Stimulation of surface IgM of chronic lymphocytic leukemia cells induces an unfolded protein response dependent on BTK and SYK.

B-cell receptor (BCR) signaling plays a key role in the behavior of chronic lymphocytic leukemia (CLL). However, cellular consequences of signaling are incompletely defined. Here we explored possible links between BCR signaling and the unfolded protein response (UPR), a stress response pathway that can promote survival of normal and malignant cells. Compared with normal B cells, circulating CLL cells expressed increased, but variable, levels of UPR components. Higher expression of CHOP and XBP1 RNAs was associated with more aggressive disease. UPR activation appeared due to prior tissue-based antigenic stimulation because elevated expression of UPR components was detected within lymph node proliferation centers. Basal UPR activation also correlated closely with surface immunoglobulin M (sIgM) signaling capacity in vitro in both IGHV unmutated CLL and within mutated CLL. sIgM signaling increased UPR activation in vitro with responders showing increased expression of CHOP and XBP1 RNAs, and PERK and BIP proteins, but not XBP1 splicing. Inhibitors of BCR-associated kinases effectively prevented sIgM-induced UPR activation. Overall, this study demonstrates that sIgM signaling results in activation of some components the UPR in CLL cells. Modulation of the UPR may contribute to variable clinical behavior, and its inhibition may contribute to clinical responses to BCR-associated kinase inhibitors.

Identification in CLL of circulating intraclonal subgroups with varying B-cell receptor expression and function.

Chronic lymphocytic leukemia (CLL) is a tumor of circulating B cells, variably stimulated and anergized following exposure to antigen in lymphoid tissues. Down-modulation of surface IgM (sIgM) occurs, but expression and signal capacity can recover in vitro and apparently in vivo during recirculation. We have now dissected individual circulating clones of CLL cases according to sIgM expression level by differential binding to bead-bound anti-IgM. Four clear subgroups (SG1-4) with increasing sIgM were identified in 37/37 cases. Engagement of sIgM induced phosphorylation of PLCγ2 and ERK1/2 at levels ranging from very low in SG1 to high in SG4. Phosphorylation was suppressed by the BTK inhibitor ibrutinib. Expression of CXCR4 also increased from SG1 to SG4, but markers of previous activation and proliferation were dominant in SG1. Incubation of whole CLL populations in vitro led to striking increases in CXCR4 expression as well as recovery of sIgM. Clonal analysis reveals dynamic SGs following presumed antigen stimulation in tissues. SG4 represents a fully recovered, potentially dangerous population equipped to migrate to tissue and receive a proliferative stimulus. SG1 likely represents a postmitotic unresponsive "resting" population. The effect of ibrutinib on the small SG4 population may be the critical factor in therapeutic success.

Glycosylation of surface Ig creates a functional bridge between human follicular lymphoma and microenvironmental lectins.

Surface Ig (sIg) of follicular lymphoma (FL) is vital for tumor cell survival. We found previously that the Ig in FL is unusual, because the variable region genes carry sequence motifs for N-glycan addition. These are introduced by somatic mutation and are tumor specific. Unexpectedly, added glycans terminate at high mannose, suggesting a potentially important interaction of FL cells with mannose-binding lectins of the innate immune system. We have now identified mannosylated IgM at the surface of primary lymphoma cells. Recombinant lectin domains of the mannose receptor (MR) or DC-SIGN bind mannosylated Igs in vitro and bind to FL cells, signaling sIgM-associated increases in intracellular Ca(2+). Lectins also bind to normal B cells but fail to signal. In contrast, anti-Ig signaled similarly in both FL and normal B cells. Mannosylation patterns were mimicked by FL Ig-derived single-chain Fvs (scFv), providing probes for potential receptors. Mannosylated scFv bound specifically to the lectin domains of the MR and DC-SIGN and blocked signaling. Mannosylated scFv also bound to DC-SIGN on the surface of dendritic cells. This unique lymphoma-specific interaction of sIg with lectins of innate immunity reveals a potential route for microenvironmental support of tumor cells, mediated via the key B-cell receptor.

Surface IgM of CLL cells displays unusual glycans indicative of engagement of antigen in vivo.

Surface IgM (sIgM) has a key influence on the clinical behavior of chronic lymphocytic leukemia (CLL). We now report that it exists in 2 forms with different N-glycosylation patterns in the mu-constant region. One glycoform is similar to normal B cells in bearing mature complex glycans common to most cell-surface glycoproteins. The other is an immature mannosylated form more characteristic of mu chains in the endoplasmic reticulum. Unmutated CLL (U-CLL) expresses a higher proportion of mannosylated surface mu chains than mutated CLL. Normal B cells express only the mature glycoform but can express the immature form after persistent engagement of sIgM, suggesting that glycan modification is a consequence of antigen exposure. CLL cells express variable proportions of the mannosylated form and can revert to the mature form after incubation in vitro. Both glycoforms are able to signal after sIgM engagement in vitro, leading to enhanced tyrosine phosphorylation. These findings support the concept that CLL cells are continuously exposed to antigen in vivo, driving the N-glycosylation pattern of expressed sIgM toward a mannosylated form, especially in U-CLL. Strikingly, this is reminiscent of follicular lymphoma, where mannosylated Ig is expressed constitutively via N-glycosylation sites in the variable region, suggesting a functional asset for this glycoform.

