Pleiotrophin-Neuregulin1 promote axon regeneration and sorting in conduit repair of critical nerve gap injuries.

Significant challenges remain in the treatment of critical nerve gap injuries using artificial nerve conduits. We previously reported successful axon regeneration across a 40 mm nerve gap using a biosynthetic nerve implant (BNI) with multi-luminal synergistic growth factor release. However, axon sorting, remyelination, and functional recovery were limited. Neuregulin1 (NRG1) plays a significant role in regulating the proliferation and differentiation of Schwann cells (SCs) during development and after injury. We hypothesize that the release of NRG1 type III combined with pleiotrophin (PTN) in the BNI will enhance axon growth, remyelination, and function of regenerated nerves across a critical gap. A rabbit 40 mm peroneal gap injury model was used to investigate the therapeutic efficacy of BNIs containing either NRG1, PTN, or PTN+NRG1 growth factor release. We found that NRG1 treatment doubled the number of regenerated axons (1276±895) compared to empty controls (633±666) and PTN tripled this number (2270±989). NRG1 also significantly increased the number of SOX10+ Schwann cells in mid-conduit (20.42%±11.78%) and reduced the number of abnormal Remak axon bundles. The combination of PTN+NRG1 increased axon diameter (1.70±1.06) vs control (1.21±0.77) (p<0.01), with 15.35% of axons above 3 µm, comparable to autograft. However, the total number of remyelinated axons was not increased by the added NRG1 release, which correlated with absence of axonal NRG1 type III expression in the regenerated axons. Electrophysiological evaluation showed higher muscle force recruitment (23.8±16.0 mN vs 17.4±1.4 mN) and maximum evoked compound motor action potential (353 µV vs 37 µV) in PTN-NRG1 group versus control, which correlated with the improvement in the toe spread recovery observed in PTN-NRG1 treated animals (0.64±0.02) vs control (0.50±0.01). These results revealed the need of a combination of pro-regenerative and remyelinating growth factor combination therapy for the repair of critical nerve gaps.

12-OH-17,18-Epoxyeicosatetraenoic acid alleviates eosinophilic airway inflammation in murine lungs.

BACKGROUND: Asthma is characterized by airway inflammation and obstruction with eosinophil infiltration into the airway. Arachidonic acid, an omega-6 fatty acid, is metabolized into cysteinyl leukotriene with pro-inflammatory properties for allergic inflammation, whereas the omega-3 fatty acid eicosapentaenoic acid (EPA) and its downstream metabolites are known to have anti-inflammatory effects. In this study, we investigated the mechanism underlying the counter-regulatory roles of EPA in inflamed lungs. METHODS: Male C57BL6 mice were sensitized and challenged by ovalbumin (OVA). After EPA treatment, we evaluated the cell count of Bronchoalveolar lavage fluid (BALF), mRNA expressions in the lungs by q-PCR, and the amounts of lipid mediators by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics. We investigated the effect of the metabolite of EPA by in vivo and in vitro studies. RESULTS: Eicosapentaenoic acid treatment reduced the accumulation of eosinophils in the airway and decreased mRNA expression of selected inflammatory mediators in the lung. Lipidomics clarified the metabolomic profile in the lungs. Among EPA-derived metabolites, 12-hydroxy-17,18-epoxyeicosatetraenoic acid (12-OH-17,18-EpETE) was identified as one of the major biosynthesized molecules; the production of this molecule was amplified by EPA administration and allergic inflammation. Intravenous administration of 12-OH-17,18-EpETE attenuated airway eosinophilic inflammation through downregulation of C-C chemokine motif 11 (CCL11) mRNA expression in the lungs. In vitro, this molecule also inhibited the release of CCL11 from human airway epithelial cells stimulated with interleukin-4. CONCLUSION: These results demonstrated that EPA alleviated airway eosinophilic inflammation through its conversion into bioactive metabolites. Additionally, our results suggest that 12-OH-17,18-EpETE is a potential therapeutic target for the management of asthma.

