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Background: Kinesin family member C1 (KIFC1) is an essential member of the motor protein family, which is critically involved in various cellular events, such as mitosis, meiosis, and macromolecular transport, but also in carcinogenesis, malignant progression, and tumor recurrence., Methods: The analysis determined the relationship between KIFC1 expression, prognosis significance, immune characteristics landscape, and genetic alterations in pan-cancer with the data extracted from web-based platforms and databases, including but not limited to UCSC, NCBI, GEPIA2, HPA, cBioPortal, SangerBox, UALCAN, GEO and TCGA. Additionally, the expression of KIFC1 in pancreatic cancer tumor tissues and adjacent normal tissues was evaluated through immunohistochemistry. In vitro Edu, colony formation, wound healing, and Transwell assay were done to elucidate the biological functions of KIFC1 in pancreatic cancer cells., Results: The analysis revealed that KIFC1 is upregulated in most cancers, and its increased expression is significantly associated with reduced overall survival and disease-free survival in multiple cancer types. Additionally, strong correlations between KIFC1 expression and tumor immunotherapy were observed across various malignancies. Through univariate and multivariate Cox regression analyses using TCGA data, KIFC1 was identified as an independent predictor of prognosis in pancreatic cancer cases. Furthermore, cellular experiments demonstrated that knockdown of KIFC1 resulted in the suppression of cell proliferation, migration, and invasive ability., Conclusions: Our study indicated that KIFC1 harbors the potential to be a prognostic and immunotherapeutic biomarker of tumors, and it can have an impact on the metastasis and the cell cycle of pancreatic cancer cells.

Autophagy induction by starvation has been shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of the Mycobacterium tuberculosis reference strain H37Rv. In contrast to H37Rv, our previous study showed that strains belonging to the notorious M. tuberculosis Beijing genotype could evade autophagic elimination. Our recent RNA-Seq analysis also discovered that the autophagy-resistant M. tuberculosis Beijing strain (BJN) evaded autophagic control by upregulating the expression of Kxd1, a BORC complex component, and Plekhm2, both of which function in lysosome positioning towards the cell periphery in host macrophages, thereby suppressing enhanced lysosomal delivery to its phagosome and sparing the BJN from elimination as a result. In this work, we further characterised the other specific components of the BORC complex, BORC5-8, and Kinesin proteins in autophagy resistance by the BJN. Depletion of BORCS5-8 and Kinesin-1, but not Kinesin-3, reverted autophagy avoidance by the BJN, resulting in increased lysosomal delivery to the BJN phagosomes. In addition, the augmented lysosome relocation towards the perinuclear region could now be observed in the BJN-infected host cells depleted in BORCS5-8 and Kinesin-1 expressions. Taken together, the data uncovered new roles for BORCS5-8 and Kinesin-1 in autophagy evasion by the BJN., (© 2023. The Author(s).)

Abstract: Cholangiocarcinoma (CCA) is one of the most common malignant tumors. Although gene-targeted therapies have significantly improved the outcome of many cancers, the results are still not satisfactory for patients with CCA. Owing to the lack of an effective biomarker for guiding clinical treatment and monitoring prognosis in patients with CCA, the purpose of this study was to identify a new biomarker that could help predict the outcome of patients with CCA using bioinformatics tools.Gene expression data were collected from three publicly available datasets, comprising 263 patients with CCA and 22 healthy controls. Differentially expressed genes were obtained using the limma package (FDR < 0.05, |Log2FC|>1), and the respective protein-protein interaction revealed five relevant genes in the STRING dataset (TOP2A, BUB1, RRM2, TYMS, and KIF4A). The immunohistochemistry and PCR were used to analyze the difference in KIF4A expression in CCA.Kinesin Family Member 4A (KIF4A) was the only gene significantly associated with overall patient survival (P .035), with higher KIF4A expression being associated with poor survival rates. Moreover, KIF4A was significantly correlated with the infiltration of activated memory T cells (P = .0198) and activated mast cells (P = .008) in the tumor microenvironment. Increase in KIF4A expression affected the infiltration degree of the immune cells, which may be involved in the regulation of immune tolerance by CCA cells. The results indicated that the expression of KIF4A in CCA was higher than that in paracancerous tissues.Taken together, these findings suggest that KIF4A could be a potential new biomarker in CCA for predicting the response of patients to targeted immunotherapies., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: As the prognosis of biliary tract cancer (BTC) is extremely poor and treatment options are limited, new treatment modalities are urgently needed. We designed a phase II clinical trial to investigate the immune responses and clinical benefits of OCV-C01, an HLA-A*24:02-restricted three-peptide cancer vaccine targeting VEGFR1, VEGFR2, and KIF20A., Patients and Methods: Participants were patients with advanced BTC who had unresectable tumours and were refractory to standard chemotherapy. OCV-C01 was injected weekly until the discontinuance criteria were met., Results: Six participants, including four patients positive for HLA-A*24:02, were enrolled in this study for assessment of efficacy. Four out of six patients exhibited vaccine-specific T-cell responses to one or more of three antigens. Log-rank tests revealed that vaccine-specific T cell responses contributed significantly to overall survival., Conclusion: The cancer vaccine had positive effects on survival, indicating that this approach warrants further clinical studies., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Pediatric refractory solid tumors are aggressive malignant diseases, resulting in an extremely poor prognosis. KOC1, FOXM1, and KIF20A are cancer antigens that could be ideal targets for anticancer immunotherapy against pediatric refractory solid tumors with positive expression for these antigens. This nonrandomized, open-label, phase I clinical trial evaluated the safety and efficacy of the NCCV Cocktail-1 vaccine, which is a cocktail of cancer peptides derived from KOC1, FOXM1, and KIF20A, in patients with pediatric refractory solid tumors. Twelve patients with refractory pediatric solid tumors underwent NCCV Cocktail-1 vaccination weekly by intradermal injections. The primary endpoint was the safety of the NCCV Cocktail-1 vaccination, and the secondary endpoints were the immune response, as measured by interferon-r enzyme-linked immunospot assay, and the clinical outcomes including tumor response and progression-free survival. The NCCV Cocktail-1 vaccine was well tolerated. The clinical response of this trial showed that 4 patients had stable disease after 8 weeks and 2 patients maintained remission for >11 months. In 4, 8, and 5 patients, the NCCV Cocktail-1 vaccine induced the sufficient number of peptide-specific CTLs for KOC1, FOXM1, and KIF20A, respectively. Patients with high peptide-specific CTL frequencies for KOC1, FOXM1, and KIF20A had better progression-free survival than those with low frequencies. The findings of this clinical trial showed that the NCCV Cocktail-1 vaccine could be a novel therapeutic strategy, with adequate effects against pediatric refractory solid tumors. Future large-scale trials should evaluate the efficacy of the NCCV Cocktail-1 vaccination., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Kinesin-like protein KIF20B, originally named M-phase phosphoprotein 1 (MPP1), is a plus-end-directed kinesin-related protein that exhibits in vitro microtubule-binding and -bundling properties as well as microtubule-stimulated ATPase activity. It has been characterized as a slow molecular motor that moves toward the plus-end of microtubules. Human autoantibodies directed against KIF20B have been described in up to 25% of patients with idiopathic ataxia and less commonly in other neuropathies and autoinflammatory conditions. One of the limitations of research into the structure and function of KIF20B has been a reliable monoclonal antibody that can be used in a variety of applications. To establish a reference standard for anti-KIF20B immunoassays and facilitate studies on the role of KIF20B in developmental cell biology, we developed an IgG1 monoclonal antibody, 10C7, which reacts with the cognate KIF20B protein in Western immunoblots and in addressable laser bead immunoassays. In HEp2 cells, leptomeningeal pericytes, and transfected HEK293T cells, indirect immunofluorescence studies showed that reactivity was mainly localized to a proportion of interphase nuclei, but during metaphase, it was redistributed throughout the cytoplasm and perichromatin mass. Later in telophase/anaphase, KIF20B was localized to the stem body and midzone of the midbody. 10C7 also showed remarkable staining of a subset of cells in the cerebellum, ovary, and testis tissues. KIF20B was shown to have extensive coiled-coil domains. The monoclonal antibody, 10C7, will be of value to diagnostic laboratory scientists interested in having a reliable reference standard for anti-KIF20B immunoassays as well as cell, molecular, and developmental biology researchers.