Biodistribution and efficacy of [131I]A33scFv::CDy, a recombinant antibody-enzyme protein for colon cancer.

In antibody-directed enzyme-prodrug therapy (ADEPT), an antibody-bound enzyme localizes to tumor tissue, where it selectively converts a subsequently administered non-toxic prodrug into a cytotoxic drug. A33scFv::CDy is a bifunctional fusion construct comprising a single chain antibody against the gpA33 antigen and the prodrug-converting enzyme cytosine deaminase. gpA33 is highly and homogeneously expressed in >95% of all colorectal cancers. Here we describe the biodistribution and tumor-targeting capacity of 131I labeled A33scFv::CDy. 131I labeling of A33scFv::CDy was performed by the chloramine-T method, and the properties of the resulting [131I]A33scFv::CDy conjugate were determined in vivo and in vitro, including biodistribution studies in nude mice bearing human LIM1215 colon carcinoma xenografts. The [131I]A33scFv::CDy conjugate bound specifically to colorectal cancer cells in vitro with KD = 15.8 nM as determined by a saturation assay. in vivo, the tumor uptake of [131I]A33scFv::CDy peaked at 87% injected dose/g 47 h post injection. Normal tissue uptake was low, and activity in blood was lower than in tumor at all time-points studied (6-92 h). The tumor-to-blood ratio increased over time with a maximum of 8.1 at 67 h post injection. [131I]A33scFv::CDy thus shows a biodistribution that makes it attractive for both radioimmunotherapy (RIT) and ADEPT. Preliminary therapeutic experiments showed a significant reduction of tumor size in mice treated with the A33scFv::CDy-5-fluorocytosine/5-fluorouracil ADEPT system. This work demonstrates the feasibility of ADEPT and RIT based on the A33scFv::CDy recombinant construct.

Production of bifunctional single-chain antibody-based fusion proteins in Pichia pastoris supernatants.

Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.

Surface expression of gpA33 is dependent on culture density and cell-cycle phase and is regulated by intracellular traffic rather than gene transcription.

The cell-surface marker, gpA33, a new member of the immunoglobulin superfamily, is expressed by gastrointestinal cells and by 95% of colon cancers. It has become a promising target of immunologic therapy strategies, but its biologic function and potential role in tumorigenesis are unknown. In this study, we have investigated the expression of gpA33 on the mRNA and cell-surface protein levels by quantitative reverse transcriptase polymerase chain reaction and flow cytometry, respectively, in response to cell density in the culture and to cell-cycle arrest in the G1, S, or G2/M phases. As internalization of the surface protein had previously been reported, we also investigated the binding and intracellular migration of an anti-gpA33 fluobody with green fluorescent protein (A33scFv::GFP) by laser confocal microscopy. Contrary to intuition, we found that gpA33 surface expression and mRNA levels do only partly correlate under the conditions tested. Dependent on cell density in culture, gpA33 surface expression peaked at the point of confluence. Dependent on cell-cycle phase, it peaked in the G2/M phase but was lowest in the S phase, whereas mRNA levels were highest in S, but almost absent in G1. Laser confocal microscopy clearly demonstrated the intracellular uptake of A33scFv::GFP and showed the migration of microvesicles over time. These findings are, in part, concordant with the putative role of gpA33 as an adhesion molecule. However, intracellular traffic and recycling to the cell surface appear to play a major role in its function and to have an influence on its surface density superseding translational regulation.

A33scFv-green fluorescent protein, a recombinant single-chain fusion protein for tumor targeting.

Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for diagnostic purposes and experimental therapeutic approaches. Recombinant technology allows for the design and expression of tailored genuine fusion proteins, providing defined molecules as to size, molar ratios of the functional components and stability. The production of functional protein, however, is often limited or impossible due to refolding and solubility problems. Here, we report on the production of a soluble recombinant fusion construct, A33scFv-green fluorescent protein (A33scFv::GFP) in Pichia pastoris. A33scFv is a single-chain antibody recognizing the A33 antigen, which is expressed by approximately 95% of colorectal carcinomas and has become a focus of pre-clinical and clinical investigation. The fusion partner GFP was selected both as an experimental tool for functional studies of the A33 antigen and as a potential diagnostic for colon cancer detection and therapy planning. Pichia pastoris yeast strains were transformed with A33scFv::GFP cDNA under the methanol-inducible AOX1 promotor. The construct was properly expressed and secreted into culture supernatants as a soluble protein, which was bifunctional without additional renaturation or solubilization steps. The crude protein solution was purified by affinity chromatography. Surface plasmon resonance, flow cytometry and fluorescence microscopy on sections of normal and cancerous colon tissue revealed specific binding and the applicability of this fusion protein for diagnostic purposes. In addition, the biodistribution of A33scFv::GFP was analyzed in mice bearing A33-positive tumor xenografts, confirming specific tumor targeting.

