cells are more chemosensitive than diploid cells in human osteosarcomas.

Time-dependent potentiation of the beta-cell is a Ca2+-independent phenomenon.

When isolated rat pancreatic islets are treated with 16.7 mM glucose, a time-dependent potentiation (TDP) of insulin release occurs that can be detected by subsequent treatment with 50 mM KCl. It has been thought that TDP by glucose is a Ca2+-dependent phenomenon and only occurs when exposure to glucose is carried out in the presence of Ca2+. In contrast to this, we now demonstrate TDP under stringent Ca2+-free conditions (Ca2+-free buffer containing 1 mM EGTA). In fact, under these Ca2+-free conditions glucose caused an even stronger TDP than in the presence of Ca2+. TDP induced by glucose in the absence of extracellular Ca2+ was unaffected by inhibitors of protein kinase C (PKC). However, cerulenin or tunicamycin, two inhibitors of protein acylation, eradicated TDP without affecting glucose metabolism. The TDP by glucose was not associated with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) during subsequent treatment with high K+. Exposure of islets to forskolin under Ca(2+)-free conditions did not cause TDP despite a large increase in the cellular cAMP levels. In conclusion, glucose alone induces TDP under stringent Ca2+-free conditions when [Ca2+]i was significantly lowered. Protein acylation is implicated in the underlying mechanism of TDP.

Peptide mimics of monocyte chemoattractant protein-1 (MCP-1) with an antagonistic activity.

In this study, we attempted to analyze the peptide motifs recognized by 24822.111 and F9, monoclonal antibodies (mAbs) that inhibit the chemotactic activity of monocyte chemoattractant protein-1 (MCP-1), a member of the CC subfamily of chemokines. We isolated phage clones from a phage display library and identified six peptide motifs. One of these clones, C27, was strongly and specifically recognized by 24822.111 mAb, while another, G25, was similarly recognized by F9 mAb. Both the C27 motif and the G25 motif contain two cysteines in their sequences and have little homology to the primary amino acid sequence of MCP-1. These clones, however, bound to THP-1 cells, and the binding was competitively inhibited by MCP-1. The clones strongly inhibited the MCP-1-induced chemotaxis of human monocytes. The synthetic and intramolecularly disulfide-linked peptides of C27 and G25 (sC27 and sG25) also inhibited the chemotaxis induced by MCP-1, while their derivatives with serine in place of cysteine did not, suggesting the importance of the loop structure for the inhibition. These results suggest that sC27 and sG25 may mimic the MCP-1-binding domain to the MCP-1 receptor.

Primary progressive aphasia presenting as conduction aphasia.

We report a case of a woman with primary progressive aphasia (PPA) who presented with conduction aphasia. A 60-year-old, right-handed, Japanese female suffering from progressive aphasia had difficulty in repeating words and phrases. She displayed phonemic paraphasias but had preserved comprehension and had no cognitive or behavior disorder for more than 6 years after the onset of the condition. She was able to continue to work successfully and to perform all her normal daily activities. T1-weighted magnetic resonance imaging revealed minute dilatation of the left inferior horn and sulci in the left hemisphere, and positron emission tomography revealed mild hypometabolism in the left supramarginal gyrus and its surrounding areas. Therefore, she was diagnosed as suffering from PPA presenting as conduction aphasia. We believe that the progressive conduction aphasia of the patient belongs to one of the fluent forms of PPA, and the ability to continue normal work along with the clinical portrayal of preserved memory and cognition skills may be features of a form of PPA presenting as conduction aphasia.

Effectiveness of activated vitamin D3 on improving prognosis of osteosarcoma patients.

We examined the survival prognosis of patients with osteosarcoma after orally administering the active form of vitamin D3, 1alpha (OH) D3 (alphaD3). The 29 patients, ranging in age from 9 to 58 years (mean, 19 years), with osteosarcoma were treated in our department between 1983 and 1995. The surgical stage was IIB in all patients. Among the 29 patients, alphaD3 was administered to 18 patients, and the remaining 11 patients served as controls. These patients underwent chemotherapy mainly with adriamycin and cisplatin, and wide tumor resection. In the alphaD3-treated group, the 5- or 10-year survival rate was 61.1%, while in the untreated group, the 5-year survival rate was 63.6%, and 10-year survival rate was 33.9%. Prognosis of the patients treated with alphaD3 tended to be better than that of untreated patients, but there was no significant difference in the survival rate between the groups (p=0.3823). The survival rate in the group treated with a total dose of alphaD3 more than 1,500 microg was also higher than that in the untreated group, but the difference was not significant (p=0.0740). Therefore, we concluded that alphaD3 at the dose used in this study was not effective in improving the prognosis of osteosarcoma patients.