Visceral leishmaniasis (VL) or kala-azar, the most severe form of leishmaniasis, can lead to death if not properly diagnosed and treated. Correct identification of infected patients and reservoirs is vital for controlling the spread of leishmaniasis. Current diagnostic kits for leishmaniasis show high sensitivity and specificity, but can also result in false negatives and cross reactions with related parasitic infections. New diagnostic methods with greater accuracy are urgently needed for diagnosis of leishmaniasis. In this study, we aimed to uncover a new highly effective antigen for the diagnosis of visceral leishmaniasis in dogs and humans, aiming to improve the accuracy compared with those of current methods of diagnosis. Initially, in-silico epitope prediction analyses identified several potential B-cell epitopes in the repetitive region of Leishmania infantum kinesin, which co-localized with predicted structural disordered regions, suggesting high potential for antigenicity. Based on this analysis, 8.5 genomic motifs, which encode the repetitive sequence of 39 degenerate amino acids, were selected for recombinant expression. BLASTn analysis of this repetitive region indicated that it is absent in the T. cruzi parasite, which is closely related to Leishmania, indicating the specificity of this region. This potentially antigenic protein, named recombinant kinesin degenerated derived repeat (rKDDR), was recombinantly expressed in Escherichia coli BL21-Star using the pET28a-TEV expression vector. We then evaluated the performance of rKDDR in correctly diagnosing Leishmania infection and compared this new assay with currently used diagnostic tests for leishmaniasis. rKDDR showed greater sensitivity and specificity in correctly diagnosing leishmaniasis both in human (sensitivity 92.86% and specificity 100%) and canine (sensitivity 88.54% and specificity 97.30%) sera compared with those of rK39 (human: sensitivity 90.48% and specificity 97.92%; canine: sensitivity 78.13% and specificity 90.09%). In addition, the rKDDR-ELISA outperformed the EIE-LVC kit, which is the serologic kit recommended by the Brazilian Ministry of Health for the diagnosis of canine visceral leishmaniasis. These results indicate that rKDDR is a highly promising candidate for diagnosis of visceral leishmaniasis, and is more accurate than the currently used gold-standard antigens., Competing Interests: The authors have declared that no competing interests exist.

Purpose: A phase I study using two peptide vaccines derived from M phase phosphoprotein 1 (MPHOSPH1) and DEP domain containing 1 (DEPDC1) demonstrated promising results for the treatment of advanced bladder cancer. Therefore, we further tested the ability of these peptides to prevent recurrence after transurethral resection of the bladder tumor in patients with non-muscle invasive bladder cancer (NMIBC)., Materials and Methods: 127 patients were enrolled in a multicenter, non-randomized phase II clinical trial. The primary endpoint was recurrence-free survival (RFS) rate, and secondary endpoints were safety and immunological response. HLA-A24-restricted peptides were subcutaneously administered in addition to intravesical BCG therapy. The exploratory endpoint evaluated differences of RFS rate between HLA-A*2402-positive (A24(+)) and -negative (A24(-)) groups., Results: A 2-year RFS rate in all patients was 74.0%. The RFS rate in the A24(+) group (n = 75) and in the A24(-) group (n = 52) were 76.0 and 71.2%, respectively. This vaccine therapy was well-tolerated and feasible. MPHOSPH1 and DEPDC1 peptide-specific cytotoxic T lymphocyte responses were observed in 75.8 and 77.5% of the A24(+) group, respectively. Patients having both peptide-specific CTL responses showed significantly better RFS than patients without CTL response (P = 0.014). In the A24(+) group, patients who had positive reaction at the injection sites (RAI) had significantly lower rates of recurrence than RAI-negative patients (P = 0.0019)., Conclusions: Cancer peptide vaccines in combination with intravesical BCG therapy demonstrated good immunogenicity and safety, and may provide benefit for preventing recurrence of NMIBC.

We performed a clinical trial using HLA-A24-binding peptide vaccines containing a combination of novel cancer-testis antigens and anti-angiogenic peptides for advanced gastric cancer (GC). Thirty-five GC patients who had shown resistance to the standard therapy were enrolled in this clinical trial using vaccinations with a mixture of multiple peptides derived from DEPDC1, URLC10, FoxM1, Kif20A and VEGFR1. The safety, the overall survival (OS), and the immunological responses based on an ELISPOT assay were determined to assess differences in patients who were HLA-A24-positive [24(+)] and HLA-A24-negative [24(-)]. No severe adverse effects were observed except for severe skin reactions in 4 patients. The differences in OS were not significant between patients who were 24(+) and 24(-). In the 24(+) group, patients who showed T cell responses specific to antigen peptides had a tendency towards better survival than those who showed no response, especially to the DEPDC1 peptide. The patients with local skin reactions had significantly better OS than the others. Peptide vaccine therapy was found to be safe and is expected to induce specific T cell responses in patients with advanced GC. The survival benefit of peptide vaccine monotherapy may not have been shown and further trials are needed to confirm these results.

