Oral manifestations of human T‐cell lymphotropic virus infection in adult patients from Brazil

Oral Diseases (2010) 16 , 167–171 Objective: Human T‐cell lymphotropic virus type 1 (HTLV‐1) was the first human retrovirus discovered and its pathogenesis is related to T cells infection. This study aimed to verify the presence of oral manifestations in a Brazilian population of patients who was seropositive for HTLV, and identify risk factors for oral manifestations. Subjects and methods: An assessment was made of 139 patients at the Emilio Ribas Institute of Infectious Diseases. Results: A total of 112 (80.5%) patients were HTLV‐1, 26 (18.7%) were HTLV‐2+. About 35.2% of patients had myelopathy/tropical spastic paraparesis (HAM/TSP), with 48 of them being HTLV‐1+ and one patient was seropositive for HTLV‐1 and ‐2. The most common oral manifestations were: xerostomia (26.8%), candidiasis (20.8%), fissured tongue (17.9%), and loss of tongue papillae (10.0%). A multivariate logistic regression analysis showed that HAM/TSP is an independent risk factor for xerostomia (P = 0.02). The patients who were HAM/TSP+ were three times more likely to develop xerostomia when compared with patients without HAM/TSP (odds ratio = 2.69, 95% confidence interval = 1.17–6.17). Conclusion: Despite the fact that the findings of this study suggest a relationship between xerostomia and HAM/TSP, more studies should be developed to show what the association would be between xerostomia presented by HTLV patients and pathogenesis of the virus.     

Kaposi's sarcoma (KS)-associated herpesvirus-like DNA sequences in peripheral blood mononuclear cells: association with KS and persistence in patients receiving anti-herpesvirus drugs

Herpesvirus-like DNA sequences (KSHV/HHV-8) have recently been described in AIDS-associated Kaposi's sarcoma (KS) lesions. Many questions remain regarding the role of this virus in KS and the therapeutic implications of this finding. In the current study, KSHV/HHV-8 DNA was detected in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus (HIV)-infected patients with KS (34/98) more often than in HIV-infected individuals without KS (12/64, P = .03). The detection of KSHV/HHV-8 DNA did not correlate with the CD4 lymphocyte count. Five patients demonstrated KSHV/HHV-8 DNA in their PBMCs during administration of intravenous foscarnet and/or ganciclovir. The continued detection of KSHV/HHV-8 DNA in the PBMCs of patients receiving these anti-herpesvirus drugs has potential implications regarding the virus-cell relationship of KSHV/HHV-8, as well as for the value of these drugs in treating or preventing KS, but additional studies are needed.     

A complex of distal appendage–associated kinases linked to human disease regulates ciliary trafficking and stability

Cilia biogenesis is a complex, multistep process involving the coordination of multiple cellular trafficking pathways. Despite the importance of ciliogenesis in mediating the cellular response to cues from the microenvironment, we have only a limited understanding of the regulation of cilium assembly. We previously identified Tau tubulin kinase 2 (TTBK2) as a key regulator of ciliogenesis. Here, using CRISPR kinome and biotin identification screening, we identify the CK2 catalytic subunit CSNK2A1 as an important modulator of TTBK2 function in cilia trafficking. Superresolution microscopy reveals that CSNK2A1 is a centrosomal protein concentrated at the mother centriole and associated with the distal appendages. Csnk2a1 mutant cilia are longer than those of control cells, showing instability at the tip associated with ciliary actin cytoskeleton changes. These cilia also abnormally accumulate key cilia assembly and SHH-related proteins. De novo mutations of Csnk2a1 were recently linked to the human genetic disorder Okur-Chung neurodevelopmental syndrome (OCNDS). Consistent with the role of CSNK2A1 in cilium stability, we find that expression of OCNDS-associated Csnk2a1 variants in wild-type cells causes ciliary structural defects. Our findings provide insights into mechanisms involved in ciliary length regulation, trafficking, and stability that in turn shed light on the significance of cilia instability in human disease.