Abnormal asymmetries in subcortical brain volume in schizophrenia.

Subcortical structures, which include the basal ganglia and parts of the limbic system, have key roles in learning, motor control and emotion, but also contribute to higher-order executive functions. Prior studies have reported volumetric alterations in subcortical regions in schizophrenia. Reported results have sometimes been heterogeneous, and few large-scale investigations have been conducted. Moreover, few large-scale studies have assessed asymmetries of subcortical volumes in schizophrenia. Here, as a work completely independent of a study performed by the ENIGMA consortium, we conducted a large-scale multisite study of subcortical volumetric differences between patients with schizophrenia and controls. We also explored the laterality of subcortical regions to identify characteristic similarities and differences between them. T1-weighted images from 1680 healthy individuals and 884 patients with schizophrenia, obtained with 15 imaging protocols at 11 sites, were processed with FreeSurfer. Group differences were calculated for each protocol and meta-analyzed. Compared with controls, patients with schizophrenia demonstrated smaller bilateral hippocampus, amygdala, thalamus and accumbens volumes as well as intracranial volume, but larger bilateral caudate, putamen, pallidum and lateral ventricle volumes. We replicated the rank order of effect sizes for subcortical volumetric changes in schizophrenia reported by the ENIGMA consortium. Further, we revealed leftward asymmetry for thalamus, lateral ventricle, caudate and putamen volumes, and rightward asymmetry for amygdala and hippocampal volumes in both controls and patients with schizophrenia. Also, we demonstrated a schizophrenia-specific leftward asymmetry for pallidum volume. These findings suggest the possibility of aberrant laterality in neural pathways and connectivity patterns related to the pallidum in schizophrenia.

A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2.

Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.

[Combination chemotherapy of carboplatin and docetaxel for advanced non-small cell lung cancer (NSCLC)].

Since mid-1997, we have treated 60 patients with advanced NSCLC with carboplatin (CBDCA) plus docetaxel (TXT). CBDCA (300-400 mg/m2) and TXT (60 mg/m2) were given on day 1 every 3 weeks. The mean treatment cycle was 2.3 +/- 1.4 (range 1-7). Fifty-four patients had measurable tumors, of whom 2 patients achieved a complete response and 19 patients achieved a partial response (37.0%, 95% CI: 24.3-51.3%) (Ad 12/29, Sq 7/23, Lar 1/2). Median survival time was 12.8 months and 1 year survival was 53.6%. AUC of CBDCA was not related to response (AUC of responders and non-responders was 3.29). Myelosuppression was moderate (WBC 2,284 mm3, range 800-4,700, PLT 16.4 x 10(4) mm3, range 6.6-41.5 x 10(4), Hb 10.9 g/dl, range 6.4-15.8). Leukocytopenia was related to AUC of CBDCA (R2 = 0.1093) but thrombocytopenia was not related to AUC of CBDCA (R2 = 0.0553). Gastrointestinal toxicity was mild (grade 0-1: 57%, grade 2: 35%, grade 3: 8%, grade 4: 0%). Treatment with CBDCA plus TXT combination is safe and effective in patients with NSCLC.

Retrospective analysis of administration of a combination of docetaxel and carboplatin for advanced non-small cell lung cancer.