The serodiagnosis for tegumentary leishmaniasis (TL) presents problems related to the sensitivity and/or specificity of the tests. In the present study, an enzyme-linked immunosorbent assay (ELISA) technique was used to evaluate the performance from a Leishmania braziliensis hypothetical protein, LbHyM, in an attempt to compare its serological reactivity with a soluble Leishmania antigenic preparation (SLA) for the serodiagnosis of cutaneous (CL) and mucosal (ML) leishmaniasis. LbHyM was predicted to be a kinesin-like protein by bioinformatics tools. Serum samples were collected from both CL and ML patients, as well as from those with Chagas disease and from healthy subjects living in endemic or non-endemic areas of TL. Also, sera were collected from patients before and after the treatments, seeking to evaluate their serological follow-up in relation to the anti-protein and anti-parasite antibody levels. When an ELISA-rLbHyM assay was performed, it proved to be significantly more sensitive than ELISA-L. braziliensis SLA in detecting both CL and ML patients. Also, when using sera from Chagas disease patients, the ELISA-rLbHyM proved to be more specific than ELISA-SLA. The anti-protein and anti-parasite antibody levels were also evaluated 6 months after the treatments, and treated patients showed significantly lower levels of specific-rLbHyM antibodies, when compared to the anti-parasite antibody levels. In conclusion, the ELISA-rLbHyM assay can be considered a confirmatory serological technique for the serodiagnosis of L. braziliensis infection and can also be used in the serological follow-up of treated patients, aiming to correlate the low anti-protein antibody levels with the improvement of the healthy state of the patients.

We investigated peptide cocktail vaccine OCV-C01 containing epitope peptides derived from KIF20A, vascular endothelial growth factor receptor (VEGFR)1 and VEGFR2 combined with gemcitabine in the adjuvant treatment for resected pancreatic cancer patients. A single-arm multicenter phase II study was performed on 30 patients with pancreatic ductal carcinoma who underwent pancreatectomy. At each 28-day treatment cycle, patients received weekly subcutaneous injection of OCV-C01 for 48 weeks and gemcitabine was administered intravenously at 1,000 mg/m 2 on days 1, 8 and 15 for 24 weeks. Patients were followed for 18 months. The primary endpoint was disease-free survival (DFS) and secondary endpoints included safety, overall survival (OS) and immunological assays on peptide-specific cytotoxic T lymphocyte (CTL) activity and KIF20A expression in resected pancreatic cancer. The median DFS was 15.8 months [95% confidence interval (CI), 11.1-20.6] and the DFS rate at 18 months was 34.6% (95% CI, 18.3-51.6). The median OS was not reached and the OS rate at 18 months was 69.0% (95% CI, 48.8-82.5). The administration of OCV-C01 was well tolerated. In the per protocol set, there were significant differences in DFS between patients with KIF20A-specific CTL responses and without (p = 0.027), and between patients with KIF20A expression and without (p = 0.014). In addition, all four patients who underwent R0 resection with KIF20A expression had no recurrence of pancreatic cancer with KIF20A-specific CTL responses. OCV-C01 combined with gemcitabine was tolerable with a median DFS of 15.8 months, which was favorable compared with previous data for resected pancreatic cancer., (© 2016 UICC.)

We previously conducted a phase I clinical trial combining the HLA-A*2402-restricted KIF20A-derived peptide vaccine with gemcitabine for advanced pancreatic cancer (PC) and confirmed its safety and immunogenicity in cancer patients. In this study, we conducted a multicenter, single-armed, phase II trial using two antiangiogenic cancer vaccines targeting VEGFR1 and VEGFR2 in addition to the KIF20A peptide. We attempted to evaluate the clinical benefit of the cancer vaccination in combination with gemcitabine. Chemotherapy naïve PC patients were enrolled to evaluate primarily the 1-year survival rate, and secondarily overall survival (OS), progression free survival (PFS), response rate (RR), disease control rate (DCR) and the peptide-specific immune responses. All enrolled patients received therapy without the HLA-A information, and the HLA genotypes were used for classification of the patients. Between June 2012 and May 2013, a total of 68 patients were enrolled. No severe systemic adverse effects of Grade 3 or higher related to these three peptides were observed. The 1-year survival rates between the HLA-A*2402-matched and -unmatched groups were not significantly different. In the HLA-A*2402 matched group, patients showing peptide-specific CTL induction for KIF20A or VEGFR1 showed a better prognosis compared to those without such induction (P = 0.023, P = 0.009, respectively). In the HLA-A*2402-matched group, the patients who showed a strong injection site reaction had a better survival rate (P = 0.017) compared to those with a weak or no injection site reaction. This phase II study demonstrated that this therapeutic peptide cocktail might be effective in patients who demonstrate peptide-specific immune reactions although predictive biomarkers are needed for patient selection in its further clinical application., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

KIF3A, the gene encoding kinesin family member 3A, is a susceptibility gene locus associated with asthma; however, mechanisms by which KIF3A might influence the pathogenesis of the disorder are unknown. In this study, we deleted the mouse Kif3a gene in airway epithelial cells. Both homozygous and heterozygous Kif3a gene-deleted mice were highly susceptible to aeroallergens from Aspergillus fumigatus and the house dust mite, resulting in an asthma-like pathology characterized by increased goblet cell metaplasia, airway hyperresponsiveness, and Th2-mediated inflammation. Deletion of the Kif3a gene increased the severity of pulmonary eosinophilic inflammation and expression of cytokines (Il-4, Il-13, and Il-17a) and chemokine (Ccl11) RNAs following pulmonary exposure to Aspergillus extract. Inhibition of Kif3a disrupted the structure of motile cilia and impaired mucociliary clearance, barrier function, and epithelial repair, demonstrating additional mechanisms by which deficiency of KIF3A in respiratory epithelial cells contributes to pulmonary pathology. Airway epithelial KIF3A suppresses Th2 pulmonary inflammation and airway hyperresponsiveness following aeroallergen exposure, implicating epithelial microtubular functions in the pathogenesis of Th2-mediated lung pathology., (Copyright © 2016 by The American Association of Immunologists, Inc.)