Extra-articular wide tumor resection and limb reconstruction in malignant bone tumors invading the knee joint.

We demonstrate our procedure of extra-articular wide tumor resection and its clinical outcome. The knee joint including the femorotibial, patellofemoral and the proximal tibiofibular joints was totally resected without any view of articular cartilage and synovium. The resected limb was reconstructed with endoprosthesis of the knee joint. The average score of the limb function evaluated by ISOLS criteria was 69.3% in all patients. The limb function of these patients was almost the same as that of the patients who received the intra-articular procedure. The procedure of extra-articular resection was theoretically necessary and clinically practical.

Extraabdominal desmoid tumor with dissemination detected by thallium-201 scintigraphy.

Extraabdominal desmoid tumor is a locally aggressive tumor despite being histologically benign. To avoid local recurrence, it is important to preoperatively detect the exact localization and extension of the infiltrating or disseminating lesion in this tumor. We report a case of recurrent extraabdominal desmoid tumor, which arose in the posterior thigh region, detected with Tl-201 (Tl) scintigraphy. In this case, Tl accumulated in the small disseminating lesion and to the recurrent tumor. This lesion was not identified by palpation because of its small size, deep localization and absence of symptoms, although MR imaging, which was performed after the Tl scintigraphy, clearly showed the lesion. After tumor resection, Tl did not accumulate in any region. These results suggest that Tl scintigraphy may be useful, not only for the diagnosis of extraabdominal desmoid tumor, but also for the detection of the exact localization or extension of small infiltrating or disseminating lesions before treatment.

Case report of secondary chondrosarcoma showing spontaneous regression after frequent recurrences.

We report a case of secondary chondrosarcoma arising in the ilium showing spontaneous regression after frequent local tumor recurrences followed by multiple surgeries of marginal or intralesional excision. The patient was a 16-year-old boy who had been diagnosed as having multiple exostosis from 9 years of age. He experienced an increasing abdominal tumor mass that formed a huge tumor. Although marginal resection of the tumor was attempted, intraperitoneal dissemination was caused by rupture of the tumor capsule and the peritoneum, as a result of severe tumor adhesion to the peritoneum. During the 5 years after the initial operation, local recurrences occurred seven times in various areas of the intra- or retro-peritoneum and marginal or intralesional excision was performed every time for a total of 14 tumors. However, since the seventh operation, the patient has had no evidence of recurrence or metastasis of the disease for more than 10 years. Therefore, we considered that the cancer might have spontaneously regressed.

Assessment of chemosensitivity in patients with malignant bone and soft tissue tumors using thallium-201 scintigraphy and doxorubicin binding assay.

This study was performed to compare the accumulation of thallium (Tl)-201 which is correlated with malignancy and the doxorubicin binding ability, which is correlated with chemosensitivity, in nine patients who received preoperative chemotherapy with doxorubicin and cisplatin. Tl-201 scintigraphy was performed at 15 minutes (early image) and 3 hours (delayed image) after injection of 111 MBq of Tl-201. The change of degree of the radionuclide uptake between the early and delayed images was evaluated before and after preoperative chemotherapy. The doxorubicin binding ability (%DB) to nuclear DNA in living tumor cells isolated from biopsy materials was assessed by doxorubicin binding assay. The histologic response to preoperative chemotherapy was evaluated by the percentage of tumor necrosis. Before preoperative chemotherapy no changes of Tl-201 uptake between the early and delayed images was detected in any tumors. Five patients, who had no change of Tl-201 uptake after preoperative chemotherapy, showed a poor histologic response and had a %DB ranging from 10% to 70% (mean: 36.0%). The other four patients, who had a %DB greater than 90%, showed a good histologic response. All of these four patients had decreased Tl-201 uptake after preoperative chemotherapy. This study demonstrated that doxorubicin binding assay and midcourse Tl-201 scintigraphy are useful methods to assess the response to chemotherapy early in malignant bone and soft tissue tumors.