Retrospective analysis was performed for 60 patients with advanced non-small-cell lung cancer (NSCLC) who had been treated with combination therapy combining 60 mg/m2 of docetaxel with carboplatin in the range of 200 to 360 mg/m2 (average: 290 mg/m2) every 3 weeks. Considering the patients' performance status, the dose of carboplatin was lowered accordingly, being equivalent to AUC 1.9 to 6.1 (average: 3.26) by the Chatelut formula. The mean treatment cycle was 2.3 (range 1 to 7). Complete response and partial response were observed in 2 and 18 (37.0%) of the 54 evaluable patients, respectively, with a median survival time of 12.8 months and 1-year survival of 56.4%. The calculated AUC of carboplatin was not proportional to the response rate. Moderate myelosuppression was exhibited. The severity of leukopenia increased in relation to the AUC of carboplatin (R2=0.1093), whereas the relation between the platelet count and the AUC of carboplatin was relatively disproportional (R2=0.0553). Although gastrointestinal toxicity was slight, its severity increased dependent on the AUC of carboplatin. No occurrence of neurotoxicity was observed. Treatment with a combination of docetaxel and low-dose carboplatin seemed to be effective and safer in patients with advanced NSCLC.

[A trial with single-agent vinorelbine in taxane-resistant non-small cell lung cancer].

It is reported that the single-agent administration of vinorelbine (VNR) is improper in salvage therapy for non-small cell lung cancer. However, there are few reports on its use as second line in taxane-containing chemotherapy. We used single-agent VNR administration for nine cases of taxane-resistant non-small cell lung cancer, and an antitumor effect was seen in four cases. We present three of these cases. A factor for the high response rate is considered to be that vinca alkaloid is not used as a pre-treatment. Moreover, VNR may be effective even if there is a gene mutation for beta tubulin, which causes taxane resistance.

Identification and characterization of the miniature pig Huntington's disease gene homolog: evidence for conservation and polymorphism in the CAG triplet repeat.

Huntington's disease (HD) is associated with a significant expansion of a CAG trinucleotide repeat, which results in a lengthened polyglutamine tract in the single gene product, huntingtin, on human 4p16.3. We isolated cDNA clones that encompassed the entire coding sequence of the miniature pig HD gene (Sus HD) from two porcine testis cDNA libraries. The cDNA contig revealed a 12,749-nucleotide transcript coding for a 345-kDa protein (3139 amino acid residues), which exhibited 96% peptide sequence homology to human huntingtin. Northern blot analysis revealed that the Sus HD gene was ubiquitously expressed as two large transcripts of approximately 11 and 13 kb in size in all the tested tissues, much like the human HD gene. The CAG trinucleotide repeat was found to be interrupted by CAA triplets and to encode 17 or 18 consecutive glutamine residues. In our laboratory stock of miniature pig, three allotypes in the triplet repeat sequence were found. Thus, the Sus HD gene closely resembles its human counterpart in terms of sequence and expression pattern. In particular, human-miniature pig similarities in the normal length of the CAG triplet repeat as well as its repeat-number polymorphism may indicate that miniature pig would provide a good animal model for Huntington's disease.

The RS447 human megasatellite tandem repetitive sequence encodes a novel deubiquitinating enzyme with a functional promoter.

We have recently identified a tandem repetitive DNA sequence that we designated the RS447 megasatellite. In this study, we describe a functional novel deubiquitinating enzyme (USP17, 60 kDa) gene that is intronless and encoded by the RS447 repeating unit. Northern blot analysis in conjunction with 5' and 3' rapid amplification of cDNA ends confirmed the presence of poly(A)(+) containing RS447 RNA in normal cells. We also identified a functional promoter sequence as well as an open reading frame within every RS447 repeat. When USP17 was expressed in Escherichia coli, it exhibited deubiquitinating activity in vivo. An antibody against USP17 detected USP17 protein in human cells. Our results indicate that the RS447 repeating unit on this megasatellite repeat codes for and actively expresses a functional deubiquitinating enzyme. Although it is expressed ubiquitously in human tissues, USP17 exhibited a unique expression pattern in that its complementary strand is transcribed as an antisense transcript that may modulate the level of USP17 expression in the human brain.