KIF20A (RAB6KIFL) belongs to the kinesin superfamily of motor proteins, which play critical roles in the trafficking of molecules and organelles during the growth of pancreatic cancer. Immunotherapy using a previously identified epitope peptide for KIF20A is expected to improve clinical outcomes. A phase I clinical trial combining KIF20A-derived peptide with gemcitabine (GEM) was therefore conducted among patients with advanced pancreatic cancer who had received prior therapy such as chemotherapy and/or radiotherapy. GEM was administered at a dose of 1000 mg/m(2) on days 1, 8, and 15 in a 28-day cycle. The KIF20A-derived peptide was injected subcutaneously on a weekly basis in a dose-escalation manner (doses of 0.5, 1, and 3 mg/body; 3 patients/cohort). Safety and immunologic parameters were assessed. No severe adverse effects of grade 3 or higher related to KIF20A-derived peptide were observed. Of the 9 patients who completed at least one course of treatment, interferon-γ (IFN-γ)-producing cells were induced in 4 of 9 patients (P2, P3, P6, and P7), and IFN-γ-producing cells were increased in 4 of the 9 patients (P1, P5, P8, and P9). Four of the 9 patients achieved stable disease. The disease control rate was 44%. The median survival time after first vaccination was 173 days and 1-year survival rate was 11.1%. IFN-γ-producing cells were induced by the KIF20A-derived peptide vaccine at a high rate, even in combination with GEM. These results suggest that this combination therapy will be feasible and promising for the treatment of advanced pancreatic cancer.

Background: We previously developed an immunotherapy treatment utilizing a cancer vaccine reagent KIF20A-66 in order to treat pancreatic cancer. KIF20A-66 is HLA-A24-restricted epitope peptide derived from KIF20A, a member of kinesin super family protein 20A that is significantly transactivated in pancreatic cancer. In this report, we further demonstrated non-randomized, open-label, single centered phase I/II clinical trial of immunotherapy using the KIF20A-66 peptide for the patients with advanced pancreatic cancer., Methods: Vaccination was performed to the patients with metastatic pancreatic cancer, in whom gemcitabine-based therapy had failed. In phase I study, KIF20A-66 peptide was subcutaneously injected weekly in a dose-escalation manner (doses of 1.0 and 3.0 mg/body, 6 patients/1 cohort). After safety was assessed, phase II study was conducted using 3.0 mg of KIF20A-66 peptide., Results: KIF20A-66 peptide vaccination was well tolerated in the doses we examined and tumor responses after 1 month of the treatment were evaluated. Among 29 patients who completed one course of the treatment at least, stable disease (SD) was found in 21 cases, while progressive disease (PD) was found in 8 cases, indicating that the disease control rate was 72%. Objective tumor shrinkage was observed in 8 cases, including 1 case of complete response (CR). The median survival time (MST) and progression free survival time (PFS) were 142 days and 56 days, respectively. These results clearly demonstrate that overall survival of the patients was significantly prolonged, compared to the historical controls of 9 cases with unmatched HLA in the same hospital (MST: 83 days), as well as 81 cases in our and other hospitals (MST: 63 days)., Conclusion: The patients vaccinated with KIF20A-66 peptide had better prognosis than the control group with best supportive care (BSC). Thus, we concluded that KIF20A-66 vaccination is significantly effective as an immunotherapy against advanced pancreatic cancer. KIF20A-66 peptide was well tolerable in the dose of either 1.0 mg or 3.0 mg/body, and effectively induced peptide-specific response of cytotoxic T lymphocyte (CTL). Further clinical study using this peptide is a promising approach for advanced pancreatic cancer to achieve high potential benefit for better prognosis., Clinical Trial Registration: UMIN-CTR, number UMIN000004919.

Background: In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells., Methodology/principal Findings: By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time., Conclusion: A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.

The spontaneous T-cell responses to the KIF20A cancer-associated antigen found by Tomita and colleagues among peripheral blood mononuclear cells of patients with cancer, but not healthy people, involve both CD4 and CD8 T cells. Synthetic long peptides of KIF20A stimulate synergy between these two T-cell types to promote cancer cell killing., (©2013 AACR.)

Purpose: To identify long peptides (LP) derived from a novel tumor-associated antigen (TAA), kinesin family member 20A (KIF20A), which induce tumor-specific T-helper type 1 (TH1) cells and CTLs., Experimental Design: We combined information from a recently developed computer algorithm predicting HLA class II-binding peptides with KIF20A-derived CTL-epitope sequences presented by HLA-A2 (A*02:01) or HLA-A24 (A*24:02) to select candidate promiscuous TH1-cell epitopes containing CTL epitopes. Peripheral blood mononuclear cells (PBMC) derived from healthy donors or patients with head-and-neck malignant tumor (HNMT) were used to study the immunogenicity of KIF20A-LPs, and the in vitro cross-priming potential of KIF20A-LPs bearing CTL epitopes. We used HLA-A24 transgenic mice to address whether vaccination with KIF20A-LP induces efficient cross-priming of CTLs in vivo. The TH1-cell response to KIF20A-LPs in HNMT patients receiving immunotherapy with TAA-derived CTL-epitope peptides was analyzed using IFN-γ enzyme-linked immunospot assays., Results: We identified promiscuous KIF20A-LPs bearing naturally processed epitopes recognized by CD4(+) T cells and CTLs. KIF20A-specific CTLs were induced by vaccination with a KIF20A-LP in vivo. KIF20A expression was detected in 55% of HNMT by immunohistochemistry, and significant frequencies of KIF20A-specific TH1 cell responses were detected after short-term in vitro stimulation of PBMCs with KIF20A-LPs in 50% of HNMT patients, but not in healthy donors. Furthermore, these responses were associated with KIF20A expression in HNMT tissues., Conclusions: These are the first results showing the presence of KIF20A-specific TH1 cell responses in HNMT patients and underline the possible utility of KIF20A-LPs for propagation of TH1 cells and CTLs., (©2013 AACR.)

Objective: The field of cancer vaccine therapy is currently expected to become the fourth option in the treatment of cancer after surgery, chemotherapy and radiation therapy. We developed a novel cancer peptide vaccine therapy for bladder cancer through a genome-wide expression profile analysis., Methods: Among a number of oncoproteins that are transactivated in cancer cells, we focused on M phase phosphoprotein 1 and DEP domain containing 1, both of which are cancer-testis antigens playing critical roles in the growth of bladder cancer cells, as candidate molecules for the development of drugs for bladder cancer. In an attempt to identify the peptide epitope from these oncoantigens, we conducted a clinical trial using these peptides for patients with advanced bladder cancer., Results: We identified HLA-A24-restricted peptide epitopes corresponding to parts of M phase phosphoprotein 1 and DEP domain containing 1 proteins, which could induce peptide-specific cytotoxic T lymphocytes. Using these peptides, we found that M phase phosphoprotein 1- and DEP domain containing 1-derived peptide vaccines could be well tolerated without any serious adverse events, and effectively induced peptide-specific cytotoxic T lymphocytes in vivo., Conclusions: The novel approach adopted in the treatment with peptide vaccines is considered to be a promising therapy for bladder cancer.