Design, construction, and in vitro analysis of A33scFv::CDy, a recombinant fusion protein for antibody-directed enzyme prodrug therapy in colon cancer.

Antibody-directed enzyme-prodrug therapy (ADEPT) aims at improving the specificity of conventional chemotherapy by employing artificial antibody-enzyme constructs to convert a non-toxic prodrug into a cytotoxic agent specifically localized to the tumor site. The gpA33 antigen is a promising target for ADEPT in colon cancer, as it is expressed by >95% of human colon cancers, but is absent in all non-gastrointestinal tissues. We designed a recombinant fusion construct of a phage display-generated anti-gpA33 single chain fragment, A33scFv, with cytosine deaminase from yeast (CDy), which converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). The resulting construct, A33scFv::CDy, was overexpressed in Pichia pastoris and secreted into culture supernatant. The fusion protein was purified by affinity chromatography on protein L. Silver-staining after SDS-polyacrylamide gel electrophoresis confirmed molecular mass and purity. Antibody binding and specificity were quantified by flow cytometry. The complete ADEPT system was applied in vitro on gpA33-positive LIM1215 cells, assessing cell survival by a fluorescein diacetate assay. Cytotoxicity of the prodrug 5-FC after A33scFv::CDy binding was equimolar to that of 5-FU, and this effect depended specifically on both antibody and enzyme function. These results demonstrate bifunctional activity of the heterogeneous Pichia-produced A33scFv::CDy fusion protein and proof of principle for the ADEPT system proposed herein.

Association of the A870G cyclin D1 gene polymorphism with genetic susceptibility to nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal cancer (NPC) is multifactorial, and the genetic background may be a crucial etiologic factor. Cyclin D1 (CCND1) is a key regulator of the cell cycle, and its altered activity is associated with the development of cancer. METHODS: We analyzed the A870G CCND1 polymorphism by polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) in 281 individuals, including 94 patients with NPC and 187 healthy individuals. RESULTS: Our results indicate that individuals carrying two G alleles have a 2.17-fold increase in the risk for the development of NPC (odds ratio [OR], 2.17; 95% confidence interval [CI], 1.19-3.98; p = .016). Age-adjusted logistic regression analysis confirmed this association (adjusted odds ratio [aOR], 2.14; 95% CI, 1.14-4.04; p = .018). Multivariate analysis demonstrates an independent association between GG CCND1 genotype (aOR, 2.06), male sex (aOR, 2.66), and age at diagnosis (aOR, 2.02) regarding the development of undifferentiated NPC. The proportion of NPC cases attributable to the GG CCND1 genotype was 14.76%. CONCLUSIONS: Our results may be important in the definition of a biologic predictive profile for the development of NPC within our population.

Acolhimento em saúde mental na unidade básica: uma revisão teórica

A Organização Mundial de Saúde (2001) acredita que os transtornos mentais serão a segunda causa de adoecimento da população em 2020. Em virtude disso, observa-se a importância da melhoria dos serviços de saúde dedicados aos pacientes com transtornos mentais nas diversos âmbitos da saúde. O objetivo desse trabalho consiste em realizar uma revisão teórica sobre saúde mental na atenção básica, identificando a importância do acolhimento como porta de entrada das demandas em saúde mental na atenção básica e contextualizando o critério de responsabilização da equipe de saúde da família nas ações em saúde mental. A metodologia utilizada para esse trabalho foi a seleção de artigos de interesse a partir de levantamentos bibliográfico em acervos do sistema informatizado: Bireme, Scielo, Lilacs, nos sites com dados estatísticos oficiais do governo - Datasus e IBGE e na Biblioteca Joaquim Baeta Vianna (UFMG) em Belo Horizonte. A partir da elaboração dessa revisão foi observado que o acolhimento em saúde pressupõe dar atenção ao individuo de maneira que o mesmo se sinta confortável e apto a receber um atendimento ímpar, por meio de parâmetros humanitários, éticos, técnicos e solidários. Sendo assim, o profissional capacitado em prestar um bom acolhimento tem condições de ouvir o paciente de uma forma receptiva, atenciosa e solidária, sendo capaz de promover maior efetividade e eficiência no decorrer do seu trabalho. Concluímos que a inclusão das ações de saúde mental na atenção básica são características essenciais na unidade básica de saúde no país, sendo imprescindível que a equipe de saúde tenha conhecimento e motivação para atuar frente aos pacientes com transtornos mentais. Para que isso ocorra as equipes de saúde da família devem estar em constante treinamento e interlocução com as equipes de saúde mental.