Clinical Characteristics Associated with the Development of Diabetic Ketoacidosis in Patients with Type 2 Diabetes.

Objective This study analyzed the clinical and laboratory parameters that might influence the clinical outcomes of patients with type 2 diabetes who develop diabetic ketoacidosis (DKA), which has not been well investigated. Methods We reviewed the clinical and laboratory data of 158 patients who were hospitalized due to DKA between January 2006 and June 2019 and compared the data of patients stratified by the type of diabetes. In addition, the patients with type 2 diabetes were subdivided according to age, and their clinical and laboratory findings were evaluated. Results Patients with type 2 diabetes had a longer symptom duration associated with DKA, higher body mass index (BMI), and higher C-peptide levels than those with type 1 diabetes (p<0.05). Among patients with type 2 diabetes, elderly patients (≥65 years old) had a longer duration of diabetes, higher frequency of DKA onset under diabetes treatment, higher effective osmolarity, lower BMI, and lower urinary C-peptide levels than nonelderly patients (<65 years old) (p<0.05). A correlation analysis showed that age was significantly negatively correlated with the index of insulin secretory capacity. Conclusion Patients with DKA and type 2 diabetes had a higher BMI and insulin secretion capacity than those with type 1 diabetes. However, elderly patients with type 2 diabetes, unlike younger patients, were characterized by a lean body, impaired insulin secretion, and more frequent DKA development while undergoing treatment for diabetes.

Myristoyl group-aided protein import into the mitochondrial intermembrane space.

The MICOS complex mediates formation of the crista junctions in mitochondria. Here we analyzed the mitochondrial import pathways for the six yeast MICOS subunits as a step toward understanding of the assembly mechanisms of the MICOS complex. Mic10, Mic12, Mic26, Mic27, and Mic60 used the presequence pathway to reach the intermembrane space (IMS). In contrast, Mic19 took the TIM40/MIA pathway, through its CHCH domain, to reach the IMS. Unlike canonical TIM40/MIA substrates, presence of the N-terminal unfolded DUF domain impaired the import efficiency of Mic19, yet N-terminal myristoylation of Mic19 circumvented this effect. The myristoyl group of Mic19 binds to Tom20 of the TOM complex as well as the outer membrane, which may lead to "entropy pushing" of the DUF domain followed by the CHCH domain of Mic19 into the import channel, thereby achieving efficient import.

Inhibition of tumor formation and metastasis by a monoclonal antibody against lymphatic vessel endothelial hyaluronan receptor 1.

Although cancer metastasis is associated with poor prognosis, the mechanisms of this event, especially via lymphatic vessels, remain unclear. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is expressed on lymphatic vessel endothelium and is considered to be a specific marker of lymphatic vessels, but it is unknown how LYVE-1 is involved in the growth and metastasis of cancer cells. We produced rat monoclonal antibodies (mAb) recognizing the extracellular domain of mouse LYVE-1, and investigated the roles of LYVE-1 in tumor formation and metastasis. The mAb 38M and 64R were selected from hybridoma clones created by cell fusion between spleen cells of rats immunized with RH7777 rat hepatoma cells expressing green fluorescent protein (GFP)-fused mouse LYVE-1 proteins and mouse myeloma cells. Two mAb reacted with RH7777 and HEK293F human embryonic kidney cells expressing GFP-fused mouse LYVE-1 proteins in a GFP expression-dependent manner, and each recognized a distinct epitope. On immunohistology, the 38M mAb specifically stained lymphatic vessels in several mouse tissues. In the wound healing assay, the 64R mAb inhibited cell migration of HEK293F cells expressing LYVE-1 and mouse lymphatic endothelial cells (LEC), as well as tube formation by LEC. Furthermore, this mAb inhibited primary tumor formation and metastasis to lymph nodes in metastatic MDA-MB-231 xenograft models. This shows that LYVE-1 is involved in primary tumor formation and metastasis, and it may be a promising molecular target for cancer therapy.

Mitochondrial hyperfusion causes neuropathy in a fly model of CMT2A.