We here identified human leukocyte antigen-(HLA-)A(∗)2402-restricted epitope peptides from Cadherin 3, type 1, P-cadherin (CDH3) and kinesin family member 20A (KIF20A) that were found to be specifically expressed in cancer cells through genome-wide expression profile analysis. CDH3-10-807 peptide and KIF20A-10-66 peptide successfully induced specific CTL clones, and these selectively responded to COS7 cells expressing both HLA-A(∗)2402 and respective protein while did not respond to parental cells or COS7 cells expressing either HLA-A(∗)2402 or respective protein. Furthermore, CTL clones responded to cancer cells that endogenously express HLA-A(∗)2402 and respective protein, suggesting that CDH3-10-807 peptide and KIF20A-10-66 peptide are naturally presented on HLA-A(∗)2402 molecule of human cancer cells. Our results demonstrated that CDH3-10-807 peptide and KIF20A-10-66 peptide are novel HLA-A24-restricted tumor-associated antigens and would be applicable for CTL-inducing cancer therapies.

Regulation of microtubule (MT) dynamics is essential for proper spindle assembly and organization. Kinesin-8 family members are plus-end-directed motors that modulate plus-end MT dynamics by acting as MT depolymerases or as MT plus-end capping proteins. In this paper, we show that the human kinesin-8 Kif18B functions during mitosis to control astral MT organization. Kif18B is a MT plus-tip-tracking protein that localizes to the nucleus in interphase and is enriched at astral MT plus ends during early mitosis. Knockdown of Kif18B caused spindle defects, resulting in an increased number and length of MTs. A yeast two-hybrid screen identified an interaction of the C-terminal domain of Kif18B with the plus-end MT-binding protein EB1. EB1 knockdown disrupted Kif18B targeting to MT plus ends, indicating that EB1/Kif18B interaction is physiologically important. This interaction is direct, as the far C-terminal end of Kif18B is sufficient for binding to EB1 in vitro. Overexpression of this domain is sufficient for plus-end MT targeting in cells; however, targeting is enhanced by the motor domain, which cooperates with the tail to achieve proper Kif18B localization at MT plus ends. Our results suggest that Kif18B is a new MT dynamics regulatory protein that interacts with EB1 to control astral MT length.

Cancer immunotherapy is a potential therapeutic strategy, in addition to surgical treatment, radiotherapy, and chemotherapy. Cancer-specific immunotherapy, such as the MAGE peptide vaccine, has been utilized clinically. How-ever, there are inherent limits to the effectiveness of vaccinotherapy using a single antigen because of the expression frequency of cancer-specific antigens on tumor cells. Thus, identification of a new cancer-specific antigen is needed. In this study, we examined the possibility of using cancer-specific immunotherapy based upon mitotic centromere-associated kinesin (MCAK) which was previously identified as a novel cancer/testis antigen. To evaluate the feasibility of developing cancer immunotherapy using MCAK peptides, we studied HLA-A*0201 and *2402 as targets for CTLs in the context of HLA class I molecules. By using a peptide with a sequence of AINPELLQL (amino acid positions 63-71 in MCAK, HLA-A*0201) and FFEIYNGKL (amino acid positions 401-409 in MCAK, HLA-A*2402), CTL responses could be induced from unseparated PBMCs by stimulation of freshly isolated, peptide-pulsed PBMCs as antigen-presenting cells (APCs) and also by using interleukin-7 and keyhole limpet hemocyanin in primary culture. The induced CTLs could lyse HLA-A-*0201/*2402 colon and gastric cancer cells expressing MCAK, as well as the peptide-pulsed target cells, in an HLA class l, and CD8 restricted manner. The identification of the MCAK/HLA-A*0201 and *2402 peptides suggests the possibility of designing peptide-based immunotherapeutic approaches that might prove effective in treating patients with MCAK-positive cancer.

Background: Identification of tumour-associated antigens (TAAs) that induce cytotoxic T lymphocytes (CTLs) specific to cancer cells is critical for the development of anticancer immunotherapy. In this study, we aimed at identifying a novel TAA of pancreatic cancer for immunotherapy., Methods: On the basis of the genome-wide cDNA microarray analysis, we focused on KIF20A (also known as RAB6KIFL/MKlp2) as a candidate TAA in pancreatic cancer cells. The HLA-A2 (A*02:01)-restricted CTL epitopes of KIF20A were identified using HLA-A2 transgenic mice (Tgm) and the peptides were examined to check whether they could generate human CTLs exhibiting cytotoxic responses against KIF20A(+), HLA-A2(+) tumour cells in vitro., Results: KIF20A was overexpressed in pancreatic cancer and in some other malignancies, but not in their non-cancerous counterparts and many normal adult tissues. We found that KIF20A-2 (p12-20, LLSDDDVVV), KIF20A-8 (p809-817, CIAEQYHTV), and KIF20A-28 (p284-293, AQPDTAPLPV) peptides could induce HLA-A2-restricted CTLs in HLA-A2 Tgm without causing autoimmunity. Peptide-reactive human CTLs were generated from peripheral blood mononuclear cells of HLA-A2(+) healthy donors by in vitro stimulation with the three peptides, and those CTLs successfully exhibited cytotoxic responses to cancer cells expressing both KIF20A and HLA-A2., Conclusion: KIF20A is a novel promising candidate for anticancer immunotherapeutic target for pancreatic cancers.

At least two hypotheses have been proposed to explain the mechanism of clonal expansion of mutant cells in paroxysmal nocturnal haemoglobinuria (PNH). One hypothesis assumes an immune escape mechanism and another proposes an intrinsic second mutational event within clonal cells. We hypothesised that autoantibodies detected in PNH patients could identify antigens that might play a role in the pathophysiology of this disease and screened a human fetal liver cDNA library for serological reactivity against haematopoietic stem/progenitor cells antigens using the SEREX approach. Two antigens were identified that are constitutively expressed in CD34(+) cells. Three and four of 10 PNH patients showed antibody responses against kinesin family member 20B (KIF20B) (previously termed M-phase phosphoprotein 1, MPP1) and desmoplakin (DSP) respectively. We also found an antibody response in one of 20 healthy volunteers against desmoplakin, yet at a much lower titre than in PNH patients. No response to KIF20B or desmoplakin was detected in five patients with aplastic anaemia without a glycosyl phosphatidyl inositol -deficient clone. We conclude that KIF20B and desmoplakin have been shown to be the first known auto-antigens to be recognised by the immune system of patients with PNH. The analysis of the mechanisms underlying the autoimmune response might contribute to our understanding of the clonal expansion in PNH., (© 2010 Blackwell Publishing Ltd.)