Primary cilia (PC) are microtubule-based cellular projections that function as vital cellular-signaling organelles, transducing signals through a variety of important developmental pathways. In addition to their roles in embryonic development, cilia also persist on the cells of most adult tissues (1–4). PC are dynamic organelles, and bidirectional trafficking of proteins and other materials within cilia by the process of intraflagellar transport (IFT) is vital for the maintenance of ciliary structure and signaling (5–7). However, little is known about the processes and pathways upstream of IFT that control the presence or absence of a cilium. In prior work, we identified a kinase, TTBK2, that is required for ciliogenesis. TTBK2 mediates both the recruitment of IFT proteins to the mother centriole and removal of centrosomal proteins that suppress cilium assembly (8). TTBK2 is also important for maintaining the stability of the ciliary axoneme (9), and both of these requirements are linked to a role for TTBK2 in a human neurodegenerative disease (4, 10). A limited number of interactors and effectors of TTBK2 in ciliogenesis have been identified, including the distal appendage proteins CEP164 (11, 12) and CEP83 (13), and a suppressor of ciliogenesis, M-phase phosphoprotein 9 (14). However, the pathway through which TTBK2 functions to mediate cilium assembly and stability is largely undefined.Here, to uncover additional players in this pathway, we undertook two distinct unbiased approaches to identify previously unknown physical and functional interactors of TTBK2: proximity-dependent biotin identification (BioID) to identify TTBK2-proximate proteins, and a CRISPR-based screen to uncover genetic modifiers of a hypomorphic Ttbk2 phenotype (9). We identified Casein kinase II subunit alpha (CSNK2A1), the main catalytic subunit of casein kinase II (CK2) (15), in both screens, making it an attractive candidate as a modulator of TTBK2 function in cilia regulation.CK2 is a widely expressed serine-threonine kinase involved in many cellular processes, including cell-cycle progression, circadian-clock regulation, and WNT signaling (16–20). Mutations in Csnk2a1 are associated with Okur-Chung neurodevelopmental syndrome (OCNDS), a dominantly inherited disorder characterized by dysmorphic facial features and neurological impairments (21–26). We show that CSNK2A1 acts in opposition to TTBK2 in establishing and maintaining ciliary structure, with knockout of Csnk2a1 partially rescuing ciliary phenotypes of Ttbk2 hypomorphic mutant cells. We also demonstrate that CSNK2A1 mainly localizes to the mother centriole and is required for cilia structure and stability. Csnk2a1-knockout cells have long cilia that exhibit a variety of defects in trafficking and actin dynamics. Additionally, overexpression of Csnk2a1 variants associated with OCNDS causes ciliary structural abnormalities. Thus, we reveal a previously undescribed role for CSNK2A1 in cilia homeostasis, define a kinase network that regulates the stability of PC, and identify links between cilia integrity and human disease.     

Molecular evidence for a single clonal origin in biphenotypic concomitant chronic lymphocytic leukemia and multiple myeloma

To establish the clonal origin of a case of concomitant B-cell chronic lymphocytic leukemia (IgM kappa) and multiple myeloma (IGA lambda), we analyzed the immunoglobulin (Ig) gene rearrangements in the patient's blood and bone marrow. Despite the different isotypes, pretreatment investigation of the heavy chain gene (JH) revealed a germline fragment and two identical rearrangements in the blood and marrow. Both kappa and lambda light-chain genes were rearranged in the blood, suggesting peripheral blood lymphocyte involvement in the myeloma. Analysis of the Ig genes after chemotherapy demonstrated no change in the JH or CK rearrangements; however, the lambda genes were now in a germline configuration. Our results suggest that in this patient both chronic lymphocytic leukemia and myeloma originated from the same B-cell progenitor.     

Characterization of a new non-Hodgkin's lymphoma cell line (NCEB-1) with a chromosomal (11:14) translocation [t(11:14)(q13;q32)]

A new cell line, NCEB-1, was established by Epstein-Barr virus (EBV) transformation of peripheral blood mononuclear cells from a patient with centroblastic-centrocytic diffuse lymphoma expressing IgM lambda. The transformed cells were lymphoblastoid, with many cells showing a plasmacytoid morphology. The NCEB-1 cells had cytoplasmic Ig (CyIg), with loss of the surface Ig (SIg) expression. Cytogenetic analysis of the cell line demonstrated two clones with variations: a hypodiploid clone, with a complex karyotype including a t(11;14)(q13;q32) similar to the original tumor cells, and a near tetraploid clone with the same markers. Southern blot analysis of DNA from the patient's neoplastic cells and NCEB-1 demonstrated identical Ig heavy chain gene rearrangement, confirming the origin of the cell line. The cell line was not tumorigenic when tested in an in vitro assay using immunosuppressed mice. NCEB-1 has been in continuous culture for 9 months and will be valuable for the in vivo study of non-Hodgkin's lymphoma and EBV transformation.     

Transformation-associated alterations in interactions between pre-B cells and fibronectin

Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2-10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2-10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2-10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.     

Characterization and functional analysis of adult human bone marrow cell subsets in relation to B-lymphoid development

To study the frontiers between pluripotent stem cells and committed progenitors and to further define the B-cell pathway in adult bone marrow (BM), CD34+ subpopulations and CD34- B-lineage cells were analyzed by multiparameter flow cytometry, studied by light and electron microscopy, and in short-term and long-term cultures (LTC). While the total CD34+ cells represent 4.9% +/- 0.8 of BM mononuclear cells within the lymphoid-blast window, 73.8 +/- 3.5%, 14.4 +/- 1.8% and 8.8 +/- 2.9% of them were CD34+ CD10- CD19-, CD34+ CD10+ CD19+, and CD34+ CD10+ CD19-, respectively. CD34+ CD10+ CD19+ cells represent a smal homogeneous TdT4 c micro-blast population. Although expressing CD38 and high level of HLA-DR antigens, like myeloid committed progenitors, they did not generate LTC, myeloid, and T lymphoid colonies suggesting that the CD34+ CD10+ CD19+ population represents exclusively B lymphoid committed progenitors. By contrast, all myeloid progenitors and LTC-initiating cells were found in the CD34+ CD10- CD19- cell fraction. This fraction appeared more heterogeneous and contained CD38- HLA-DRlow small cells, larger blasts, and promonocyte-like cells exhibiting small peroxidase-positive granules. Interestingly, CD10 was also present on CD34+ CD19- cells. This population mainly coexpressed CD33 and gave rise to macrophagic colonies.