Asymmetric synthesis of 4-deoxyverrucarol via two types of ring expansion reactions

Asymmetric synthesis of a trichothecane analogue, 4-deoxyverrucarol (2), was carried out through two types of ring expansion reactions. First, synthesis of the racemate of 2 was investigated. Thus, 1-[1-(tert-butyldimethylsiloxy)-ethyl]-1-methoxycarbonyl-2-hexen-4-on e (10), prepared by Diels-Alder reaction, was converted into the cyclopropylidene 15. The cyclobutanone (+/-)-18 was obtained from 15 via dihydroxylation, followed by successive treatments with SO(2)Cl(2) in the presence of imidazole and Florisil. After transformation of (+/-)-18 into the vinylcyclobutanol (+/-)-19, the second ring expansion reaction was performed with Pd(OAc)(2) to provide the cyclopentanone (+/-)-20. The product was converted into the racemate of 4-deoxyverrucarol (2) through the cyclohexenone (+/-)-22, but the diastereoselectivity during the introduction of the double bond was unsatisfactory. The selectivity was improved in the case of the asymmetric synthesis. The optically active cyclobutanone (+)-18 was prepared via AD reaction of 15 with 73% ee. After the transformation of (+)-18 into the cyclohexanone (-)-30 through the palladium-mediated ring expansion reaction, (-)-30 was subjected to the diastereoselective deprotonation reaction using the chiral amide. The key synthetic intermediate (-)-25 of 4-deoxyverrucarol (2) was synthesized in an optically pure form by taking advantage of a kind of kinetic resolution that occurred during the deprotonation step.

[Clinicopathologic study of adult-onset membranoproliferative glomerulonephritis].

We analyzed clinicopathologic characteristics of adult-onset membranoproliferative glomerulonephritis (MPGN) by comparing two tentatively-classified subgroups. Group A consisted of 9 patients in whom more than 50% of glomeruli showed mixed segmental and global duplication of glomerular basement membrane (GBM), and Group B 9 patients with global duplication of GBM. Group A showed a tendency for more favorable clinical course and outcome than Group B. Nephrotic syndrome was present in 33% of Group A and 100% in Group B, hypertension in 22% and 44%, hypocomplementemia in 44% and 67% at the time of renal biopsy. Deterioration of renal function, at comparable durations of follow-up of 62 +/- 12 (M +/- SE) and 53 +/- 13 months, was observed in 22% of Group A and 56% in Group B, respectively. Histologically, mesangial proliferation and tubulointerstitial change were more pronounced and frequency of sclerosing glomeruli was greater in Group B. There was also a negative correlation between the extent of global double contour and renal function as assessed by creatinine clearance at the time of renal biopsy (r = -0.55, P less than 0.05). These results indicate that the high incidence of global double contour, in addition to the presence of marked tubulointerstitial change and sclerosing glomeruli, may relate to progressive deterioration of renal function in MPGN. That outcomes for Group A and Group B may be different when clinical parameters, including the durations from onset of symptoms to the time of biopsy and length of follow-up periods, are comparable also indicates that these two groups should be considered separate entities, at least on the basis of clinical results.

[Changes in plasma, erythrocytes and tissue polyamine levels in patients with colorectal cancer].

Polyamines (putrescine, spermidine, spermine) are closely linked to cellular synthesis of DNA, RNA and protein, and are thought to be an indicator of cell proliferation. Plasma, erythrocytes and tissue polyamine levels in 58 patients with colorectal cancer were measured to survey the relationship between polyamines and stage classification. The polyamine levels of plasma, erythrocytes and tissue in patients were increased significantly compared with those of controls. Plasma spermine and erythrocytes spermidine and spermine levels were increased with the advance of stage. In plasma and erythrocytes, spermidine/spermine ratios were decreased in accordance with the stages. On the contrary, the polyamine levels and the ratio in cancer tissue were not varied in all stages. These results present the following conclusions. Although cancer tissue has higher proliferative activity than normal mucosa, these activities of main tumors in each stage are not fluctuated. The polyamine levels in plasma and erythrocytes are possibly influenced by tumor burden and therefore those could be an useful marker for indicating the stage of colorectal cancer.