The human genome contains genes encoding for over 40 different types of kinesin and kinesin-like proteins. Of these, the functions of 13 kinesins remain uncharacterized. In this study, we constructed a plasmid containing the ORF of KIF18B and revealed that the KIF18B message of approximately 3kb is expressed in a tissue- and cell type-specific manners. A polypeptide of 842 amino acids was deduced from the ORF sequence. We identified another form of 873 amino acids which arises from alternative splicing at the C-terminal end. We also generated an anti-KIF18B antibody which detects a protein band of 120kDa. Western analyses showed that the protein level of KIF18B is elevated at late G(2) through metaphase, very similar to cyclin B1. Immunocytochemical staining revealed that the KIF18B protein is present predominantly in the nucleus and to a lesser extent in the cytoplasm of interphase cells. During mitosis, most KIF18B was found to be closely associated with astral microtubules emanating from the spindle pole during prometaphase and metaphase. Meanwhile, KIF18B was not detected at anaphase and telophase, consistent with the Western blotting data. The nuclear localization signal was roughly determined by using several EGFP-tagged deletion mutants of KIF18B. Together, the expression of KIF18B is regulated in a cell cycle-dependent manner and therefore may play an important role(s) in cell division., (2010 Elsevier B.V. All rights reserved.)

Mycobacterium tuberculosis and Leishmania donovani are important intracellular pathogens, especially in Indian context. In India and other South East Asian countries, both these infections are highly endemic and in about 20% cases co-infection of these pathogens is reported. For both these pathogens cell mediated immunity plays most important role. The available treatment of these infections is either prolonged or cumbersome or it is ineffective in controlling the outbreaks and spread. Therefore, potentiation of a common host defense mechanism can be used to prevent both the infections simultaneously. In this study we have developed a novel chimeric DNA vaccine candidate comprising the esat-6 gene of M. tuberculosis and kinesin motor domain gene of L. donovani. After developing this novel chimera, its immunogenicity was studied in mouse model. The immune response was compared with individual constructs of esat-6 and kinesin motor domain. The results showed that immunization with chimeric DNA vaccine construct resulted in stronger IFN-gamma and IL-2 response against kinesin (3012+/-102 and 367.5+/-8.92pg/ml) and ESAT-6 (1334+/-46.5 and 245.1+/-7.72pg/ml) in comparison to the individual vaccine constructs. The reciprocal immune response (IFN-gamma and IL-2) against individual construct was lower (kinesin motor domain: 1788+/-36.48 and 341.8+/-9.801pg/ml and ESAT-6: 867.0+/-47.23 and 170.8+/-4.578pg/ml, respectively). The results also suggest that using the chimeric construct both proteins yielded a reciprocal adjuvant affect over each other as the IFN-gamma production against chimera vaccination is statistically significant (p<0.0001) than individual construct vaccination. From this pilot study we could envisage that the chimeric DNA vaccine construct may offer an attractive strategy in controlling co-infection of leishmaniasis and tuberculosis and have important implication in future vaccine design.

Antibodies against the nuclear mitotic spindle apparatus protein (NuMA) are infrequently detected during antinuclear antibodies testing on HEp-2 cells. In a series of 428 psoriatic patients anti-NuMA antibodies were found only in a patient, at a titer of 1:640, without any apparent clinical relevance. The significance of anti-NuMA is not yet known and is briefly reviewed, also in consideration of potential therapeutic implications. Although biologic drugs targeting tumor necrosis factor-alpha have been associated with the development of non-organ specific autoantibodies and rare reports of autoimmune phenomena, infliximab was well tolerated in this patient and caused no changes in autoantibody titers.

Introduction: The clinical associations of NuMA and HsEg5 antibodies, the main anti-mitotic spindle apparatus autoantibodies, remain unclear due to their extremely low prevalence., Patients and Methods: We have analysed the clinical data of 40 anti-NuMA- (0.87 per thousand) and 7 anti-HsEg5- (0.15 per thousand) positive patients detected during routine immunofluorescence examination of 45,804 sera. NuMA reactivity was further confirmed by immunoblotting., Results: Antibodies to HsEg5 did not associate with any specific pathology. NuMA positivity associated with a diagnosis of connective tissue disease (CTD) in 18 patients (45%), primary Sjögren or sicca syndrome and undifferentiated connective tissue disease being the most represented. Seven patients (17.5%) were diagnosed with different organ-specific autoimmune diseases, whereas in the other 15 patients (37.5%), no autoimmune pathology could be documented., Conclusions: Therefore, although both anti-mitotic spindle apparatus antibodies are not associated to a defined autoimmune pathology, the presence of NuMA antibodies, mainly at high titers, may be an indication for a more extensive screening of CTD.

Visceral leishmaniasis (VL) is one of the important parasitic diseases, with approximately 350 million people at risk. Due to the nonavailability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. The present study was carried out to examine the immunological potential of kinesin protein from the microtubule locus of Leishmania donovani as a suitable vaccine candidate. In silico analysis of this region revealed clusters of major histocompatibility complex class I and II binding epitopes in its motor domain region. A recombinant protein was expressed from this region and named rLvacc. The antigenicity and immunogenicity studies of this protein by Western blot analysis revealed that rLvacc is strongly recognized by sera from acute VL patients. To evaluate its immunogenicity, peripheral blood mononuclear cells from cured VL patients were separated, and a lymphocyte proliferation assay was carried out in the presence of rLvacc. After lymphocyte proliferation, the pooled culture supernatant was assayed for anti-rLvacc antibody titers using an enzyme-linked immunosorbent assay. The results showed that immunoglobulin G2 (IgG2) subtype antibodies were predominant, while IgG1 subtype antibodies were produced in very low titers. On the basis of these ex vivo preliminary findings, its immunogenicity was studied in BALB/c mice. Vaccination with the DNA construct generated a good cellular immune response with significant increases in gamma interferon and interleukin-2 (IL-2) cytokine levels (Th1), but no increase in IL-4 levels (Th2). Taken together, our findings suggest the kinesin motor domain region of L. donovani as a potential vaccine candidate against visceral leishmaniasis.

Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations.