Mitochondria undergo frequent fusion and fission events, which are essential to maintain a functional mitochondrial network. A disruption of these processes can cause severe neurodegeneration. Charcot-Marie-Tooth disease type 2A (CMT2A) is a neuropathy that is caused by mutations in the fusion factor Mfn2. It is generally assumed that impaired mitochondrial fusion causes CMT2A. However, the detailed molecular mechanism underlying the pathophysiology of CMT2A is only incompletely understood. In this issue of EMBO Reports, El Fissi et al established a fly model to analyze the consequence of frequently occurring MFN2 mutations on locomotor behavior, mitochondrial morphology, and function and find that some pathogenic mutants enhance fusion activity, indicating that increased mitochondrial fusion can drive CMT2A-like pathology [1]. Moreover, this novel assay system will be a useful tool to analyze CMT2A pathogenesis in vivo.

Anti-tumor effect against human cancer xenografts by a fully human monoclonal antibody to a variant 8-epitope of CD44R1 expressed on cancer stem cells.

BACKGROUND: CD44 is a major cellular receptor for hyaluronic acids. The stem structure of CD44 encoded by ten normal exons can be enlarged by ten variant exons (v1-v10) by alternative splicing. We have succeeded in preparing MV5 fully human IgM and its class-switched GV5 IgG monoclonal antibody (mAb) recognizing the extracellular domain of a CD44R1 isoform that contains the inserted region coded by variant (v8, v9 and v10) exons and is expressed on the surface of various human epithelial cancer cells. METHODS AND PRINCIPAL FINDINGS: We demonstrated the growth inhibition of human cancer xenografts by a GV5 IgG mAb reshaped from an MV5 IgM. The epitope recognized by MV5 and GV5 was identified to a v8-coding region by the analysis of mAb binding to various recombinant CD44 proteins by enzyme-linked immunosorbent assay. GV5 showed preferential reactivity against various malignant human cells versus normal human cells assessed by flow cytometry and immunohistological analysis. When ME180 human uterine cervix carcinoma cells were subcutaneously inoculated to athymic mice with GV5, significant inhibition of tumor formation was observed. Furthermore, intraperitoneal injections of GV5markedly inhibited the growth of visible established tumors from HSC-3 human larynx carcinoma cells that had been subcutaneously transplanted one week before the first treatment with GV5. From in vitro experiments, antibody-dependent cellular cytotoxicity and internalization of CD44R1 seemed to be possible mechanisms for in vivo anti-tumor activity by GV5. CONCLUSIONS: CD44R1 is an excellent molecular target for mAb therapy of cancer, possibly superior to molecules targeted by existing therapeutic mAb, such as Trastuzumab and Cetuximab recognizing human epidermal growth factor receptor family.

Mutations in the NS5B region of the hepatitis C virus genome correlate with clinical outcomes of interferon-alpha plus ribavirin combination therapy.

BACKGROUND AND AIM: Combination treatments of interferon-alpha (IFN) and ribavirin (RBV) are more effective than those of IFN alone in hepatitis C virus (HCV) infection. However, mechanisms of the action of the combination regimen are not well understood. To elucidate the viral genetic basis of IFN plus RBV combination therapy, genetic variabilities of HCV-1b were analyzed. METHODS: We performed pair-wise comparisons of full-length HCV genomic sequences in three patients' sera before and after initiation of IFN plus RBV treatment. Subsequently, we analyzed amino acid sequences of the NS5B region, which codes for the viral RNA-dependent RNA polymerase, and compared these with the outcomes of the therapy in 81 patients. RESULTS: Analysis of the entire HCV sequence in patients who received IFN plus RBV therapy did not show consistent amino acid changes between before and after the initiation of the therapy. NS5B sequence analyses revealed that mutations at positions 300-358 of NS5B, including polymerase motif B to E, occurred more frequently in a group of patients exhibiting a sustained viral response (SVR) or an end-of-treatment response (ETR) compared with a group of patients exhibiting a non-response (NR). Closer examination revealed that mutations at aa 309, 333, 338 and 355 of NS5B occurred significantly more frequently in the SVR plus ETR group than in the NR group (P = 0.0004). Multivariate analysis showed that the number of mutations at these four sites was an independent predictor of SVR plus ETR versus NR. CONCLUSIONS: Particular amino acid changes in the NS5B region of HCV may correlate with outcomes of IFN plus RBV combination therapy.