Effect of hyperlipidemia on glomerular sclerosis in unilateral nephrectomized rats.

The development of focal segmental glomerular sclerosis (FSGS) and its relation to hypertriglycemia were studied in unilateral nephrectomized rats. Group A (n = 6), fed standard rat chow supplemented with 20% beef tallow and 0.6% cholic acid for 25 weeks, showed evidence of hypertriglycemia (109.4 +/- 4.3 mg/dl). Group B (n = 7) was given the same rat chow as group A, but they did not have high serum levels of TG (66.4 +/- 2.3 mg/dl). Group C (n = 6) were the controls and their serum TG levels were 53.0 +/- 3.8 mg/dl. The incidence of FSGS and body weight was significantly higher in group A than in groups B (p less than 0.01) and C (p less than 0.05). In all three groups, rats with over a 4% FSGS revealed significantly high serum TG levels, proteinuria, and body weight, as compared with rats with less than 1% of FSGS. The serum cholesterol levels did not correlate with the incidence of FSGS. We tentatively conclude that hypertriglycemia induced by a diet rich in saturated fatty acid may play an important role in the production and progression of FSGS.

Hyperamylasemia associated with endometrioid carcinoma of the ovary: case report and immunohistochemical study.

Hyperamylasemia was noted in a patient with a stage I well-differentiated endometrioid adenocarcinoma of the ovary. Serum levels of amylase decreased rapidly after removal of the ovarian tumor. Immunohistochemical study revealed intracytoplasmic localization of amylase in many tumor cells. Although it was strongly suspected that the tumor cells had produced amylase in the present patient, further studies are required to be sure that amylase can be considered an important tumor marker for the endometrioid type of ovarian tumors.

Beneficial effects of polyunsaturated fatty acids in partially nephrectomized rats.

Evening primrose oil, safflower oil, and salmon oil, all with high polyunsaturated fatty acid content, were fed to partially nephrectomized rats; the effects were compared to those of feeding beef tallow. All three oils had favorable effects on progression of renal failure, salmon oil on kidney histology as well. The changes induced in platelet production of thromboxane A2, and in the renal production of various eicosanoids may explain the protective role of these oils.

Evaluation and correlation of clinical and histological features of focal segmental glomerulosclerosis.

Clinico-histological features in 32 patients with nephrotic syndrome (NS) due to focal segmental glomerulosclerosis (FSGS) were examined. Thirteen (group A1) were diagnosed as cases of FSGS within 2 years of the onset of NS, and 8 (61%) showed progressive renal dysfunction. Ten (group A2) developed FSGS more than 2 years after the onset of NS and had a favorable prognosis. Nine (group B) differed from groups A1 and A2 in that the remaining nonsclerosed glomeruli showed slight mesangial proliferation. All but 1 patient of group B developed FSGS within 2 years of the onset of NS, and the prognosis was poor. No patient studied showed a transition between groups A and B. In some patients, lipoid nephrosis preceded FSGS, in group A2. Thus, for an accurate prediction of the prognosis, FSGS should be divided into three subclasses, based on clinico-histological features.

The strong association of HLA-DR4 with spherical mesangial dense deposits in IgA nephropathy.

41 Japanese patients with primary IgA nephropathy (IgA GN) were determined for HLA-A, -B, -C and -DR antigens. The frequency of HLA-DR4 increased slightly in total patients compared with 63 normal control persons, but this increase was not statistically significant. However, 24 patients with spherical mesangial dense deposits (SMDD) in electron micrograph out of 41 patients with IgA GN were found to have markedly increased incidence of HLA-DR4 (chi 2 = 11.52, Relative risk = 6.45, Corrected p less than 0.03). In contrast to this finding, 17 patients without SMDD had no association with any particular HLA antigens. These results suggest that IgA GN might be subdivided by the association of HLA and electron microscopic findings into at least 2 types.