During spermatogenesis, spermatids undergo a "down and up" translocation event in the seminiferous epithelium. This event has been proposed to result from the movement of ectoplasmic specializations, which are formed in Sertoli cells at sites of adhesion to spermatids, along adjacent microtubule tracts. To test the hypothesis that a kinesin is associated with ectoplasmic specializations, we generated antibodies to conserved kinesin sequences and detected kinesins on fixed frozen testis sections and fixed seminiferous epithelial fragments. The antibodies reacted with ectoplasmic specializations related to spermatids, in addition to reacting with other structures in the epithelium known to contain kinesins. At the electron microscopy level, the antibodies reacted with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. Based on mRNA transcript screens using mouse GeneChip arrays of testis and Sertoli cells, we identified KIF20 as a candidate kinesin at ectoplasmic specializations. Antibodies generated against a peptide sequence unique to this kinesin reacted at ectoplasmic specializations in testis sections and epithelial fragments, as well as with the endoplasmic reticulum component of ectoplasmic specializations when analyzed by electron microscopy. The antibody reacted on Western blots with full-length KIF20. On Western blots of testis lysates, the antibody reacted with a protein that is not present in other tissues and which migrates at a higher molecular weight than that predicted for KIF20. Our results demonstrate that a kinesin is associated with apical ectoplasmic specializations in Sertoli cells and that the motor may be an isoform of KIF20.

Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins have been suggested to play an important role as growth stimulators in the pathogenesis of these neoplasms. To identify such targets, we screened cDNA libraries from human testis, lung and breast cancer, bovine and porcine muscle and wheat germ for reactivity with paraproteins in the sera from 115 patients with MGUS and MM. Of >6 x 10(8) paraprotein-antigen interactions screened, an IgA paraprotein from a female patient bound to sperm-specific cylicin-2, and 3 IgG paraproteins bound to tripeptidyl-peptidase-II (TPP-2), insulin-like growth-factor binding-protein-2 (IGFBP-2) and porcine kinesin. Specificity was confirmed by reverse Western blots using recombinant antigens. The broad spectrum of auto-, allo- and heteroantigens as targets of human paraproteins in patients without signs of chronic antigenic stimulation renders a causal role of the antigenic stimulus in the pathogenesis of MGUS and MM unlikely., ((c) 2007 Wiley-Liss, Inc.)

To detect IgG antibody in the serodiagnosis of visceral leishmaniasis (VL), a recombinant antigen rK39, which is part of a Leishmania chagasi kinesin-related protein, has been used successfully and showed high sensitivity and specificity. We report production of a recombinant protein rKRP42, which is part of an L. donovani kinesin-related protein and a homolog of rK39, and its application in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of VL. When rKRP42 and rK39 were compared, amino acid sequence analysis showed 89.3% identity and 98.7% homology, with rKRP42 having 39 more amino acids than rK39. The ELISA using rKRP42 showed a sensitivity of 94.6% (70 positive samples among 74 from VL patients) and a specificity of 99.3% (148 negative samples among 149 samples from Japanese controls), whereas the sensitivity of the commercial rK39 dipstick test was 93.2% (69 positive samples among 74 from patients with VL). The rKRP42 is a promising new antigen in developing immunodiagnostic methods for VL.

One promising modality in the treatment of malignant glioma is specific immunotherapy. However, this modality requires information about target antigens and their epitope peptides that are recognized by T cells. In this study, we searched for new target candidates in specific immunotherapy for malignant glioma by utilizing cDNA microarray technology to compare gene expressions in malignant glioma tissues to those in benign glioma and a panel of normal tissues. The selected genes included three members of the kinesin superfamily proteins (KIFs): KIF1C, KIF3C, and KIF21B. RT-PCR showed that these three genes were expressed in the majority of glioma cell lines. These antigen-derived 25 peptides, which had the ability to bind to human leukocyte antigen (HLA)-A24 molecules, were first screened for their ability to be recognized by the immunoglobulin G of glioma patients, and then tested for their potential to induce peptide-specific and glioma-reactive cytotoxic T lymphocytes (CTLs) from the peripheral blood mononuclear cells of HLA-A24+ glioma patients. The results showed that the KIF1C149-158 and KIF3C512-520 peptides efficiently induced HLA-A24-restricted and glioma-reactive CD8+ T cells. These results suggest the existence of KIF-reactive CTL precursors in glioma patients, and should facilitate the development of specific immunotherapies for malignant glioma.

Chromokinesins are chromosome-bound proteins during mitosis that play multiple important roles in chromosome segregation. The chromokinesin Kid has been shown to be involved in chromosome congression during mitosis and meiosis. Here we have generated a monoclonal antibody specific for the human chromokinesin hKid by immunizing BALB/c mice with a recombinant protein fragment corresponding to the C-terminal 250-amino acid residues of hKid. All five immunized mice responded excellently and gave high, nearly monospecific, antibody titers. One hybridoma, 8C12, was generated from spleen cells of a selected mouse, which recognized hKid on immunoblots and in immunofluorescence experiments. As hKid is an important regulator of chromosome segregation, this monoclonal antibody will be a useful tool for further analysis of this chromokinesin.

The isolation of the cDNA sequence encoding the human neuronal kinesin (a force-generating motor protein which transports various membrane organelles along microtubules in an ATP-dependent manner) heavy chain (nKHC) and the construction of expression vectors to produce the full-length nKHC and its domains in Escherichia coli is described. By tuning up the conditions for the expression of nKHC, a sufficient amount of the soluble protein intragenously tagged with 6xHis tag was obtained and purified by nickel chromatography. The recombinant structural domains of nKHC, including the motor domain (FKHC1--amino acids 1-330), the microtubule binding domain (FKHC2--amino acids 174-315) and the coiled-coil stalk domain (FKHC3--amino acids 331-906) were used to determine the epitope location for monoclonal antibodies KN-01, KN-02, and IB II raised against different kinesin heavy chains. The antibodies were shown to recognize epitopes located in the stalk domain of nKHC and represent thus useful probes for this domain.

The present paper describes two new monoclonal antibodies (MAbs) KN-02 and KN-03 against the heavy chain of conventional kinesin. The kinesin was purified from porcine brain by a combined procedure of ion exchange chromatography, tripolyphosphate-supported microtubule affinity-binding, and gel filtration. Hybridoma cell lines producing antibodies were obtained after immunization of a Balb/c mouse with kinesin and subsequent fusion of the spleen cells with Sp2/0 myeloma cells. The specificity was verified by enzyme-linked immunosorbent assay (ELISA) and further confirmed by immunoblotting and immunoprecipitation analysis. The antibodies recognize different epitopes on the heavy chain of the kinesin molecule as demonstrated by chymotryptic cleavage of kinesin followed by immunoblotting. Differential location of relevant epitopes was also documented by in vitro binding experiments with purified kinesin and taxol-stabilized microtubules. While the KN-03 antibody decorated microtubules, no such staining was observed with KN-02 antibody. The antibodies have a lower affinity to sodium dodecyl sulfate (SDS)-denatured kinesin, but immunofluorescence on fixed cells gave strong dot-like staining characteristic for localization of kinesin on vesicles. The same staining pattern was observed in different cell types. Double-label fluorescence with polyclonal anti-tubulin antibody revealed a co-distribution of stained vesicles with microtubules on the cell periphery. The antibodies KN-02 and KN-03 are therefore valuable tools for localization of kinesins in cells of different tissue origin.