Changes of HCV quasispecies during combination therapy with interferon and ribavirin.

Treatment of chronic hepatitis C virus (HCV) infection with interferon (IFN) and ribavirin improves the rate of eradication of HCV, but only about 13-14% of non-responders (NR) with HCV of genotype 1b previously treated with IFN achieve a sustained virological response (SVR). To determine whether HCV quasispecies diversity correlates with the outcome of therapy with IFN and ribavirin, we studied 13 patients undergoing combination therapy with IFN-alpha2b and ribavirin after failure of IFN monotherapy. HCV quasispecies diversity was assessed by cloning and sequencing before and during combination therapy. During therapy, quasispecies diversity diminished in NR patients, both in the hypervariable region (HVR) 1 of the envelope 2 (E2) domain and in the interferon sensitivity-determining region (ISDR) in the NS5A. Pre-treatment nucleotide quasispecies diversity was lower in SVR and end-of-therapy viral response (ETR) patients than in NR patients. Resistance to ribavirin was associated with high pre-treatment heterogeneity and the selection of quasispecies of the HCV genome. HVR quasispecies may be a predictor of efficacy of combination therapy with IFN and ribavirin.

Synergistic inhibition of intracellular hepatitis C virus replication by combination of ribavirin and interferon- alpha.

Treatment of hepatitis C virus (HCV) infection with interferon (IFN)- alpha and ribavirin combination therapy results in superior clinical antiviral responses than does monotherapy with IFN. To explore the virological basis of the effects of combination therapy, we analyzed the effects of IFN- alpha and ribavirin, singly and in combination, on intracellular HCV replication by use of an HCV replicon system. A new replicon that expressed a selectable chimeric reporter protein comprising firefly luciferase and neomycin phosphotransferase was constructed. The replicon was highly sensitive to IFN-alpha (50% inhibitory concentration [IC(50)], 0.5 U/mL). Therapy with ribavirin showed weak suppression of HCV replication at a lower concentration (IC(50), 126 mu mol/L). The nucleotide sequence diversity of the replicon was increased significantly by therapy with ribavirin, suggesting that error-prone HCV replication was induced by the drug. Importantly, use of a clinically achievable concentration of ribavirin (approximately 10 mu mol/L) in combination with IFN showed strong synergistic inhibitory effects on HCV replication. Our results suggest that the direct effects of ribavirin on the genetic stability of the HCV subgenome and its synergistic action combined, with IFN-alpha, may explain the improved clinical responses to combination therapy.

Hepatitis C virus nonstructural protein 5A inhibits tumor necrosis factor-alpha-mediated apoptosis in Huh7 cells.

To analyze the influence of hepatitis C virus nonstructural protein 5A (NS5A) on apoptosis, we established Huh7 cells that stably express NS5A, and induced apoptosis using tumor necrosis factor (TNF)-alpha. The viability of control Huh7 cells was reduced to 40%, compared with untreated cells, after TNF-alpha treatment, whereas that of Huh7-NS5A cells was reduced only to 80%. DNA fragmentation also decreased to <50% in Huh7-NS5A compared with control cells. Nuclear factor-kappaB activation was the same in both cell types, whereas caspase-8, -9, and -3 activity was decreased in Huh7-NS5A cells, compared with control cells, which indicates that the inhibition of caspase-8 activation is responsible for the antiapoptotic effect of the NS5A protein. The coexpression of NS5A did not inhibit apoptosis induced by caspase-8 or Fas-associating death domain protein expression. These findings suggest that the NS5A protein inhibits the apoptotic effect of TNF-alpha upstream of caspase-8 in the apoptosis cascade.