Objective: To determine the clinical significance of anti-NuMA and anti-HsEg5 antibodies in a group of patients affected with rheumatic diseases., Materials and Methods: Indirect immunofluorescence on HEp-2000 cells at serum dilution of 1:40 was used to examine 26 sera which had previously showed a "mitotic spindle" fluoroscopic pattern type during laboratory routine., Results: 21 sera (80,7%) were identified with NuMA and 5 (19,3%) with HsEg5 patterns alone or associated with other ANA patterns. However only patients with isolated positivity and that is 15 with NuMA and 4 with HsEg5 stainings were included in this study. Of the NuMA positive patients 5 were affected with arthropathies associated to different forms of thyroiditis, 2 with seronegative arthritis, 2 with antiphospholipid syndrome, 1 with systemic lupus erythematosus (SLE), 1 with rheumatoid arthritis, 1 with sicca syndrome, 1 with undifferentiated connective tissue disease, 1 with Mycoplasma pneumoniae infection and 1 with retinal thrombosis. Of the HsEg5 positive patients 3 were affected with SLE and 1 with seronegative arthritis., Conclusions: NuMA does not prevail in any defined rheumatic disease, while HsEg5 staining were more frequent (75%) in patients affected with SLE all of whom showing high antibody titres.

Cadherin-mediated cell-cell adhesion is a dynamic process that is regulated during embryonic development, cell migration, and differentiation. Different cadherins are expressed in specific tissues consistent with their roles in cell type recognition. In this study, we examine the formation of N-cadherin-dependent cell-cell contacts in fibroblasts and myoblasts. In contrast to E-cadherin, both endogenous and ectopically expressed N-cadherin shuttles between an intracellular and a plasma membrane pool. Initial formation of N-cadherin-dependent cell-cell contacts results from the recruitment of the intracellular pool of N-cadherin to the plasma membrane. N-cadherin also localizes to the Golgi apparatus and both secretory and endocytotic vesicles. We demonstrate that the intracellular pool of N-cadherin is tightly associated with the microtubule (MT) network and that junction formation requires MTs. In addition, localization of N-cadherin to the cortex is dependent on an intact F-actin cytoskeleton. We show that N-cadherin transport requires the MT network as well as the activity of the MT-associated motor kinesin. In conclusion, we propose that N-cadherin distribution is a regulated process promoted by cell-cell contact formation, which controls the biogenesis and turnover of the junctions through the MT network.

A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface.

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression.

Using pan-kinesin antibodies to screen a differentiating C2C12 cell library, we identified the kinesin proteins KIF3A, KIF3B, and conventional kinesin heavy chain to be present in differentiating skeletal muscle. We compared the expression and subcellular localization characteristics of these kinesins in myogenic cells to others previously identified in muscle, neuronal, and mitotic systems (KIF1C, KIF3C, and mitotic-centromere-associated kinesin). Because members of the KIF3 subfamily of kinesin-related proteins showed altered subcellular fractionation characteristics in differentiating cells, we focused our study of kinesins in muscle on the function of kinesin-II. Kinesin-II is a motor complex comprised of dimerized KIF3A and KIF3B proteins and a tail-associated protein, KAP. The Xenopus homologue of KIF3B, Xklp3, is predominantly localized to the region of the Golgi apparatus, and overexpression of motorless-Xklp3 in Xenopus A6 cells causes mislocalization of Golgi components (). In C2C12 myoblasts and myotubes, KIF3B is diffuse and punctate, and not primarily associated with the Golgi. Overexpression of motorless-KIF3B does not perturb localization of Golgi components in myogenic cells, and myofibrillogenesis is normal. In adult skeletal muscle, KIF3B colocalizes with the excitation-contraction-coupling membranes. We propose that these membranes, consisting of the transverse-tubules and sarcoplasmic reticulum, are dynamic structures in which kinesin-II may function to actively assemble and maintain in myogenic cells.

The Rab6-binding kinesin, Rab6-KIFL, was identified in a two-hybrid screen for proteins that interact with Rab6, a small GTPase involved in membrane traffic through the Golgi apparatus. We find that Rab6-KIFL accumulates in mitotic cells where it localizes to the midzone of the spindle during anaphase, and to the cleavage furrow and midbody during telophase. Overexpression of Rab6-KIFL causes a cell division defect resulting in cell death. Microinjection of antibodies to Rab6-KIFL results in the cells becoming binucleate after one cell cycle, and time-lapse microscopy reveals that this is due to a defect in cleavage furrow formation and thus cytokinesis. These data show that endogenous Rab6-KIFL functions in cell division during cleavage furrow formation and cytokinesis, in addition to its previously described role in membrane traffic.

Background & Aims: Kinesin has recently been localized to zymogen granules of pancreatic acini and is suggested to participate in exocytosis of exocrine pancreas. We examined the function of kinesin in regulated exocytosis of pancreatic acini in this study., Methods: Kinesin function in exocytosis was examined by introducing hexahistidine-tagged recombinant kinesin protein and antikinesin monoclonal antibody into streptolysin-O-permeabilized acini. Intracellular localization of introduced recombinant kinesin was investigated by immunohistochemistry. Interaction between recombinant kinesin and the microtubule network was confirmed by nocodazole pretreatment of acini. Kinesin regulation by secretagogues was investigated by examining their effect on adenosine triphosphatase (ATPase) activity of endogenous kinesin., Results: Recombinant kinesin enhanced calcium-stimulated amylase release from streptolysin-O-permeabilized acini. Introduced recombinant kinesin was localized to both the microtubule network and zymogen granule. Nocodazole pretreatment of acini abolished the enhancing effect of recombinant kinesin on calcium-stimulated amylase release. Antikinesin antibody inhibited amylase release stimulated by the combination of calcium and cyclic adenosine monophosphate (cAMP) but not that stimulated by calcium alone. Secretin and 8-bromo-cAMP increased ATPase activity of endogenous kinesin., Conclusions: Kinesin plays a stimulatory role in regulated exocytosis of pancreatic acini and is involved in stimulus-secretion coupling through a cAMP-dependent pathway.